Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Jiro Nakayama Last modified date:2023.11.27

Professor / Division of Systems Bioengineering / Department of Bioscience and Biotechnology / Faculty of Agriculture


Papers
1. Kota Ikari, Junichiro Tezuka, Masafumi Sanefuji, Jiro Nakayama, Daisuke Nishima, Yuri Sonoda, Masanobu Ogawa, Masayuki Shimono, Reiko Suga, Satoshi Honjo, Koichi Kusuhara, Shouichi Ohga, The association between early formula and reduced risk of cow's milk allergy during the first three year of life: a Japanese cohort study., Allergy, asthma, and clinical immunology : official journal of the Canadian Society of Allergy and Clinical Immunology, 10.1186/s13223-022-00712-z, 18, 1, 71-71, 2022.08, BACKGROUND: Our recent observational study showed that regular consumption of cow's milk (CM) formula during early infancy (3-6 months old) was associated with a reduced risk of CM allergy (CMA) at 12 months old. However, the long-term association is unclear. The present study was aimed to examine how long this inverse association persists after 12 months old. METHODS: This study used the dataset of an ongoing nationwide prospective cohort, the Japan Environment and Children's Study, in which participants were registered between January 2011 and March 2014. We analyzed 65,568 children followed-up until 36 months old. The exposure factors were the consumption statuses of formula milk from 0-3, 3-6, and 6-12 months old. The primary outcome was the prevalence of CMA at 6, 12, 18, 24 and 36 months old. CMA was defined as an allergic reaction and sensitization to CM protein in an individual with no or limited intake of this protein at the evaluation time, combined with physician-diagnosed food allergy. Multivariable regression models were used to estimate the association between the periods of formula consumption and the prevalence of CMA. RESULTS: The prevalence of CMA increased with a peak of 1.51% at 18 months old and then declined to 0.79% at 36 months old. Formula milk from 3-6 months old was associated with a reduced risk of CMA throughout the first 3 years of life, although the extent of the reduction was mitigated with age (adjusted relative risk: [95% confidence interval]: 0.19 [0.10-0.34] at 12 months old, 0.23 [0.16-0.33] at 18 months old, 0.41 [0.26-0.64] at 24 months old, and 0.47 [0.26-0.80] at 36 months old). The association between early formula and CMA were observed in both children with and without eczema, but more prominent and long-lasting in the former than the latter. CONCLUSIONS: Regular exposure to CM protein during infancy was associated with a reduced prevalence of CMA during early childhood. At present, however, this observational study does not necessarily encourage formula feeding, and randomized controlled trials are warranted to confirm the findings and their significance..
2. Jun-Ichi Nagao, Sari Kishikawa, Honami Tanaka, Kenji Toyonaga, Yuka Narita, Kanae Negoro-Yasumatsu, Sonoko Tasaki, Ken-Ichi Arita-Morioka, Jiro Nakayama, Yoshihiko Tanaka, Pathobiont-responsive Th17 cells in gut-mouth axis provoke inflammatory oral disease and are modulated by intestinal microbiome., Cell reports, 10.1016/j.celrep.2022.111314, 40, 10, 111314-111314, 2022.09, Host immune response via Th17 cells against oral pathobionts is a key mediator in periodontitis development. However, where and how the Th17-type immune response is induced during the development of periodontitis is not well understood. Here, we demonstrate that gut translocation of the oral pathobiont Porphyromonas gingivalis (Pg) exacerbates oral pathobiont-induced periodontitis with enhanced Th17 cell differentiation. The oral pathobiont-responsive Th17 cells are differentiated in Peyer's patches and translocated systemically in the peripheral immune tissues. They are also capable of migrating to and accumulating in the mouth upon oral infection. Development of periodontitis via the oral pathobiont-responsive Th17 cells is regulated by the intestinal microbiome, and altering the intestinal microbiome composition with antibiotics affects the development of periodontitis. Our study highlights that pathobiont-responsive Th17 cells in the gut-mouth axis and the intestinal microbiome work together to provoke inflammatory oral diseases, including periodontitis..
3. Riko Mishima, Masaru Tanaka, Rie Momoda, Masafumi Sanefuji, Seiichi Morokuma, Masanobu Ogawa, Kiyoko Kato, Jiro Nakayama, Longitudinal gut mycobiota changes in Japanese infants during first three years of life., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2023.01.007, 135, 4, 266-273, 2023.04, Although fungi can have a large impact on host health through the stimulation of the immune system and toxin production, few studies have investigated the gut mycobiota during infancy, a period during which sensitivity to internal and external stimuli is high. To capture the trend in fungal colonization during infancy, we evaluated the gut mycobiota of ten Japanese infants during the first 3 years of life. Infants had two major phyla, Ascomycota (68.9%) and Basidiomycota (29.6%), and the most abundant genus was Saccharomyces (26.8%), followed by Malassezia (18.5%), Candida (12.3%), Meyerozyma (8.5%), and Penicillium (8.3%). Alpha diversity analysis revealed a significant decrease in fungal richness and evenness with age, suggesting adaptive selection of the colonizing species in the gut environment. Beta diversity analysis divided infant mycobiota into age-related clusters and showed discrete separation before and after weaning, suggesting shift in microenvironment via weaning. In the initial stage, a variety of fungal species that likely originated from an environment, such as Malassezia spp., was highly colonized and were replaced by yeasts, such as Saccharomyces, after weaning. Further studies are needed to shed light on how the passage of the series of fungal colonizations in infancy affects the development of the host immune system and the other homeostasis involved in health later in life..
4. Huanghuang Dai, Akira Otsuka, Kurumi Tanabe, Teruyoshi Yanagita, Jiro Nakayama, Hiroshi Kitagaki, Glucosylceramide Changes Bacterial Metabolism and Increases Gram-Positive Bacteria through Tolerance to Secondary Bile Acids In Vitro., International journal of molecular sciences, 10.3390/ijms23105300, 23, 10, 2022.05, Glucosylceramide is present in many foods, such as crops and fermented foods. Most glucosylceramides are not degraded or absorbed in the small intestine and pass through the large intestine. Glucosylceramide exerts versatile effects on colon tumorigenesis, skin moisture, cholesterol metabolism and improvement of intestinal microbes in vivo. However, the mechanism of action has not yet been fully elucidated. To gain insight into the effect of glucosylceramide on intestinal microbes, glucosylceramide was anaerobically incubated with the dominant intestinal microbe, Blautia coccoides, and model intestinal microbes. The metabolites of the cultured broth supplemented with glucosylceramide were significantly different from those of broth not treated with glucosylceramide. The number of Gram-positive bacteria was significantly increased upon the addition of glucosylceramide compared to that in the control. Glucosylceramide endows intestinal microbes with tolerance to secondary bile acid. These results first demonstrated that glucosylceramide plays a role in the modification of intestinal microbes..
5. Dieter M Tourlousse, Koji Narita, Takamasa Miura, Akiko Ohashi, Masami Matsuda, Yoshifumi Ohyama, Mamiko Shimamura, Masataka Furukawa, Ken Kasahara, Keishi Kameyama, Sakae Saito, Maki Goto, Ritsuko Shimizu, Riko Mishima, Jiro Nakayama, Koji Hosomi, Jun Kunisawa, Jun Terauchi, Yuji Sekiguchi, Hiroko Kawasaki, Characterization and Demonstration of Mock Communities as Control Reagents for Accurate Human Microbiome Community Measurements., Microbiology spectrum, 10.1128/spectrum.01915-21, 10, 2, e0191521, 2022.04, Standardization and quality assurance of microbiome community analysis by high-throughput DNA sequencing require widely accessible and well-characterized reference materials. Here, we report on newly developed DNA and whole-cell mock communities to serve as control reagents for human gut microbiota measurements by shotgun metagenomics and 16S rRNA gene amplicon sequencing. The mock communities were formulated as near-even blends of up to 20 bacterial species prevalent in the human gut, span a wide range of genomic guanine-cytosine (GC) contents, and include multiple strains with Gram-positive type cell walls. Through a collaborative study, we carefully characterized the mock communities by shotgun metagenomics, using previously developed standardized protocols for DNA extraction and sequencing library construction. Further, we validated fitness of the mock communities for revealing technically meaningful differences among protocols for DNA extraction and metagenome/16S rRNA gene amplicon library construction. Finally, we used the mock communities to reveal varying performance of metagenome-based taxonomic profilers and the impact of trimming and filtering of sequencing reads on observed species profiles. The latter showed that aggressive preprocessing of reads may result in substantial GC-dependent bias and should thus be carefully evaluated to minimize unintended effects on species abundances. Taken together, the mock communities are expected to support a myriad of applications that rely on well-characterized control reagents, ranging from evaluation and optimization of methods to assessment of reproducibility in interlaboratory studies and routine quality control. IMPORTANCE Application of high-throughput DNA sequencing has greatly accelerated human microbiome research and its translation into new therapeutic and diagnostic capabilities. Microbiome community analyses results can, however, vary considerably across studies or laboratories, and establishment of measurement standards to improve accuracy and reproducibility has become a priority. The here-developed mock communities, which are available from the NITE Biological Resource Center (NBRC) at the National Institute of Technology and Evaluation (NITE, Japan), provide well-characterized control reagents that allow users to judge the accuracy of their measurement results. Widespread and consistent adoption of the mock communities will improve reproducibility and comparability of microbiome community analyses, thereby supporting and accelerating human microbiome research and development..
6. Yuichiro Nishihara, Haruei Ogino, Masaru Tanaka, Eikichi Ihara, Keita Fukaura, Kei Nishioka, Takatoshi Chinen, Yoshimasa Tanaka, Jiro Nakayama, Dongchon Kang, Yoshihiro Ogawa, Mucosa-associated gut microbiota reflects clinical course of ulcerative colitis., Scientific reports, 10.1038/s41598-021-92870-0, 11, 1, 13743-13743, 2021.07, This longitudinal study was designed to elucidate whether gut microbiota is associated with relapse and treatment response in ulcerative colitis (UC) patients. Fifty-one patients with UC were enrolled between 2012 and 2017, and followed up through 2020. Colon mucosal biopsy were obtained at enrollment, and 16S ribosomal RNA sequencing was performed using extracted RNA. Of the 51 patients, 24 were in remission and 27 had active UC at enrollment. Of the 24 patients in remission, 17 maintained remission and 7 developed relapse during follow-up. The 7 patients with relapse showed lower diversity, with a lower proportion of Clostridiales (p = 0.0043), and a higher proportion of Bacteroides (p = 0.047) at enrollment than those without relapse. The 27 patients with active UC were classified into response (n = 6), refractory (n = 13), and non-response (n = 8) groups according to their treatment response in 6 months. The refractory and non-response groups showed lower diversity with a lower proportion of Prevotella (p = 0.048 and 0.043) at enrollment than the response group. This study is the first demonstration that reduced diversity and particular microbes are associated with the later clinical course of relapse events and treatment response in UC..
7. Mugihito Oshiro, Masaru Tanaka, Rie Momoda, Takeshi Zendo, Jiro Nakayama, Mechanistic Insight into Yeast Bloom in a Lactic Acid Bacteria Relaying-Community in the Start of Sourdough Microbiota Evolution., Microbiology spectrum, 10.1128/Spectrum.00662-21, 9, 2, e0066221, 2021.10, The spontaneous microbiota of wheat sourdough, often comprising one yeast species and several lactic acid bacteria (LAB) species, evolves over repeated fermentation cycles, which bakers call backslopping. The final product quality largely depends on the microbiota functions, but these fluctuate sometimes during the initial months of fermentation cycles due to microbiota evolution in which three phases of LAB relay occur. In this study, the understanding of yeast-LAB interactions in the start of the evolution of the microbiota was deepened by exploring the timing and trigger interactions when sourdough yeast entered a preestablished LAB-relaying community. Monitoring of 32 cycles of evolution of 6 batches of spontaneous microbiota in wheat sourdoughs revealed that sourdough yeasts affected the LAB community when the 2nd- or 3rd-relaying types of LAB genera emerged. In in vitro pairwise cocultures, all 12 LAB strains containing the 3 LAB-relaying types arrested the growth of a Saccharomyces cerevisiae strain, a frequently found species in sourdoughs, to various extents by sugar-related interactions. These findings suggest competition due to different affinities of each LAB and a S. cerevisiae strain for each sugar. In particular, maltose was the driver of S. cerevisiae growth in all pairwise cocultures. The functional prediction of sugar metabolism in sourdough LAB communities showed a positive correlation between maltose degradation and the yeast population. Our results suggest that maltose-related interactions are key factors that enable yeasts to enter and then settle in the LAB-relaying community during the initial part of evolution of spontaneous sourdough microbiota. IMPORTANCE Unpredictable evolution of spontaneous sourdough microbiota sometimes prevents bakers from making special-quality products because the unstable microbiota causes the product quality to fluctuate. Elucidation of the evolutionary mechanisms of the sourdough community, comprising yeast and lactic acid bacteria (LAB), is fundamental to control fermentation performance. This study investigated the mechanisms by which sourdough yeasts entered and settled in a bacterial community in which a three-phase relay of LAB occurred. Our results showed that all three layers of LAB restricted the cohabiting yeast population by competing for the sugar sources, particularly maltose. During the initial evolution of spontaneous sourdough microbiota, yeasts tended to grow synchronously with the progression of the lactic acid bacterial relay, which was predictably associated with changes in the maltose degradation functions in the bacterial community. Further study of ≥3 species' interactions while considering yeast diversity can uncover additional interaction mechanisms driving the initial evolution of sourdough microbiota..
8. Mai Watanabe, Abraham Sianoya, Riko Mishima, Phatthanaphong Therdtatha, Abigail Rodriguez, Donna Christene Ramos, Yuan Kun Lee, Leslie Michelle Dalmacio, Jiro Nakayama, Gut microbiome status of urban and rural Filipino adults in relation to diet and metabolic disorders., FEMS microbiology letters, 10.1093/femsle/fnab149, 368, 20, 2021.12, Here, we aim to understand the condition of the gut microbiome of Filipino adults in relation to their diet and metabolic status. Compared to rural Albay (n = 67), the gut microbiome of subjects living in urban Manila (n = 25) was more colonized by the order Clostridiales, which was negatively correlated with host carbohydrate consumption. Principal component analysis using the genus composition of the 92 total subjects indicated four microbiome types: one type driven by Prevotella, which was associated with high rice consumption and mainly consisted of healthy Albay subjects, one Clostridiales-driven group containing a number of type 2 diabetes mellitus (T2D) subjects from both Manila and Albay who showed lower butyrate levels in association with a decrease in Mediterraneibacter faecis, and the other two types showing dysbiosis-like microbiomes with Lactobacillus and Bifidobacterium overgrowth, with a high ratio of T2D and obese subjects. Multivariate logistic regression analysis suggested high dietary energy intake, and two Veillonellaeae genera, Dialister and Megasphaera, as T2D risk factors, while Prevotella and M. faecis as anti-T2D factors. In conclusion, low-carbohydrate diets restructured the Prevotella-driven gut microbiome, which may predispose Filipino people with high energy diet to T2D..
9. Sakura Onizuka, Masaru Tanaka, Riko Mishima, Jiro Nakayama, Cultivation of Spore-Forming Gut Microbes Using a Combination of Bile Acids and Amino Acids., Microorganisms, 10.3390/microorganisms9081651, 9, 8, 2021.08, Spores of certain species belonging to Firmicutes are efficiently germinated by nutrient germinators, such as amino acids, in addition to bile acid. We attempted to culture difficult-to-culture or yet-to-be cultured spore-forming intestinal bacteria, using a combination of bile acids and amino acids. The combination increased the number of colonies that formed on agar medium plated with ethanol-treated feces. The operational taxonomic units of these colonized bacteria were classified into two types. One type was colonized only by the bile acid (BA) mixture and the other type was colonized using amino acids, in addition to the BA mixture. The latter contained 13 species, in addition to 14 species of the former type, which mostly corresponds to anaerobic difficult-to-culture Clostridiales species, including several new species candidates. The use of a combination of BAs and amino acids effectively increased the culturability of spore-forming intestinal bacteria..
10. Mohamed Abdelfattah Maky, Naoki Ishibashi, Jiro Nakayama, Takeshi Zendo, Characterization of the Biosynthetic Gene Cluster of Enterocin F4-9, a Glycosylated Bacteriocin., Microorganisms, 10.3390/microorganisms9112276, 9, 11, 2021.11, Enterocin F4-9 belongs to the glycocin family having post-translational modifications by two molecules of N-acetylglucosamine β-O-linked to Ser37 and Thr46. In this study, the biosynthetic gene cluster of enterocin F4-9 was cloned and expressed in Enterococcus faecalis JH2-2. Production of glycocin by the JH2-2 expression strain was confirmed by expression of the five genes. The molecular weight was greater than glycocin secreted by the wild strain, E. faecalis F4-9, because eight amino acids from the N-terminal leader sequence remained attached. This N-terminal extension was eliminated after treatment with the culture supernatant of strain F4-9, implying an extracellular protease from E. faecalis F4-9 cleaves the N-terminal sequence. Thus, leader sequences cleavage requires two steps: the first via the EnfT protease domain and the second via extracellular proteases. Interestingly, the long peptide, with N-terminal extension, demonstrated advanced antimicrobial activity against Gram-positive and Gram-negative bacteria. Furthermore, enfC was responsible for glycosylation, a necessary step prior to secretion and cleavage of the leader peptide. In addition, enfI was found to grant self-immunity to producer cells against enterocin F4-9. This report demonstrates specifications of the minimal gene set responsible for production of enterocin F4-9, as well as a new biosynthetic mechanism of glycocins..
11. Said E Desouky, Mohammed Abu-Elghait, Eman A Fayed, Samy Selim, Basit Yousuf, Yasuhiro Igarashi, Basel A Abdel-Wahab, Amnah Mohammed Alsuhaibani, Kenji Sonomoto, Jiro Nakayama, Secondary Metabolites of Actinomycetales as Potent Quorum Sensing Inhibitors Targeting Gram-Positive Pathogens: In Vitro and In Silico Study., Metabolites, 10.3390/metabo12030246, 12, 3, 2022.03, Anti-virulence agents are non-bacteriostatic and non-bactericidal emerging therapeutic options which hamper the production of virulence factors in pathogenic flora. In Staphylococcus aureus and Enterococcus faecalis, regulation of virulence genes' expression occurs through the cyclic peptide-mediated accessory gene regulator (agr) and its ortholog fsr quorum sensing systems, respectively. In the present study, we screened a set of 54 actinomycetales secondary metabolites as novel anti-virulence compounds targeting quorum sensing system of the Gram-positive bacteria. The results indicated that four compounds, Phenalinolactones A-D, BU-4664LMe, 4,5-dehydrogeldamycin, and Questinomycin A, potentially inhibit the agr quorum sensing system and hemolytic activity of S. aureus. On the other hand, Decatromicin A and B, Okilactomycin, Rishirilide A, Abyssomicin I, and Rebeccamycin selectively blocked the fsr quorum sensing system and the gelatinase production in E. faecalis at sub-lethal concentrations. Interestingly, Synerazol uniquely showed the capability to inhibit both fsr and agr quorum sensing systems. Further, in silico molecular docking studies were performed which provided closer insights into the mode of action of these compounds and proposed that the inhibitory activity of these compounds could be attributed to their potential ability to bind to the ATP-active site of S. aureus AgrA. Taken together, our study highlights the potential of actinomycetales secondary metabolites with diverse structures as anti-virulence quorum sensing inhibitors..
12. Takako Inoue, Yui Funatsu, Masaya Ohnishi, Masanori Isogawa, Keigo Kawashima, Masaru Tanaka, Kei Moriya, Hideto Kawaratani, Rie Momoda, Etsuko Iio, Hidewaki Nakagawa, Yutaka Suzuki, Kentaro Matsuura, Kei Fujiwara, Atsushi Nakajima, Hitoshi Yoshiji, Jiro Nakayama, Yasuhito Tanaka, Bile acid dysmetabolism in the gut-microbiota-liver axis under hepatitis C virus infection., Liver international : official journal of the International Association for the Study of the Liver, 10.1111/liv.15041, 42, 1, 124-134, 2022.01, BACKGROUND & AIMS: We recently analysed and reported the features of the micro biome under hepatitis C virus (HCV) infection, but the effect of HCV infection on bile acid (BA) metabolism in the gut-liver axis remains poorly understood. The aim of this study was to clarify the characteristics of the gut-liver axis in HCV-infected patients. METHODS: The faecal BAs composition and gut microbiota from 100 chronic hepatitis C (CHC) patients were compared with those from 23 healthy individuals. For transcriptional analysis of the liver, 22 mild CHC (fibrosis stages [F] 0-2) and 42 advanced CHC (F3-4) cases were compared with 12 healthy individuals. The findings were confirmed using chimeric mice with human hepatocytes infected with HCV HCR6. RESULTS: Chronic hepatitis C patients, even at earlier disease stages, showed BA profiles distinct from healthy individuals, in which faecal deoxycholic acid (DCA) was significantly reduced and lithocholic acid or ursodeoxycholic acid became dominant. The decrease in faecal DCA was correlated with reduction in commensal Clostridiales and increase in oral Lactobacillales. Impaired biosynthesis of cholic acid (CA) was observed as a reduction in the transcription level of cytochrome P450 8B1 (CYP8B1), a key enzyme in CA biosynthesis. The reductions in faecal DCA and liver CYP8B1 were also observed in HCV-infected chimeric mice. CONCLUSIONS: Chronic hepatitis C alters the intestinal BA profile, in association with the imbalance of BA biosynthesis, which differs from the pattern in NAFLD. These imbalances appear to drive disease progression through the gut-microbiome-liver axis..
13. Phatthanaphong Therdtatha, Yayi Song, Masaru Tanaka, Mariyatun Mariyatun, Maisaroh Almunifah, Nancy Eka Putri Manurung, Siska Indriarsih, Yi Lu, Koji Nagata, Katsuya Fukami, Tetsuo Ikeda, Yuan-Kun Lee, Endang Sutriswati Rahayu, Jiro Nakayama, Gut Microbiome of Indonesian Adults Associated with Obesity and Type 2 Diabetes: A Cross-Sectional Study in an Asian City, Yogyakarta., Microorganisms, 10.3390/microorganisms9050897, 9, 5, 2021.04, Indonesia is a developing country facing the national problem of the growing obesity and diabetes in its population due to recent drastic dietary and lifestyle changes. To understand the link between the gut microbiome, diet, and health of Indonesian people, fecal microbiomes and metabolomes of 75 Indonesian adults in Yogyakarta City, including obese people (n = 21), type 2 diabetes (T2D) patients (n = 25), and the controls (n = 29) were characterized together with their dietary and medical records. Variations of microbiomes showed a triangular distribution in the principal component analysis, driven by three dominant bacterial genera, namely Bacteroides, Prevotella, and Romboutsia. The Romboutsia-driven microbiome, characterized by low bacterial diversity and high primary bile acids, was associated with fat-driven obesity. The Bacteroides-driven microbiome, which counteracted Prevotella but was associated with Ruminococcaceae concomitantly increased with high-carbohydrate diets, showed positive correlation with T2D indices but negative correlation with body mass index. Notably, Bacteroides fragilis was increased in T2D patients with a decrease in fecal conjugated bile acids, particularly tauroursodeoxycholic acid (TUDCA), a farnesoid X receptor (FXR) antagonist with anti-diabetic activity, while these features disappeared in patients administered metformin. These results indicate that the gut microbiome status of Indonesian adults is differently associated with obesity and T2D under their varied dietary habits..
14. Mugihito Oshiro, Takeshi Zendo, Jiro Nakayama, Diversity and dynamics of sourdough lactic acid bacteriota created by a slow food fermentation system., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2020.11.007, 131, 4, 333-340, 2021.04, Sourdough is a naturally fermented dough that is used worldwide to produce a variety of baked foods. Various lactic acid bacteria (LAB), which can determine the quality of sourdough baked foods by producing metabolites, have been found in the sourdough ecosystem. However, spontaneous fermentation of sourdough leads to unpredictable growth of various micro-organisms, which result in unstable product quality. From an ecological perspective, many researchers have recently studied sourdough LAB diversity, particularly the elucidation of LAB community interactions and the dynamic mechanisms during the fermentation process, in response to requests for the control and design of a desired sourdough microbial community. This article reviews recent advances in the study of sourdough LAB diversity and its dynamics in association with unique characteristics of the fermentation system; it also discusses future perspectives for better understanding of the complex sourdough microbial ecosystem, which can be attained efficiently by both in vitro and in situ experimental approaches..
15. Tawatchai Chumponsuk, Lucsame Gruneck, Eleni Gentekaki, Paiboon Jitprasertwong, Niwed Kullawong, Jiro Nakayama, Siam Popluechai, The salivary microbiota of Thai adults with metabolic disorders and association with diet., Archives of oral biology, 10.1016/j.archoralbio.2020.105036, 122, 105036-105036, 2021.02, OBJECTIVE: This study aimed to investigate abundance of specific bacterial taxa in the saliva of 105 Thai adults with different BMI (lean, overweight, and obese) and T2DM subjects using qPCR targeting the 16S rRNA gene of various bacteria taxa. DESIGN: We employed qPCR targeting 16S rRNA genes to explore the bacterial profiles and abundances in the saliva of Thai adult subjects with different BMI and T2DM. Multivariate statistical analyses (multiple factor analysis (MFA) and sparse Partial Least Squares Discriminant Analysis (sPLS-DA) were performed to assess the associations of salivary bacteria with diet, blood profile, gender, age, and use of antibiotics. RESULTS: We found that abundance profiles of the examined salivary bacteria were similar across the four groups. When diet, blood profile, and gender, age, and use of antibiotics were considered, significant differences were noted between subgroups. A positive correlation was also found between consumption of carbonate soft drinks and Bacteroidetes, Gamma-proteobacteria, Veillonella, Fusobacterium and Fusobacterium nucleatum. CONCLUSIONS: This is the first study demonstrating the relative abundance of salivary bacteria in adult Thai subjects with different levels of BMI and T2DM. Regardless of the similar pattern of bacterial profiles across groups, sPLS-DA analysis highlighted the influence of host variables (gender, age, and use of antibiotics) on the abundance of salivary microbiota. Our findings pave the way for further hypothesis testing to gain insight into the association between host factors and salivary microbiome..
16. Naoki Ishibashi, Naho Matsumoto, Rodney Honrada Perez, Shun Iwatani, Haruki Sugino, Takeshi Zendo, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Molecular characterization of the possible regulation of multiple bacteriocin production through a three-component regulatory system in Enterococcus faecium NKR-5-3, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2020.09.019, 131, 2, 131-138, 2021.02, Enterococcus faecium NKR-5-3 produces multiple-bacteriocins, enterocins NKR-5-3A, B, C, D, and Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). However, the biosynthetic mechanisms on how their productions are regulated are yet to be fully understood. In silico analysis revealed putative promoters and terminators in the enterocin NKR-5-3ACDZ gene cluster, and the putative direct repeats (5′-ATTTTAGGATA-3′) were conserved upstream of each promoter. Transcriptional analysis by quantitative real-time polymerase chain reaction (PCR) of the biosynthetic genes for the enterocins NKR-5-3 suggested that an inducing peptide (Ent53D) regulates the transcription of the structure genes and corresponding biosynthetic genes of enterocins NKR-5-3, except for Ent53B (a circular bacteriocin), thus consequently regulating their production. Moreover, transcriptional analysis of some knock-out mutants showed that the production of Ent53A, C, D and Z is controlled by a three-component regulatory system (TCS) consisting of Ent53D, EnkR (response regulator), and EnkK (histidine kinase). The production of the circular bacteriocin Ent53B appeared to be independent from this TCS. Nevertheless, disrupting the TCS by deletion of a single component (enkD, enkR and enkK) resulted in a slight increase of enkB transcription and consequently the production of Ent53B, presumably, as an indirect consequence of the increase of available energy to the strain NKR-5-3. Here, we demonstrate the regulatory control of the multiple bacteriocin production of strain NKR-5-3 likely through the TCS consisting of Ent53D, EnkR, and EnkK. The information of the sharing of the regulatory machinery between bacteriocins in strain NKR-5-3 can be useful in its future application such as designing strategies to effectively dispense its multiple bacteriocin arsenal..
17. Rafli Zulfa Kamil, Agnes Murdiati, Mohammad Juffrie, Jiro Nakayama, Endang Sutriswati Rahayu, Gut Microbiota and Short-Chain Fatty Acid Profile between Normal and Moderate Malnutrition Children in Yogyakarta, Indonesia., Microorganisms, 10.3390/microorganisms9010127, 9, 1, 1-15, 2021.01, Malnutrition has been associated with the gut microbiota composition and the gastrointestinal environment. This study aimed to evaluate whether there is a difference in the gut microbiota profile between the normal and undernutrition (considered moderate malnutrition) children and evaluate the gastrointestinal environment observed from the short-chain fatty acid (SCFA) profile. Ten days' observations were done between normal (n:13) and undernutrition (n:15) children. The subject's diet was recorded using a food record. Analysis of the gut microbiota was performed using 16S rRNA gene sequencing targeting the V3-V4 variables region, while the SCFA profile was analyzed using gas chromatography. The result shows that the undernutrition group's energy intake was lower than in the normal group. Although there was no difference in diversity index and overall gut composition, overexpression of the genera Methanobrevibacter, Anaerococcus, Eubacterium, and Succinivibrio was observed in the undernutrition group. Meanwhile, in the normal group, Ruminococcus and Fusobacterium were found. In both groups, there was also the dominant of Prevotella enterotype. Gastrointestinal conditions in the normal group tended to be more acidic compared to the undernutrition group. It occurs due to the high concentration of propionate and butyric acids..
18. Hirotoshi Sushida, Miyuki Sakei, Rodney H Perez, Naoki Ishibashi, Takeshi Zendo, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Processing and secretion of non-cognate bacteriocins by EnkT, an ABC transporter from a multiple-bacteriocin producer, Enterococcus faecium NKR-5-3., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2020.07.017, 130, 6, 596-603, 2020.12, EnkT is an ATP-binding cassette (ABC) transporter produced by Enterococcus faecium NKR-5-3, which is responsible for the secretion of multiple bacteriocins; enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z). EnkT has been shown to possess a tolerant recognition mechanism that enables it to secrete the mature Ent53C from a chimeric precursor peptide containing the leader peptide moieties that are derived from different heterologous bacteriocins. In this study, to further characterize EnkT, we aimed to investigate the capacity of EnkT to recognize, process, and secrete non-cognate bacteriocins, which belong to different subclasses of class II. For this, the non-cognate bacteriocin precursor peptides, including enterocin A, pediocin PA-1, lactococcin Q, lactococcin A, and lacticin Q were co-expressed with EnkT, and thereafter, the production of the mature forms of these non-cognate bacteriocins was assessed. Our results revealed that EnkT could potentially recognize, process, and secrete the non-cognate bacteriocins with an exception of the leaderless bacteriocin, lacticin Q. Moreover, the processing and secretion efficiencies of these heterologous non-cognate bacteriocins by EnkT were further enhanced when the leader peptide moiety was replaced with the Ent53C leader peptide (derived from a native NKR-5-3 bacteriocin). The findings of this study describe the wide substrate tolerance of this ABC transporter, EnkT, that can be exploited in the future in establishing effective bacteriocin production systems adaptive to complex fermentation conditions common in many food systems..
19. Wei Wei Thwe Khine, Endang Sutriswati Rahayu, Ting Yi See, Sherwin Kuah, Seppo Salminen, Jiro Nakayama, Yuan-Kun Lee, Indonesian children fecal microbiome from birth until weaning was different from microbiomes of their mothers., Gut microbes, 10.1080/19490976.2020.1761240, 12, 1, 1761240-1761240, 2020.11, Gastrointestinal (GI) microbiota play an important role in human health and wellbeing and the first wave of gut microbes arrives mostly through vertical transmission from mother to child. This study has undertaken to understand the microbiota profile of healthy Southeast Asian mother-infant pairs. Here, we examined the fecal, vaginal and breast milk microbiota of Indonesian mothers and the fecal microbiota of their children from less than 1 month to 48 months old. To determine the immune status of children and the effect of diet at different ages, we examined the level of cytokines, bile acids in the fecal water and weaning food frequency. The fecal microbiota of the children before weaning contained mainly Bacteroides and Bifidobacterium, which presented at low abundance in the samples of mothers. After weaning, the fecal microbiome of children was mainly of the Prevotella type, with decreasing levels of Bifidobacterium, thus becoming more like the fecal microbiome of the mother. The abundance of infant fecal commensals generally correlated inversely with potential pathogens before weaning. The fecal Bifidobacterium in children correlated inversely with the consumption of complex carbohydrates and fruits after weaning. The specific cytokines related to the proliferation and maturation of immunity were found to increase after weaning. A decreasing level of primary bile acids and an increase of secondary bile acids were observed after weaning. This study highlights the change in the GI microbiota of infants to adult-type microbiota after weaning and identifies diet as a major contributing factor..
20. T. Ikeda, M. Aida, Y. Yoshida, S. Matsumoto, M. Tanaka, J. Nakayama, Y. Nagao, R. Nakata, E. Oki, T. Akahoshi, S. Okano, M. Nomura, M. Hashizume, Y. Maehara, Alteration in faecal bile acids, gut microbial composition and diversity after laparoscopic sleeve gastrectomy, British Journal of Surgery, 10.1002/bjs.11654, 107, 12, 1673-1685, 2020.11, Background: Laparoscopic sleeve gastrectomy (LSG) is a well established treatment for severe obesity and type 2 diabetes. Although the gut microbiota is linked to the efficacy of LSG, the underlying mechanisms remain elusive. The effect of LSG for morbid obesity on the gut microbiota and bile acids was assessed here. Methods: Severely obese subjects who were candidates for LSG were included and followed until 6 months after surgery. The composition and abundance of the microbiota and bile acids in faeces were assessed by 16S ribosomal RNA sequencing, quantitative PCR and liquid chromatography–mass spectrometry. Results: In total, 28 patients with a mean(s.d.) BMI of 44·2(6·6) kg/m were enrolled. These patients had achieved excess weight loss of 53·2(19·0) per cent and showed improvement in metabolic diseases by 6 months after LSG, accompanied by an alteration in the faecal microbial community. The increase in α-diversity and abundance of specific taxa, such as Rikenellaceae and Christensenellaceae, was strongly associated with reduced faecal bile acid levels. These changes had a significant positive association with excess weight loss and metabolic alterations. However, the total number of faecal bacteria was lower in patients before (mean(s.d.) 10·26(0·36) log cells per g faeces) and after (10·39(0·29) log cells per g faeces) operation than in healthy subjects (10·83(0·27) log cells per g faeces). Conclusion: LSG is associated with a reduction in faecal bile acids and greater abundance of specific bacterial taxa and α-diversity that may contribute to the metabolic changes. 2 10 10 10.
21. Masaru Tanaka, Sakura Onizuka, Riko Mishima, Jiro Nakayama, Cultural isolation of spore-forming bacteria in human feces using bile acids., Scientific reports, 10.1038/s41598-020-71883-1, 10, 1, 15041-15041, 2020.09, Structurally-diversified bile acids (BAs) are involved in shaping of intestinal microbiota as well as absorption of dietary lipids. Taurocholic acid, a conjugated form of BA, has been reported to be a factor triggering germination of a wide range of spore-forming bacteria in intestine. To test a hypothesis that other BAs also promote germination of intestinal bacteria, we attempted culture of bacteria from ethanol-treated feces by using a series of BAs. It was found that conjugated-BAs, notably three glycine-conjugated BAs, glycodeoxycholic acid and glycochenodeoxycholic acid, significantly increased the number and the species variety of colonies formed on the agar plate. These colonized bacteria mostly belonged to class Clostridia, mainly consisting of families Lachnospiraceae, Clostridiaceae, and Peptostreptococcaceae. There were several types of bacteria associated with different sensitivity to each BA. Eventually, we isolated 72 bacterial species of which 61 are known and 11 novel. These results demonstrate that the culturable range of bacteria in intestine can be widened using the germination-inducing activity of BAs. This approach would advance the research on spore-forming Clostridia that contains important but difficult-to-cultured bacteria associate with host health and diseases..
22. Mugihito OSHIRO, Masaru TANAKA, Takeshi ZENDO, Jiro NAKAYAMA, Impact of pH on succession of sourdough lactic acid bacteria communities and their fermentation properties, Bioscience of Microbiota, Food and Health, 10.12938/bmfh.2019-038, 39, 3, 152-159, 2020.07.
23. Masanori Furukawa, Kei Moriya, Jiro Nakayama, Takako Inoue, Rie Momoda, Hideto Kawaratani, Tadashi Namisaki, Shinya Sato, Akitoshi Douhara, Kosuke Kaji, Mitsuteru Kitade, Naotaka Shimozato, Yasuhiko Sawada, Soichiro Saikawa, Hiroaki Takaya, Koh Kitagawa, Takemi Akahane, Akira Mitoro, Junichi Yamao, Yasuhito Tanaka, Hitoshi Yoshiji, Gut dysbiosis associated with clinical prognosis of patients with primary biliary cholangitis., Hepatology research : the official journal of the Japan Society of Hepatology, 10.1111/hepr.13509, 50, 7, 840-852, 2020.07, AIM: Although some relationships between gut microbiota and liver diseases have been reported, it remains uncertain whether changes in gut microbiota owing to differences in race, food and living environment have similar effects. Response to ursodeoxycholic acid (UDCA) may predict the long-term prognosis of patients with primary biliary cholangitis (PBC); however, little is known about the significance of the gut microbiome in patients with PBC. We elucidated the relationships among clinical profiles, biochemical response to UDCA and gut microbiome composition in patients with PBC. METHODS: Fecal samples from 76 patients with PBC treated at our hospital were collected; patients whose UDCA intake period was
24. Orawan La-Ongkham, Massalin Nakphaichit, Jiro Nakayama, Suttipun Keawsompong, Sunee Nitisinprasert, Age-related changes in the gut microbiota and the core gut microbiome of healthy Thai humans., 3 Biotech, 10.1007/s13205-020-02265-7, 10, 6, 276-276, 2020.06, The gut microbial diversity of Thai people was investigated between two large cohorts, adult and elderly subjects, from the middle region of Thailand; the cohorts were divided into different age groups of healthy adult (73) and elderly subjects (47). The diversities of the groups were characterized using a pyrosequencing technique with primers targeting the V6-V8 region of the 16S rRNA gene, and a significant decrease in the Firmicutes and Bacteroidetes ratio from 7.3 to 4.5 was observed with increased age. The microbiota of the adult and elderly groups had a significantly higher abundance of the phylum Actinobacteria, including the three species Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium pseudocatenulatum, and the phylum Bacteroidetes containing the four species Bacteroides uniformis, Bacteroides ovatus, Bacteroides caccae and Bacteroides thetaiotaomicron. Firmicutes showed no significant differences between the two groups. Eleven species belonging to Firmicutes, Bacteroidetes and Proteobacteria were shared by at least 90% of all subjects and defined as core gut microbiota of healthy Thai, among which a high abundance of Escherichia coli was particularly characterized in Thai elderly individuals. Multiple linear regression analysis of age, gender, BMI and diet consumption frequency showed the correlation of age with Bacteroides and Bifidobacterium. Rice consumption frequency showed a significant positive correlation with Bacteroides, while no correlation was found for other factors. Taken together, in the gut of Thai adults, Bifidobacterium decreased and Bacteroides increased with age, while rice consumption increased the abundance of Bacteroides. These link of age and food, especially rice carbohydrate, to gut microbiota and health could be ultimately proposed as the Thai feature..
25. Masaru Tanaka, Masafumi Sanefuji, Seiichi Morokuma, Misako Yoden, Rie Momoda, Kenji Sonomoto, Masanobu Ogawa, Kiyoko Kato, Jiro Nakayama, The association between gut microbiota development and maturation of intestinal bile acid metabolism in the first 3 y of healthy Japanese infants, Gut Microbes, 10.1080/19490976.2019.1650997, 2, 11, 205-216, 2020.02, The gut microbial community greatly changes in early life, influencing infant health and subsequent host physiology, notably through its collective metabolism, including host–microbiota interplay of bile acid (BA) metabolism. However, little is known regarding how the development of the intestinal microbial community is associated with maturation of intestinal BA metabolism. To address this, we monitored the succession of gut bacterial community and its association with fecal BA profile in the first 3 y of ten healthy Japanese infants. The BA profiles were classified into four types, defined by high content of conjugated primary BA (Con type), unconjugated primary BA (chenodeoxycholic acid and cholic acid) (Pri type), ursodeoxycholic acid (Urs type), and deoxycholic and lithocholic acid (Sec type). Most subjects begun with Con type or Pri type profiles during lactation and eventually transited to Sec type through Urs type after the start of solid food intake. Con type and Pri type were associated with Enterobacteriaceae-dominant microbiota corresponding to the neonatal type or Bifidobacterium-dominant microbiota corresponding to lactation type, respectively. Urs type subjects were strongly associated with Ruminococcus gnavus colonization, mostly occurring between Pri type and Sec type. Sec type was associated with adult-type complex microbiota dominated by a variety of Firmicutes and Bacteroidetes species. Addressing the link of the common developmental passage of intestinal BA metabolism with infant’s health and subsequent host physiology requires further study..
26. Miki Fujiwara, Daichi Kuwahara, Masahiro Hayashi, Takeshi Zendo, Masao Sato, Jiro Nakayama, Kenji Sonomoto, Lowering effect of viable Pediococcus pentosaceus QU 19 on the rise in postprandial glucose, Bioscience of Microbiota, Food and Health, 10.12938/BMFH.19-041, 39, 2, 57-64, 2020.01, In the present study, we investigated the glucose-decreasing action of lactic acid bacteria (LAB). The finding of this study could be helpful for people in controlling their blood sugar levels. The LAB candidate was isolated from a Japanese fermented food and identified as Pediococcus pentosaceus by an analysis of its genome sequence. Postprandial blood glucose elevation was investigated using oral starch tolerance tests in mice. Normal mice were fed starch and lyophilized cells of P. pentosaceus QU 19 at the same time. Even without pre-administration of P. pentosaceus QU 19, elevation of the blood glucose level was significantly suppressed by the intake of P. pentosaceus QU 19 at the same time as oral administration of starch. According to the results for its survival in simulated digestive juice and the reduction of blood glucose level in mice, P. pentosaceus QU 19 has potential hypoglycemic activity. In vitro measurements revealed that the glucose-decreasing action of P. pentosaceus QU 19 is probably caused by the glucose assimilation of the strain, not the inhibition of carbohydrate-splitting enzymes which has been reported for other LABs previously. These findings indicate that specific strains of LAB, especially P. pentosaceus QU 19, and foods fermented by LAB may be beneficial for people who must manage glucose ingestion..
27. Orawan La-Ongkham, Massalin Nakphaichit, Jiro Nakayama, Suttipun Keawsompong, Sunee Nitisinprasert, Age-related changes in the gut microbiota and the core gut microbiome of healthy Thai humans., 3 Biotech, 10.1007/s13205-020-02265-7, 10, 6, 276-276, 2020.06, The gut microbial diversity of Thai people was investigated between two large cohorts, adult and elderly subjects, from the middle region of Thailand; the cohorts were divided into different age groups of healthy adult (73) and elderly subjects (47). The diversities of the groups were characterized using a pyrosequencing technique with primers targeting the V6-V8 region of the 16S rRNA gene, and a significant decrease in the Firmicutes and Bacteroidetes ratio from 7.3 to 4.5 was observed with increased age. The microbiota of the adult and elderly groups had a significantly higher abundance of the phylum Actinobacteria, including the three species Bifidobacterium adolescentis, Bifidobacterium longum and Bifidobacterium pseudocatenulatum, and the phylum Bacteroidetes containing the four species Bacteroides uniformis, Bacteroides ovatus, Bacteroides caccae and Bacteroides thetaiotaomicron. Firmicutes showed no significant differences between the two groups. Eleven species belonging to Firmicutes, Bacteroidetes and Proteobacteria were shared by at least 90% of all subjects and defined as core gut microbiota of healthy Thai, among which a high abundance of Escherichia coli was particularly characterized in Thai elderly individuals. Multiple linear regression analysis of age, gender, BMI and diet consumption frequency showed the correlation of age with Bacteroides and Bifidobacterium. Rice consumption frequency showed a significant positive correlation with Bacteroides, while no correlation was found for other factors. Taken together, in the gut of Thai adults, Bifidobacterium decreased and Bacteroides increased with age, while rice consumption increased the abundance of Bacteroides. These link of age and food, especially rice carbohydrate, to gut microbiota and health could be ultimately proposed as the Thai feature..
28. Masanori Fukao, Takeshi Zendo, Takuro Inoue, Nobuo Fuke, Tomoo Moriuchi, Yasuhiro Yamane, Jiro Nakayama, Kenji Sonomoto, Tetsuya Fukaya, Relation between cell-bound exopolysaccharide production via plasmid-encoded genes and rugose colony morphology in the probiotic Lactobacillus brevis KB290., Animal science journal = Nihon chikusan Gakkaiho, 10.1111/asj.13297, 90, 12, 1575-1580, 2019.12, The probiotic Lactobacillus brevis KB290 is a natural producer of cell-bound exopolysaccharide (EPS), and the plasmid-encoded glycosyltransferase genes are responsible for this EPS production. KB290 forms unique rugose colonies inside an agar medium; this characteristic is useful for detecting and enumerating KB290 in the gut or feces. However, the genetic elements associated with this morphology remain unclear. Here, we aimed to investigate the relation between the plasmid eps genes and rugose colony morphology in KB290. The plasmid-cured mutants formed smooth colonies, and the rugose colony morphology was restored after complementation with the eps genes. The eps genes were successfully cloned and expressed in other L. brevis and L. plantarum strains. In these transformant strains, the presence of the EPS, consisting of glucose and N-acetylglucosamine, correlated with rugose colonies, indicating that EPS is responsible for rugose colony formation. To the best of our knowledge, this is the first report identifying the genetic factors influencing rugose colonies in Lactobacillus strains. This rugose colony formation may serve as a useful selective marker for KB290 in routine laboratory and research settings and can be used to detect the spontaneous loss of plasmids in this strain..
29. Masanori Fukao, Takeshi Zendo, Takuro Inoue, Jiro Nakayama, Shigenori Suzuki, Tetsuya Fukaya, Nobuhiro Yajima, Kenji Sonomoto, Plasmid-encoded glycosyltransferase operon is responsible for exopolysaccharide production, cell aggregation, and bile resistance in a probiotic strain, Lactobacillus brevis KB290., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2019.04.008, 128, 4, 391-397, 2019.10, We demonstrate here that exopolysaccharide (EPS) production, cell aggregation, and bile resistance in Lactobacillus brevis KB290 are conferred by three eps genes (gtf27, gtf28, and orf29) located on the 42.4-kb plasmid pKB290-1. The predicted products of gtf27 and gtf28 belong to the membrane-bound glycosyltransferase family whereas the orf29 gene product showed homology with the ABC transporter. On in silico analysis, these genes were found to be widely distributed among lactobacilli from publicly available genomes and metagenomes, and their function is not yet elucidated. RT-PCR analysis showed that the eps genes were organised in an operon and their expression was markedly lower in arabinose- and xylose-containing media than in a glucose-containing medium. The three eps genes were cloned and expressed in homologous and heterologous strains. Considerably less EPS was produced by the plasmid-cured KB1802 strain than by the parental KB290 strain, whereas a similar amount was produced by the KB1802 strain expressing the three eps genes. The KB1802 strain expressing gtf27 and gtf28 but not orf29 did not produce EPS. Cell aggregation and bile resistance were also decreased in KB1802 strains but were complemented by eps genes. Moreover, the three eps genes conferred these phenotypes to a Lactobacillus plantarum strain. In conclusion, the three eps genes in pKB290-1 were sufficient for EPS biosynthesis with glucose and N-acetylglucosamine, and were responsible for cell aggregation and bile resistance. We consider these phenotypes to be at least partly responsible for KB290-specific properties..
30. Sean Littlewood, Helena Tattersall, Charlotte S. Hughes, Rohanah Hussain, Pikyee Ma, Stephen E. Harding, Jiro Nakayama, Mary K. Phillips-Jones, The gelatinase biosynthesis-activating pheromone binds and stabilises the FsrB membrane protein in Enterococcus faecalis quorum sensing, FEBS Letters, 10.1002/1873-3468.13634, 594, 3, 553-563, 2019.10, Quorum-sensing mechanisms regulate gene expression in response to changing cell-population density detected through pheromones. In Enterococcus faecalis, Fsr quorum sensing produces and responds to the gelatinase biosynthesis-activating pheromone (GBAP). Here we establish that the enterococcal FsrB membrane protein has a direct role connected with GBAP by showing that GBAP binds to purified FsrB. Far-UV CD measurements demonstrated a predominantly α-helical protein exhibiting a small level of conformational flexibility. Fivefold (400 μm) GBAP stabilised FsrB (80 μm) secondary structure. FsrB thermal denaturation in the presence and absence of GBAP revealed melting temperatures of 70.1 and 60.8 °C, respectively, demonstrating GBAP interactions and increased thermal stability conferred by GBAP. Addition of GBAP also resulted in tertiary structural changes, confirming GBAP binding..
31. Hiroshi Hamajima, Masaru Tanaka, Miyuki Miyagawa, Mayuko Sakamoto, Tsuyoshi Nakamura, Teruyoshi Yanagita, Megumi Nishimukai, Susumu Mitsutake, Jiro Nakayama, Koji Nagao, Hiroshi Kitagaki, Koji glycosylceramide commonly contained in Japanese traditional fermented foods alters cholesterol metabolism in obese mice., Bioscience, biotechnology, and biochemistry, 10.1080/09168451.2018.1562877, 83, 8, 1514-1522, 2019.08, Koji, which is manufactured by proliferating non-pathogenic fungus Aspergillus oryzae on steamed rice, is the base for Japanese traditional fermented foods. We have revealed that koji and related Japanese fermented foods and drinks such as amazake, shio-koji, unfiltered sake and miso contain abundant glycosylceramide. Here, we report that feeding of koji glycosylceramide to obese mice alters the cholesterol metabolism . Liver cholesterol was significantly decreased in obese mice fed with koji glycosylceramide. We hypothesized that their liver cholesterol was decreased because it was converted to bile acids. Consistent with the hypothesis, many bile acids were increased in the cecum and feces of obese mice fed with koji glycosylceramide. Expressions of CYP7A1 and ABCG8 involved in the metabolism of cholesterol were significantly increased in the liver of mice fed with koji glycosylceramide. Therefore, it was considered that koji glycosylceramide affects the cholesterol metabolism in obese mice..
32. Mugihito Oshiro, Rie Momoda, Masaru Tanaka, Takeshi Zendo, Jiro Nakayama, Dense tracking of the dynamics of the microbial community and chemicals constituents in spontaneous wheat sourdough during two months of backslopping., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2019.02.006, 128, 2, 170-176, 2019.08, Wheat sourdough is a common traditional fermented food that is produced worldwide. However, product quality of spontaneous sourdough is not easy to control because it depends on natural fermentation and backslopping, about which little is known, notably after ten backslopping steps. To this end, we tracked the spontaneous fermentation of three sourdoughs made from wheat flours during 32 backslopping steps for 60 days. At 24 time points, the microbial community was analyzed by both culture-dependent and culture-independent methods and its chemical constituents were assessed. Dynamic changes were observed in the microbial community, which showed a common succession pattern among the three sourdoughs at the bacterial family level and differences at the species level. The bacterial communities evolved through three phases that were driven by different groups of lactic acid bacteria (LAB) species. The dynamism among the metabolites also differed, depending on the species composition of the LAB and yeast communities. In one sourdough, the growth of Saccharomyces cerevisiae was detected along with a concentration of increased ethanol, while in the other two sourdoughs, Wickerhamomyces anomalus was detected without ethanol production. Regarding the LAB communities, two sourdoughs were eventually co-dominated by Lactobacillus plantarum and L. brevis, while the other sourdough was eventually dominated solely by the heterolactic fermentative bacterium Lactobacillus fermentum, and ethanol was produced at the same level as lactic acid. Further research is needed to determine the bacterial and yeast species involved in the fermentation of sourdough, to help improve the design and quality control of the final product..
33. M. Nakphaichit, S. Sobanbua, S. Siemuang, W. Vongsangnak, Jiro Nakayama, S. Nitisinprasert, Protective effect of lactobacillus reuteri KUB-AC5 against salmonella enteritidis challenge in chickens, Beneficial Microbes, 10.3920/BM2018.0034, 10, 1, 43-54, 2019.01, Poultry is an important high-quality food and protein source for humans. However, chicken is considered a primary source of foodborne diseases, especially Salmonella Enteritidis infection. Reducing Salmonella contamination in live poultry will thus lower the risk to consumers. Our previous studies reported that Lactobacillus reuteri KUB-AC5 can produce a substance with antimicrobial activity against pathogenic bacteria, especially Salmonella. In vivo testing revealed that this strain greatly influenced the ileal microbiota by improving chicken gastrointestinal health and inhibiting certain pathogenic bacteria. However, its activity against Salmonella in chicken is unknown. This study investigated the effects of the probiotic L. reuteri KUB-AC5 at various concentrations against Salmonella and the microbiota status in the gastrointestinal tract of broiler chickens. Four treatments groups were used: negative-control group (no Salmonella challenge), positive-control group (Salmonella challenge), and 5 or 7 log cfu probiotic supplementation to Salmonella-challenged chickens. The resultant microbial diversities at the growing and finisher stages were not significantly different among the groups (P>0.05). However, a high dosage of KUB-AC5 maintained similar microbial diversity in Salmonella-challenged chickens as observed in the non-challenged group in the early stage. The exposure Salmonella can affect the microbial diversity that consequently contributes to the disease progression in chicken. Low and high dosages of KUB-AC5 eliminated S. Enteritidis from the ileum and caecum at 14, 21 and 35 days of age. A high-dose of KUB-AC5 also enhanced Lactobacillaceae levels in the growing stage in both the ileum and caecum and suppressed Enterobacteriaceae levels in the finisher stage on day 35, whereas these effects were not observed in the low dose of KUB-AC5 or control groups. These results support the potential value of high-dose L. reuteri KUB-AC5 supplementation for three days after hatching in preventing Salmonella infection in chickens..
34. Juma M. Kisuse, Orawan La-ongkham, Massalin Nakphaichit, Phatthanaphong Therdtatha, Rie Momoda, Masaru Tanaka, Shinji Fukuda, Siam Popluechai, Kongkiat Kespechara, Kenji Sonomoto, Yuan-Kun Lee, Sunee Nitisinprasert and Jiro Nakayama, Urban Diets Linked to Gut Microbiome and Metabolome Alterations in Children: A Comparative Cross-sectional Study in Thailand, Front. Microbiol., 10.3389/fmicb.2018.01345, 2018.08.
35. Hirotoshi Sushida, Naoki Ishibashi, Takeshi Zendo, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Evaluation of leader peptides that affect the secretory ability of a multiple bacteriocin transporter, EnkT., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2018.01.015, 126, 1, 23-29, 2018.07, EnkT is a novel ATP-binding cassette (ABC) transporter responsible for secretion of four bacteriocins, enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z), produced by Enterococcus faecium NKR-5-3. It is generally recognized that the secretion of a bacteriocin requires a dedicated ABC transporter, although molecular mechanisms of this secretion are yet to be revealed. In order to characterize the unique ability of EnkT to secrete multiple bacteriocins, the role of N-terminal leader peptides of bacteriocin precursors was evaluated using Ent53C precursor as a model. The 18-amino acid leader peptide of Ent53C (Lc) was modified by site-directed mutagenesis to generate various point mutations, truncations, or extensions, and substitutions with other leader peptides. The impact of these Lc mutations on Ent53C secretion was evaluated using a quantitative antimicrobial activity assay. We observed that Ent53C production increased with Ala substitution of the highly conserved C-terminal double glycine residues that are recognized as the cleavage site. In contrast, Ent53C antimicrobial activity decreased, with decrease in the length of the putative α-helix-forming region of Lc. Furthermore, EnkT recognized and transported Ent53C of the transformants possessing heterologous leader peptides of enterocin A, pediocin PA-1, brochocins A and B, and lactococcins Qα and Qβ. These results indicated that EnkT shows significant tolerance towards the sequence and length of leader peptides, to secrete multiple bacteriocins. This further demonstrates the functional diversity of bacteriocin ABC transporters and the importance of leader peptides as their recognition motif..
36. Keika Adachi, Kaori Ohtani, Michio Kawano, Ravindra Pal Singh, Basit Yousuf, Kenji Sonomoto, Tohru Shimizu, Jiro Nakayama, Metabolic dependent and independent pH-drop shuts down VirSR quorum sensing in Clostridium perfringens., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2017.12.019, 125, 5, 525-531, 2018.05, Clostridium perfringens produces various exotoxins and enzymes that cause food poisoning and gas gangrene. The genes involved in virulence are regulated by the agr-like quorum sensing (QS) system, which consists of a QS signal synthesis system and a VirSR two-component regulatory system (VirSR TCS) which is a global regulatory system composed of signal sensor kinase (VirS) and response regulator (VirR). We found that the perfringolysin O gene (pfoA) was transiently expressed during mid-log phase of bacterial growth; its expression was rapidly shut down thereafter, suggesting the existence of a self-quorum quenching (sQQ) system. The sQQ system was induced by the addition of stationary phase culture supernatant (SPCS). Activity of the sQQ system was heat stable, and was present following filtration through the ultrafiltration membrane, suggesting that small molecules acted as sQQ agents. In addition, sQQ was also induced by pure acetic and butyric acids at concentrations equivalent to those in the stationary phase culture, suggesting that organic acids produced by C. perfringens were involved in sQQ. In pH-controlled batch culture, sQQ was greatly diminished; expression level of pfoA extended to late-log growth phase, and was eventually increased by one order of magnitude. Furthermore, hydrochloric acid induced sQQ at the same pH as was used in organic acids. SPCS also suppressed the expression of genes regulated by VirSR TCS. Overall, the expression of virulence factors of C. perfringens was downregulated by the sQQ system, which was mediated by primary acidic metabolites and acidic environments. This suggested the possibility of pH-controlled anti-virulence strategies..
37. Basit Yousuf, Keika Adachi, Jiro Nakayama, Developing anti-virulence chemotherapies by exploiting the diversity of microbial quorum sensing systems, Biotechnological Applications of Quorum Sensing Inhibitors, 10.1007/978-981-10-9026-4_9, 151-208, 2018.05, Quorum sensing is a process of chemical communication that adjusts the genetic expression of certain important biological functions in a cell-density- dependent manner. Quorum sensing (QS) regulates important bacterial behaviors, such as production of virulence factors, formation of biofilm, and antibiotic resistance, via signaling molecules called autoinducers (AIs). Gram-negative bacteria typically use N-acyl homoserine lactones (AHLs) and S-adenosyl methionine (SAM) molecules as signaling molecules for communication with neighboring cells, while Gram-positive bacteria use oligopeptides for the same purpose. Autoinducer-2 (AI-2) is used as a signaling molecule by both Gram-negative and Gram-positive bacteria. These QS molecules are often involved in the expression of pathogenicity in different bacterial species. Because of the rapid emergence and dissemination of drug-resistant pathogens worldwide, antivirulence chemotherapies may be potential alternatives to the use of antibiotics, which kill bacteria but allow the emergence of resistance. Recently, successful strategies, employed for inhibition/ manipulation of QS signaling, has brought real excitement for identifying novel advances to combat these life-threatening pathogens. This review highlights the QS systems used by Gram-negative and Gram-positive bacterial species and discusses promising QS inhibitor (QSI) molecules that may aid in designing novel antimicrobial therapeutics. Utilization of different QS components in the design and development of novel biotechnological products, such as biosensors, engineered microbial consortia, and anticancer molecules, is also addressed..
38. Inoue T, Nakayama J, Moriya K, Kawaratani H, Momoda R, Ito K, Iio E, Nojiri S, Fujiwara K, Yoneda M, Yoshiji H, Tanaka Y, Gut dysbiosis associated with hepatitis C virus infection, Clin Infect Dis, 10.1093/cid/ciy205, 67, 6, 869-877, 2018.05, Background:Little is known about the effect of hepatitis C virus (HCV) infection on gut microbiota and the relationship between alteration of gut microbiota and chronic hepatitis C (CHC) progression. We performed a comparative study of gut microbiota composition between CHC patients and healthy individuals.

Methods:Fecal samples from 166 CHC patients were compared with those from 23 healthy individuals; the gut microbiota community was analyzed using 16S ribosomal RNA gene sequencing. CHC patients were diagnosed with persistently normal serum alanine aminotransferase without evidence of liver cirrhosis (LC) (PNALT, n = 18), chronic hepatitis (CH, n = 84), LC (n = 40), and hepatocellular carcinoma in LC (n = 24).

Results:Compared with healthy individuals, bacterial diversity was lower in persons with HCV infection, with a decrease in the order Clostridiales and an increase in Streptococcus and Lactobacillus. Microbiota dysbiosis already appeared in the PNALT stage with the transient increase in Bacteroides and Enterobacteriaceae. Predicted metagenomics of microbial communities showed an increase in the urease gene mainly encoded by viridans streptococci during CHC progression, consistent with a significantly higher fecal pH in CH and LC patients than in healthy individuals or those in the PNALT stage.

Conclusions:HCV infection is associated with gut dysbiosis, even in patients with mild liver disease. Additionally, overgrowth of viridans streptococci can account for hyperammonemia in CH and LC. Further studies would help to propose a novel treatment strategy because the gut microbiome can be therapeutically altered, potentially reducing the complications of chronic liver disease..
39. Basit Yousuf, Keika Adachi, Jiro Nakayama, Developing anti-virulence chemotherapies by exploiting the diversity of microbial quorum sensing systems, Biotechnological Applications of Quorum Sensing Inhibitors, 10.1007/978-981-10-9026-4_9, 151-208, 2018.05, Quorum sensing is a process of chemical communication that adjusts the genetic expression of certain important biological functions in a cell-density- dependent manner. Quorum sensing (QS) regulates important bacterial behaviors, such as production of virulence factors, formation of biofilm, and antibiotic resistance, via signaling molecules called autoinducers (AIs). Gram-negative bacteria typically use N-acyl homoserine lactones (AHLs) and S-adenosyl methionine (SAM) molecules as signaling molecules for communication with neighboring cells, while Gram-positive bacteria use oligopeptides for the same purpose. Autoinducer-2 (AI-2) is used as a signaling molecule by both Gram-negative and Gram-positive bacteria. These QS molecules are often involved in the expression of pathogenicity in different bacterial species. Because of the rapid emergence and dissemination of drug-resistant pathogens worldwide, antivirulence chemotherapies may be potential alternatives to the use of antibiotics, which kill bacteria but allow the emergence of resistance. Recently, successful strategies, employed for inhibition/ manipulation of QS signaling, has brought real excitement for identifying novel advances to combat these life-threatening pathogens. This review highlights the QS systems used by Gram-negative and Gram-positive bacterial species and discusses promising QS inhibitor (QSI) molecules that may aid in designing novel antimicrobial therapeutics. Utilization of different QS components in the design and development of novel biotechnological products, such as biosensors, engineered microbial consortia, and anticancer molecules, is also addressed..
40. Hirotoshi Sushida, Naoki Ishibashi, Takeshi Zendo, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Evaluation of leader peptides that affect the secretory ability of a multiple bacteriocin transporter, EnkT, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2018.01.015, 2018.01, EnkT is a novel ATP-binding cassette (ABC) transporter responsible for secretion of four bacteriocins, enterocins NKR-5-3A, C, D, and Z (Ent53A, C, D, and Z), produced by Enterococcus faecium NKR-5-3. It is generally recognized that the secretion of a bacteriocin requires a dedicated ABC transporter, although molecular mechanisms of this secretion are yet to be revealed. In order to characterize the unique ability of EnkT to secrete multiple bacteriocins, the role of N-terminal leader peptides of bacteriocin precursors was evaluated using Ent53C precursor as a model. The 18-amino acid leader peptide of Ent53C (Lc) was modified by site-directed mutagenesis to generate various point mutations, truncations, or extensions, and substitutions with other leader peptides. The impact of these Lc mutations on Ent53C secretion was evaluated using a quantitative antimicrobial activity assay. We observed that Ent53C production increased with Ala substitution of the highly conserved C-terminal double glycine residues that are recognized as the cleavage site. In contrast, Ent53C antimicrobial activity decreased, with decrease in the length of the putative α-helix-forming region of Lc. Furthermore, EnkT recognized and transported Ent53C of the transformants possessing heterologous leader peptides of enterocin A, pediocin PA-1, brochocins A and B, and lactococcins Qα and Qβ. These results indicated that EnkT shows significant tolerance towards the sequence and length of leader peptides, to secrete multiple bacteriocins. This further demonstrates the functional diversity of bacteriocin ABC transporters and the importance of leader peptides as their recognition motif..
41. Masaru Tanaka, Jiro Nakayama, Development of the gut microbiota in infancy and its impact on health in later life., Allergology international : official journal of the Japanese Society of Allergology, 10.1016/j.alit.2017.07.010, 66, 4, 515-522, 2017.10, Gut microbial ecology and function are dynamic in infancy, but are stabilized in childhood. The 'new friends' have a great impact on the development of the digestive tract and host immune system. In the first year of life, especially, the gut microbiota dramatically changes through interactions with the developing immune system in the gut. The process of establishing the gut microbiota is affected by various environmental factors, with the potential to be a main determinant of life-long health. In this review, we summarize recent findings regarding gut microbiota establishment, including the importance of various factors related to the development of the immune system and allergic diseases later in life..
42. Kohei Kitazaki, Shoko Koga, Kohei Nagatoshi, Koichi Kuwano, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Hitoshi Ano, Hiromu Katamoto, In vitro synergistic activities of cefazolin and nisin A against mastitis pathogens., The Journal of veterinary medical science, 10.1292/jvms.17-0180, 79, 9, 1472-1479, 2017.09, First-generation cephalosporins such as cefazolin (CEZ) have been widely used for mastitis treatment in dairy cattle. However, the use of antibiotics results in the presence of antibiotic residues in milk, which is used for human consumption. Nisin A, a bacteriocin produced by Lactococcus lactis, has been used as a broad-spectrum food preservative for over 50 years. Therefore, a combination of CEZ and nisin A might provide an extended activity spectrum against mastitis pathogens and reduce the antibiotic dose for mastitis treatment. This study aimed to evaluate the combined effect of CEZ and nisin A against mastitis pathogens using the checkerboard and time-kill assays. In the checkerboard assay, the CEZ-nisin A combination exhibited a synergistic effect against Staphylococcus aureus (n=20/20) and Enterococcus faecalis (n=13/18), and meanwhile exhibited a mostly additive effect against Staphylococcus intermedius (n=12/20), Streptococcus agalactiae (n=10/10), Streptococcus dysgalactiae (n=18/18), and Escherichia coli (n=14/18). There were no indifferent or antagonistic effects between CEZ and nisin A. In the time-kill assay, the CEZ-nisin A combination at 0.5 × or 1 × minimum inhibitory concentration exhibited synergistic reduction of bacterial growth by over 3 log10 colony forming units per ml relative to that observed with either antimicrobial substance alone. These results suggest that the CEZ-nisin A combination can be used for developing an intramammary infusion for mastitis treatment, with lower antibiotic concentrations than normal..
43. Juma Kisuse, Jiro Nakayama, 16S rRNA Metagenomics of Asian Gut Microbiota, Understanding Host-Microbiome Interactions - An Omics Approach Omics of Host-Microbiome Association, 10.1007/978-981-10-5050-3_6, 71-81, 2017.09, To characterize the diversity of gut microbial community structures of Asian people, Asian Microbiome Project (AMP) has been established. AMP notably aims to understand the linkage of their gut microbiota with diets and its impact on their health. To that end, AMP began with phase I which focused on the gut microbiota of school-age children who must follow the regional dietary habit. Stool samples were collected from 303 school-age children living in urban or rural regions in five countries spanning temperate and tropical areas of Asia. Bacterial compositions of those samples were determined by using the hypervariable sequences of 16S rRNA V6-V8 region analyzed by 454 pyrosequencing platform. Their community profiles were characterized into two enterotype-like clusters, each driven by Prevotella (P-type) or Bifidobacterium/Bacteroides (BB-type), respectively. Moreover, random forest analysis marked the participant country of residence through fecal species analysis by demonstrating accumulating gut microbiota. The predicted metagenomics using PICRUSt program has suggested overrepresentation of certain enzymes which may reflect their intestinal environment, such as amylase for nondigestible starch in P-type subjects and choloylglycine hydrolase for bile acid metabolism in BB-type subjects. Following this pilot study using 454 sequencing platform, MiSeq pair-end sequencing platform has been introduced into AMP. The MiSeq platform covered more than 99% of gut microbial community profile. Enterotyping was reproduced regardless of the read regions and taxonomy levels. Further study using the MiSeq 16S rRNA metagenomics is promising to gain deep insight of gut microbial community of Asian people..
44. Masaru Tanaka, Yuki Korenori, Masakazu Washio, Takako Kobayashi, Rie Momoda, Chikako Kiyohara, Aki Kuroda, Yuka Saito, Kenji Sonomoto, Jiro Nakayama, Signatures in the gut microbiota of Japanese infants who developed food allergies in early childhood., FEMS microbiology ecology, 10.1093/femsec/fix099, 93, 8, 1-11, 2017.08, Bacterial colonization in infancy is considered crucial for the development of the immune system. Recently, there has been a drastic increase in childhood allergies in Japan. Therefore, we conducted a prospective study with 56 infants on the relationship between gut microbiota in the first year of life and the development of allergies during the first 3 years. In the lactation period, organic acid producers such as Leuconostoc, Weissella and Veillonella tended to be underrepresented in subjects who developed food allergies (FA, n = 14) within the first two years. In the weaning period, children in the FA group were highly colonized by unclassified Enterobacteriaceae and two Clostridium species closely related to Clostridium paraputrificum and C. tertium, and the whole tree phylogenetic diversity index was significantly lower in the FA group. All of these differences in the weaning period were statistically significant, even after adjusting for potential confounding factors. A higher abundance of unclassified Enterobacteriaceae was also found in the other allergic group (n = 15), whereas the two Clostridium species were highly specific to the FA group. The mode of action of these Clostridium species in childhood food allergies remains unknown, warranting further investigation..
45. Jiro Nakayama, Heping Zhang, Yuan Kun Lee, Asian gut microbiome, Science Bulletin, 10.1016/j.scib.2017.04.001, 62, 12, 816-817, 2017.06.
46. Rodney H Perez, Haruki Sugino, Naoki Ishibashi, Takeshi Zendo, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis., Microbiology (Reading, England), 10.1099/mic.0.000435, 163, 4, 431-441, 2017.04, Enterocin NKR-5-3B (Ent53B) is a 64-residue novel circular bacteriocin synthesized from an 87-residue prepeptide. Albeit through a still unknown mechanism, the EnkB1234 biosynthetic enzyme complex processes the prepeptide to yield its mature active, circular form. To gain insights into the key region/residue that plays a role in Ent53 maturation, several mutations near the cleavage site on the precursor peptide were generated. The interaction of the precursor peptide and EnkB1234 appeared to be hydrophobic in nature. At the Leu1 position, only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative. In this study, we also highlight the possible conformation-stabilizing role of the Ent53B leader peptide on the precursor peptide for its interaction with its biosynthetic enzyme complex. Any truncations of the leader peptide moiety interfered in the processing of the prepeptide. However, when propeptides of other circular bacteriocins (circularin A, leucocyclicin Q or lactocyclicin Q) were cloned at the C-terminus of the leader peptide, EnkB1234 could not process them to yield a mature bacteriocin. Taken together, these findings offer new perspectives in our understanding of the possible molecular mechanism of the biosynthesis of this circular bacteriocin. These new perspectives will help advance our current understanding to eventually elucidate circular bacteriocin biosynthesis. Understanding the biosynthetic mechanism of circular bacteriocins will materialize their application potential..
47. Jiro Nakayama, Azusa Yamamoto, Ladie A. Palermo-Conde, Kanako Higashi, Kenji Sonomoto, Julie Tan, Yuan Kun Lee, Impact of westernized diet on gut microbiota in children on Leyte island, Frontiers in Microbiology, 10.3389/fmicb.2017.00197, 8, FEB, 2017.02, Urbanization has changed life styles of the children in some towns and cities on Leyte island in the Philippines. To evaluate the impact of modernization in dietary habits on gut microbiota, we compared fecal microbiota of 7 to 9-year-old children from rural Baybay city (n = 24) and urban Ormoc city (n = 19), and assessed the correlation between bacterial composition and diet. A dietary survey indicated that Ormoc children consumed fast food frequently and more meat and confectionary than Baybay children, suggesting modernization/westernization of dietary habits. Fat intake accounted for 27.2% of the total energy intake in Ormoc children; this was remarkably higher than in their Baybay counterparts (18.1%) and close to the upper limit (30%) recommended by the World Health Organization. Their fecal microbiota were analyzed by high-throughput 16S rRNA gene sequencing in conjunction with a dataset from five other Asian countries. Their microbiota were classified into two enterotype-like clusters with the other countries' children, each defined by high abundance of either Prevotellaceae (P-type) or Bacteroidaceae (BB-type), respectively. Baybay and Ormoc children mainly harbored P-type and BB-type, respectively. Redundancy analysis showed that P-type favored carbohydrates whereas BB-type preferred fats. Fat intake correlated positively with the Firmicutes-to-Bacteroidetes (F/B) ratio and negatively with the relative abundance of the family Prevotellaceae/genus Prevotella. A species-level analysis suggested that dietary fat positively correlated with an Oscillibacter species as well as a series of Bacteroides/Parabacteroides species, whereas dietary carbohydrate positively correlated with Dialister succinatiphilus known as succinate-utilizing bacteria and some succinate-producing species of family Prevotellaceae, Veillonellaceae, and Erysipelotrichaceae. We also found that a Succinivibrio species was overrepresented in the P-type community, suggesting the syntroph via hydrogen and succinate. Predicted metagenomics suggests that BB-type microbiota is well nourished and metabolically more active with simple sugars, amino acids, and lipids, while P-type community is more involved in digestion of complex carbohydrates. Overweight and obese children living in Ormoc, who consumed a high-fat diet, harbored microbiota with higher F/B ratio and low abundance of Prevotella. The altered gut microbiota may be a sign of a modern diet-associated obesity among children in developing areas..
48. K. Nobutani, D. Sawada, S. Fujiwara, Y. Kuwano, K. Nishida, J. Nakayama, H. Kutsumi, T. Azuma, K. Rokutan, The effects of administration of the Lactobacillus gasseri strain CP2305 on quality of life, clinical symptoms and changes in gene expression in patients with irritable bowel syndrome, Journal of Applied Microbiology, 10.1111/jam.13329, 122, 1, 212-224, 2017.01, Aims: To clarify the effects of Lactobacillus gasseri CP2305 (CP2305) on quality of life and clinical symptoms and its functional mechanisms in patients with irritable bowel syndrome (IBS). Methods and Results: After the patients were administered CP2305 daily for 4 weeks, the IBS-severity index score was significantly improved compared with that of the placebo group, and this improvement was accompanied by a reduction in health-related worry and changes in intestinal microbiota. The gene expression profiling of the peripheral blood leucocytes showed that CP2305 treatment significantly up-regulated genes related to eukaryotic initiation factor 2 (EIF2) signalling. Eighty-two genes were down-regulated in IBS patients compared with healthy controls. The expression of 23 of these genes exhibited a CP2305-dependent increase associated with an improvement in IBS severity. The majority of the restored genes were related to EIF2 signalling. Conclusions: CP2305 administration is a potential candidate therapeutic option for patients with IBS. Significance and Impact of the Study: Although probiotics have been proposed to benefit IBS patients, objective clinical evidence and elucidation of the functional mechanism remain insufficient. Our study demonstrated that CP2305 administration beneficially influences IBS patients in both subjective and objective evaluations, and gene expression profiling provided insights into the functional mechanism..
49. Kohei Kitazaki, Shoko Koga, Kohei Nagatoshi, Koichi Kuwano, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Hitoshi Ano, Hiromu Katamoto, In vitro synergistic activities of cefazolin and nisin a against mastitis pathogens, Journal of Veterinary Medical Science, 10.1292/jvms.17-0180, 79, 9, 1472-1479, 2017.01, First-generation cephalosporins such as cefazolin (CEZ) have been widely used for mastitis treatment in dairy cattle. However, the use of antibiotics results in the presence of antibiotic residues in milk, which is used for human consumption. Nisin A, a bacteriocin produced by Lactococcus lactis, has been used as a broad-spectrum food preservative for over 50 years. Therefore, a combination of CEZ and nisin A might provide an extended activity spectrum against mastitis pathogens and reduce the antibiotic dose for mastitis treatment. This study aimed to evaluate the combined effect of CEZ and nisin A against mastitis pathogens using the checkerboard and time-kill assays. In the checkerboard assay, the CEZ-nisin A combination exhibited a synergistic effect against Staphylococcus aureus (n=20/20) and Enterococcus faecalis (n=13/18), and meanwhile exhibited a mostly additive effect against Staphylococcus intermedius (n=12/20), Streptococcus agalactiae (n=10/10), Streptococcus dysgalactiae (n=18/18), and Escherichia coli (n=14/18). There were no indifferent or antagonistic effects between CEZ and nisin A. In the time-kill assay, the CEZ-nisin A combination at 0.5 × or 1 × minimum inhibitory concentration exhibited synergistic reduction of bacterial growth by over 3 log10 colony forming units per ml relative to that observed with either antimicrobial substance alone. These results suggest that the CEZ-nisin A combination can be used for developing an intramammary infusion for mastitis treatment, with lower antibiotic concentrations than normal..
50. Yuhzo Fujita, Haruo Tsuno, Jiro Nakayama, Fermented papaya preparation restores age- related reductions in peripheral blood mononuclear cell cytolytic activity in tube- fed patients, PLoS One, 10.1371/journal.pone.0169240, 12, 1, 2017.01, Tube-fed elderly patients are generally supplied with the same type of nutrition over long periods, resulting in an increased risk for micronutrient deficiencies. Dietary polyphenols promote immunity and have anti-inflammatory, anti-carcinogenic, and anti-oxidative properties. Carica papaya Linn. is rich in several polyphenols; however, these polyphenols are poorly absorbed from the digestive tract in their original polymerized form. Therefore, we determined the molecular components of a fermented Carica papaya Linn. preparation, as well as its effects on immunity and the composition of gut microbiota in tube-fed patients. Different doses of the fermented C. papaya L. preparation were administered to three groups of tube-fed patients for 30 days. Its effects on fecal microbiota composition and immunity were assessed by 16S rRNA gene sequencing and immune-marker analysis, respectively. The chemical composition of the fermented C. papaya L. preparation was analyzed by capillary electrophoresisand liquid chromatography- time of flight mass spectrometry. The fermented C. papaya L. preparation restored peripheral blood mononuclear cell (PBMC) cytolytic activity; however, no other biomarkers of immunity were observed. Treatment with the preparation (9 g/day) significantly reduced the abundance of Firmicutes in the fecal microbiota. In particular, treatment reduced Clostridium scindens and Eggerthella lenta in most patients receiving 9 g/day. Chemical analysis identified low-molecular-weight phenolic acids as polyphenol metabolites; however, no polymerized, large-molecular-weight molecules were detected. Our study indicates that elderly patients who are tube-fed over the long-term have decreased PBMC cytolytic activity. In addition, low-molecular-weight polyphenol metabolites fermented from polymerized polyphenols restore PBMC cytolytic activity and modulate the composition of gut microbiota in tube-fed patients..
51. Hiroshi Hamajima, Haruka Matsunaga, Ayami Fujikawa, Tomoya Sato, Susumu Mitsutake, Teruyoshi Yanagita, Koji Nagao, Jiro Nakayama, Hiroshi Kitagaki, Japanese traditional dietary fungus koji Aspergillus oryzae functions as a prebiotic for Blautia coccoides through glycosylceramide
Japanese dietary fungus koji is a new prebiotic, SpringerPlus, 10.1186/s40064-016-2950-6, 5, 1, 2016.12, Background: The Japanese traditional cuisine, Washoku, considered to be responsible for increased longevity among the Japanese, comprises various foods fermented with the non-pathogenic fungus Aspergillus oryzae (koji). We have recently revealed that koji contains an abundant amount of glycosylceramide. Intestinal microbes have significant effect on health. However, the effects of koji glycosylceramide on intestinal microbes have not been studied. Materials and methods: Glycosylceramide was extracted and purified from koji. C57BL/6N mice were fed a diet containing 1 % purified koji glycosylceramide for 1 week. Nutritional parameters and faecal lipid constituents were analyzed. The intestinal microbial flora of mice on this diet was investigated. Results: Ingested koji glycosylceramide was neither digested by intestinal enzymes nor was it detected in the faeces, suggesting that koji glycosylceramide was digested by the intestinal microbial flora. Intestinal microbial flora that digested koji glycosylceramide had an increased ratio of Blautia coccoides. Stimulation of B. coccoides growth by pure koji glycosylceramide was confirmed in vitro. Conclusions: Koji functions as a prebiotic for B. coccoides through glycosylceramide. Since there are many reports of the effects of B. coccoides on health, an increase in intestinal B. coccoides by koji glycosylceramide might be the connection between Japanese cuisine, intestinal microbial flora, and longevity..
52. Supatjaree Ruengsomwong, Orawan La-Ongkham, Jiahui Jiang, Bhusita Wannissorn, Jiro Nakayama, Sunee Nitisinprasert, Microbial Community of Healthy Thai Vegetarians and Non-Vegetarians, Their Core Gut Microbiota, and Pathogen Risk., Journal of microbiology and biotechnology, 10.4014/jmb.1603.03057, 26, 10, 1723-1735, 2016.10, Pyrosequencing analysis of intestinal microflora from healthy Thai vegetarians and non-vegetarians exhibited 893 OTUs covering 189 species. The strong species indicators of vegetarians and non-vegetarians were Prevotella copri and Bacteroides vulgatus as well as bacteria close to Escherichia hermanii with % relative abundance of 16.9 and 4.5-4.7, respectively. Core gut microbiota of the vegetarian and non-vegetarian groups consisted of 11 and 20 different bacterial species, respectively, belonging to Actinobacteria, Firmicutes, and Proteobacteria commonly found in both groups. Two species, Faecalibacterium prausnitzii and Gemmiger formicilis, had a prevalence of 100% in both groups. Three species, Clostridium nexile, Eubacterium eligens, and P. copri, showed up in most vegetarians, whereas more diversity of Collinsella aerofaciens, Ruminococcus torques, various species of Bacteroides, Parabacteroides, Escherichia, and different species of Clostridium and Eubacterium were found in most non-vegetarians. Considering the correlation of personal characters, consumption behavior, and microbial groups, the age of non-vegetarians showed a strong positive correlation coefficient of 0.54 (p = 0.001) to Bacteroides uniformis but exhibited a moderate one to Alistipes finegoldii and B. vulgatus. Only a positive moderate correlation of body mass index and Parabacteroides distasonis appeared. Based on the significant abundance of potential pathogens, the microbiota of the non-vegetarian group showed an abundance of potential pathogen varieties of Bilophila wadsworthia, Escherichia coli, and E. hermannii, whereas that of the vegetarian group served for only Klebsiella pneumoniae. These results implied that the microbiota of vegetarians with high abundance of P. copri and low potential pathogen variety would be a way to maintain good health in Thais..
53. Shun Iwatani, Naoki Ishibashi, Floirendo P Flores, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, LnqR, a TetR-family transcriptional regulator, positively regulates lacticin Q production in Lactococcus lactis QU 5., FEMS microbiology letters, 10.1093/femsle/fnw200, 363, 18, 2016.09, Lacticin Q is an unmodified leaderless bacteriocin produced by Lactococcus lactis QU 5. It has been revealed that the production and self-immunity of lacticin Q are facilitated by a gene cluster lnqQBCDEF The gene for a putative TetR-family transcriptional regulator, termed lnqR, was found nearby the lnqQBCDEF cluster, but its involvement in lacticin Q biosynthesis remained unknown. In this study, we created an LnqR-overexpressing QU 5 recombinant by using lactococcal constitutive promoter P32 The recombinant QU 5 showed enhanced production of and self-immunity to lacticin Q. RT-PCR analysis has revealed that an overexpression of LnqR increases the amounts of lnqQBCDEF transcripts, and these six genes are transcribed as an operon in a single transcriptional unit. Interestingly, LnqR expression and thus lacticin Q production by L. lactis QU 5 was found temperature dependent, while LnzR, an LnqR-homologue, in L. lactis QU 14 was expressed in a similar but not identical manner to LnqR, resulting in dissimilar bacteriocin productivities by these strains. This report demonstrates LnqR as the first TetR-family transcriptional regulator involved in LAB bacteriocin biosynthesis and that, as an exceptional case of TetR-family regulators, LnqR positively regulates the transcription of these biosynthetic genes..
54. Takeshi Terahara, Katsuhisa Yamada, Jiro Nakayama, Yasuhiro Igarashi, Takeshi Kobayashi, Chiaki Imada, Bacterial community structures of deep-sea water investigated by molecular biological techniques., Gene, 10.1016/j.gene.2015.10.027, 576, 2 Pt 1, 696-700, 2016.02, The aim of the present study was to investigate the bacterial community structures of deep-sea water (DSW) and surface seawater (SSW) samples in Japan by molecular biological techniques. DGGE analyses and pyrosequencing analysis revealed that bacterial community structures of DSW were diverse and differed from those of SSW. This is the first report on the horizontal variation of bacterial community structures of DSW throughout Japan. In addition, pyrosequencing analysis revealed that the number of phyla in DSW was larger than that in SSW, and specific phyla, such as Firmicutes and Planctomycetes, were characterized by a higher proportion of the bacterial community structure in DSW than in SSW. Taken together, these results indicate that a variety of bacteria that are specifically adapted to the DSW environments can be expected to be found in DSW, and DSW would thus be a potential resource for novel or unique microorganisms and compounds..
55. Rodney H Perez, Naoki Ishibashi, Tomoko Inoue, Kohei Himeno, Yoshimitsu Masuda, Narukiko Sawa, Takeshi Zendo, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Functional Analysis of Genes Involved in the Biosynthesis of Enterocin NKR-5-3B, a Novel Circular Bacteriocin., Journal of bacteriology, 10.1128/JB.00692-15, 198, 2, 291-300, 2016.01, UNLABELLED: A putative biosynthetic gene cluster of the enterocin NKR-5-3B (Ent53B), a novel circular bacteriocin, was analyzed by sequencing the flanking regions around enkB, the Ent53B structural gene, using a fosmid library. A region approximately 9 kb in length was obtained, and the enkB1, enkB2, enkB3, and enkB4 genes, encoding putative biosynthetic proteins involved in the production, maturation, and secretion of Ent53B, were identified. We also determined the identity of proteins mediating self-immunity against the effects of Ent53B. Heterologous expression systems in various heterologous hosts, such as Enterococcus faecalis and Lactococcus lactis strains, were successfully established. The production and secretion of the mature Ent53B required the cooperative functions of five genes. Ent53B was produced only by those heterologous hosts that expressed protein products of the enkB, enkB1, enkB2, enkB3, and enkB4 genes. Moreover, self-immunity against the antimicrobial action of Ent53B was conferred by at least two independent mechanisms. Heterologous hosts harboring the intact enkB4 gene and/or a combination of intact enkB1 and enkB3 genes were immune to the inhibitory action of Ent53B. IMPORTANCE: In addition to their potential application as food preservatives, circular bacteriocins are now considered possible alternatives to therapeutic antibiotics due to the exceptional stability conferred by their circular structure. The successful practical application of circular bacteriocins will become possible only if the molecular details of their biosynthesis are fully understood. The results of the present study offer a new perspective on the possible mechanism of circular bacteriocin biosynthesis. In addition, since some enterococcal strains are associated with pathogenicity, virulence, and drug resistance, the establishment of the first multigenus host heterologous production of Ent53B has very high practical significance, as it widens the scope of possible Ent53B applications..
56. Chinatsu Shimafuji, Megumi Noguchi, Mami Nishie, Jun-Ichi Nagao, Kouki Shioya, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, In vitro catalytic activity of N-terminal and C-terminal domains in NukM, the post-translational modification enzyme of nukacin ISK-1., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2015.03.020, 120, 6, 624-629, 2015.12, Lantibiotics are antibacterial peptides containing unique thioether cross-links termed lanthionine and methyllanthionine. NukM, the modifying enzyme of nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the dehydration of specific Ser/Thr residues in a precursor peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor peptide of nisin is modified by 2 enzymes, NisB and NisC, which mediate dehydration and cyclization, respectively. While the C-terminal domain of NukM is homologous to NisC, the N-terminal domain has no homology with other known proteins. We expressed and characterized the N- and C-terminal domains of NukM, NukMN, and NukMC, separately. In vitro reconstitution revealed that full-length NukM fully modified the substrate peptide NukA. NukMN partially phosphorylated, dehydrated, and cyclized NukA. By contrast, NukMC did not catalyze dehydration, phosphorylation, or cyclization reactions. Interaction studies using surface plasmon resonance analysis indicated that NukM and NukMN can bind NukA with high affinity, whereas NukMC has low substrate-recognition activity. These results suggest that NukMN is mainly responsible for substrate recognition and dehydration and that the whole NukM structure, including the C-terminal domain, is required for the complete modification of NukA. To the best of our knowledge, this is the first report providing insights into the in vitro catalytic activity of individual domains of a LanM-type modification enzyme..
57. Ravindra Pal Singh, Ken-Ichi Okubo, Kaori Ohtani, Keika Adachi, Kenji Sonomoto, Jiro Nakayama, Rationale design of quorum-quenching peptides that target the VirSR system of Clostridium perfringens., FEMS microbiology letters, 10.1093/femsle/fnv188, 362, 22, 2015.11, In Clostridium perfringens, a 5-membered thiolactone peptide acts as an autoinducing peptide (AIPCp) to activate the VirSR two-component signal transduction system, which in turn controls the expression of genes encoding multiple toxins, including α, θ and κ. To develop anti-pathogenic agents against virulent C. perfringens, quorum-quenching peptides were rationally designed based on the structure-activity relationship (SAR) data on AIPCp. Alanine scanning study of AIPCp suggested that Trp(3) and Phe(4) are involved in receptor binding and activation, respectively. On the basis of the SAR, we designed two quorum-quenching peptides with different modes of action: Z-AIPCp-L2A/T5A (partial agonist) and Z-AIPCp-F4A/T5S (partial antagonist). Both peptides significantly attenuated transcription of θ toxin gene (pfoA) in a virulent strain of C. perfringens with IC50 = 0.32 and 0.72 μM, respectively..
58. Hidehiro Toh, Hidetoshi Morita, Hiroyuki Tsuji, Kazuhiro Iwashita, Nami Goto, Jiro Nakayama, Mitsuo Sekine, Yumiko Kato, Ken-ichiro Suzuki, Nobuyuki Fujita, Complete genome sequence of Lactobacillus acetotolerans RIB 9124 (NBRC 13120) isolated from putrefied (hiochi) Japanese sake., Journal of biotechnology, 10.1016/j.jbiotec.2015.09.006, 214, 214-5, 2015.11, Lactobacillus acetotolerans RIB 9124 (NBRC 13120) was isolated from putrefied (hiochi) Japanese sake. Here we report the complete genome sequence of this organism. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a L. acetotolerans strain..
59. Yasuhiro Igarashi, Fumiya Gohda, Taito Kadoshima, Takao Fukuda, Tomoaki Hanafusa, Akane Shojima, Jiro Nakayama, Gerald F Bills, Stephen Peterson, Avellanin C, an inhibitor of quorum-sensing signaling in Staphylococcus aureus, from Hamigera ingelheimensis., The Journal of antibiotics, 10.1038/ja.2015.50, 68, 11, 707-710, 2015.11.
60. Yasuhiro Igarashi, Kazuki Yamamoto, Takao Fukuda, Akane Shojima, Jiro Nakayama, Lorena Carro, Martha E Trujillo, Arthroamide, a Cyclic Depsipeptide with Quorum Sensing Inhibitory Activity from Arthrobacter sp., Journal of natural products, 10.1021/acs.jnatprod.5b00540, 78, 11, 2827-2831, 2015.11, Nonfilamentous actinobacteria have been less studied as secondary metabolite producers than their filamentous counterparts such as Streptomyces. From our collection of nonfilamentous actinobacteria isolated from sandstone, an Arthrobacter strain was found to produce a new cyclic peptide arthroamide (1) together with the known compound turnagainolide A (2). These compounds inhibited the quorum sensing signaling of Staphylococcus aureus in the submicromolar to micromolar range..
61. Hidehiro Toh, Hidetoshi Morita, Hiroyuki Tsuji, Kazuhiro Iwashita, Nami Goto, Jiro Nakayama, Mitsuo Sekine, Yumiko Kato, Ken Ichiro Suzuki, Nobuyuki Fujita, Complete genome sequence of Lactobacillus acetotolerans RIB 9124 (NBRC 13120) isolated from putrefied (hiochi) Japanese sake, Journal of Biotechnology, 10.1016/j.jbiotec.2015.09.006, 214, 214-215, 2015.11, Lactobacillus acetotolerans RIB 9124 (NBRC 13120) was isolated from putrefied (hiochi) Japanese sake. Here we report the complete genome sequence of this organism. This paper is the first report demonstrating the fully sequenced and completely annotated genome of a L. acetotolerans strain..
62. Kohei Himeno, K Johan Rosengren, Tomoko Inoue, Rodney H Perez, Michelle L Colgrave, Han Siean Lee, Lai Y Chan, Sónia Troeira Henriques, Koji Fujita, Naoki Ishibashi, Takeshi Zendo, Pongtep Wilaipun, Jiro Nakayama, Vichien Leelawatcharamas, Hiroyuki Jikuya, David J Craik, Kenji Sonomoto, Identification, Characterization, and Three-Dimensional Structure of the Novel Circular Bacteriocin, Enterocin NKR-5-3B, from Enterococcus faecium., Biochemistry, 10.1021/acs.biochem.5b00196, 54, 31, 4863-4876, 2015.08, Enterocin NKR-5-3B, one of the multiple bacteriocins produced by Enterococcus faecium NKR-5-3, is a 64-amino acid novel circular bacteriocin that displays broad-spectrum antimicrobial activity. Here we report the identification, characterization, and three-dimensional nuclear magnetic resonance solution structure determination of enterocin NKR-5-3B. Enterocin NKR-5-3B is characterized by four helical segments that enclose a compact hydrophobic core, which together with its circular backbone impart high stability and structural integrity. We also report the corresponding structural gene, enkB, that encodes an 87-amino acid precursor peptide that undergoes a yet to be described enzymatic processing that involves adjacent cleavage and ligation of Leu(24) and Trp(87) to yield the mature (circular) enterocin NKR-5-3B..
63. Said E Desouky, Akane Shojima, Ravindra Pal Singh, Takahisa Matsufuji, Yasuhiro Igarashi, Takashi Suzuki, Tohru Yamagaki, Ken-Ichi Okubo, Kaori Ohtani, Kenji Sonomoto, Jiro Nakayama, Cyclodepsipeptides produced by actinomycetes inhibit cyclic-peptide-mediated quorum sensing in Gram-positive bacteria., FEMS microbiology letters, 10.1093/femsle/fnv109, 362, 14, 2015.07, Cyclic peptides are commonly used as quorum-sensing autoinducers in Gram-positive Firmicutes bacteria. Well-studied examples of such molecules are thiolactone and lactone, used to regulate the expression of a series of virulence genes in the agr system of Staphylococcus aureus and the fsr system of Enterococcus faecalis, respectively. Three cyclodepsipeptides WS9326A, WS9326B and cochinmicin II/III were identified as a result of screening actinomycetes culture extracts for activity against the agr/fsr system. These molecules are already known as receptor antagonists, the first two for tachykinin and the last one for endothelin. WS9326A also inhibited the transcription of pfoA regulated by the VirSR two-component system in Clostridium perfringens. Receptor-binding assays using a fluorescence-labeled autoinducer (FITC-GBAP) showed that WS9326A and WS9326B act as receptor antagonists in this system. In addition, an ex vivo assay showed that WS9326B substantially attenuated the toxicity of S. aureus for human corneal epithelial cells. These results suggest that these three natural cyclodepsipeptides have therapeutic potential for targeting the cyclic peptide-mediated quorum sensing of Gram-positive pathogens..
64. Jiro Nakayama, Koichi Watanabe, Jiahui Jiang, Kazunori Matsuda, Shiou-Huei Chao, Pri Haryono, Orawan La-Ongkham, Martinus-Agus Sarwoko, I Nengah Sujaya, Liang Zhao, Kang-Ting Chen, Yen-Po Chen, Hsueh-Hui Chiu, Tomoko Hidaka, Ning-Xin Huang, Chikako Kiyohara, Takashi Kurakawa, Naoshige Sakamoto, Kenji Sonomoto, Kousuke Tashiro, Hirokazu Tsuji, Ming-Ju Chen, Vichai Leelavatcharamas, Chii-Cherng Liao, Sunee Nitisinprasert, Endang S Rahayu, Fa-Zheng Ren, Ying-Chieh Tsai, Yuan-Kun Lee, Diversity in gut bacterial community of school-age children in Asia., Scientific reports, 10.1038/srep08397, 5, 8397-8397, 2015.02, Asia differs substantially among and within its regions populated by diverse ethnic groups, which maintain their own respective cultures and dietary habits. To address the diversity in their gut microbiota, we characterized the bacterial community in fecal samples obtained from 303 school-age children living in urban or rural regions in five countries spanning temperate and tropical areas of Asia. The microbiota profiled for the 303 subjects were classified into two enterotype-like clusters, each driven by Prevotella (P-type) or Bifidobacterium/Bacteroides (BB-type), respectively. Majority in China, Japan and Taiwan harbored BB-type, whereas those from Indonesia and Khon Kaen in Thailand mainly harbored P-type. The P-type microbiota was characterized by a more conserved bacterial community sharing a greater number of type-specific phylotypes. Predictive metagenomics suggests higher and lower activity of carbohydrate digestion and bile acid biosynthesis, respectively, in P-type subjects, reflecting their high intake of diets rich in resistant starch. Random-forest analysis classified their fecal species community as mirroring location of resident country, suggesting eco-geographical factors shaping gut microbiota. In particular, children living in Japan harbored a less diversified microbiota with high abundance of Bifidobacterium and less number of potentially pathogenic bacteria, which may reflect their living environment and unique diet..
65. Ravindra Pal Singh, Jiro Nakayama, Development of quorum-sensing inhibitors targeting the fsr system of enterococcus faecalis, Quorum Sensing vs Quorum Quenching: A Battle with no end in Sight, 10.1007/978-81-322-1982-8__25, 319-326, 2015.01, Enterococcus spp. can cause illnesses such as bacteremia, endocarditis, urinary tract infections, posttreatment endophthalmitis, and endodontic infections (Murray 2000; Marothi et al. 2005). Medical treatment of these infectious diseases depends largely on bactericidal or bacteriostatic antibiotics. However, frequent use of such antibiotics has led to the development of drug-resistant bacterial strains, which are difficult to treat (Marothi et al. 2005; Murray 2000). As a result, the blockage of bacterial quorum-sensing (QS) systems has attracted attention owing to its potential to attenuate bacterial virulence without inducing bactericidal pressures that lead to drug resistance. This approach, called quorum quenching (QQ), can be undertaken partially or completely independently of antibiotic treatment. Among several QQ strategies, the use of QS inhibitors (QSIs), which are small molecules that have no adverse effects on bacteria, offers advantages in terms of drug delivery and decreased damage to commensal microbiota..
66. Ravindra Pal Singh, Ken Ichi Okubo, Kaori Ohtani, Keika Adachi, Kenji Sonomoto, Jiro Nakayama, Rationale design of quorum-quenching peptides that target the VirSR system of Clostridium perfringens, FEMS Microbiology Letters, 10.1093/femsle/fnv188, 362, 22, 2015.01, In Clostridium perfringens, a 5-membered thiolactone peptide acts as an autoinducing peptide (AIPCp) to activate the VirSR two-component signal transduction system, which in turn controls the expression of genes encoding multiple toxins, including α, θ and κ. To develop anti-pathogenic agents against virulent C. perfringens, quorum-quenching peptides were rationally designed based on the structure-activity relationship (SAR) data on AIPCp. Alanine scanning study of AIPCp suggested that Trp3 and Phe4 are involved in receptor binding and activation, respectively. On the basis of the SAR, we designed two quorum-quenching peptides with different modes of action: Z-AIPCp-L2A/T5A (partial agonist) and Z-AIPCp-F4A/T5S (partial antagonist). Both peptides significantly attenuated transcription of θ toxin gene (pfoA) in a virulent strain of C. perfringens with IC50 = 0.32 and 0.72 μM, respectively..
67. Ravindra Pal Singh, Jiro Nakayama, Quorum-sensing systems in enterococci, Quorum Sensing vs Quorum Quenching: A Battle with no end in Sight, 10.1007/978-81-322-1982-8__14, 155-163, 2015.01, Enterococcus is a genus of Gram-positive bacteria that is ubiquitous in natural ecosystems, plants, and animals. Some species of Enterococcus are present in the normal gastrointestinal bacterial community. However, others notably Enterococcus faecalis and Enterococcus faecium often cause opportunistic infections such as bacteremia, endocarditis, urinary tract infections, posttreatment endodontic infections, and endophthalmitis (Arias et al. 2010). Bacteria often use quorum sensing (QS) systems to control the expression of certain virulence genes and establish infection efficiently (Waters and Bassler 2005)..
68. Ravindra Pal Singh, Jiro Nakayama, Development of quorum-sensing inhibitors targeting the fsr system of enterococcus faecalis, Quorum Sensing vs Quorum Quenching: A Battle with no end in Sight, 10.1007/978-81-322-1982-8__25, 319-326, 2015.01, Enterococcus spp. can cause illnesses such as bacteremia, endocarditis, urinary tract infections, posttreatment endophthalmitis, and endodontic infections (Murray 2000; Marothi et al. 2005). Medical treatment of these infectious diseases depends largely on bactericidal or bacteriostatic antibiotics. However, frequent use of such antibiotics has led to the development of drug-resistant bacterial strains, which are difficult to treat (Marothi et al. 2005; Murray 2000). As a result, the blockage of bacterial quorum-sensing (QS) systems has attracted attention owing to its potential to attenuate bacterial virulence without inducing bactericidal pressures that lead to drug resistance. This approach, called quorum quenching (QQ), can be undertaken partially or completely independently of antibiotic treatment. Among several QQ strategies, the use of QS inhibitors (QSIs), which are small molecules that have no adverse effects on bacteria, offers advantages in terms of drug delivery and decreased damage to commensal microbiota..
69. Said E. Desouky, Akane Shojima, Ravindra Pal Singh, Takahisa Matsufuji, Yasuhiro Igarashi, Takashi Suzuki, Tohru Yamagaki, Ken Ichi Okubo, Kaori Ohtani, Kenji Sonomoto, Jiro Nakayama, Cyclodepsipeptides produced by actinomycetes inhibit cyclic-peptide-mediated quorum sensing in Gram-positive bacteria, FEMS Microbiology Letters, 10.1093/femsle/fnv109, 362, 14, 2015.01, Cyclic peptides are commonly used as quorum-sensing autoinducers in Gram-positive Firmicutes bacteria. Well-studied examples of such molecules are thiolactone and lactone, used to regulate the expression of a series of virulence genes in the agr system of Staphylococcus aureus and the fsr system of Enterococcus faecalis, respectively. Three cyclodepsipeptides WS9326A,WS9326B and cochinmicin II/III were identified as a result of screening actinomycetes culture extracts for activity against the agr/fsr system. These molecules are already known as receptor antagonists, the first two for tachykinin and the last one for endothelin. WS9326A also inhibited the transcription of pfoA regulated by the VirSR two-component system in Clostridium perfringens. Receptor-binding assays using a fluorescence-labeled autoinducer (FITC-GBAP) showed that WS9326A and WS9326B act as receptor antagonists in this system. In addition, an ex vivo assay showed that WS9326B substantially attenuated the toxicity of S. aureus for human corneal epithelial cells. These results suggest that these three natural cyclodepsipeptides have therapeutic potential for targeting the cyclic peptide-mediated quorum sensing of Gram-positive pathogens..
70. Tadao Enomoto, Masanori Sowa, Keiji Nishimori, Shinichiro Shimazu, Akira Yoshida, Kazuko Yamada, Fukumi Furukawa, Takemasa Nakagawa, Naotake Yanagisawa, Noriyuki Iwabuchi, Toshitaka Odamaki, Fumiaki Abe, Jiro Nakayama, Jin-Zhong Xiao, Effects of bifidobacterial supplementation to pregnant women and infants in the prevention of allergy development in infants and on fecal microbiota., Allergology international : official journal of the Japanese Society of Allergology, 10.2332/allergolint.13-OA-0683, 63, 4, 575-85, 2014.12, BACKGROUND: Probiotic administration may be a useful method for preventing allergies in infants; however, there have been controversial results about the efficacy. We investigated the effects of bifidobacterial supplementation on the risk of developing allergic diseases in the Japanese population. METHODS: In an open trial, we gave Bifidobacterium breve M-16V and Bifidobacterium longum BB536 prenatally to 130 mothers beginning 1 month prior to delivery and postnatally to their infants for 6 months. Another 36 mother-infant pairs served as controls and did not receive the bifidobacterial supplementation. Development of allergic symptoms in the infants was assessed at 4, 10 and 18 months of age. Fecal samples were collected from the mothers and infants. RESULTS: The risk of developing eczema/atopic dermatitis (AD) during the first 18 months of life was significantly reduced in infants in the probiotic group (OR: 0.231 [95% CI: 0.084-0.628] and 0.304 [0.105-0.892] at 10 and 18 months of age, respectively). Pyrosequencing analyses indicated an altered composition of the fecal microbiota at 4 months for infants who developed eczema/AD at 4 and 10 months of age. The proportion of Proteobacteria was significantly lower (P = 0.007) in mothers at the time of delivery who received the supplementation when compared with the control group and was positively correlated (r = 0.283, P = 0.024) with that of infants at 4 months of age. No adverse effects were related to the use of probiotics. CONCLUSIONS: These data suggest that the prenatal and postnatal supplementation of bifidobacteria is effective in primary preventing allergic diseases. Some limited changes in the composition of fecal microbiota by the bifidobacterial supplementation were observed..
71. Hiroshi Ono, Shoko Nishio, Jun Tsurii, Tetsuhiro Kawamoto, Kenji Sonomoto, Jiro Nakayama, Monitoring of the microbiota profile in nukadoko, a naturally fermented rice bran bed for pickling vegetables., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2014.04.017, 118, 5, 520-5, 2014.11, Nukadoko is a fermented rice bran mash traditionally used for pickling vegetables in Japan. To date, the production of both homemade and commercial nukadoko depends on natural fermentation without using starter cultures. Here, we monitored chemical and microbiological changes in the initial batch fermentation of nukadoko. Nukadoko samples were prepared by spontaneous fermentation of four different brands of rice bran, and microbiome dynamics were analyzed for 2 months. In the first week, non-Lactobacillales lactic acid bacteria (LAB) species, which differed among the samples, grew proportionally to pH decrease and lactate increase. Thereafter, Lactobacillus plantarum started growing and consumed residual sugars, causing further lactate increase in nukadoko. Finally, microbial communities in all tested nukadoko samples were dominated by L. plantarum. Taken together, our results suggest that the mixture of the fast-growing LAB species and slow-growing L. plantarum may be used as a suitable starter culture to promote the initial fermentation of nukadoko..
72. Naoki Ishibashi, Kohei Himeno, Yoshimitsu Masuda, Rodney Honrada Perez, Shun Iwatani, Takeshi Zendo, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Gene cluster responsible for secretion of and immunity to multiple bacteriocins, the NKR-5-3 enterocins., Applied and environmental microbiology, 10.1128/AEM.02312-14, 80, 21, 6647-55, 2014.11, Enterococcus faecium NKR-5-3, isolated from Thai fermented fish, is characterized by the unique ability to produce five bacteriocins, namely, enterocins NKR-5-3A, -B, -C, -D, and -Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). Genetic analysis with a genome library revealed that the bacteriocin structural genes (enkA [ent53A], enkC [ent53C], enkD [ent53D], and enkZ [ent53Z]) that encode these peptides (except for Ent53B) are located in close proximity to each other. This NKR-5-3ACDZ (Ent53ACDZ) enterocin gene cluster (approximately 13 kb long) includes certain bacteriocin biosynthetic genes such as an ABC transporter gene (enkT), two immunity genes (enkIaz and enkIc), a response regulator (enkR), and a histidine protein kinase (enkK). Heterologous-expression studies of enkT and ΔenkT mutant strains showed that enkT is responsible for the secretion of Ent53A, Ent53C, Ent53D, and Ent53Z, suggesting that EnkT is a wide-range ABC transporter that contributes to the effective production of these bacteriocins. In addition, EnkIaz and EnkIc were found to confer self-immunity to the respective bacteriocins. Furthermore, bacteriocin induction assays performed with the ΔenkRK mutant strain showed that EnkR and EnkK are regulatory proteins responsible for bacteriocin production and that, together with Ent53D, they constitute a three-component regulatory system. Thus, the Ent53ACDZ gene cluster is essential for the biosynthesis and regulation of NKR-5-3 enterocins, and this is, to our knowledge, the first report that demonstrates the secretion of multiple bacteriocins by an ABC transporter..
73. H. Ono, S. Nishio, J. Tsurii, T. Kawamoto, K. Sonomoto and J. Nakayama, Effects of Japanese pepper and red pepper on the microbial community during nukadoko fermentation, Biosci Microbiota Food Health, doi: 10.12938/bmfh, 34, 1, 1-9., 2014.09.
74. Supatjaree Ruengsomwong, Yuki Korenori, Naoshige Sakamoto, Bhusita Wannissorn, Jiro Nakayama, Sunee Nitisinprasert, Senior Thai fecal microbiota comparison between vegetarians and non-vegetarians using PCR-DGGE and real-time PCR., Journal of microbiology and biotechnology, 10.4014/jmb.1310.10043, 24, 8, 1026-33, 2014.08, The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets..
75. Keita Kato, Hidehiro Toh, Naoshige Sakamoto, Kazuki Mori, Kosuke Tashiro, Naruhiro Hibi, Kenji Sonomoto, Jiro Nakayama, Draft Genome Sequence of Lactobacillus namurensis Chizuka 01, Isolated from Nukadoko, a Pickling Bed of Fermented Rice Bran., Genome announcements, 10.1128/genomeA.01263-13, 2, 1, 2014.02, Lactobacillus namurensis Chizuka 01 was isolated from nukadoko, which is a fermented rice bran bed traditionally used in Japan for pickling vegetables. Here, we report the first draft of an annotated genome sequence of this organism. This paper is the first published report of the genomic sequence of L. namurensis..
76. Fuqin Mu, Yoshimitsu Masuda, Takeshi Zendo, Hiroshi Ono, Hiroshi Kitagawa, Haruo Ito, Jiro Nakayama, Kenji Sonomoto, Biological function of a DUF95 superfamily protein involved in the biosynthesis of a circular bacteriocin, leucocyclicin Q., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2013.06.023, 117, 2, 158-164, 2014.02, Biological functions of a DUF95 superfamily protein in the biosynthesis gene cluster of a novel circular bacteriocin, leucocyclicin Q (LcyQ), were characterized in this paper. Sequence analysis and database search of the regions flanking the LcyQ structural gene lcyQ revealed four open reading frames (lcyR, lcyB, lcyC, and lcyD) related to bacteriocin biosynthesis. LcyD shares some similarity to the DUF95 superfamily proteins, often found in the biosynthetic gene clusters of circular bacteriocins. Mass spectrometry analysis showed accumulation of active mature LcyQ inside lcyD knockout cells. Heterologous expression of lcyD demonstrated that it confers robust immunity against LcyQ. Peptide release/binding assay revealed that the immunity could be attributed to the secretion of LcyQ to the cell exterior. Thus, the DUF95 superfamily protein has a dual function in the biosynthesis of LcyQ, as an immunity-associated transporter and as a secretion-aiding agent. Accumulation of mature LcyQ inside the cell in lcyD knockout strains, further implied that cyclization occurs within the cell. To the best of our knowledge, this is the first report on LcyQ cyclization inside the cell and the dual role of a DUF95 superfamily protein in circular bacteriocin biosynthesis..
77. Tânia Ribeiro, Neuza Teixeira, Ryoji Yokohata, Jiro Nakayama, Michael S. Gilmore, Maria de Fátima S Lopes, Transcriptomic study reveals new pathways and genes involved in Enterococcus faecalis V583 response to a therapeutic dose of vancomycin, Archives of Clinical Microbiology, 10.3823/277, 5, 1, 2014.01, Background: An enterococcal strain carrying the VanB resistance type can become susceptible if impaired in other genes unrelated to the vanB operon. This fact alone illustrates the lack of knowledge on the vancomycin mode of action. This antibiotic is still usable to treat serious infections caused by multiresistant enterococcal strains, but may not be so for long. This work was thus set up to gather a body of knowledge that can be used in the future to increase efficacy against both vancomycin resistant (VRE) and susceptible enterococci (VSE). Methods and Findings: Microarrays were used to detect the genetic response of the VanB carrying strain Enterococcus faecalis V583 to a therapeutic dose (10 mg/ml) of vancomycin. Besides the vanRS genes, two other two-component systems were induced. The therapeutic dose of vancomycin was found to act as an anti-virulence agent, by turning-off the Fsr quorum-sensing system. Key regulators and metabolic enzymes, involved in trafficking carbon sources into glycolysis and isoprenoid synthesis and utilization, were also affected in order to support cell-wall synthesis. Also, cell-wall modification involving lipotheicoic acid synthesis, DNA repair and protein folding were highly responsive functions to the vancomycin dose tested. Conclusions: Overall, our results provide clues on the ability of a VRE strain to stand vancomycin and on the mode of action of the antibiotic. VRE response to a vancomycin therapeutic dose involves an intricate regulatory network and metabolic adjustment which is worth solving as it can help finding new targets to fight both VRE and VSE infections..
78. Akane Shojima, Jiro Nakayama, Quorum sensing in gram-positive bacteria
Assay protocols for staphylococcal agr and enterococcal fsr systems, Methods in Molecular Biology, 10.1007/978-1-4939-0467-9_3, 1147, 33-41, 2014.01, A thiolactone/lactone peptide-mediated quorum sensing (QS) system is commonly employed in gram- positive bacteria to control the expression of a variety of phenotypes, including the production of virulence factors and biofilm formation. Here, we describe assay protocols for the well-studied QS systems (agr and fsr) of two representative gram-positive pathogens, Staphylococcus aureus and Enterococcus faecalis . These convenient assay systems are useful for the screening of QS inhibitors as well as for basic research to address the mechanism of these QS systems..
79. Hiroshi Ono, Shoko Nishio, Jun Tsurii, Tetsuhiro Kawamoto, Kenji Sonomoto, Jiro Nakayama, Monitoring of the microbiota profile in nukadoko, a naturally fermented rice bran bed for pickling vegetables, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2014.04.017, 118, 5, 520-525, 2014.01, Nukadoko is a fermented rice bran mash traditionally used for pickling vegetables in Japan. To date, the production of both homemade and commercial nukadoko depends on natural fermentation without using starter cultures. Here, we monitored chemical and microbiological changes in the initial batch fermentation of nukadoko. Nukadoko samples were prepared by spontaneous fermentation of four different brands of rice bran, and microbiome dynamics were analyzed for 2 months. In the first week, non-Lactobacillales lactic acid bacteria (LAB) species, which differed among the samples, grew proportionally to pH decrease and lactate increase. Thereafter, Lactobacillus plantarum started growing and consumed residual sugars, causing further lactate increase in nukadoko. Finally, microbial communities in all tested nukadoko samples were dominated by L.plantarum. Taken together, our results suggest that the mixture of the fast-growing LAB species and slow-growing L.plantarum may be used as a suitable starter culture to promote the initial fermentation of nukadoko..
80. Naoki Ishibashi, Kohei Himeno, Yoshimitsu Masuda, Rodney Honrada Perez, Shun Iwatani, Takeshi Zendo, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Gene cluster responsible for secretion of and immunity to multiple bacteriocins, the NKR-5-3 enterocins, Applied and Environmental Microbiology, 10.1128/AEM.02312-14, 80, 21, 6647-6655, 2014.01, Enterococcus faecium NKR-5-3, isolated from Thai fermented fish, is characterized by the unique ability to produce five bacteriocins, namely, enterocins NKR-5-3A, -B, -C, -D, and -Z (Ent53A, Ent53B, Ent53C, Ent53D, and Ent53Z). Genetic analysis with a genome library revealed that the bacteriocin structural genes (enkA [ent53A], enkC [ent53C], enkD [ent53D], and enkZ [ent53Z]) that encode these peptides (except for Ent53B) are located in close proximity to each other. This NKR-5-3ACDZ (Ent53ACDZ) enterocin gene cluster (approximately 13 kb long) includes certain bacteriocin biosynthetic genes such as an ABC transporter gene (enkT), two immunity genes (enkIaz and enkIc), a response regulator (enkR), and a histidine protein kinase (enkK). Heterologous- expression studies of enkT and ΔenkT mutant strains showed that enkT is responsible for the secretion of Ent53A, Ent53C, Ent53D, and Ent53Z, suggesting that EnkT is a wide-range ABC transporter that contributes to the effective production of these bacteriocins. In addition, EnkIaz and EnkIc were found to confer self-immunity to the respective bacteriocins. Furthermore, bacteriocin induction assays performed with the ΔenkRK mutant strain showed that EnkR and EnkK are regulatory proteins responsible for bacteriocin production and that, together with Ent53D, they constitute a three-component regulatory system. Thus, the Ent53ACDZ gene cluster is essential for the biosynthesis and regulation of NKR-5-3 enterocins, and this is, to our knowledge, the first report that demonstrates the secretion of multiple bacteriocins by an ABC transporter..
81. Tadao Enomoto, Masanori Sowa, Keiji Nishimori, Shinichiro Shimazu, Akira Yoshida, Kazuko Yamada, Fukumi Furukawa, Takemasa Nakagawa, Naotake Yanagisawa, Noriyuki Iwabuchi, Toshitaka Odamaki, Fumiaki Abe, Jiro Nakayama, Jin Zhong Xiao, Effects of bifidobacterial supplementation to pregnant women and infants in the prevention of allergy development in infants and on fecal microbiota, Allergology International, 10.2332/allergolint.13-OA-0683, 63, 4, 575-585, 2014.01, Background: Probiotic administration may be a useful method for preventing allergies in infants; however, there have been controversial results about the efficacy. We investigated the effects of bifidobacterial supplementation on the risk of developing allergic diseases in the Japanese population.
Methods: In an open trial, we gave Bifidobacterium breve M-16V and Bifidobacterium longum BB536 prenatally to 130 mothers beginning 1 month prior to delivery and postnatally to their infants for 6 months. Another 36 mother-infant pairs served as controls and did not receive the bifidobacterial supplementation. Development of allergic symptoms in the infants was assessed at 4, 10 and 18 months of age. Fecal samples were collected from the mothers and infants.
Results: The risk of developing eczema[1]atopic dermatitis (AD) during the first 18 months of life was significantly reduced in infants in the probiotic group (OR: 0.231 [95% CI: 0.084-0.628] and 0.304 [0.105-0.892] at 10 and 18 months of age, respectively). Pyrosequencing analyses indicated an altered composition of the fecal microbiota at 4 months for infants who developed eczema[1]AD at 4 and 10 months of age. The proportion of Proteobacteria was significantly lower (P = 0.007) in mothers at the time of delivery who received the supplementation when compared with the control group and was positively correlated (r = 0.283, P = 0.024) with that of infants at 4 months of age. No adverse effects were related to the use of probiotics.
Conclusions: These data suggest that the prenatal and postnatal supplementation of bifidobacteria is effective in primary preventing allergic diseases. Some limited changes in the composition of fecal microbiota by the bifidobacterial supplementation were observed..
82. Urmi Roy, Mohammad Riazul Islam, Jun Ichi Nagao, Hiroshi Iida, Abdullah Al Mahin, Mengqi Li, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Bactericidal activity of nukacin ISK-1
An alternative mode of action, Bioscience, Biotechnology and Biochemistry, 10.1080/09168451.2014.918485, 78, 7, 1270-1273, 2014.01, We previously reported bacteriostatic action of nukacin ISK-1 against Bacillus subtilis JCM 1465T. Here, we found its bactericidal activity against Micrococcus luteus DSM 1790 and Staphylococcus simulans 22, showing decrease in cell viability, cell lysis, and dissipation of the membrane potential. Moreover, leakage of small molecules such as K+, suggested the formation of small-sized or specific K+-conducting-pores by nukacin ISK-1..
83. Mengqi Li, Fuminori Yoneyama, Nayu Toshimitsu, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Lethal hydroxyl radical accumulation by a lactococcal bacteriocin, lacticin Q., Antimicrobial agents and chemotherapy, 10.1128/AAC.00638-13, 57, 8, 3897-3902, 2013.08, The antimicrobial mechanism of a lactococcal bacteriocin, lacticin Q, can be described by the toroidal pore model without any receptor. However, lacticin Q showed different degrees of activity (selective antimicrobial activity) against Gram-positive bacteria even among related species. The ability of lacticin Q to induce pore formation in liposomes composed of lipids from different indicator strains indicated that its selective antimicrobial activity could not be attributed only to membrane lipid composition. We investigated the accumulation of deleterious hydroxyl radicals after exposure to lacticin Q as a contributing factor to cell death in the indicator strains. When lacticin Q of the same concentration as the MIC or minimum bactericidal concentration was added to the indicator cultures, high levels of hydroxyl radical accumulation were detected. Treatment with hydroxyl radical scavengers, thiourea and 2,2'-bipyridyl, decreased the levels of hydroxyl radical accumulation and recovered cell viability. These results suggest that, with or without pore formation, the final antimicrobial mechanism of lacticin Q is the accumulation of hydroxyl radicals, which varies by strain, resulting in the selective antimicrobial activity of lacticin Q..
84. Said E. Desouky, Kenzo Nishiguchi, Takeshi Zendo, Yasuhiro Igarashi, Paul Williams, Kenji Sonomoto, Jiro Nakayama, High-throughput screening of inhibitors targeting Agr/Fsr quorum sensing in staphylococcus aureus and enterococcus faecalis, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.120769, 77, 5, 923-927, 2013.06, Staphylococcus aureus and Enterococcus faecalis employ cyclic peptide-mediated quorum sensing (QS) systems, termed agr and fsr respectively, to regulate the expression of a series of virulence genes. To identify quorum sensing inhibitors (QSIs) that target agr/fsr systems, an efficient screening system was established. In addition to the gelatinase-induction assay to examine E. faecalis fsr QS, the use of an S. aureus agr reporter strain that carries luciferase and green fluorescence protein genes under the agr P3 promoter facilitated the development of a high-throughput screen (HTS) for QSIs. As a result of screening of 906 actinomycetes culture extracts, four showed QSI activity against the agr and fsr systems without growth inhibitory activity. The extracts were purified on a small scale, and three HPLC peaks were obtained with obvious QSI activity. In sum, the established HTS system is a promising strategy for the discovery of anti-pathogenic agents targeting cyclic peptide-mediated QS in Gram-positive pathogens..
85. Nakayama, J., Jiang, J., Watanabe, K., Chen, K., Ninxin, H., Matsuda, K., Kurakawa, T., Tsuji, H., Sonomoto, K., and Lee, Y.-K., Up to species-level community analysis of human gut microbiota by 16S rRNA amplicon pyrosequencing, Bioscience, Microflora, Food, Health, 32, 69-76, 2013.05.
86. Neuza Teixeira, Sriram Varahan, Matthew J. Gorman, Kelli L. Palmer, Anna Zaidman-Remy, Ryoji Yokohata, Jiro Nakayama, Lynn E. Hancock, António Jacinto, Michael S. Gilmore, Maria de Fátima Silva Lopes, Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors, PLoS One, 10.1371/journal.pone.0064740, 8, 5, 2013.05, Enterococcus faecalis V583 is a vancomycin-resistant clinical isolate which belongs to the hospital-adapted clade, CC2. This strain harbours several factors that have been associated with virulence, including the fsr quorum-sensing regulatory system that is known to control the expression of GelE and SprE proteases. To discriminate between genes directly regulated by Fsr, and those indirectly regulated as the result of protease expression or activity, we compared gene expression in isogenic mutants of V583 variously defective in either Fsr quorum sensing or protease expression. Quorum sensing was artificially induced by addition of the quorum signal, GBAP, exogenously in a controlled manner. The Fsr regulon was found to be restricted to five genes, gelE, sprE, ef1097, ef1351 and ef1352. Twelve additional genes were found to be dependent on the presence of GBAP-induced proteases. Induction of GelE and SprE by GBAP via Fsr resulted in accumulation of mRNA encoding lrgAB, and this induction was found to be lytRS dependent. Drosophila infection was used to discern varying levels of toxicity stemming from mutations in the fsr quorum regulatory system and the genes that it regulates, highlighting the contribution of LrgAB and bacteriocin EF1097 to infection toxicity. A contribution of SprE to infection toxicity was also detected. This work brought to light new players in E. faecalis success as a pathogen and paves the way for future studies on host tolerance mechanisms to infections caused by this important nosocomial pathogen..
87. Jiro Nakayama, Ryoji Yokohata, Mami Sato, Takashi Suzuki, Takahisa Matsufuji, Kenzo Nishiguchi, Takeshi Kawai, Yosuke Yamanaka, Koji Nagata, Masaru Tanokura, Kenji Sonomoto, Development of a peptide antagonist against fsr quorum sensing of Enterococcus faecalis., ACS chemical biology, 10.1021/cb300717f, 8, 4, 804-811, 2013.04, Enterococcus faecalis fsr quorum sensing (QS) involves an 11-residue cyclic peptide named gelatinase biosynthesis-activating pheromone (GBAP) that autoinduces two pathogenicity-related extracellular proteases in a cell density-dependent fashion. To identify anti-pathogenic agents that target fsr QS signaling, peptide antagonists of GBAP were created by our unique drug design approach based on reverse alanine scanning. First of all, a receptor-binding scaffold (RBS), [Ala(4,5,6,8,9,11)]Z-GBAP, was created, in which all amino acids within the ring region of GBAP, except for two essential aromatic residues, were substituted to alanine. Next, the substituted alanine residues were changed back to the original amino acid one by one, permitting selection of those peptide combinations exhibiting increased antagonist activity. After three cycles of this reverse alanine scan, [Ala(5,9,11)]Z-GBAP was obtained as a maximally reverted peptide (MRP) holding the strongest antagonist activity. Then, the fifth residue in MRP, which is one of the critical residues to determine agonist/antagonist activity, was further modified by substituting with different types of amino acids including unnatural amino acids. As a result, [Tyr(Bzl)(5), Ala(9,11)]Z-GBAP, named ZBzl-YAA5911, showed the strongest antagonist activity [IC(50) = 26.2 nM and Kd against GBAP receptor (FsrC) = 39.4 nM]. In vivo efficacy of this peptide was assessed with an aphakic rabbit endophthalmitis model. ZBzl-YAA5911 suppressed the translocation of E. faecalis from the aqueous humor into the vitreous cavity by more than 1 order of magnitude and significantly reduced retinal damage. We propose that ZBzl-YAA5911 or its derivatives would be useful as anti-infective agents to attenuate virulence expression in this opportunistic pathogen..
88. Shun Iwatani, Yuko Horikiri, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Bifunctional gene cluster lnqBCDEF mediates bacteriocin production and immunity with differential genetic requirements., Applied and environmental microbiology, 10.1128/AEM.03783-12, 79, 7, 2446-2449, 2013.04, A comprehensive gene disruption of lacticin Q biosynthetic cluster lnqQBCDEF was carried out. The results demonstrated the necessity of the complete set of lnqQBCDEF for lacticin Q production, whereas immunity was flexible, with LnqEF (ABC transporter) being essential for and LnqBCD partially contributing to immunity..
89. Mary K Phillips-Jones, Simon G Patching, Shalini Edara, Jiro Nakayama, Rohanah Hussain, Giuliano Siligardi, Interactions of the intact FsrC membrane histidine kinase with the tricyclic peptide inhibitor siamycin I revealed through synchrotron radiation circular dichroism., Physical chemistry chemical physics : PCCP, 10.1039/c2cp43722h, 15, 2, 444-447, 2013.01, The suitability of synchrotron radiation circular dichroism spectroscopy (SRCD) for studying interactions between the tricyclic peptide inhibitor siamycin I and the intact FsrC membrane sensor kinase in detergent micelles has been established. In the present study, tertiary structural changes demonstrate that inhibitor binding occurs at a different, non-overlapping site to the native ligand, GBAP..
90. Shun Iwatani, Fuminori Yoneyama, Shiho Miyashita, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Identification of the genes involved in the secretion and self-immunity of lacticin Q, an unmodified leaderless bacteriocin from Lactococcus lactis QU 5., Microbiology (Reading, England), 10.1099/mic.0.062943-0, 158, 12, 2927-2935, 2012.12, Lacticin Q (LnqQ) produced by Lactococcus lactis QU 5 is an unmodified linear bacteriocin, which is synthesized without an N-terminal leader peptide. In vitro synthesis and in vivo expression of LnqQ have revealed the intracellular toxicity of this leaderless peptide, as well as the necessity of a dedicated secretion and self-immunity system of producer cells. Further DNA sequencing and analysis have discovered 11 putative orf genes at the LnqQ locus. None of the orf genes showed similarities to any of the bacteriocin biosynthetic genes characterized to date; however, six orf genes (orf2q-7q), not including the structural gene (lnqQ), were highly conserved at the lacticin Z locus (orf2z-7z), which is a LnqQ homologue produced by L. lactis QU 14. ORF2q (ORF2z), the gene of which is located upstream of the structural gene, is a putative transcriptional regulator, whereas ORF6q and ORF7q (ORF6z and ORF7z) form a putative ATP-binding cassette transporter. The ORF3q-5q (ORF3z-5z) are all predicted to be membrane proteins with no clear functions. Co-expression of LnqQ and ORF3q-7q in a heterologous host allowed the extracellular production of LnqQ; additionally, the expression of ORF3q-7q rendered the host cells immune to LnqQ. This self-immunity was facilitated possibly by two means; firstly, by secreting the active LnqQ peptides, thus reducing the intracellular toxicity, and secondly, by protecting the host cells from extracellularly released LnqQ. This is the first report, to our knowledge, that describes intracellular toxicity of a leaderless bacteriocin and provides a rare example of biosynthetic genes that are required for bacteriocin secretion and immunity..
91. Rodney H Perez, Kohei Himeno, Naoki Ishibashi, Yoshimitsu Masuda, Takeshi Zendo, Koji Fujita, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Monitoring of the multiple bacteriocin production by Enterococcus faecium NKR-5-3 through a developed liquid chromatography and mass spectrometry-based quantification system., Journal of bioscience and bioengineering, 10.1016/j.jbiosc.2012.06.003, 114, 5, 490-496, 2012.11, Enterococcus faecium NKR-5-3 produces four antimicrobial peptides referred here as enterocins NKR-5-3A, B, C and D. A two-step electrospray ionization-liquid chromatography and mass spectrometry (ESI-LC/MS)-based quantification system was developed to monitor its multiple bacteriocin production profiles, which is essential in understanding the complex production regulation mechanism of strain NKR-5-3. The developed ESI-LC/MS-based quantification system can easily monitor the multiple bacteriocin production of this strain. Using the developed system, the production of enterocin NKR-5-3B was found to be not as variable as those of the other enterocins in different cultivation media. Production of enterocin NKR-5-3B was also found to have a wider optimum incubation temperature (20-30°C) than enterocins NKR-5-3A, C and D (25°C). Furthermore, at least 2 nM of the bacteriocin-like inducing peptide, enterocin NKR-5-3D, regulated the production of NKR-5-3 enterocins except enterocin NKR-5-3B. These findings taken together suggest that enterocin NKR-5-3B has an independent production regulation mechanism from the other NKR-5-3 enterocins. The developed system could effectively pin-point the production profiles of the multiple bacteriocins of E. faecium NKR-5-3 under different fermentation conditions..
92. Simon G. Patching, Shalini Edara, Pikyee Ma, Jiro Nakayama, Rohanah Hussain, Giuliano Siligardi, Mary K. Phillips-Jones, Interactions of the intact FsrC membrane histidine kinase with its pheromone ligand GBAP revealed through synchrotron radiation circular dichroism, Biochimica et Biophysica Acta - Biomembranes, 10.1016/j.bbamem.2012.02.015, 1818, 7, 1595-1602, 2012.07, FsrC is the membrane-bound histidine kinase component of the Fsr two-component signal transduction system involved in quorum sensing in the hospital-acquired infection agent Enterococcus faecalis. Synchrotron radiation circular dichroism spectroscopy was used here to study the intact purified protein solubilised in detergent micelles. Conditions required for FsrC stability in detergent were firstly determined and tested by prolonged exposure of stabilised protein to far-ultraviolet radiation. Using stabilised purified protein, far-ultraviolet synchrotron radiation circular dichroism revealed that FsrC is 61% α-helical and that it is relatively thermostable, retaining at least 57% secondary structural integrity at 90 °C in the presence or absence of gelatinase biosynthesis-activating pheromone (GBAP). Whilst binding of the quorum pheromone ligand GBAP did not significantly affect FsrC secondary structure, near-ultraviolet spectra revealed that the tertiary structure in the regions of the Tyr and Trp residues was significantly affected. Titration experiments revealed a calculated k value of 2 μM indicative of relatively loose binding of gelatinase biosynthesis-activating pheromone to FsrC. Although use of synchrotron radiation circular dichroism has been applied to membrane proteins previously, to our knowledge this is the first report of its use to determine a k value for an intact membrane protein. Based on our findings, we suggest that synchrotron radiation circular dichroism will be a valuable technique for characterising ligand binding by other membrane sensor kinases and indeed other membrane proteins in general. It further provides a valuable screening tool for membrane protein stability under a range of detergent conditions prior to downstream structural methods such as crystallisation and NMR experiments particularly when lower detergent concentrations are used. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. d d.
93. Kohei Himeno, Koji Fujita, Takeshi Zendo, Pongtep Wilaipun, Naoki Ishibashi, Yoshimitsu Masuda, Fuminori Yoneyama, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Identification of enterocin NKR-5-3C, a novel class IIa bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.120089, 76, 6, 1245-1247, 2012.06, The structure of enterocin NKR-5-3C, an antilisterial bacteriocin produced by a multiple bacteriocin producer, Enterococcus faecium NKR-5-3, was determined. Enterocin NKR-5-3C is a novel class IIa bacteriocin that possesses an YGNGL motif sequence and two disulfide bridges in its structure. It is encoded on gene ent53C together with an 18-amino-acid-residue double glycine leader peptide..
94. Naoki Ishibashi, Kohei Himeno, Koji Fujita, Yoshimitsu Masuda, Rodney Honrada Perez, Takeshi Zendo, Pongtep Wilaipun, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Purification and characterization of multiple bacteriocins and an inducing peptide produced by Enterococcus faecium NKR-5-3 from Thai fermented fish, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.110972, 76, 5, 947-953, 2012.05, Enterocins NKR-5-3A, B, C, and D were purified from the culture supernatant of Enterococcus faecium NKR-5-3 and characterized. Among the four purified peptides, enterocin NKR-5-3A (5242.3 Da) was identical to brochocin A, produced by Brochothrix campestris ATCC 43754, in mature peptides, and its putative synergistic peptide, enterocin NKR-5-3Z, was found to be encoded in ent53Z downstream of ent53A, encoding enterocin NKR-5-3A. Enterocin NKR-5-3B (6316.4Da) showed a broad antimicrobial spectrum, and enterocin NKR-5-3C (4512.8 Da) showed high activity against Listeria. Enterocin NKR-5-3D (2843.5 Da), showing high homology to an inducing peptide produced by Lactobacillus sakei 5, induced the production of the enterocins. The enterocins showed different antimicrobial spectra and intensities. E. faecium NKR-5-3 concomitantly produced enterocins NKR-5-3A, B, C, and D which probably belong to different classes of bacteriocins. Furthermore, NKR-5-3 production was induced by enterocin NKR-5-3D..
95. Neuza Teixeira, Sofia Santos, Paulo Marujo, Ryoji Yokohata, Vijayalakshmi S Iyer, Jiro Nakayama, Lynn E Hancock, Pascale Serror, Maria de Fátima Silva Lopes, The incongruent gelatinase genotype and phenotype in Enterococcus faecalis are due to shutting off the ability to respond to the gelatinase biosynthesis-activating pheromone (GBAP) quorum-sensing signal., Microbiology (Reading, England), 10.1099/mic.0.055574-0, 158, 2, 519-528, 2012.02, The concomitant presence of a complete fsr quorum-sensing system and gelE-sprE operons in Enterococcus faecalis is known to be essential for the detection of gelatinase activity. However, there are reports of the absence of gelatinase activity despite the presence of complete fsr and gelE loci. In order to understand this incongruence between genotype and phenotype we sequenced fsr and gelE loci of the E. faecalis LN68 strain, which was previously found to carry both operons but to lack gelatinase activity. Of the 59 nucleotide differences detected compared with the gelatinase-positive V583 strain, we found a nonsense mutation (a premature STOP codon) predicted to truncate the ATPase sensor domain of the FsrC protein, responsible for sensing and transducing the signal from the quorum-sensing molecule. Strain LN68 was highly affected in the expression of the gelE and sprE genes, further supporting the lack of Fsr-dependent gelE induction. When we constructed a V583 mutant with the same premature stop mutation in the fsrC gene the resulting strain was no longer able to degrade gelatin. We conclude that the reduced ability to transduce the quorum-sensing signal of the prematurely truncated FsrC protein is sufficient to explain the negative gelatinase phenotype. As the incongruent genotype and phenotype is detected in natural isolates, we believe that the silencing of the quorum-sensing system Fsr may be beneficial for some E. faecalis strains..
96. Mohammad R Islam, Mami Nishie, Jun-ichi Nagao, Takeshi Zendo, Sandro Keller, Jiro Nakayama, Daisuke Kohda, Hans-Georg Sahl, Kenji Sonomoto, Ring A of nukacin ISK-1: a lipid II-binding motif for type-A(II) lantibiotic., Journal of the American Chemical Society, 10.1021/ja300007h, 134, 8, 3687-3690, 2012.02, Ring A of nukacin ISK-1, which is also present in different type-A(II) lantibiotics, resembles a lipid II-binding motif (TxS/TxD/EC, x denotes undefined residues) similar to that present in mersacidin (type-B lantibiotics), which suggests that nukacin ISK-1 binds to lipid II as a docking molecule. Results from our experiments on peptidoglycan precursor (UDP-MurNAc-pp) accumulation and peptide antagonism assays clearly indicated that nukacin ISK-1 inhibits cell-wall biosynthesis, accumulating lipid II precursor inside the cell, and the peptide activity can be repressed by lipid I and lipid II. Interaction analysis of nukacin ISK-1 and different ring A variants with lipid II revealed that nukacin ISK-1 and nukacin D13E (a more active variant) have a high affinity (K(D) = 0.17 and 0.19 μM, respectively) for lipid II, whereas nukacin D13A (a less active variant) showed a lower affinity, and nukacin C14S (a negative variant lacking the ring A structure) exhibited no interaction. Therefore, on the basis of the structural similarity and positional significance of the amino acids in this region, we concluded that nukacin ISK-1 binds lipid II via its ring A region and may lead to the inhibition of cell-wall biosynthesis..
97. Naruhiko Sawa, Pongtep Wilaipun, Seisuke Kinoshita, Takeshi Zendo, Vichien Leelawatcharamas, Jiro Nakayama, Kenji Sonomoto, Isolation and characterization of enterocin W, a novel two-peptide lantibiotic produced by Enterococcus faecalis NKR-4-1., Applied and environmental microbiology, 10.1128/AEM.06497-11, 78, 3, 900-903, 2012.02, Enterococcus faecalis NKR-4-1 isolated from pla-ra produces a novel two-peptide lantibiotic, termed enterocin W, comprising Wα and Wβ. The structure of enterocin W exhibited similarity with that of plantaricin W. The two peptides acted synergistically, and their order of binding to the cell membrane was important for their inhibitory activity..
98. Neuza Teixeira, Sofia Santos, Paulo Marujo, Ryoji Yokohata, Vijayalakshmi S. Iyer, Jiro Nakayama, Lynn E. Hancock, Pascale Serror, Maria de Fátima Silva Lopes Lopes, The incongruent gelatinase genotype and phenotype in Enterococcus faecalis are due to shutting off the ability to respond to the gelatinase biosynthesis-activating pheromone (GBAP) quorum-sensing signal, Microbiology, 10.1099/mic.0.055574-0, 158, 2, 519-528, 2012.02, The concomitant presence of a complete fsr quorum-sensing system and gelE-sprE operons in Enterococcus faecalis is known to be essential for the detection of gelatinase activity. However, there are reports of the absence of gelatinase activity despite the presence of complete fsr and gelE loci. In order to understand this incongruence between genotype and phenotype we sequenced fsr and gelE loci of the E. faecalis LN68 strain, which was previously found to carry both operons but to lack gelatinase activity. Of the 59 nucleotide differences detected compared with the gelatinase-positive V583 strain, we found a nonsense mutation (a premature STOP codon) predicted to truncate the ATPase sensor domain of the FsrC protein, responsible for sensing and transducing the signal from the quorum-sensing molecule. Strain LN68 was highly affected in the expression of the gelE and sprE genes, further supporting the lack of Fsrdependent gelE induction. When we constructed a V583 mutant with the same premature stop mutation in the fsrC gene the resulting strain was no longer able to degrade gelatin. We conclude that the reduced ability to transduce the quorum-sensing signal of the prematurely truncated FsrC protein is sufficient to explain the negative gelatinase phenotype. As the incongruent genotype and phenotype is detected in natural isolates, we believe that the silencing of the quorum-sensing system Fsr may be beneficial for some E. faecalis strains..
99. Tijo Varghese Puramattathu, Mohammad R Islam, Mami Nishie, Sae Yanagihara, Jun-ichi Nagao, Ken-ichi Okuda, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Enhanced production of nukacin D13E in Lactococcus lactis NZ9000 by the additional expression of immunity genes., Applied microbiology and biotechnology, 10.1007/s00253-011-3563-1, 93, 2, 671-678, 2012.01, Nukacin D13E (D13E) is a variant of type-A(II) lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1. D13E exhibited a twofold higher specific antimicrobial activity than nukacin ISK-1 against a number of Gram-positive bacteria. We previously reported the heterologous production of D13E in Lactococcus lactis NZ9000 under the control of nisin-controlled gene expression system. In this study, we demonstrated enhanced production of D13E by the additional expression of immunity genes, nukFEG. The nukacin ISK-1 immunity, conferred by the ABC transporter complex, NukFEG, and the lantibiotic-binding protein, NukH, was not overwhelmed by D13E. The additional NukFEG resulted in a fourfold increase in the immunity level of the strain and a 5.2-fold increase in D13E production. The additional NukFEGH-expressing strain with the highest D13E immunity showed reduced level of production. Further improvement in D13E production was achieved by using pH-controlled batch fermentation..
100. Y. Masuda, T. Zendo, N. Sawa, R. H. Perez, J. Nakayama, K. Sonomoto, Characterization and identification of weissellicin Y and weissellicin M, novel bacteriocins produced by Weissella hellenica QU 13, Journal of Applied Microbiology, 10.1111/j.1365-2672.2011.05180.x, 112, 1, 99-108, 2012.01, Aims: To identify and characterize novel bacteriocins from Weissella hellenica QU 13. Methods and Results: Weissella hellenica QU 13, isolated from a barrel used to make Japanese pickles, produced two novel bacteriocins termed weissellicin Y and weissellicin M. The primary structures of weissellicins Y and M were determined, and their molecular masses were determined to be 4925·12 and 4968·40Da, respectively. Analysis of the DNA sequence encoding the bacteriocins revealed that they were synthesized and secreted without N-terminal extensions such as leader sequences or sec signal peptides. Weissellicin M showed significantly high and characteristic homology with enterocins L50A and L50B, produced by Enterococcus faecium L50, while weissellicin Y showed no homology with any other known bacteriocins. Both bacteriocins showed broad antimicrobial spectra, with especially high antimicrobial activity against species, which contaminate pickles, such as Bacillus coagulans, and weissellicin M showed relatively higher activity than weissellicin Y. Furthermore, the stability of weissellicin M against pH and heat was distinctively higher than that of weissellicin Y. Conclusions: Weissella hellenica QU 13 produced two novel leaderless bacteriocins, weissellicin Y and weissellicin M, and weissellicin M exhibited remarkable potency that could be employed by pickle-producing industry. Significance and Impact of the Study:  This study is the first report, which represents a complete identification and characterization of novel leaderless bacteriocins from Weissella genus. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology..
101. M. Nakphaichit, S. Thanomwongwattana, C. Phraephaisarn, N. Sakamoto, S. Keawsompong, J. Nakayama, S. Nitisinprasert, The effect of including Lactobacillus reuteri KUB-AC5 during post-hatch feeding on the growth and ileum microbiota of broiler chickens, Poultry Science, 10.3382/ps.2011-01637, 90, 12, 2753-2765, 2011.12, The probiotic strain Lactobacillus reuteri KUB-AC5, which was originally isolated from chicken intestine, was fed to newborn broiler chicks for the first week post-hatch. The growth and ileum microbiota of the chickens were carefully monitored for 6 wk. The inclusion of 5 log cfu/g of feed statistically increased the BW gain in the first week compared with that of the control group, but this effect did not continue thereafter. Significant effects on host feed consumption and the feed-to-growth conversion ratio were not detected. The total amount and composition of ileum bacteria were investigated by quantitative PCR and pyrosequencing of the 16S rRNA gene (rDNA), respectively, and were compared between the control and the probiotic-treated groups. The amount of total bacterial 16S rDNA in ileum samples at d 42 was 5 times higher in the probiotic group than in the control, whereas no significant difference was observed at d 21. A composition analysis revealed the establishment of lactobacillienriched microbiota in the probiotic-treated chickens at d 42. At this point, the population level and species diversity of lactobacilli were significantly enhanced compared with those of the control group. In addition, Actinobacteria, mainly genera Corynebacterium and Dietzia, were also statistically higher in the probiotic group. However, Proteobacteria, including those of the family Campylobacterales and some other nonbeneficial bacterial groups, were decreased in the probiotic group at the growing stage. Therefore, with probiotic supplementation, it was demonstrated that Lactobacillus reuteri KUB-AC5 in the early post-hatching period had a delayed effect on ileum microbiota, which resulted in the enrichment of potentially beneficial lactobacilli and the suppression of Proteobacteria, including nonbeneficial bacterial groups. © 2011 Poultry Science Association Inc..
102. Jiro Nakayama, Takako Kobayashi, Shigemitsu Tanaka, Yuki Korenori, Atsushi Tateyama, Naoshige Sakamoto, Chikako Kiyohara, Taro Shirakawa, Kenji Sonomoto, Aberrant structures of fecal bacterial community in allergic infants profiled by 16S rRNA gene pyrosequencing., FEMS immunology and medical microbiology, 10.1111/j.1574-695X.2011.00872.x, 63, 3, 397-406, 2011.12, We investigated the correlation between fecal bacteria composition in early infancy and the prevalence of allergic diseases in late infancy. The fecal microbiota in the first 2 months was profiled using the 16S rRNA V6 short-tag sequences in the community and statistically compared between two groups of subjects who did and did not show allergic symptoms in the first 2 years (n = 11 vs. 11). In the allergic group, genus Bacteroides at 1 month and genera Propionibacterium and Klebsiella at 2 months were more abundant, and genera Acinetobacter and Clostridium at 1 month were less abundant than in the nonallergic group. Allergic infants who showed high colonization of Bacteroides and/or Klebsiella showed less colonization of Clostridium perfringens/butyricum, suggesting antagonism between these bacterial groups in the gastrointestinal tract. It was also remarkable that the relative abundance of total Proteobacteria, excluding genus Klebsiella, was significantly lower in the allergic than in the nonallergic group at the age of 1 month. These results indicate that pyrosequence-based 16S rRNA gene profiling is valid to find the intestinal microbiotal disorder that correlates with allergy development in later life..
103. Jun Ichi Nagao, Kouki Shioya, Yoshitaka Harada, Ken Ichi Okuda, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Engineering unusual amino acids into peptides using lantibiotic synthetase, Heterologous Gene Expression in E.coli Methods and Protocols, 10.1007/978-1-61737-967-3_13, 705, 225-236, 2011.12, Alteration of protein structure and function by introducing unusual amino acids has great potential to develop new biological tool and to produce novel therapeutic agents. Lantibiotics produced by Gram-positive bacteria are ribosomally synthesized and post-translationally modified antimicrobial peptides. The modification enzyme involved in lantibiotic biosynthesis can catalyze the formation of unusual amino acids in the nascent lantibiotic prepeptide. Here, a novel methodology on the lantibiotic nukacin ISK-1 is described for engineering unusual amino acid residues into hexa-histidine-tagged (His-tagged) prepeptide NukA by the modification enzyme NukM in Escherichia coli. Co-expression of His-tagged NukA and NukM, purification of the resulting His-tagged prepeptide by affinity chromatography, and subsequent mass spectrometry analysis show that the prepeptide is converted into a postulated peptide with decrease in mass which results from the formation of unusual amino acids such as dehydrated amino acid, lanthionine, or 3-methyl lanthionine at the expected positions. The modified prepeptide can be readily obtained by one-step purification. This strategy will thus be a simple and powerful tool for introducing unusual amino acid residues aimed at peptide engineering..
104. Akira Tsujimura, Tetsuya Takao, Yasushi Miyagawa, Hidenobu Okuda, Keisuke Yamamoto, Shinichiro Fukuhara, Jiro Nakayama, Tomohiro Ueda, Hiroshi Kiuchi, Toshiaki Hirai, Yuichi Tsujimoto, Hidenobu Miura, Nobuteru Kanno, Makoto Higashino, Yoshihiro Nakamura, Kenji Nishimura, Norio Nonomura, Survey of Overactive Bladder Symptoms Influencing Bother Before and After Treatment With Tamsulosin Hydrochloride in Japanese Patients With Benign Prostatic Hyperplasia, UROLOGY, 10.1016/j.urology.2011.05.032, 78, 5, 1058-1062, 2011.11, OBJECTIVE To evaluate the relation between bother and overactive bladder (OAB) symptoms in patients with benign prostatic hyperplasia (BPH) patients using questionnaires: the BPH Impact Index (BII) and the OAB symptom score (OABSS). Annoyance from BPH usually provides the basis for a patient's decision to seek medical treatment. However, a study investigating the bother caused by OAB symptoms in patients with BPH and OAB has not been fully conducted.
METHODS The present study included 100 male patients who were diagnosed with BPH and OAB according to questionnaire criteria. All patients were instructed to take tamsulosin for 28 days. The relation between the BII and OABSS was assessed to determine the factors influencing OAB symptoms on the BII before and after treatment.
RESULTS The BII correlated positively with the OABSS, and multivariate analysis showed that the subscore of urgency was the only independent factor influencing the BII. Even after treatment, lower urinary tract symptoms were diagnosed as OAB using the OABSS criteria in 45 (45.0%) of the 100 patients. In these patients, the BII still correlated positively with the OABSS. However, multivariate analysis showed that the subscore of urgency incontinence, not urgency, was the only independent factor influencing the BII, although the subscore of urgency incontinence was significantly decreased with tamsulosin treatment.
CONCLUSION The degree of bother correlated with the degree of OAB symptoms in patients with BPH and OAB at baseline and after treatment with tamsulosin. The OAB symptom causing the bother was altered by treatment with tamsulosin in these patients. UROLOGY 78: 1058-1062, 2011. (C) 2011 Elsevier Inc..
105. Pikyee Ma, Kenzo Nishiguchi, Hayley M Yuille, Lianne M Davis, Jiro Nakayama, Mary K Phillips-Jones, Anti-HIV siamycin I directly inhibits autophosphorylation activity of the bacterial FsrC quorum sensor and other ATP-dependent enzyme activities., FEBS letters, 10.1016/j.febslet.2011.07.026, 585, 17, 2660-2664, 2011.09, Siamycin I disrupts growth and quorum sensing in Enterococcus faecalis. Using purified intact protein, we demonstrate here that quorum membrane sensor kinase FsrC is a direct target of siamycin I, reducing pheromone-stimulated autophosphorylation activity by up to 91%. Inhibition was non-competitive with ATP as substrate. Other ATP-binding enzymes were also inhibited, including nine other membrane sensor kinases of E. faecalis, Rhodobacter sphaeroides PrrB, porcine Na(+)-dependent ATPase and the catalytic subunit of bovine protein kinase A, but not bacterial β-galactosidase, confirming targeted inhibition of a wide range of ATP dependent reactions, and elucidating a likely mechanism underlying the lethality of the inhibitor..
106. Shoichiro Horita, Yosuke Yamanaka, Akihiro Yamamura, Akitoshi Okada, Jiro Nakayama, Koji Nagata, Masaru Tanokura, Crystallization and preliminary X-ray analysis of a putative sensor histidine kinase domain: the C-terminal domain of HksP4 from Aquifex aeolicus VF5., Acta crystallographica. Section F, Structural biology and crystallization communications, 10.1107/S1744309111018434, 67, 7, 803-807, 2011.07, The histidine kinase domain of the cytoplasmic protein HksP4 from the hyperthermophilic bacterium Aquifex aeolicus VF5, located in the C-terminal half of the protein, was expressed, purified and crystallized. Diffraction-quality crystals were obtained in the presence of adenosine triphosphate (ATP) or adenosine 5'-(β,γ-imido)triphosphate (AMPPNP) by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals obtained in the presence of ATP and AMPPNP diffracted X-rays to 3.1 and 2.9 Å resolution, respectively, on BL-5A at Photon Factory (Ibaraki, Japan) and were found to belong to the same space group P2(1)2(1)2(1), with unit-cell parameters a=80.2, b=105.5, c=122.0 Å and a=81.5, b=105.5, c=130.9 Å, respectively. Their Matthews coefficients (VM=2.74 and 2.51 Å3 Da(-1), respectively) indicated that both crystals contained four protein molecules per asymmetric unit..
107. Fuminori Yoneyama, Kanako Ohno, Yuichi Imura, Mengqi Li, Takeshi Zendo, Jiro Nakayama, Katsumi Matsuzaki, Kenji Sonomoto, Lacticin Q-mediated selective toxicity depending on physicochemical features of membrane components, Antimicrobial Agents and Chemotherapy, 10.1128/AAC.00808-10, 55, 5, 2446-2450, 2011.05, Lacticin Q, a lactococcal pore-forming bacteriocin, shows activity toward Gram-positive bacteria but not Gram-negative bacteria. Lacticin Q did not induce permeability of the outer membrane of Gram-negative bacteria. Experiments using model membranes containing outer membrane components suggested that lacticin Q binds to the outer membrane of Gram-negative bacteria but is unable to penetrate it. The lack of activity of lacticin Q was attributed to physicochemical features of the outer membrane components..
108. Mami Nishie, Makoto Sasaki, Jun Ichi Nagao, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Lantibiotic transporter requires cooperative functioning of the peptidase domain and the ATP binding domain, Journal of Biological Chemistry, 10.1074/jbc.M110.212704, 286, 13, 11163-11169, 2011.04, Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics that contain unusual amino acids such as dehydro and lanthionine residues. Nukacin ISK-1 is a class II lantibiotic, whose precursor peptide (NukA) is modified by NukM to form modified NukA. ATP-binding cassette (ABC) transporter NukT is predicted to cleave off the N-terminal leader peptide of modified NukA and secrete the mature peptide. Multiple sequence alignments revealed that NukT has an N-terminal peptidase domain (PEP) and a C-terminal ATP binding domain (ABD). Previously, in vitro reconstitution of NukT has revealed that NukT peptidase activity depends on ATP hydrolysis. Here, we constructed a series of NukT mutants and investigated their transport activity in vivo and peptidase activity in vitro. Most of the mutations of the conserved residues of PEP or ABD resulted in failure of nukacin ISK-1 production and accumulation of modified NukA inside the cells. NukT(N106D) was found to be the only mutant capable of producing nukacin ISK-1. Asn106 is conserved as Asp in other related ABC transporters. Additionally, an in vitro peptidase assay of NukT mutants demonstrated that PEP is on the cytosolic side and all of the ABD mutants as well as PEP (with the exception of NukT(N106D)) did not have peptidase activity in vitro. Taken together, these observations suggest that the leader peptide is cleaved off inside the cells before peptide secretion; both PEP and ABD are important for NukT peptidase activity, and cooperation between these two domains inside the cells is indispensable for proper functioning of NukT..
109. Naoshige Sakamoto, Shigemitsu Tanaka, Kenji Sonomoto, Jiro Nakayama, 16S rRNA pyrosequencing-based investigation of the bacterial community in nukadoko, a pickling bed of fermented rice bran., International journal of food microbiology, 10.1016/j.ijfoodmicro.2010.10.017, 144, 3, 352-359, 2011.01, Nukadoko is a naturally fermented rice bran mash traditionally used for pickling vegetables in Japan; its refreshment and fermentation cycles sometimes continue for many years. Here, we investigated the structure and dynamics of the bacterial community in nukadoko by conducting pyrosequencing and quantitative polymerase chain reaction (PCR) analyses of 16S ribosomal RNA genes (rDNA). Of the 16 different samples studied, 13 showed Lactobacillus-dominated microbiota, suggesting that aged nukadoko samples tend to realize a niche, favorable Lactobacillus species. The lactic acid bacterial community of each of the 16 samples was classified into 3 types according to the presence or absence of 2 predominant species, Lactobacillus namurensis and Lactobacillus acetotolerans. The dynamics of the bacterial community during fermentation and the subsequent ripening process were examined using a laboratory model of nukadoko inoculated with an aged nukadoko sample (inoculated model). Lb. namurensis grew rapidly in the first 2 days, accompanied with a rapid decrease in pH and an increase in lactate levels, while Lb. acetotolerans grew with a longer doubling time and slow acidification during the 20 days after inoculation. On the other hand, spontaneous fermentation of the nukadoko model prepared from fresh rice bran without the nukadoko inoculation (inoculant-free model), showed the growth of some non-Lactobacillus species such as staphylococci and bacilli within the first 10 days; thereafter, Lb. namurensis was dominant, while Lb. acetotolerans was not detected during the 20-day experimental period. These results suggest that the naturally established Lactobacillus community in aged nukadoko is effectively involved in the biocontrol of the microbial community of nukadoko during the refreshment and fermentation cycles..
110. Akira Tsujimura, Tetsuya Takao, Yasushi Miyagawa, Keisuke Yamamoto, Shinichiro Fukuhara, Jiro Nakayama, Hiroshi Kiuchi, Nakamori Suganuma, Tadashi Nakamura, Takayuki Kumano-go, Yoshiro Sugita, Norio Nonomura, Akihiko Okuyama, Urgency Is an Independent Factor for Sleep Disturbance in Men with Obstructive Sleep Apnea, UROLOGY, 10.1016/j.urology.2010.01.070, 76, 4, 967-970, 2010.10, OBJECTIVES The relationship between overactive bladder (OAB) symptoms, other than nocturia, and sleep, has not been fully evaluated, although a close relationship between nocturia and sleep disturbance has been reported. In the present study, we evaluated the relationship between OAB symptoms and several polysomnography (PSG) parameters in middle-age men with sleep disturbance, especially to clarify whether urgency as the hallmark symptom of an OAB is independently associated with sleep quality.
METHODS A total of 32 men >40 years of age (mean age 58.0 +/- 12.6), who had been diagnosed with obstructive sleep apnea syndrome by PSG, were included in the present study. Their OAB symptoms were evaluated using the OAB symptom score (OABSS) before PSG. The relationship between the OABSS and several parameters, such as sleeping time, sleeping efficiency, sleep latency, percentage of rapid eye movement during sleeping time, and apnea/hypopnea index obtained from PSG, was evaluated.
RESULTS Multivariate analysis showed that only sleeping efficiency was an influencing factor on the total OABSS. Of the 4 subscores of OABSS, including frequency, nocturia, urgency, and urgency incontinence, multivariate analysis showed that the subscores of nocturia and urgency were independent influencing factors on sleeping efficiency. Nocturia correlated negatively with sleeping efficiency (Pearson's correlation 0.533, P <.01 and urgency also correlated negatively with sleeping efficiency correlation p>CONCLUSIONS We found that urgency and nocturia were factors that independently affected sleep or were affected by sleep quality, although only the association of nocturia with sleep disturbance has been the focus of previous studies. UROLOGY 76: 967-970, 2010. (C) 2010 Elsevier Inc..
111. N. Sawa, K. Okamura, T. Zendo, K. Himeno, J. Nakayama, K. Sonomoto, Identification and characterization of novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15, Journal of Applied Microbiology, 10.1111/j.1365-2672.2009.04653.x, 109, 1, 282-291, 2010.07, Aim: To characterize novel multiple bacteriocins produced by Leuconostoc pseudomesenteroides QU 15. Methods and Results: Leuconostoc pseudomesenteroides QU 15 isolated from Nukadoko (rice bran bed) produced novel bacteriocins. By using three purification steps, four antimicrobial peptides termed leucocin A (δC7), leucocin A-QU 15, leucocin Q and leucocin N were purified from the culture supernatant. The amino acid sequences of leucocin A (δC7) and leucocin A-QU 15 were identical to that of leucocin A-UAL 187 belonging to class IIa bacteriocins, but leucocin A (δC7) was deficient in seven C-terminal residues. Leucocin Q and leucocin N are novel class IId bacteriocins. Moreover, the DNA sequences encoding three bacteriocins, leucocin A-QU 15, leucocin Q and leucocin N were obtained. Conclusions: These bacteriocins including two novel bacteriocins were identified from Leuc. pseudomesenteroides QU 15. They showed similar antimicrobial spectra, but their intensities differed. The C-terminal region of leucocin A-QU 15 was important for its antimicrobial activity. Leucocins Q and N were encoded by adjacent open reading frames (ORFs) in the same operon, but leucocin A-QU 15 was not. Significance and Impact of Study: These leucocins were produced concomitantly by the same strain. Although the two novel bacteriocins were encoded by adjacent ORFs, a characteristic of class IIb bacteriocins, they did not show synergistic activity. © 2010 The Authors..
112. Chih-Bo Hu, Wanna Malaphan, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Enterocin X, a novel two-peptide bacteriocin from Enterococcus faecium KU-B5, has an antibacterial spectrum entirely different from those of its component peptides., Applied and environmental microbiology, 10.1128/AEM.02264-09, 76, 13, 4542-4545, 2010.07, Enterocin X, composed of two antibacterial peptides (Xalpha and Xbeta), is a novel class IIb bacteriocin from Enterococcus faecium KU-B5. When combined, Xalpha and Xbeta display variably enhanced or reduced antibacterial activity toward a panel of indicators compared to each peptide individually. In E. faecium strains that produce enterocins A and B, such as KU-B5, only one additional bacteriocin had previously been known..
113. Ken-ichi Okuda, Sae Yanagihara, Tomomichi Sugayama, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Functional significance of the E loop, a novel motif conserved in the lantibiotic immunity ATP-binding cassette transport systems., Journal of bacteriology, 10.1128/JB.00003-10, 192, 11, 2801-2808, 2010.06, Lantibiotics are peptide-derived antibacterial substances produced by some Gram-positive bacteria and characterized by the presence of unusual amino acids, like lanthionines and dehydrated amino acids. Because lantibiotic producers may be attacked by self-produced lantibiotics, they express immunity proteins on the cytoplasmic membrane. An ATP-binding cassette (ABC) transport system mediated by the LanFEG protein complex is a major system in lantibiotic immunity. Multiple-sequence alignment analysis revealed that LanF proteins contain the E loop, a variant of the Q loop, which is a well-conserved motif in the nucleotide-binding domains (NBDs) of general ABC transporters. To elucidate E loop function, we introduced a mutation in the NukF protein, which is involved in the nukacin-ISK-1 immunity system. Amino acid replacement of glutamic acid in the E loop with glutamine (E85Q) resulted in slight decreases in the immunity level and transport activity. Additionally, the E85A mutation severely impaired the immunity level and transport activity. On the other hand, ATPase activities of purified E85Q and E85A mutants were almost similar to that of the wild type. These results suggested that the E loop found in ABC transporters involved in lantibiotic immunity plays a significant role in the function of these transporters, especially in the structural change of transmembrane domains..
114. Kouki Shioya, Yoshitaka Harada, Jun-ichi Nagao, Jiro Nakayama, Kenji Sonomoto, Characterization of modification enzyme NukM and engineering of a novel thioether bridge in lantibiotic nukacin ISK-1., Applied microbiology and biotechnology, 10.1007/s00253-009-2334-8, 86, 3, 891-899, 2010.04, The lantibiotic nukacin ISK-1 is an antimicrobial peptide containing unusual amino acids such as lanthionine and dehydrobutyrine. The nukacin ISK-1 prepeptide (NukA) undergoes posttranslational modifications, such as the dehydration and cyclization reactions required to form the unusual amino acids by the modification enzyme NukM. We have previously constructed a system for the introduction of unusual amino acids into NukA by coexpression of NukM in Escherichia coli. Using this system, we describe the substrate specificity of NukM by the coexpression of a series of NukA mutants. Our results revealed the following characteristics of NukM: (1) its dehydration activity is not coupled to its cyclization activity; (2) its dehydration activity is site-specific; (3) the length of the substrate is important for its dehydration activity. Furthermore, we succeeded in introducing a novel thioether bridge in NukA by replacing an unmodified Ser at position 27 with a Cys residue..
115. Fuminori Yoneyama, Kouki Shioya, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Effect of a negatively charged lipid on membrane-lacticin Q interaction and resulting pore formation, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.90666, 74, 1, 218-221, 2010.02, Lacticin Q is an antimicrobial peptide that forms pores on membranes. We investigated effects of negatively charged lipids on the binding and pore formation of lacticin Q with liposomes by surface plasmon resonance analysis and fluorescence dye leakage experiments respectively. Negatively charged lipids accelerated the binding of lacticin Q on the membranes and the resulting pore formation. However, the acceleration was not an essential factor in the killing activity of lacticin Q, since pore-forming activities against electrically neutral and negatively charged liposomes occurred similarly..
116. Akira Tsujimura, Shingo Takada, Yasuhiro Matsuoka, Jiro Nakayama, Tetsuya Takao, Yasushi Miyagawa, Mina Sonoda, Hitoshi Nishizawa, Hiromi Iwahashi, Tohru Funahashi, Norio Nonomura, Akihiko Okuyama, Adiponectin and testosterone in patients with symptoms of late-onset hypogonadism: Is there a link?, INTERNATIONAL JOURNAL OF UROLOGY, 10.1111/j.1442-2042.2009.02376.x, 16, 10, 830-835, 2009.10, Objectives:
To examine the correlation between testosterone and adiponectin in symptomatic late-onset hypogonadism (LOH) patients.
Methods:
The study included 174 patients (> 40 years old) with at least one LOH symptom and an Aging Male Symptoms score > 26. The correlation between serum adiponectin levels and various factors was investigated by simple and multiple regression analyses. Serum adiponectin levels before and after testosterone replacement therapy (TRT) in 43 patients with serum free testosterone Results:
Serum adiponectin levels increased with increased age (P Conclusions:
Serum adiponectin is not related to serum testosterone in symptomatic LOH patients, suggesting that TRT in these subjects does not pose a higher risk of inducing a metabolic syndrome..
117. Fuminori Yoneyama, Yuichi Imura, Kanako Ohno, Takeshi Zendo, Jiro Nakayama, Katsumi Matsuzaki, Kenji Sonomoto, Peptide-lipid huge toroidal pore, a new antimicrobial mechanism mediated by a lactococcal bacteriocin, lacticin Q., Antimicrobial agents and chemotherapy, 10.1128/AAC.00209-09, 53, 8, 3211-3217, 2009.08, Lacticin Q is a pore-forming bacteriocin produced by Lactococcus lactis QU 5, and its antimicrobial activity is in the nanomolar range. Lacticin Q induced calcein leakage from negatively charged liposomes. However, no morphological changes in the liposomes were observed by light scattering. Concomitantly with the calcein leakage, lacticin Q was found to translocate from the outer to the inner leaflet of the liposomes, after it initially bound to the membrane within 2 s. Lacticin Q also induced lipid flip-flop. These results reveal that the antimicrobial mechanism of lacticin Q can be described by the toroidal pore model. This is the first report of a bacteriocin of gram-positive bacteria that forms a toroidal pore. From liposomes, lacticin Q leaked fluorescence-labeled dextran with a diameter of 4.6 nm. In addition, lacticin Q caused the leakage of small proteins, such as the green fluorescent protein, from live bacterial cells. There are no other reports of antimicrobial peptides that exhibit protein leakage properties. The proposed pore formation model of lacticin Q is as follows: (i) quick binding to outer membrane leaflets; (ii) the formation of at least 4.6-nm pores, causing protein leakage with lipid flip-flop; and (iii) the migration of lacticin Q molecules from the outer to the inner membrane leaflets. Consequently, we termed the novel pore model in the antimicrobial mechanism of lacticin Q a "huge toroidal pore.".
118. Sikder M Asaduzzaman, Jun-ichi Nagao, Hiroshi Iida, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Nukacin ISK-1, a bacteriostatic lantibiotic., Antimicrobial agents and chemotherapy, 10.1128/AAC.01623-08, 53, 8, 3595-8, 2009.08, We determined the mode of action of nukacin ISK-1. It did not cause membrane potential dissipation or the efflux of ATP or K(+) ions from the cells of a sensitive bacterial strain; however, it blocked the membrane depolarization activity of nisin. Nukacin ISK-1-treated cells had single arrangements of cells without the formation of a complete septum. A remarkable reduction in cell wall width was also observed, but cytoplasmic content was unaffected. We concluded that nukacin ISK-1 is bacteriostatic..
119. Jun-Ichi Nagao, Yoshiko Morinaga, Mohammad R Islam, Sikder M Asaduzzaman, Yuji Aso, Jiro Nakayama, Kenji Sonomoto, Mapping and identification of the region and secondary structure required for the maturation of the nukacin ISK-1 prepeptide., Peptides, 10.1016/j.peptides.2009.05.021, 30, 8, 1412-1420, 2009.08, The prepeptide NukA of the lantibiotic nukacin ISK-1 consists of an N-terminal leader peptide followed by a propeptide moiety that undergoes post-translational modifications, that is, formation of unusual amino acids by NukM, cleavage of the leader peptide and transport by NukT to yield a mature peptide. To identify the region and conformation required for the maturation of prepeptide, we expressed a series of NukA mutants, mutants with the N-terminus-truncated leader peptide and site-directed mutants with conserved residues in the leader peptide of type A(II) lantibiotics, which were evaluated on the basis of the production of nukacin ISK-1. In addition, the secondary structure data of NukA mutants or fragments were obtained by circular dichroism spectra. The results indicated the importance of the alpha-helical leader peptide with hydrophobic and hydrophilic orientation consisting of the conserved residues in type A(II) lantibiotics. The expression data from various combinations of the chimeric prepeptides consisting of NukA and LctA (the prepeptide of lacticin 481, which shows high identity with NukA) further revealed that the amino acid difference at the C-terminus of the propeptide moiety between NukA and LctA, especially His at position 15 and Phe at position 19, was important for the maturation processes by the nukacin ISK-1 biosynthetic enzymes. Our findings suggest that the determinants in NukA were critically involved in the biosynthesis of nukacin ISK-1 and would thus be important for recognition by the nukacin ISK-1 biosynthetic enzymes..
120. Mohammad R. Islam, K. Shioya, J. Nagao, M. Nishie, H. Jikuya, T. Zendo, J. Nakayama, K. Sonomoto, Evaluation of essential and variable residues of nukacin ISK-1 by NNK scanning, Molecular Microbiology, 10.1111/j.1365-2958.2009.06733.x, 72, 6, 1438-1447, 2009.06, Nukacin ISK-1, a type-A(II) lantibiotic, comprises 27 amino acids with a distinct linear N-terminal and a globular C-terminal region. To identify the positional importance or redundancy of individual residues responsible for nukacin ISK-1 antimicrobial activity, we replaced the native codons of the parent peptide with NNK triplet oligonucleotides in order to generate a bank of nukacin ISK-1 variants. The bioactivity of each peptide variant was evaluated by colony overlay assay, and hence we identified three Lys residues (Lys1, Lys2 and Lys3) that provided electrostatic interactions with the target membrane and were significantly variable. The ring structure of nukacin ISK-1 was found to be crucially important as replacing the ring-forming residues caused a complete loss of bioactivity. In addition to the ring-forming residues, Gly5, His12, Asp13, Met16, Asn17 and Gln20 residues were found to be essential for antimicrobial activity; Val6, Ile7, Val10, Phe19, Phe21, Val22, Phe23 and Thr24 were relatively variable; and Ser4, Pro8, His15 and Ser27 were extensively variable relative to their positions. We obtained two variants, Asp13Glu and Val22Ile, which exhibited a twofold higher specific activity compared with the wild-type and are the first reported type-A(II) lantibiotic mutant peptides with increased potency. © 2009 Blackwell Publishing Ltd..
121. Jiro Nakayama, Keisuke Yamamoto, Shinichiro Fukuhara, Toshiaki Hirai, Tomohiro Ueda, Hiroshi Kiuchi, Tetsuya Takao, Yasushi Miyagawa, Akira Tsujimura, Akihiko Okuyama, TRANSPLANTATION OF OLFACTORY MUCOSA FOLLOWING SPINAL CORD INJURY IMPROVES VOIDING EFFICIENCY IN RAT, JOURNAL OF UROLOGY, 10.1016/S0022-5347(09)60960-5, 181, 4, 337-337, 2009.04.
122. Naruhiko Sawa, Takeshi Zendo, Junko Kiyofuji, Koji Fujita, Kohei Himeno, Jiro Nakayama, Kenji Sonomoto, Identification and characterization of lactocyclicin Q, a novel cyclic bacteriocin produced by Lactococcus sp. strain QU 12., Applied and environmental microbiology, 10.1128/AEM.02299-08, 75, 6, 1552-1558, 2009.03, Lactococcus sp. strain QU 12, which was isolated from cheese, produced a novel cyclic bacteriocin termed lactocyclicin Q. By using cation-exchange chromatography, hydrophobic interaction chromatography, and reverse-phase high-performance liquid chromatography, lactocyclicin Q was purified from culture supernatant, and its molecular mass was determined to be 6,062.8 Da by mass spectrometry. Lactocyclicin Q has been characterized by its unique antimicrobial spectrum, high level of protease resistance, and heat stability compared to other reported bacteriocins of lactic acid bacteria. The amino acid sequence of lactocyclicin Q was determined chemically, and this compound is composed of 61 amino acid residues that have a cyclic structure with linkage between the N and C termini by a peptide bond. It showed no homology to any other antimicrobial peptide, including cyclic bacteriocins. On the basis of the amino acid sequences obtained, the sequence of the gene encoding the prepeptide lactocyclicin Q was obtained. This is the first report of a cyclic bacteriocin purified from a strain belonging to the genus Lactococcus..
123. Jiro Nakayama, Yumi Uemura, Kenzo Nishiguchi, Norito Yoshimura, Yasuhiro Igarashi, Kenji Sonomoto, Ambuic acid inhibits the biosynthesis of cyclic peptide quormones in gram-positive bacteria., Antimicrobial agents and chemotherapy, 10.1128/AAC.00995-08, 53, 2, 580-586, 2009.02, Quorum sensing is a cell-density-dependent regulatory system in gram-positive bacteria and is often regulated by cyclic peptides called "quormones," which function as extracellular communication signals. With an aim to discover an antipathogenic agent targeting quorum sensing in gram-positive bacteria, we screened 153 samples of fungal butanol extracts with the guidance of the inhibition of quorum-sensing-mediated gelatinase production in Enterococcus faecalis. Following the screenings, we found that ambuic acid, a known secondary fungal metabolite, inhibited the quorum-sensing-mediated gelatinase production without influencing the growth of E. faecalis. We further demonstrated that ambuic acid targeted the biosynthesis of a cyclic peptide quormone called gelatinase biosynthesis-activating pheromone. Furthermore, ambuic acid also inhibited the biosynthesis of the cyclic peptide quormones of Staphylococcus aureus and Listeria innocua. These results suggest the potential use of ambuic acid as a lead compound of antipathogenic drugs that target the quorum-sensing-mediated virulence expression of gram-positive bacteria..
124. Kenzo Nishiguchi, Koji Nagata, Masaru Tanokura, Kenji Sonomoto, Jiro Nakayama, Structure-activity relationship of gelatinase biosynthesis-activating pheromone of Enterococcus faecalis., Journal of bacteriology, 10.1128/JB.01029-08, 191, 2, 641-650, 2009.01, The expression of pathogenicity-related extracellular proteases, namely, gelatinase and serine protease, in Enterococcus faecalis is positively regulated by a quorum-sensing system mediated by an autoinducing peptide called gelatinase biosynthesis-activating pheromone (GBAP). GBAP is an 11-amino-acid-residue cyclic peptide containing a lactone linkage. To study the structure-activity relationship of GBAP, we synthesized a series of GBAP analogues and evaluated their activities by a gelatinase-inducing assay and newly developed receptor-binding assays in which fluorescence-labeled peptides bound onto the FsrC-overexpressing Lactococcus lactis cell surface were observed by fluorescent microscopy and quantified by using a fluorophotometer. Alanine-scanning analysis of GBAP showed that the entire ring region was involved in the GBAP agonist activity, while side chains of the tail region were not strictly recognized. The alanine substitution of Phe(7) or Trp(10) almost abolished their receptor-binding abilities and GBAP agonist activities, suggesting that these two aromatic side chains are strongly involved in receptor interaction and activation. Furthermore, the Trp(10) substitution with natural and unnatural aromatic amino acids, except pentafluorophenylalanine, caused no loss of agonist activity. This suggested the importance of a negative electrostatic potential created by an pi-electron cloud on the aromatic ring surface. Structural analysis of GBAP with nuclear magnetic resonance spectroscopy revealed that the ring region adopted a hairpin-like fold and was tightly packed into a compact form. The side chain of Trp(10) was partially buried in the core structure, contributing to the stabilization of the compact form, while that of Phe(7) was extended from the core structure into the solvent and was probably directly involved in receptor binding..
125. Fuminori Yoneyama, Yuichi Imura, Shiro Ichimasa, Koji Fujita, Takeshi Zendo, Jiro Nakayama, Katsumi Matsuzaki, Kenji Sonomoto, Lacticin Q, a lactococcal bacteriocin, causes high-level membrane permeability in the absence of specific receptors., Applied and environmental microbiology, 10.1128/AEM.01827-08, 75, 2, 538-541, 2009.01, To characterize the mode of action of lacticin Q (LnqQ), its membrane-permeabilizing activity was compared with that of nisin A because of the similar antimicrobial features of these compounds. Lipid II, the receptor for nisin A, was not required for LnqQ activity. LnqQ induced high-level membrane permeability in the absence of specific receptors..
126. Toshio Fujii, Colin Ingham, Jiro Nakayama, Marke Beerthuyzen, Ryoko Kunuki, Douwe Molenaar, Mark Sturme, Elaine Vaughan, Michiel Kleerebezem, Willem de Vos, Two homologous Agr-like quorum-sensing systems cooperatively control adherence, cell morphology, and cell viability properties in Lactobacillus plantarum WCFS1., Journal of bacteriology, 10.1128/JB.01489-07, 190, 23, 7655-65, 2008.12, A two-component regulatory system of Lactobacillus plantarum, encoded by genes designated lamK and lamR (hpk10 and rrp10), was studied. The lamK and lamR genes encode proteins which are highly homologous to the quorum-sensing histidine kinase LamC and the response regulator LamA, respectively. Transcription analysis of the lamKR operon and the lamBDCA operon and liquid chromatography-mass spectrometry analysis of production of the LamD558 autoinducing peptide were performed for DeltalamA, DeltalamR, DeltalamA DeltalamR deletion mutants and a wild-type strain. The results suggested that lamA and lamR are cooperating genes. In addition, typical phenotypes of the DeltalamA mutant, such as reduced adherence to glass surfaces and filamentous cell morphology, were enhanced in the DeltalamA DeltalamR mutant. Microarray analysis suggested that the same cell wall polysaccharide synthesis genes, stress response-related genes, and cell wall protein-encoding genes were affected in the DeltalamA and DeltalamA DeltalamR mutants. However, the regulation ratio was more significant for the DeltalamA DeltalamR mutant, indicating the cooperative effect of LamA and LamR..
127. F. Yoneyama, M. Fukao, T. Zendo, J. Nakayama, K. Sonomoto, Biosynthetic characterization and biochemical features of the third natural nisin variant, nisin Q, produced by Lactococcus lactis 61-14, Journal of Applied Microbiology, 10.1111/j.1365-2672.2008.03958.x, 105, 6, 1982-1990, 2008.12, Aims: To characterize the genetic and biochemical features of nisin Q. Methods and Results: The nisin Q gene cluster was sequenced, and 11 putative orfs having 82% homology with the nisin A biosynthesis gene cluster were identified. Nisin Q production was confirmed from the nisQ-introduced nisin Z producer. In the reporter assay, nisin Q exhibited an induction level that was threefold lower than that of nisin A. Nisin Q demonstrated an antimicrobial spectrum similar to those of the other nisins. Under oxidizing conditions, nisin Q retained a higher level of activity than nisin A. This higher oxidative tolerance could be attributed to the presence of only one methionine residue in nisin Q, in contrast to other nisins that contain two. Conclusions: The 11 orfs of the nisin producers were identical with regard to their functions. The antimicrobial spectra of the three natural nisins were similar. Nisin Q demonstrated higher oxidative tolerance than nisin A. Significance and Impact of the Study: Genetic and biochemical features of nisin Q are similar to those of other variants. Moreover, owing to its higher oxidative tolerance, nisin Q is a potential alternative for nisin A. © 2008 The Authors..
128. Ken-ichi Okuda, Sae Yanagihara, Kouki Shioya, Yoshitaka Harada, Jun-ichi Nagao, Yuji Aso, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Binding specificity of the lantibiotic-binding immunity protein NukH., Applied and environmental microbiology, 10.1128/AEM.00789-08, 74, 24, 7613-7619, 2008.12, NukH is a lantibiotic-binding immunity protein that shows strong binding activity against type A(II) lantibiotics. In this study, the binding specificity of NukH was analyzed by using derivatives of nukacin ISK-1, which is a type A(II) lantibiotic produced by Staphylococcus warneri ISK-1. Interactions between cells of Lactococcus lactis transformants expressing nukH and nukacin ISK-1 derivatives were analyzed by using a quantitative peptide-binding assay. Differences in the cell-binding rates of each nukacin ISK-1 derivative suggested that three lysine residues at positions 1 to 3 of nukacin ISK-1 contribute to the effective binding of nukacin ISK-1 to nukH-expressing cells. The binding levels of mutants with lanthionine and dehydrobutyrine substitutions (S11A nukacin(4-27) and T24A nukacin(4-27), respectively) to nukH-expressing cells were considerably lower than those of nukacin(4-27), suggesting that unusual amino acids play a decisive role in NukH recognition. Additionally, it was suggested that T9A nukacin(4-27), a mutant with a 3-methyllanthionine substitution, binds to NukH via an intermolecular disulfide bond after it is weakly recognized by NukH. We succeeded in the detection of specific type A(II) lantibiotics from the culture supernatants of various bacteriocin producers by using the binding specificity of nukH-expressing cells..
129. Toshio Fujii, Colin Ingham, Jiro Nakayama, Marke Beerthuyzen, Ryoko Kunuki, Douwe Molenaar, Mark Sturme, Elaine Vaughan, Michiel Kleerebezem, Willem De Vos, Two homologous agr-like quorum-sensing systems cooperatively control adherence, cell morphology, and cell viability properties in Lactobacillus plantarum WCFS1, Journal of Bacteriology, 10.1128/JB.01489-07, 190, 23, 7655-7665, 2008.12, A two-component regulatory system of Lactobacillus plantarum, encoded by genes designated lamK and lamR (hpk10 and rrp10), was studied. The lamK and lamR genes encode proteins which are highly homologous to the quorum-sensing histidine kinase LamC and the response regulator LamA, respectively. Transcription analysis of the lamKR operon and the lamBDCA operon and liquid chromatography-mass spectrometry analysis of production of the LamD558 autoinducing peptide were performed for ΔlamA, ΔlamR, ΔlamA ΔlamR deletion mutants and a wild-type strain. The results suggested that lamA and lamR are cooperating genes. In addition, typical phenotypes of the ΔlamA mutant, such as reduced adherence to glass surfaces and filamentous cell morphology, were enhanced in the ΔlamA ΔlamR mutant. Microarray analysis suggested that the same cell wall polysaccharide synthesis genes, stress response-related genes, and cell wall protein-encoding genes were affected in the ΔlamA and ΔlamA ΔlamR mutants. However, the regulation ratio was more significant for the ΔlamA ΔlamR mutant, indicating the cooperative effect of LamA and LamR..
130. F. Yoneyama, M. Fukao, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Biosynthetic characterization and biochemical features of the third natural nisin variant, nisin Q, produced by Lactococcus lactis 61-14, Journal of Applied Microbiology, 10.1111/j.1365-2672.2008.03958.x, 105, 6, 1982-1990, 2008.12, Aims: To characterize the genetic and biochemical features of nisin Q. Methods and Results: The nisin Q gene cluster was sequenced, and 11 putative orfs having 82% homology with the nisin A biosynthesis gene cluster were identified. Nisin Q production was confirmed from the nisQ-introduced nisin Z producer. In the reporter assay, nisin Q exhibited an induction level that was threefold lower than that of nisin A. Nisin Q demonstrated an antimicrobial spectrum similar to those of the other nisins. Under oxidizing conditions, nisin Q retained a higher level of activity than nisin A. This higher oxidative tolerance could be attributed to the presence of only one methionine residue in nisin Q, in contrast to other nisins that contain two. Conclusions: The 11 orfs of the nisin producers were identical with regard to their functions. The antimicrobial spectra of the three natural nisins were similar. Nisin Q demonstrated higher oxidative tolerance than nisin A. Significance and Impact of the Study: Genetic and biochemical features of nisin Q are similar to those of other variants. Moreover, owing to its higher oxidative tolerance, nisin Q is a potential alternative for nisin A..
131. Adisorn Swetwiwathana, Yaowalak Surapantapisit, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Identification and partial characterization of pediocin PA-1 producing Pediococcus pentosaceus associated in traditional Thai fermented beef (mum), JOURNAL OF BIOTECHNOLOGY, 10.1016/j.jbiotec.2008.07.1781, 136, S748-S749, 2008.10.
132. Pikyee Ma, Hayley M Yuille, Victor Blessie, Nadine Göhring, Zsófia Iglói, Kenzo Nishiguchi, Jiro Nakayama, Peter J F Henderson, Mary K Phillips-Jones, Expression, purification and activities of the entire family of intact membrane sensor kinases from Enterococcus faecalis., Molecular membrane biology, 10.1080/09687680802359885, 25, 6-7, 449-473, 2008.09, Two-component signal transduction systems are the main mechanism by which bacteria sense and respond to their environment, and their membrane-located histidine protein kinases generally constitute the sensory components of these systems. Relatively little is known about their fundamental mechanisms and precise nature of the molecular signals sensed, because of the technical challenges of producing sufficient quantities of these hydrophobic membrane proteins. This study evaluated the heterologous production, purification and activities of the 16 intact membrane sensor kinases of Enterococcus faecalis. Following the cloning of the genes into expression plasmid pTTQ18His, all but one kinase was expressed successfully in Escherichia coli inner membranes. Purification of the hexa-histidine 'tagged' recombinant proteins was achieved for 13, and all but one were verified as intact. Thirteen intact kinases possessed autophosphorylation activity with no added signal when assayed in membrane vesicles or as purified proteins. Signal testing of two functionally-characterized kinases, FsrC and VicK, was successful examplifying the potential use of in vitro activity assays of intact proteins for systematic signal identification. Intact FsrC exhibited an approximately 10-fold increase in activity in response to a two-fold molar excess of synthetic GBAP pheromone, whilst glutathione, and possibly redox potential, were identified for the first time as direct modulators of VicK activity in vitro. The impact of DTT on VicK phosphorylation resulted in increased levels of phosphorylated VicR, the downstream response regulator, thereby confirming the potential of this in vitro approach for investigations of modulator effects on the entire signal transduction process of two-component systems..
133. C. B. Hu, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Description of durancin TW-49M, a novel enterocin B-homologous bacteriocin in carrot-isolated Enterococcus durans QU 49, Journal of Applied Microbiology, 10.1111/j.1365-2672.2008.03798.x, 105, 3, 681-690, 2008.09, Aims: To characterize the novel bacteriocin produced by Enterococcus durans. Methods and Results: Enterococcus durans QU 49 was isolated from carrot and expressed bactericidal activity over 20-43°C. Bacteriocins were purified to homogeneity using the three-step purification method, one of which, termed durancin TW-49M, was an enterocin B-homologous peptide with most identical residues occurring in the N-terminus. Durancin TW-49M was more tolerant in acidic than in alkali. DNA sequencing analysis revealed durancin TW-49M was translated as a prepeptide of the double-glycine type. Durancin TW-49M and enterocin B expressed similar antimicrobial spectra, in which no significant variation due to the diversity in their C-termini was observed. Conclusions: Durancin TW-49M, a novel nonpediocin-like class II bacteriocin, was characterized to the amino acid and genetic levels. The diverse C-terminal parts of durancin TW-49M and enterocin B were hardly to be suggested as the place determining the target cell specificity. Significance and Impact of the Study: This is the first and comprehensive study of a novel bacteriocin produced by Ent. durans. The high homology at the N-terminal halves between durancin TW-49M and enterocin B makes them suitable to study the structure-function relationship of bacteriocins and their immunity proteins. © 2008 The Authors..
134. Pongtep Wilaipun, Takeshi Zendo, Ken-ichi Okuda, Jiro Nakayama, Kenji Sonomoto, Identification of the nukacin KQU-131, a new type-A(II) lantibiotic produced by Staphylococcus hominis KQU-131 isolated from Thai fermented fish product (Pla-ra)., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.80239, 72, 8, 2232-2235, 2008.08, Staphylococcus hominis KQU-131, isolated from Thai fermented marine fish, produces a heat stable bacteriocin. Structural and genetic analysis indicated that the bacteriocin is a variant of nukacin ISK-1, a type-A(II) lantibiotic, and we termed the bacteriocin nukacin KQU-131. There were three different amino acid residues between nukacin ISK-1 and nukacin KQU-131, one residue in the leader peptide and the other two in the mature peptide..
135. Masanori Fukao, Takayuki Obita, Fuminori Yoneyama, Daisuke Kohda, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Complete covalent structure of nisin Q, new natural nisin variant, containing post-translationally modified amino acids., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.80066, 72, 7, 1750-5, 2008.07, The third member of the nisin variant, nisin Q, produced by Lactococcus lactis 61-14, is a ribosomally-synthesized antimicrobial peptide, the so-called lantibiotic containing post-translationally modified amino acids such as lanthionine and dehydroalanine. Here, we determined the complete covalent structure of nisin Q, consisting of 34 amino acids, by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy. Sequential assignment of nisin Q containing the unusual amino acids was performed by total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The observed long range nuclear Overhauser effect (NOE) in nisin Q indicated assignment of all five sets of lanthionines that intramolecularly bridge residues 3-7, 8-11, 13-19, 23-26, and 25-28. Consequently, the covalent structure of nisin Q was determined to hold the same thioether linkage formation as the other two nisins, but to harbor the four amino acid substitutions, in contrast with nisin A..
136. Shinya Sugimoto, Kozue Saruwatari, Chihana Higashi, Keigo Tsuruno, Shunsuke Matsumoto, Jiro Nakayama, Kenji Sonomoto, In vivo and in vitro complementation study comparing the function of DnaK chaperone systems from halophilic lactic acid bacterium Tetragenococcus halophilus and Escherichia coli., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.70691, 72, 3, 811-22, 2008.03, In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli..
137. T. Zendo, J. Nakayama, K. Fujita, K. Sonomoto, Bacteriocin detection by liquid chromatography/mass spectrometry for rapid identification, Journal of Applied Microbiology, 10.1111/j.1365-2672.2007.03575.x, 104, 2, 499-507, 2008.02, Aims: To establish a new system to detect and identify bacteriocins in the early stage of screening for novel bacteriocins. Methods and Results: Liquid chromatography/mass spectrometry (LC/MS) was employed for development of a new system for rapid detection and identification of bacteriocins. The system detected and identified bacteriocins such as nisin and lacticin 481 from 25 μl of culture supernatants of their producing strains by accurate mass determination coupled with simultaneous impurity removal within 40 min. Especially, the system clearly distinguished three nisin variants (A, Z, Q) in culture supernatants of their producing strains, although they have similar structures and molecular masses. Each one-step pretreatment by cell adsorption-desorption or acetone precipitation improved bacteriocin detection dramatically, especially for mundticin KS. This system could be applied for detection and molecular mass determination of novel bacteriocins by extracting bacteriocin-related ions. Conclusions: The developed system could detect and identify some kinds of bacteriocin from culture supernatants or pretreated samples. Significance and Impact of the Study: The developed system helps us to identify bacteriocins in the early stage of screening without any or with one-step pretreatment. This system is effective on not only detection of known bacteriocins but also identification of novel bacteriocins. Consequently, this system will accelerate discovery of novel bacteriocins. © 2007 The Authors..
138. Ken-ichi Okuda, Yuji Aso, Jiro Nakayama, Kenji Sonomoto, Cooperative transport between NukFEG and NukH in immunity against the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1., Journal of bacteriology, 10.1128/JB.01300-07, 190, 1, 356-62, 2008.01, Nukacin ISK-1 is a lantibiotic produced by Staphylococcus warneri ISK-1. Previous studies have reported that the self-protection system of the nukacin ISK-1 producer involves the cooperative function of the ABC transporter NukFEG and the lantibiotic-binding immunity protein NukH. In this study, the cooperative mechanism between NukFEG and NukH was characterized by using fluorescein-4-isothiocyanate (FITC)-labeled nukacin ISK-1 (FITC-nuk) to clarify the localization of nukacin ISK-1 in the immunity process. Lactococcus lactis recombinants expressing nukFEGH, nukFEG, or nukH showed immunity against FITC-nuk, suggesting that FITC-nuk was recognized by the self-protection system against nukacin ISK-1. Analysis of the interaction between FITC-nuk and energy-deprived cells of the L. lactis recombinants showed that FITC-nuk specifically bound to cells expressing nukH. The interaction between FITC-nuk and nukH-expressing cells was inhibited by the addition of unlabeled nukacin ISK-1 and its derivatives with deletions of the N-terminal tail region, but not by the addition of a synthesized N-terminal tail region. This suggests that the NukH protein recognizes the C-terminal ring region of nukacin ISK-1. The addition of glucose to nukFEGH-expressing cells treated with FITC-nuk resulted in a time-dependent decrease in fluorescence intensity, indicating that FITC-nuk was transported from the cell membrane by the NukFEG protein. These results revealed that after being captured by NukH in an energy-independent manner, nukacin ISK-1 was transported to the extracellular space by NukFEG in an energy-dependent manner..
139. Shinya Sugimoto, Kozue Saruwatari, Chihana Higashi, Keigo Tsuruno, Shunsuke Matsumoto, Jiro Nakayama, Kenji Sonomoto, In vivo and in vitro complementation study comparing the function of DnaK chaperone systems from halophilic lactic acid bacterium Tetragenococcus halophilus and Escherichia coli, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.70691, 72, 3, 811-822, 2008.01, In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli..
140. Ken Ichi Okuda, Yuji Aso, Jiro Nakayama, Kenji Sonomoto, Cooperative transport between NukFEG and NukH in immunity against the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1, Journal of Bacteriology, 10.1128/JB.01300-07, 190, 1, 356-362, 2008.01, Nukacin ISK-1 is a lantibiotic produced by Staphylococcus warneri ISK-1. Previous studies have reported that the self-protection system of the nukacin ISK-1 producer involves the cooperative function of the ABC transporter NukFEG and the lantibiotic-binding immunity protein NukH. In this study, the cooperative mechanism between NukFEG and NukH was characterized by using fluorescein-4-isothiocyanate (FITC)-labeled nukacin ISK-1 (FITC-nuk) to clarify the localization of nukacin ISK-1 in the immunity process. Lactococcus lactis recombinants expressing nukFEGH, nukFEG, or nukH showed immunity against FITC-nuk, suggesting that FITC-nuk was recognized by the self-protection system against nukacin ISK-1. Analysis of the interaction between FITC-nuk and energy-deprived cells of the L. lactis recombinants showed that FITC-nuk specifically bound to cells expressing nukH. The interaction between FITC-nuk and nukH-expressing cells was inhibited by the addition of unlabeled nukacin ISK-1 and its derivatives with deletions of the N-terminal tail region, but not by the addition of a synthesized N-terminal tail region. This suggests that the NukH protein recognizes the C-terminal ring region of nukacin ISK-1. The addition of glucose to nukFEGH-expressing cells treated with FITC-nuk resulted in a time-dependent decrease in fluorescence intensity, indicating that FITC-nuk was transported from the cell membrane by the NukFEG protein. These results revealed that after being captured by NukH in an energy-independent manner, nukacin ISK-1 was transported to the extracellular space by NukFEG in an energy-dependent manner..
141. Masanori Fukao, Takayuki Obita, Fuminori Yoneyama, Daisuke Kohda, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Complete covalent structure of nisin Q, new natural nisin variant, containing post-translationally modified amino acids, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.80066, 72, 7, 1750-1755, 2008.01, The third member of the nisin variant, nisin Q, produced by Lactococcus lactis 61-14, is a ribosomally-synthesized antimicrobial peptide, the so-called lantibiotic containing post-translationally modified amino acids such as lanthionine and dehydroalanine. Here, we determined the complete covalent structure of nisin Q, consisting of 34 amino acids, by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Sequential assignment of nisin Q containing the unusual amino acids was performed by total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The observed long range nuclear Overhauser effect (NOE) in nisin Q indicated assignment of all five sets of lanthionines that intramolecularly bridge residues 3-7, 8-11, 13-19, 23-26, and 25-28. Consequently, the covalent structure of nisin Q was determined to hold the same thioether linkage formation as the other two nisins, but to harbor the four amino acid substitutions, in contrast with nisin A..
142. Jiro Nakayama, Hiroyuki Hoshiko, Mizuki Fukuda, Hidetoshi Tanaka, Naoshige Sakamoto, Shigemitsu Tanaka, Kazutoshi Ohue, Kenji Sakai, Kenji Sonomoto, Molecular monitoring of bacterial community structure in long-aged nukadoko: pickling bed of fermented rice bran dominated by slow-growing lactobacilli., Journal of bioscience and bioengineering, 10.1263/jbb.104.481, 104, 6, 481-489, 2007.12, Nukadoko is the fermented rice bran bed traditionally used for pickling vegetables in Japan. Here, we investigate the bacterial community structure of nukadoko using several culture-independent methods. Denaturing gradient gel electrophoresis (DGGE) and sequence analysis of V2-V3 16S rRNA gene (16S rDNA) fragments amplified from a long-aged nukadoko bacterial community indicated seven predominant operational taxonomic units (OTUs) closely related to known Lactobacillus species. Phylogenetic analysis of these OTUs indicated a major cluster consisting of six OTUs including a dominant OTU closely related to Lactobacillus acidifarinae and one distinct OTU corresponding to Lactobacillus acetotolerans. L. acetotolerans was commonly detected as a dominant species in samples from different seasons. The succession of microbial community structure in the fermentation and ripening processes was investigated using a laboratory model nukadoko. The L. acidifarinae-like bacteria grew rapidly with a pH decrease in the first few days after inoculation, whereas L. acetotolerans grew slowly and became dominant after one week. Real-time quantitative polymerase chain reaction (Q-PCR) showed that the doubling time of L. acetotolerans was 12 h, while that of total bacteria was 4 h. Real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) targeting 16S rRNA showed a low metabolic activity of L. acetotolerans throughout the fermentation and ripening processes. Fluorescence in situ hybridization (FISH) showed that L. acetotolerans was a dominant bacterium in the ripening period and had a low metabolic activity. These results indicate that the slow-growing L. acetotolerans stably dominated nukadoko microbiota after the L. acidifarinae-like bacteria mainly contributed to the lactic acid fermentation of the rice bran..
143. S. Sebei, T. Zendo, A. Boudabous, J. Nakayama, K. Sonomoto, Characterization, N-terminal sequencing and classification of cerein MRX1, a novel bacteriocin purified from a newly isolated bacterium: Bacillus cereus MRX1, Journal of Applied Microbiology, 10.1111/j.1365-2672.2007.03395.x, 103, 5, 1621-1631, 2007.11, Aim: To purify and characterize the bacteriocin produced by strain MRX1. Methods and Results: A bacteriocin-producing strain was isolated and identified as Bacillus cereus. The bacteriocin, called cerein MRX1, was purified from the culture supernatant using hydrophobic interaction, cation-exchange chromatography and RP-HPLC. It could also be purified in abundance from the cell surfaces of the producer strain. Mass spectrometry revealed its molecular mass of 3137.93 Da. Sequencing of chemically modified bacteriocin identified its partial sequence: DWTCWSCLVCAACSVELL. Amino acid analysis, confirmed by H-NMR, suggested cerein MRX1 to be a class II bacteriocin. This bacteriocin was remarkably hydrophobic, heat-stable and could withstand a wide range of pH. It exhibited a bactericidal mode of action against Bacillus coagulans JCM 2257 . Cerein MRX1 was especially active against spoilage bacteria such as Bacillus subtilis and Listeria innocua (MICs in the 1 μg ml range). In contrast, lactic acid bacteria were resistant or required higher concentrations to be inhibited. Conclusions: Cerein MRX1 is similar by its N-terminal sequence to thuricin 17 recently isolated from Bacillus thuringiensis NEB17. However, the two bacteriocins are different by their molecular masses and amino acid compositions. Significance and Impact of the Study: Chemical stability of cerein MRX1 and its ability to inhibit a large number of undesirable bacteria may give an advantage to its food or clinical application as an antibacterial agent. © 2007 The Authors. 1 T -1.
144. Prapa Songjinda, Jiro Nakayama, Atsushi Tateyama, Shigemitsu Tanaka, Mina Tsubouchi, Chikako Kiyohara, Taro Shirakawa, Kenji Sonomoto, Differences in developing intestinal microbiota between allergic and non-allergic infants
A pilot study in Japan, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.70154, 71, 9, 2338-2342, 2007.11, The bacterial compositions of feces were monitored in the first 2 months for 15 infants born in Japan, including eight subjects who developed allergy by the age of 2 years. Primer sets targeting six predominant bacterial groups in the infant intestine, Bacteroidaceae, Enterobacteriaceae, bifidobacteria, enterococci, lactobacilli, and the Clostridium perfringens group, were used for real-time PCR to quantitate each population in the feces. The population of Bacteroidaceae was significantly higher in the allergic group at the ages of 1 month (P = 0.03) and 2 months (P = 0.05) than in the non-allergic group, while no statistically significant difference was observed for the other bacterial populations..
145. S. Sebei, Takeshi Zendo, A. Boudabous, Jiro Nakayama, Kenji Sonomoto, Characterization, N-terminal sequencing and classification of cerein MRX1, a novel bacteriocin purified from a newly isolated bacterium
Bacillus cereus MRX1, Journal of Applied Microbiology, 10.1111/j.1365-2672.2007.03395.x, 103, 5, 1621-1631, 2007.11, Aim: To purify and characterize the bacteriocin produced by strain MRX1. Methods and Results: A bacteriocin-producing strain was isolated and identified as Bacillus cereus. The bacteriocin, called cerein MRX1, was purified from the culture supernatant using hydrophobic interaction, cation-exchange chromatography and RP-HPLC. It could also be purified in abundance from the cell surfaces of the producer strain. Mass spectrometry revealed its molecular mass of 3137.93 Da. Sequencing of chemically modified bacteriocin identified its partial sequence: DWTCWSCLVCAACSVELL. Amino acid analysis, confirmed by 1H-NMR, suggested cerein MRX1 to be a class II bacteriocin. This bacteriocin was remarkably hydrophobic, heat-stable and could withstand a wide range of pH. It exhibited a bactericidal mode of action against Bacillus coagulans JCM 2257T. Cerein MRX1 was especially active against spoilage bacteria such as Bacillus subtilis and Listeria innocua (MICs in the 1 μg ml-1 range). In contrast, lactic acid bacteria were resistant or required higher concentrations to be inhibited. Conclusions: Cerein MRX1 is similar by its N-terminal sequence to thuricin 17 recently isolated from Bacillus thuringiensis NEB17. However, the two bacteriocins are different by their molecular masses and amino acid compositions. Significance and Impact of the Study: Chemical stability of cerein MRX1 and its ability to inhibit a large number of undesirable bacteria may give an advantage to its food or clinical application as an antibacterial agent..
146. Shun Iwatani, Takeshi Zendo, Fuminori Yoneyama, Jiro Nakayama, Kenji Sonomoto, Characterization and structure analysis of a novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.70169, 71, 8, 1984-1992, 2007.11, A novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14 isolated from a horse's intestinal tract was identified. Lacticin Z was purified through a three step procedure comprised of hydrophobic-interaction, cation-exchange chromatography, and reverse-phase HPLC. ESI-TOF MS determined the molecular mass of lacticin Z to be 5,968.9 Da. The primary structure of lacticin Z was found to consist of 53 amino acid residues without any leader sequence or signal peptide. Lacticin Z showed homology to lacticin Q from L. lactis QU 5, aureocin A53 from Staphylococcus aureus A53, and mutacin BHT-B from Streptococcus rattus strain BHT. It exhibited a nanomolar range of MICs against various Gram-positive bacteria, and the activity was completely stable up to 100°C. Unlike many of other LAB bacteriocins, the stability of lacticin Z was emphasized under alkaline conditions rather than acidic conditions. All the results indicated that lacticin Z belongs to a novel type of bacteriocin..
147. Prapa Songjinda, Jiro Nakayama, Atsushi Tateyama, Shigemitsu Tanaka, Mina Tsubouchi, Chikako Kiyohara, Taro Shirakawa, Kenji Sonomoto, Differences in developing intestinal microbiota between allergic and non-allergic infants: a pilot study in Japan., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.70154, 71, 9, 2338-42, 2007.09, The bacterial compositions of feces were monitored in the first 2 months for 15 infants born in Japan, including eight subjects who developed allergy by the age of 2 years. Primer sets targeting six predominant bacterial groups in the infant intestine, Bacteroidaceae, Enterobacteriaceae, bifidobacteria, enterococci, lactobacilli, and the Clostridium perfringens group, were used for real-time PCR to quantitate each population in the feces. The population of Bacteroidaceae was significantly higher in the allergic group at the ages of 1 month (P=0.03) and 2 months (P=0.05) than in the non-allergic group, while no statistically significant difference was observed for the other bacterial populations..
148. Shun Iwatani, Takeshi Zendo, Fuminori Yoneyama, Jiro Nakayama, Kenji Sonomoto, Characterization and structure analysis of a novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.70169, 71, 8, 1984-92, 2007.08, A novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14 isolated from a horse's intestinal tract was identified. Lacticin Z was purified through a three step procedure comprised of hydrophobic-interaction, cation-exchange chromatography, and reverse-phase HPLC. ESI-TOF MS determined the molecular mass of lacticin Z to be 5,968.9 Da. The primary structure of lacticin Z was found to consist of 53 amino acid residues without any leader sequence or signal peptide. Lacticin Z showed homology to lacticin Q from L. lactis QU 5, aureocin A53 from Staphylococcus aureus A53, and mutacin BHT-B from Streptococcus rattus strain BHT. It exhibited a nanomolar range of MICs against various Gram-positive bacteria, and the activity was completely stable up to 100 degrees C. Unlike many of other LAB bacteriocins, the stability of lacticin Z was emphasized under alkaline conditions rather than acidic conditions. All the results indicated that lacticin Z belongs to a novel type of bacteriocin..
149. Shinya Sugimoto, Chihana Higashi, Kozue Saruwatari, Jiro Nakayama, Kenji Sonomoto, A gram-negative characteristic segment in Escherichia coli DnaK is essential for the ATP-dependent cooperative function with the co-chaperones DnaJ and GrpE., FEBS letters, 10.1016/j.febslet.2007.05.055, 581, 16, 2993-2999, 2007.06, We describe importance of the characteristic segment in ATPase domain of DnaK chaperone which is present in all gram-negative bacteria but is absent in all gram-positive bacteria. In vitro studies, ATPase activity, luciferase-refolding activity, and surface plasmon resonance analyses, demonstrated that a segment-deletion mutant DnaKDelta74-96 became defective in the cooperation with the co-chaperones DnaJ and GrpE. In addition, in vivo complementation assay showed that expression of DnaKDelta74-96 could not rescue the viability of Escherichia coli DeltadnaK mutant at 43 degrees C. Consequently, we suggest evolutionary significance for this DnaK ATPase domain segment in gram-negative bacteria towards the DnaK chaperone system..
150. Koji Fujita, Shiro Ichimasa, Takeshi Zendo, Shoko Koga, Fuminori Yoneyama, Jiro Nakayama, Kenji Sonomoto, Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of gram-positive bacteria., Applied and environmental microbiology, 10.1128/AEM.02286-06, 73, 9, 2871-3877, 2007.05, Lactococcus lactis QU 5 isolated from corn produces a novel bacteriocin, termed lacticin Q. By acetone precipitation, cation-exchange chromatography, and reverse-phase high-performance liquid chromatography, lacticin Q was purified from the culture supernatant of this organism, and its molecular mass was determined to be 5,926.50 Da by mass spectrometry. Subsequent analyses of amino acid and DNA sequences revealed that lacticin Q comprised 53 amino acid residues and that its N-terminal methionine residue was formylated. In contrast to most bacteriocins produced by gram-positive bacteria, lacticin Q had no N-terminal extensions such as leader or signal sequences. It showed 66% and 48% identity to AucA, a hypothetical protein from Corynebacterium jeikeium plasmid pA501, and aureocin A53, a bacteriocin from Staphylococcus aureus A53, respectively. The characteristics of lacticin Q were determined and compared to those of nisin A. Similar to nisin A, lacticin Q exhibited antibacterial activity against various gram-positive bacteria. Lacticin Q was very stable against heat treatment and changes in pH; in particular, it was stable at alkaline pH values, while nisin A was inactivated. Moreover, lacticin Q induced ATP efflux from a Listeria sp. strain in a shorter time and at a lower concentration than nisin A, indicating that the former affected indicator cells in a different manner from that of the latter. The results described here clarified the fact that lacticin Q belongs to a new family of class II bacteriocins and that it can be employed as an alternative to or in combination with nisin A..
151. Jiro Nakayama, Emi Tanaka, Reiko Kariyama, Koji Nagata, Kenzo Nishiguchi, Ritsuko Mitsuhata, Yumi Uemura, Masaru Tanokura, Hiromi Kumon, Kenji Sonomoto, Siamycin attenuates fsr quorum sensing mediated by a gelatinase biosynthesis-activating pheromone in Enterococcus faecalis., Journal of bacteriology, 10.1128/JB.00969-06, 189, 4, 1358-1365, 2007.02, The expression of two Enterococcus faecalis virulence-related proteases, gelatinase (GelE) and serine protease (SprE), is positively regulated by a quorum-sensing system encoded by the fsr gene cluster. In this system, E. faecalis secretes an autoinducing peptide, gelatinase biosynthesis-activating pheromone (GBAP), which triggers the FsrC-FsrA two-component regulatory system controlling the expression of two transcripts, fsrBDC and gelE-sprE. In the present study, we screened actinomycete metabolites for inhibitors of fsr quorum sensing. E. faecalis was cultured with each actinomycete culture supernatant tested, and the production of gelatinase and the production of GBAP were examined using the first screening and the second screening, respectively. Culture supernatant of Streptomyces sp. strain Y33-1 had the most potent inhibitory effect on both gelatinase production and GBAP production without inhibiting E. faecalis cell growth. The inhibitor in the culture supernatant was identified as a known peptide antibiotic, siamycin I. Siamycin I inhibited both gelatinase production and GBAP production at submicromolar concentrations, and it inhibited E. faecalis cell growth at concentrations above micromolar concentrations. Quantitative analysis of fsrBDC and gelE-sprE transcripts revealed that siamycin I suppressed the expression of both transcripts at a sublethal concentration. Siamycin I attenuated gelatinase production even when an overdose of GBAP was exogenously added to the culture. These results suggested that siamycin I inhibited the GBAP signaling via the FsrC-FsrA two-component regulatory system in a noncompetitive manner. The sublethal concentrations of siamycin I also attenuated biofilm formation. Treatment with siamycin could be a novel means of treating enterococcal infections..
152. Swetwiwathana, A., L. Napha, J. Nakayama, K. Sonomoto, Maturation of Nahm ? a Thai fermented meat product. Effect of pediocin PA-1 producer (Pediococcus pentosaceus TISTR 536) as starter culture, nitrite and garlic on Salmonella anatum during Nham fermentation, Fleischwirtschaft International, 22, 3, 46-49, 22(3), 46-49, 2007.01.
153. Okuda, K., Y. Aso, J. Nakayama, K. Sonomoto, Cooperative transport mechanism between NukFEG and NukH in immunity process against the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1, J. Bacteriol.,, 190(1), 356-362, 2007.01.
154. Shinya Sugimoto, Hiroyuki Yoshida, Yoshimitsu Mizunoe, Keigo Tsuruno, Jiro Nakayama, Kenji Sonomoto, Structural and functional conversion of molecular chaperone ClpB from the gram-positive halophilic lactic acid bacterium Tetragenococcus halophilus mediated by ATP and stress., Journal of bacteriology, 10.1128/JB.00404-06, 188, 23, 8070-8, 2006.12, In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB(Tha)) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB(Tha) forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45 degrees C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB(Tha) reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE(Tha)) and ATP. Interestingly, the mixture of dimer and monomer ClpB(Tha), which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB(Tha) forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE(Tha) and ATP under poststress conditions..
155. Jiro Nakayama, Shengmin Chen, Nozomi Oyama, Kenzo Nishiguchi, Essam A Azab, Emi Tanaka, Reiko Kariyama, Kenji Sonomoto, Revised model for Enterococcus faecalis fsr quorum-sensing system: the small open reading frame fsrD encodes the gelatinase biosynthesis-activating pheromone propeptide corresponding to staphylococcal agrd., Journal of bacteriology, 10.1128/JB.00865-06, 188, 23, 8321-8326, 2006.12, Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB', an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system..
156. Shinya Sugimoto, Hiroyuki Yoshida, Yoshimitsu Mizunoe, Keigo Tsuruno, Jiro Nakayama, Kenji Sonomoto, Structural and functional conversion of molecular chaperone ClpB from the gram-positive halophilic lactic acid bacterium Tetragenococcus halophilus mediated by ATP and stress, Journal of Bacteriology, 10.1128/JB.00404-06, 188, 23, 8070-8078, 2006.12, In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpBTha) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpBTha forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45°C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpBTha reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJETha) and ATP. Interestingly, the mixture of dimer and monomer ClpBTha, which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpBTha forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJETha and ATP under poststress conditions..
157. Sikder M Asaduzzaman, Jun-Ichi Nagao, Yuji Aso, Jiro Nakayama, Kenji Sonomoto, Lysine-oriented charges trigger the membrane binding and activity of nukacin ISK-1., Applied and environmental microbiology, 10.1128/AEM.00678-06, 72, 9, 6012-7, 2006.09, The antibacterial activities and membrane binding of nukacin ISK-1 and its fragments and mutants were evaluated to delineate the determinants governing structure-function relationships. The tail region (nukacin(1-7)) and ring region (nukacin(7-27)) were shown to have no antibacterial activity and also had no synergistic effect on each other or even on nukacin ISK-1. Both a fragment with three lysines in the N terminus deleted (nukacin(4-27)) and a mutant with three lysines in the N terminus replaced with alanine (K1-3A nukacin ISK-1) imparted very low activity (32-fold lower than nukacin ISK-1) and also exhibited a similar antagonistic effect on nukacin ISK-1. Addition of two lysine residues at the N terminus (+2K nukacin ISK-1) provided no further increased antibacterial activity. Surface plasmon resonance sensorgrams and kinetic rate constants determined by a BIAcore biosensor revealed that nukacin ISK-1 has remarkably higher binding affinity to anionic model membrane than to zwitterionic model membrane. Similar trends of strong binding responses and kinetics were indicated by the high affinities of nukacin ISK-1 and +2K nukacin ISK-1, but there was no binding of tail region, ring region, nukacin(4-27), and K1-3A nukacin ISK-1 to the anionic model membrane. Our findings therefore suggest that the complete structure of nukacin ISK-1 is necessary for its full activity, in which the N-terminus three lysine residues play a crucial role in electrostatic binding to the target membrane and therefore nukacin ISK-1's ability to exert its potent antibacterial activity..
158. Jun-ichi Nagao, Sikder M Asaduzzaman, Yuji Aso, Ken-Ichi Okuda, Jiro Nakayama, Kenji Sonomoto, Lantibiotics: insight and foresight for new paradigm., Journal of bioscience and bioengineering, 10.1263/jbb.102.139, 102, 3, 139-49, 2006.09, Lantibiotics are a unique type of antimicrobial peptide produced by a large number of gram-positive bacteria that contain unusual amino acids, such as lanthionine and dehydrated amino acids. Ribosomally synthesized lantibiotic prepeptide consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events to yield a biologically active lantibiotic. Research on lantibiotics has drawn much attention in recent years and has undergone extensive progress as a step forward to the next paradigm. Unusual amino acids in lantibiotics solely contribute to their biological activity and also enhance their structural stability. Thus, enzymes involved in lantibiotic biosynthesis would have a high potential for peptide engineering by introducing unusual amino acids into desired peptides, which may establish a universal approach to advance the structural design of novel peptides, termed lantibiotic engineering. In this review, we focus on recent development with contemporary innovations and perspective of lantibiotic research..
159. Sikder M. Asaduzzaman, Jun Ichi Nagao, Yuji Aso, Jiro Nakayama, Kenji Sonomoto, Lysine-oriented charges trigger the membrane binding and activity of nukacin ISK-1, Applied and Environmental Microbiology, 10.1128/AEM.00678-06, 72, 9, 6012-6017, 2006.09, The antibacterial activities and membrane binding of nukacin ISK-1 and its fragments and mutants were evaluated to delineate the determinants governing structure-function relationships. The tail region (nukacin1-7) and ring region (nukacin7-27) were shown to have no antibacterial activity and also had no synergistic effect on each other or even on nukacin ISK-1. Both a fragment with three lysines in the N terminus deleted (nukacin4-27) and a mutant with three lysines in the N terminus replaced with alanine (K1-3A nukacin ISK-1) imparted very low activity (32-fold lower than nukacin ISK-1) and also exhibited a similar antagonistic effect on nukacin ISK-1. Addition of two lysine residues at the N terminus (+2K nukacin ISK-1) provided no further increased antibacterial activity. Surface plasmon resonance sensorgrams and kinetic rate constants determined by a BIAcore biosensor revealed that nukacin ISK-1 has remarkably higher binding affinity to anionic model membrane than to zwitterionic model membrane. Similar trends of strong binding responses and kinetics were indicated by the high affinities of nukacin ISK-1 and +2K nukacin ISK-1, but there was no binding of tail region, ring region, nukacin4-27, and K1-3A nukacin ISK-1 to the anionic model membrane. Our findings therefore suggest that the complete structure of nukacin ISK-1 is necessary for its full activity, in which the N-terminus three lysine residues play a crucial role in electrostatic binding to the target membrane and therefore nukacin ISK-1's ability to exert its potent antibacterial activity..
160. Jun-ichi Nagao, Sikder M Asaduzzaman, Yuji Aso, Ken-Ichi Okuda, Jiro Nakayama, Kenji Sonomoto, Lantibiotics: insight and foresight for new paradigm., Journal of bioscience and bioengineering, 10.1263/jbb.102.139, 102, 3, 139-49, 2006.09, Lantibiotics are a unique type of antimicrobial peptide produced by a large number of gram-positive bacteria that contain unusual amino acids, such as lanthionine and dehydrated amino acids. Ribosomally synthesized lantibiotic prepeptide consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events to yield a biologically active lantibiotic. Research on lantibiotics has drawn much attention in recent years and has undergone extensive progress as a step forward to the next paradigm. Unusual amino acids in lantibiotics solely contribute to their biological activity and also enhance their structural stability. Thus, enzymes involved in lantibiotic biosynthesis would have a high potential for peptide engineering by introducing unusual amino acids into desired peptides, which may establish a universal approach to advance the structural design of novel peptides, termed lantibiotic engineering. In this review, we focus on recent development with contemporary innovations and perspective of lantibiotic research..
161. Takeshi Zendo, Shoko Koga, Yasushi Shigeri, Jiro Nakayama, Kenji Sonomoto, Lactococcin Q, a novel two-peptide bacteriocin produced by Lactococcus lactis QU 4., Applied and environmental microbiology, 10.1128/AEM.72.5.3383-3389.2006, 72, 5, 3383-3389, 2006.05, A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, alpha and beta, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qalpha plus Qbeta) and lactococcin G (Galpha plus Gbeta) clarified that hybrid combinations (Qalpha plus Gbeta and Galpha plus Qbeta) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qalpha plus Gbeta was 32 times higher than that of Qalpha plus Qbeta, suggesting that the difference in beta peptides was important for the intensity of antibacterial activity..
162. Takeshi Zendo, N. Eungruttanagorn, S. Fujioka, Yukihiro Tashiro, K. Nomura, Y. Sera, G. Kobayashi, Jiro Nakayama, A. Ishizaki, Kenji Sonomoto, Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean, Journal of Applied Microbiology, 10.1111/j.1365-2672.2005.02704.x, 99, 5, 1181-1190, 2005.11, Aims: Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. Methods and Results: The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca 2+ (CaCO3 or CaCl2). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6.0 whereas the highest cell growth was obtained at pH 7.0. Conclusions: Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca2+ and pH) influenced the bacteriocin production. Significance and Impact of the Study: A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains..
163. Jun-Ichi Nagao, Yoshitaka Harada, Kouki Shioya, Yuji Aso, Takeshi Zendo, Jiro Nakayama, Kenji Sonomoto, Lanthionine introduction into nukacin ISK-1 prepeptide by co-expression with modification enzyme NukM in Escherichia coli., Biochemical and biophysical research communications, 10.1016/j.bbrc.2005.08.125, 336, 2, 507-513, 2005.10, We demonstrated lanthionine introduction into hexa-histidine-tagged (His-tagged) nukacin ISK-1 prepeptide NukA by modification enzyme NukM in Escherichia coli. Co-expression of nukA and nukM, purification of the resulting His-tagged prepeptide by affinity chromatography, and subsequent mass spectrometry analysis showed that the prepeptide was converted into a postulated peptide with decrease in mass of 72Da which resulted from dehydration of four amino acids. Characterization of the resultant prepeptide indicated the presence of unusual amino acids, such as dehydrated amino acid, lanthionine or 3-methyllanthionine, in its C-terminal propeptide moiety. The modified prepeptide encompassing the leader peptide attached to the post-translationally modified propeptide moiety was readily obtained by one-step purification. Our findings will thus be a powerful tool for introducing unusual amino acids aimed at peptide engineering and also helpful to provide new insight for further understanding of lanthionine-forming enzymes for lantibiotics..
164. Ken-ichi Okuda, Yuji Aso, Jun-ichi Nagao, Kouki Shioya, Youhei Kanemasa, Jiro Nakayama, Kenji Sonomoto, Characterization of functional domains of lantibiotic-binding immunity protein, NukH, from Staphylococcus warneri ISK-1., FEMS microbiology letters, 10.1016/j.femsle.2005.06.039, 250, 1, 19-25, 2005.09, The immunity to a lantibiotic, nukacin ISK-1, is conferred by NukFEG (ABC transporter) and NukH (lantibiotic-binding protein) cooperatively. The present study identifies the functional domains of NukH. The topological analysis indicated that NukH possesses two external loops and three transmembrane helices. Deletion of N or C terminus of NukH did not affect the function. Amino acids substitutions in the respective loops abolished the function. Deletion of the third transmembrane helix resulted in loss of immunity but did not affect the binding activity. These findings suggested that the whole structure of NukH, except for N and C termini, is essential for its full immunity function, and that NukH inactivates nukacin ISK-1 after binding..
165. Jun Ichi Nagao, Yuji Aso, Toshihiro Sashihara, Kouki Shioya, Asaho Adachi, Jiro Nakayama, Kenji Sonomoto, Localization and interaction of the biosynthetic proteins for the lantibiotic, nukacin ISK-1, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.69.1341, 69, 7, 1341-1347, 2005.09, Nukacin ISK-1 is a type-A(II) lantibiotic produced by Staphylococcus warneri ISK-1. In this study, we characterized NukM and NukT, which are predicted to be involved in modification of prepeptide (NukA) and cleavage of leader peptide and subsequent secretion respectively. Localization analysis of NukM and NukT in the wild-type strain indicated that both proteins were located at the cytoplasm membrane. Interestingly, NukM expressed heterologously in St. carnosus TM300 was also located at the cytoplasm membrane even in the absence of NukT. Yeast two-hybrid assay showed that a complex of at least two each of NukM and NukT was associated with NukA. In vitro interaction analysis by surface plasmon resonance biosensor further suggested that membrane-located NukM interacted with NukA. These results indicate that NukM and NukT form a membrane-located multimeric protein complex and that post-translational modification of nukacin ISK-1 would occur at the cytoplasm membrane..
166. Ken Ichi Okuda, Yuji Aso, Jun Ichi Nagao, Kouki Shioya, Youhei Kanemasa, Jiro Nakayama, Kenji Sonomoto, Characterization of functional domains of lantibiotic-binding immunity protein, NukH, from Staphylococcus warneri ISK-1, FEMS Microbiology Letters, 10.1016/j.femsle.2005.06.039, 250, 1, 19-25, 2005.09, The immunity to a lantibiotic, nukacin ISK-1, is conferred by NukFEG (ABC transporter) and NukH (lantibiotic-binding protein) cooperatively. The present study identifies the functional domains of NukH. The topological analysis indicated that NukH possesses two external loops and three transmembrane helices. Deletion of N or C terminus of NukH did not affect the function. Amino acids substitutions in the respective loops abolished the function. Deletion of the third transmembrane helix resulted in loss of immunity but did not affect the binding activity. These findings suggested that the whole structure of NukH, except for N and C termini, is essential for its full immunity function, and that NukH inactivates nukacin ISK-1 after binding..
167. Yuji Aso, Ken Ichi Okuda, Jun Ichi Nagao, Youhei Kanemasa, Nguyen Thi Bich Phuong, Hanako Koga, Kouki Shioya, Toshihiro Sashihara, Jiro Nakayama, Kenji Sonomoto, A novel type of immunity protein, NukH, for the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.69.1403, 69, 7, 1403-1410, 2005.09, Staphylococcus warneri ISK-1 produces a lantibiotic, nukacin ISK-1. The nukacin ISK-1 gene cluster consists of at least six genes, nukA, -M, -T, -F, -E, and -G, and two open reading frames, ORF1 and ORF7 (designated nukH). Sequence comparisons suggested that NukF, -E, -G, and -H contribute to immunity to nukacin ISK-1. We investigated the immunity levels of recombinant Lactococcus lactis expressing nukFEG and nukH against nukacin ISK-1. The co-expression of nukFEG and nukH resulted in a high degree of immunity. The expression of either nukFEG or nukH conferred partial immunity against nukacin ISK-1. These results suggest that NukH contributes cooperatively to self-protection with Nuk-FEG. The nukacin ISK-1 immunity system might function against another lantibiotic, lacticin 481. Western blot analysis showed that NukH expressed in Staphylococcus carnosus was localized in the membrane. Peptide release/bind assays indicated that the recombinant L. lactis expressing nukH interacted with nukacin ISK-1 and lacticin 481 but not with nisin A. These findings suggest that NukH contributes cooperatively to host immunity as a novel type of lantibiotic-binding immunity protein with NukFEG..
168. Mark H J Sturme, Jiro Nakayama, Douwe Molenaar, Yoshiko Murakami, Ryoko Kunugi, Toshio Fujii, Elaine E Vaughan, Michiel Kleerebezem, Willem M de Vos, An agr-like two-component regulatory system in Lactobacillus plantarum is involved in production of a novel cyclic peptide and regulation of adherence., Journal of bacteriology, 10.1128/JB.187.15.5224-5235.2005, 187, 15, 5224-5235, 2005.08, We have analyzed a locus on the annotated Lactobacillus plantarum WCFS1 genome that showed homology to the staphylococcal agr quorum-sensing system and designated it lam for Lactobacillus agr-like module. Production of the lamBDCA transcript was shown to be growth phase dependent. Analysis of a response regulator-defective mutant (Delta)lamA) in an adherence assay showed that lam regulates adherence of L. plantarum to a glass surface. Global transcription analysis of the wild-type and (Delta)lamA strains in early, mid-, and late log phase of growth was performed using a clone-based microarray. Remarkably, only a small set of genes showed significant differences in transcription profiles between the wild-type and lamA mutant strains. The microarray analysis confirmed that lamBDCA is autoregulatory and showed that lamA is involved in regulation of expression of genes encoding surface polysaccharides, cell membrane proteins, and sugar utilization proteins. The lamBD genes encoding the putative autoinducing peptide precursor (LamD) and its processing protein (LamB) were overexpressed using the nisin-controlled expression system, and culture supernatants were analyzed by liquid chromatography/mass spectrometry (LC/MS) to identify overproduced LamD-derived peptides. In this way, a cyclic thiolactone pentapeptide that possesses a ring structure similar to those of autoinducing peptides of the staphylococcal agr system was identified. The peptide was designated LamD558, and its sequence (CVGIW) matched the annotated precursor peptide sequence. Time course analysis of wild-type culture supernatants by LC/MS indicated that LamD558 production was increased markedly from mid-log to late log growth phase. This is the first example of an agr-like system in nonpathogenic bacteria that encodes a cyclic thiolactone autoinducing peptide and is involved in regulation of adherence..
169. Jun-ichi Nagao, Yuji Aso, Toshihiro Sashihara, Kouki Shioya, Asaho Adachi, Jiro Nakayama, Kenji Sonomoto, Localization and interaction of the biosynthetic proteins for the lantibiotic, Nukacin ISK-1., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.69.1341, 69, 7, 1341-7, 2005.07, Nukacin ISK-1 is a type-A(II) lantibiotic produced by Staphylococcus warneri ISK-1. In this study, we characterized NukM and NukT, which are predicted to be involved in modification of prepeptide (NukA) and cleavage of leader peptide and subsequent secretion respectively. Localization analysis of NukM and NukT in the wild-type strain indicated that both proteins were located at the cytoplasm membrane. Interestingly, NukM expressed heterologously in St. carnosus TM300 was also located at the cytoplasm membrane even in the absence of NukT. Yeast two-hybrid assay showed that a complex of at least two each of NukM and NukT was associated with NukA. In vitro interaction analysis by surface plasmon resonance biosensor further suggested that membrane-located NukM interacted with NukA. These results indicate that NukM and NukT form a membrane-located multimeric protein complex and that post-translational modification of nukacin ISK-1 would occur at the cytoplasm membrane..
170. Yuji Aso, Ken-ichi Okuda, Jun-ichi Nagao, Youhei Kanemasa, Nguyen Thi Bich Phuong, Hanako Koga, Kouki Shioya, Toshihiro Sashihara, Jiro Nakayama, Kenji Sonomoto, A novel type of immunity protein, NukH, for the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.69.1403, 69, 7, 1403-10, 2005.07, Staphylococcus warneri ISK-1 produces a lantibiotic, nukacin ISK-1. The nukacin ISK-1 gene cluster consists of at least six genes, nukA, -M, -T, -F, -E, and -G, and two open reading frames, ORF1 and ORF7 (designated nukH). Sequence comparisons suggested that NukF, -E, -G, and -H contribute to immunity to nukacin ISK-1. We investigated the immunity levels of recombinant Lactococcus lactis expressing nukFEG and nukH against nukacin ISK-1. The co-expression of nukFEG and nukH resulted in a high degree of immunity. The expression of either nukFEG or nukH conferred partial immunity against nukacin ISK-1. These results suggest that NukH contributes cooperatively to self-protection with NukFEG. The nukacin ISK-1 immunity system might function against another lantibiotic, lacticin 481. Western blot analysis showed that NukH expressed in Staphylococcus carnosus was localized in the membrane. Peptide release/bind assays indicated that the recombinant L. lactis expressing nukH interacted with nukacin ISK-1 and lacticin 481 but not with nisin A. These findings suggest that NukH contributes cooperatively to host immunity as a novel type of lantibiotic-binding immunity protein with NukFEG..
171. Prapa Songjinda, Jiro Nakayama, Yumiko Kuroki, Shigemitsu Tanaka, Sanae Fukuda, Chikako Kiyohara, Tetsuro Yamamoto, Kunio Izuchi, Taro Shirakawa, Kenji Sonomoto, Molecular monitoring of the developmental bacterial community in the gastrointestinal tract of Japanese infants., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.69.638, 69, 3, 638-41, 2005.03, The dynamics of the developmental bacterial community in the Japanese neonatal gastrointestinal tract were examined by monitoring 16S ribosomal RNA gene (rDNA) diversity in fecal samples by PCR and denaturing gradient gel electrophoresis (DGGE). The results showed a certain pattern common in infants without antibiotic treatment, in which aerobes, e.g., Pseudomonas, appeared first and were then immediately replaced by facultative anaerobe, Enterococcus, Streptococcus, and Enterobacteriaceae through the first month, and finally strictly anaerobic Bifidobactrerium appeared..
172. Yuji Aso, Hanako Koga, Toshihiro Sashihara, Jun-Ichi Nagao, Youhei Kanemasa, Jiro Nakayama, Kenji Sonomoto, Description of complete DNA sequence of two plasmids from the nukacin ISK-1 producer, Staphylococcus warneri ISK-1., Plasmid, 10.1016/j.plasmid.2004.08.003, 53, 2, 164-178, 2005.03, We report the whole DNA sequence of two plasmids, pPI-1 (30.2 kb) and pPI-2 (2.8 kb). These plasmids are from Staphylococcus warneri ISK-1, which produces a lantibiotic, nukacin ISK-1. Curing of pPI-1 resulted in a loss of bactericidal activity in the culture supernatant and the host's immunity to nukacin ISK-1, suggesting that the biosynthetic genes of the bacteriocin are encoded by pPI-1. Based on the results of a homology search of each open reading flame, pPI-1 is comprised of the following four distinct regions: (1) the nukacin ISK-1 biosynthesis and immunity gene cluster, (2) the thioredoxin gene cluster, (3) the replication region, and (4) a region of Staphylococcus epidermidis ATCC 12228, highly homologous to pSE-12228-05. Gene organization in the nukacin ISK-1 biosynthesis and immunity gene cluster is different from that in other lacticin-481 type gene clusters. The features of the replication protein encoded in the replicating region are somewhat different from other staphylococcus theta-replicating plasmids. pPI-2 comprised a disinfectant resistant gene, qacC, and the whole DNA sequence showed significant similarity to those of other qacC plasmids such as pSK108, suggesting that pPI-2 belongs to the qacC plasmid group..
173. Prapa Songjinda, Jiro Nakayama, Yumiko Kuroki, Shigemitsu Tanaka, Sanae Fukuda, Chikako Kiyohara, Tetsuro Yamamoto, Kunio Izuchi, Taro Shirakawa, Kenji Sonomoto, Molecular monitoring of the developmental bacterial community in the gastrointestinal tract of Japanese infants, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.69.638, 69, 3, 638-641, 2005.03, The dynamics of the developmental bacterial community in the Japanese neonatal gastrointestinal tract were examined by monitoring 16S ribosomal RNA gene (rDNA) diversity in fecal samples by PCR and denaturing gradient gel electrophoresis (DGGE). The results showed a certain pattern common in infants without antibiotic treatment, in which aerobes, e.g., Pseudomonas, appeared first and were then immediately replaced by facultative anaerobe, Enterococcus, Streptococcus, and Enterobacteriaceae through the first month, and finally strictly anaerobic Bifidobactrerium appeared..
174. Amonlaya Tosukhowong, Jiro Nakayama, Yoshimitsu Mizunoe, Shinya Sugimoto, Daisuke Fukuda, Kenji Sonomoto, Reconstitution and function of Tetragenococcus halophila chaperonin 60 tetradecamer., Journal of bioscience and bioengineering, 10.1263/jbb.99.30, 99, 1, 30-7, 2005.01, Tetragenococcus halophila originally isolated from soy sauce is a halophilic lactic acid bacterium which can grow under 4 M sodium chloride. T. halophila chaperonin composed of a core moiety of chaperonin 60 (cpn60) and a lid moiety of chaperonin 10 (cpn10), is thought to contribute to host halotolerant capability. In this report, we reconstituted and characterized the core complex of T. halophila chaperonin by using a recombinant T. halophila cpn60 (Tcpn60) overexpressed in Escherichia coli. The reconstitution of Tcpn60 was performed in the presence of 10 mM MgCl2, 2 mM ATP and 0.8 M (NH4)2SO4 and the resultant oligomer was purified by gel filtration chromatography. Electron microscopy of the reconstituted Tcpn60 revealed a double toroidal tetradecameric structure that is characteristic of bacterial cpn60. The T. halophila tetradecamer cpn60 exhibited an ATPase activity and a refolding activity of both chemically and thermally denatured enolases under wide range of salt concentrations. Furthermore, we demonstrated that heterologous expression of Tcpn60 allowed the normal growth of host Escherichia coli cells under salt stress conditions and this effect was further enhanced by co-expression with Tcpn10. These results suggested that Tcpn60 contributes to the halotolerance property of T. halophila cell as a tetradecamer complex, probably associated with the Tcpn10 complex..
175. Yuji Aso, Toshihiro Sashihara, Jun-Ichi Nagao, Youhei Kanemasa, Hanako Koga, Taku Hashimoto, Toshimasa Higuchi, Asaho Adachi, Harumi Nomiyama, Ayaaki Ishizaki, Jiro Nakayama, Kenji Sonomoto, Characterization of a gene cluster of Staphylococcus warneri ISK-1 encoding the biosynthesis of and immunity to the lantibiotic, nukacin ISK-1., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.68.1663, 68, 8, 1663-1671, 2004.08, We characterized a gene cluster in a plasmid designated pPI-1 of Staphylococcus warneri ISK-1 encoding the biosynthesis of and immunity to the lacticin-481 type lantibiotic, nukacin ISK-1. The DNA sequence suggested that the nukacin ISK-1 gene cluster consists of at least six genes, nukA (a structural gene), -M, -T, -F, -E, -G, and two open reading frames, ORF1 and ORF7. NukM and NukT were predicted to be involved in post-translational modification and secretion of nukacin ISK-1 respectively. NukF, -E, and -G were predicted to form a membrane complex which contributes to self-protection from nukacin ISK-1. Transcriptional analyses revealed that nukM through ORF7 comprises an operon, and that ORF1 is transcribed independently from downstream of nukA. The transcriptional levels of the nukA and nukM genes were enhanced by osmotic stress. The expression level of the nukA transcript was scarcely enhanced by nukacin ISK-1, suggesting that expression is not under the control of the autoregulatory circuit..
176. Wilaipun, P., T. Zendo, M. Sangjindavong, S. Nitisinprasert, V. Leelawatcharamas, J. Nakayama, and K. Sonomoto, The two-synergistic peptide bacteriocin produced by Enterococcus faecium NKR-5-3 isolated from Thai fermented fish (Plara)., ScienceAsia, 2004.01.
177. Yuji Aso, Jun Ichi Nagao, Hanako Koga, Ken Ichi Okuda, Youhei Kanemasa, Toshihiro Sashihara, Jiro Nakayama, Kenji Sonomoto, Heterologous expression and functional analysis of the gene cluster for the biosynthesis of and immunity to the lantibiotic, nukacin ISK-1, Journal of Bioscience and Bioengineering, 10.1016/S1389-1723(05)00308-7, 98, 6, 429-436, 2004.01, Nukacin ISK-1 is a lantibiotic produced by Staphylococcus warneri ISK-1. The gene cluster of nukacin ISK-1 consists of at least nukAMTFEG, ORF1 and ORF7. In this study, we demonstrated the heterologous production of nukacin ISK-1 in Lactococcus lactis by the artificial polycistronic expression of nukAMTFEG-ORF7 under the control of the nisin-controlled expression (NICE) system. Consequently, the recombinant L. lactis showed antimicrobial activity. Mass analysis clarified the presence of nukacin ISK-1 produced in the culture supernatant. These results suggested that the recombinant L. l.actis produced nukacin ISK-1 heterologously. Inactivation of nukA, -M or -T resulted in the complete loss of the nukacin ISK-1 production phenotype. This finding suggested that nukAMT are indispensably associated with the biosynthesis of nukacin ISK-1. To our knowledge, this is the first report of the heterologous production of lantibiotic using the NICE system..
178. Hiroshi Matsuura, Susumu Okamoto, Sarintip Anamnart, Qiuqi Wang, Ze-Yang Zhou, Takuya Nihira, Yasuhiro Yamada, Tomohisa Kuzuyama, Haruo Seto, Jiro Nakayama, Akinori Suzuki, Hiromichi Nagasawa, Shohei Sakuda, Nucleotide sequences of genes encoding allosamidin-sensitive and -insensitive chitinases produced by allosamidin-producing Streptomyces., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.67.2002, 67, 9, 2002-2005, 2003.09, Allosamidin is a strong inhibitor of family 18 chitinases. We previously reported the presence of allosamidin-sensitive and -insensitive chitinases (chitinase S and IS) in the culture filtrate of the allosamidin-producing strain, Streptomyces sp. AJ9463. In this study, we cloned and sequenced the genes encoding the two chitinases, which clarified that chitinase S and IS belong to the family 18 and 19 chitinase, respectively..
179. Takeshi Zendo, Masanori Fukao, Kyoko Ueda, Tomoko Higuchi, Jiro Nakayama, Kenji Sonomoto, Identification of the lantibiotic nisin Q, a new natural nisin variant produced by Lactococcus lactis 61-14 isolated from a river in Japan., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.67.1616, 67, 7, 1616-9, 2003.07, Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria. N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q. Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence..
180. Jiro Nakayama, Antoon D L Akkermans, Willem M De Vos, High-throughput PCR screening of genes for three-component regulatory system putatively involved in quorum sensing from low-G + C gram-positive bacteria., Bioscience, biotechnology, and biochemistry, 10.1271/bbb.67.480, 67, 3, 480-489, 2003.03, Quorum sensing of gram-positive bacteria is often regulated by three-component regulatory system composed of autoinducing peptide, sensor kinase and response regulator. We used PCR to study a gene cassette encoding this three-component regulatory system. Degenerate primers were designed from consensus amino acid sequences in the HPK10 subfamily, mostly involved in quorum sensing. Products amplified from genomic DNA of Lactobacillus, Enterococcus, and Clostridium species were cloned and sequenced; their deduced amino acid sequences were similar to those of members of the HPK10 subfamily. Complete genes for the putative gene cassette were cloned by inverse PCR from L. paracasei E93490 and L. plantarum WCFS6. Phylogenetic analysis grouped the cloned putative HPKs into the HPK10 subfamily. These results indicated the usefulness of this high-throughput gene screening and suggested that the three-component regulatory gene cassette are widely present..
181. Takeshi Zendo, Masanori Fukao, Kyoko Ueda, Tomoko Higuchi, Jiro Nakayama, Kenji Sonomoto, Identification of the lantibiotic nisin q, a new natural nisin variant produced by lactococcus lactis 61-14 isolated from a river in japan, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.67.1616, 67, 7, 1616-1619, 2003.01, Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria. N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q. Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence..
182. Shinya Sugimoto, Jiro Nakayama, Daisuke Fukuda, Shino Sonezaki, Maki Watanabe, Amonlaya Tosukhowong, Kenji Sonomoto, Effect of heterologous expression of molecular chaperone DnaK from Tetragenococcus halophilus on salinity adaptation of Escherichia coli, Journal of Bioscience and Bioengineering, 10.1016/S1389-1723(03)90114-9, 96, 2, 129-133, 2003.01, Molecular chaperone DnaK of halophilic Tetragenococcus halophilus JCM5888 was characterized under salinity conditions both in vitro and in vivo. The dnaK gene was cloned into an expression vector and transformed into Escherichia coli. The DnaK protein obtained from the recombinant E. coli showed a significantly higher refolding activity of denatured lactate dehydrogenase than that from non-halophilic Lactococcus lactis under NaCl concentrations higher than 1 M. E. coil without the overexpression of DnaK exhibited a growth profile with a prolonged lag phase and suppressed maximum cell density in Luria-Bertani medium containing 5% (0.86 M) NaCl. On the contrary, the overexpression of T. halophilus DnaK greatly shortened this prolonged lag phase with no effect on maximum growth, while that of L. lactis DnaK decreased maximum growth. The amount of protein aggregates was increased by salt stress in the E. coli cells, while this aggregation was greatly suppressed by the overexpression of T. halophilus DnaK. These results suggest that heterologous overexpression of T. halophilus DnaK, via its chaperone activity, promotes salinity adaptation of E. coli..
183. Takaaki Horii, Hiromichi Nagasawa, Jiro Nakayama, Functional analysis of TraA, the sex pheromone receptor encoded by pPD1, in a promoter region essential for the mating response in Enterococcus faecalis., Journal of bacteriology, 10.1128/JB.184.22.6343-6350.2002, 184, 22, 6343-50, 2002.11, Conjugative transfer of a bacteriocin plasmid, pPD1, of Enterococcus faecalis is induced in response to a peptide sex pheromone, cPD1, secreted from plasmid-free recipient cells. cPD1 is taken up by a pPD1 donor cell and binds to an intracellular receptor, TraA. Once a recipient cell acquires pPD1, it starts to produce an inhibitor of cPD1, termed iPD1, which functions as a TraA antagonist and blocks self-induction in donor cells. In this study, we discuss how TraA transduces the signal of cPD1 to the mating response. Gel mobility shift assays indicated that TraA is bound to a traA-ipd intergenic region, which is essential for cPD1 response. DNase I footprinting analysis suggested the presence of one strong (tab1) and two weak (tab2 and tab3) TraA-binding sites in the intergenic region. Primer extension analysis implied that the transcriptional initiation sites of traA and ipd were located in the intergenic region. Northern analysis showed that cPD1 upregulated and downregulated transcription of ipd and traA, respectively. The circular permutation assay showed that TraA bent a DNA fragment corresponding to the tab1 region, and its angle was changed in the presence of cPD1 or iPD1. From these data, we propose a model that TraA changes the conformation of the tab1 region in response to cPD1 and upregulates the transcription of ipd, which may lead to expression of genes required for the mating response..
184. Ayumu Fukuda, Shin-Ichi Matsuyama, Takashi Hara, Jiro Nakayama, Hiromichi Nagasawa, Hajime Tokuda, Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals., The Journal of biological chemistry, 10.1074/jbc.M206816200, 277, 45, 43512-43518, 2002.11, Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification..
185. Jiro Nakayama, Reiko Kariyama, Hiromi Kumon, Description of a 23.9-kilobase chromosomal deletion containing a region encoding fsr genes which mainly determines the gelatinase-negative phenotype of clinical isolates of Enterococcus faecalis in urine., Applied and environmental microbiology, 10.1128/AEM.68.6.3152-3155.2002, 68, 6, 3152-3155, 2002.06, Expression of virulence-related extracellular proteases, gelatinase, and serine protease of Enterococcus faecalis is regulated by a quorum-sensing system encoded by the fsr gene cluster. In this study, a 23.9-kb chromosomal deletion containing the fsr gene cluster region was found to be present in the majority (79%) of gelatinase-negative clinical isolates of E. faecalis from urine..
186. Takaaki Horii, Hiromichi Nagasawa, Jiro Nakayama, Functional analysis of TraA, the sex pheromone receptor encoded by pPD1, in a promoter region essential for the mating response in Enterococcus faecalis, Journal of Bacteriology, 10.1128/JB.184.22.6343-6350.2002, 184, 22, 6343-6350, 2002.01, Conjugative transfer of a bacteriocin plasmid, pPD1, of Enterococcus faecalis is induced in response to a peptide sex pheromone, cPD1, secreted from plasmid-free recipient cells. cPD1 is taken up by a pPD1 donor cell and binds to an intracellular receptor, TraA. Once a recipient cell acquires pPD1, it starts to produce an inhibitor of cPD1, termed iPD1, which functions as a TraA antagonist and blocks self-induction in donor cells. In this study, we discuss how TraA transduces the signal of cPD1 to the mating response. Gel mobility shift assays indicated that TraA is bound to a traA-ipd intergenic region, which is essential for cPD1 response. DNase I footprinting analysis suggested the presence of one strong (tab1) and two weak (tab2 and tab3) TraA-binding sites in the intergenic region. Primer extension analysis implied that the transcriptional initiation sites of traA and ipd were located in the intergenic region. Northern analysis showed that cPD1 upregulated and downregulated transcription of ipd and traA, respectively. The circular permutation assay showed that TraA bent a DNA fragment corresponding to the tab1 region, and its angle was changed in the presence of cPD1 or iPD1. From these data, we propose a model that TraA changes the conformation of the tab1 region in response to cPD1 and upregulates the transcription of ipd, which may lead to expression of genes required for the mating response..
187. Jiro Nakayama, Yong Cao, Takaaki Horii, Shohei Sakuda, Hiromichi Nagasawa, Chemical synthesis and biological activity of the gelatinase biosynthesis-activating pheromone of enterococcus faecalis and its analogs, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.65.2322, 65, 10, 2322-2325, 2001.10, An 11-residue peptide lactone, termed the gelatinase biosynthesis-activating pheromone (GBAP), triggers the production of the pathogenicit)-related extracellular proteases, gelatinase and serine protease, in Enterococcus faecalis. In this study, we synthesized GBAP and its analogs and examined their gelatinase biosynthesis-inducing activity. This study on the structure-activity relationship shows that a lactone ring was indispensable for the activity..
188. Jiro Nakayama, Yong Cao, Takaaki Horii, Shohei Sakuda, Antoon D L Akkermans, Willem M. De Vos, Hiromichi Nagasawa, Gelatinase biosynthesis-activating pheromone: a peptide lactone that mediates a quorum sensing in Enterococcus faecalis, Molecular Microbiology, 10.1046/j.1365-2958.2001.02486.x, 41, 1, 145-154, 2001.01, Biosynthesis of gelatinase, a virulence factor of Enterococcus faecalis, was found to be regulated in a cell density-dependent fashion in which its production is active in late log to early stationary phase. Addition of early stationary phase culture filtrate to medium shifted the onset of gelatinase production to that of mid-log phase, suggesting that E. faecalis secretes a gelatinase biosynthesis-activating pheromone (GBAP). GBAP was isolated from culture supernatant of E. faecalis OG1S-P. Structural analysis suggested GBAP to be an 11-residue cyclic peptide containing a lactone structure, in which the α-carboxyl group of the C-terminal amino acid is linked to a hydroxyl group of the serine of the third residue. A synthetic peptide possessing the deduced structure showed GBAP activity at nanomolar concentrations as did natural GBAP. Database searches revealed that GBAP corresponds to a C-terminal part of a 242-residue FsrB protein. Northern analysis showed that GBAP slowly induces the transcription of two operons, fsrB-fsrC encoding FsrB and a putative histidine kinase FsrC and gelE-sprE encoding gelatinase GelE and serine protease SprE. Strains with an insertion mutation in either fsrC or a putative response regulator gene fsrA failed to respond to GBAP, suggesting that the GBAP signal is transduced by a two-component regulatory system..
189. S. Sakuda, M. Ono, H. Ikeda, T. Nakamura, Y. Inagaki, R. Kawachi, Jiro Nakayama, A. Suzuki, A. Isogai, H. Nagasawa, Blasticidin A as an inhibitor of aflatoxin production by Aspergillus parasiticus, Journal of Antibiotics, 10.7164/antibiotics.53.1265, 53, 11, 1265-1271, 2000.01, Blasticidin A, an antibiotic, showed strong inhibitory activity toward aflatoxin production by Aspergillus parasiticus. Its structure was characterized by NMR and chemical degradation experiments as 1, which is a tetramic acid derivative with a highly oxygenated long alkyl chain similar to aflastatin A (2). Absolute configurations of the eight chiral centers at C-4, 6, 31, 32, 33, 34, 35 and 37 of 1 were chemically determined. Blasticidin A almost completely inhibited aflatoxin production at 0.5 μm..
190. Jiro Nakayama, Yuuichiro Takanami, Takaaki Horii, Shohei Sakuda, Akinori Suzuki, Molecular mechanism of peptide-specific pheromone signaling in Enterococcus faecalis: Functions of pheromone receptor TraA and pheromone- binding protein TraC encoded by plasmid pPD1, Journal of Bacteriology, 10.1128/jb.180.3.449-456.1998, 180, 3, 449-456, 1998.02, Conjugative transfer of the Enterococcus faecalis plasmid pPD1 is activated by cPD1, one of several peptide sex pheromones secreted by plasmid- free recipient cells, and is blocked by a donor-produced peptide inhibitor, iPD1. Using a tritiated pheromone, [ H]cPD1, we investigated how pPD1- harboring donor cells receive these peptide signals. Donor cells rapidly incorporated [ H]cPD1. The cell extract but not the membrane fraction of the donor strain exhibited significant [ H] cPD1-binding activity. On the basis of these data and those of tracer studies, it was demonstrated that cPD1 was internalized, where it bound to a high-molecular-weight compound. The cell extract of a strain carrying the traA-bearing multicopy plasmid (pDLHH21) also exhibited high [ H]cPD1-binding activity. A recombinant TraA exhibited a dissociation constant of 0.49 ± 0.08 nM against [ H]cPD1. iPD1 competitively inhibited [ H]cPD1 binding to TraA, whereas pheromones and inhibitors relating to other plasmid systems did not. These results show that TraA is a specific intracellular receptor for cPD1 and that iPD1 acts as an antagonist for TraA. A strain carrying the traC-bearing multicopy plasmid (pDLES23) exhibited significant [ H]cPD1-binding activity. A strain carrying traC-disrupted pPD1 (pAM351CM) exhibited lower [ H]cPD1-binding activity as well as lower sensitivity to cPD1 than a wild-type donor strain. Some of the other pheromones and inhibitors inhibited [ H]cPD1 binding to the traC transformant like cPD1 and iPD1 did. These results show that TraC, as an extracellular less-specific pheromone-binding protein, supports donor cells to receive cPD1. 3 3 3 3 3 3 3 3 3.
191. Makoto Ono, Shohei Sakuda, Hiroyuki Ikeda, Kazuo Furihata, Jiro Nakayama, Akinori Suzuki, Akira Isogai, Structures and biosynthesis of aflastatins: novel inhibitors of aflatoxin production by Aspergillus parasiticus, Journal of Antibiotics, 10.7164/antibiotics.51.1019, 51, 11, 1019-1028, 1998.01, Two novel inhibitors of aflatoxin production by Aspergillus parasiticus were isolated from the mycelial extracts of Streptomyces sp. MRI142 and termed aflastatin A and B. The structures of aflastatin A (1) and B (5) were elucidated by NMR and chemical degradation experiments. These compounds have a novel skeleton of a tetramic acid derivative with a highly oxygenated long alkyl chain. The incorporation experiments using 13C-labeled acetates, propionate, glucose and glycolate suggested that most of the C2 and C3 units involved in the alkyl chain moiety of aflastatin A were biosynthesized from acetic and propionic acids, but five C2 units in the alkyl chain originated from glycolic acid..
192. Shohei Sakuda, Ono Makoto, Hiroyuki Ikeda, Yasuhito Inagaki, Jiro Nakayama, Akinori Suzuki, Akira Isogai, Structure of blasticidin A, Tetrahedron Letters, 10.1016/S0040-4039(97)01734-6, 38, 42, 7399-7402, 1997.10, The structure of blasticidin A was characterized as 1, which is a tetramic acid derivative with a highly oxygenated long alkyl chain similar to aflastatin A 2..
193. Jiro Nakayama, Yuuichiro Takanami, Akinori Suzuki, Analysis of pheromone binding and pheromone reception by Enterococcus faecalis, Advances in Experimental Medicine and Biology, 418, 1033-1035, 1997.10.
194. Jiro Nakayama, Akinori Suzuki, Genetic analysis of plasmid-specific pheromone signaling encoded by ppd1 in enterococcus faecalis, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.61.1796, 61, 11, 1796-1799, 1997.01, Certain plasmids in Enterococcus faecalis encode a mating response to recipient-produced peptide sex pheromones. Targeted disruption of tra genes on pPD1 suggested that TraA plays a central role in the plasmid-specific pheromone signaling pathway. TraA functioned as a negative regulator for the pheromone-inducible conjugal transfer. Complementation analysis of pPD1 tra gene mutants by pAD1 suggested that the pheromone binding function of TraC was non-specific between these plasmids, but the function of TraA and the pheromone shutdown function of TraB are plasmid-specific. © 1997, Taylor & Francis Group, LLC. All rights reserved..
195. Shohei Sakuda, Makoto Ono, Kazuo Furihata, Jiro Nakayama, Akinori Suzuki, Akira Isogai, Aflastatin A, a novel inhibitor of aflatoxin production of Aspergillus parasiticus, from Streptomyces, Journal of the American Chemical Society, 10.1021/ja960899d, 118, 33, 7855-7856, 1996.08.
196. Liro Nakayama, Shigeru Igarashi, Hiromichi Nagasawa, Don B. Clewell, Florence Y. An, Akinori Suzuki, Isolation and Structure of staph-cAM373 Produced by Staphylococcus aureus That Induces Conjugal Transfer of Enterococcus faecalis Plasmid pAM373, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.60.1038, 60, 6, 1038-1039, 1996.01, Enterococus faecalis plasmid pAM373 encodes a mating response to the sex pheromone, cAM373, which is secreted from pAM373-free E. faecalis. cAM373-like activity was detected in a culture filtrate of Staphylococcus aureus. The major active substance, termed staph-cAM313 was isolated, and its structure was identified as a H-Ala-Ile-Phe-Ile-Leu-Ala-Ala-OH heptapeptide. © 1996, Taylor & Francis Group, LLC. All rights reserved..
197. Jiro Nakayama, Don B. Clewell, Akinori Suzuki, Targeted disruption of the PD78 gene (traF) reduces pheromone-inducible conjugal transfer of the bacteriocin plasmid pPD1 in Enterococcus faecalis, FEMS Microbiology Letters, 10.1016/0378-1097(95)00118-O, 128, 3, 283-288, 1995.05, Bacterial sex pheromone, cPD1, induces sexual aggregation of Enterococcus faecalis harboring the bacteriocin plasmid, pPD1, and enables pPD1 to transfer at high frequency in a liquid culture. PD78 is a cPD1-inducible cell surface protein encoded by pPD1. The PD78 gene, traF, was disrupted by homologous recombination between pPD1 and an artificial vector having a deletion in the middle portion of traF. The disruption of traF did not affect the cPD1-inducible aggregation but reduced the transfer frequency of pPD1 to 2% of the wild-type level..
198. Akira Isogai, Akira Isogai, Jiro Nakayama, Jiro Nakayama, Akinori Suzuki, Akihiko Kusai, Jon Y. Takemoto, Structural Analysis of New Syringopeptins by Tandem Mass Spectrometry, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.59.1374, 59, 7, 1374-1376, 1995.01, New syringopeptins SP(SC)-1 and -2 were isolated from culture filtrates of phytopathogenic bacterium strain SC1 of Pseudomonas syringae pv. syringae. These syringopeptins were composed of a β-hydroxy fatty acid, a long sequence of aliphatic amino acids, and a lactone moiety of eight amino acids. The amino acid sequences were deduced from a comparison of their tandem mass sepctra with those of known syringopeptins SP-22a and SP-25a. SP(SC)-1 and SP(SC)-2 resembled SP-22a, but differed from the latter by 3 amino acids..
199. Naoyuki Fukuchi, Kazuo Furihata, Jiro Nakayama, Akira Isogai, Rotihibins, Novel Plant Growth Regulators from Streptomyces graminofaciens, Journal of Antibiotics, 10.7164/antibiotics.48.1004, 48, 9, 1004-1010, 1995.01, In the course of screening search for plant growth regulators, a culture filtrate of Streptomyces graminofaciens 3C02 was found to inhibit the growth of lettuce seedlings. The active substances, named rotihibin A (1) and B (2), were revealed to be lipo-peptidal compounds. Rotihibins inhibit growth of various plants at below 1 μg/ml, but do not show lethal activity even at higher doses..
200. Jiro Nakayama, G. M. Dunny, D. B. Clewell, A. Suzuki, Quantitative analysis for pheromone inhibitor and pheromone shutdown in Enterococcus faecalis., Developments in Biological Standardization, 85, 35-38, 1995.01.
201. Jiro Nakayama, Yukitsugu Ono, Akinori Suzuki, Isolation and Structure of the Sex Pheromone Inhibitor, iAM373, of Enterococcus faecalis, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.59.1358, 59, 7, 1358-1359, 1995.01, An Enterococcus faecalis plasmid, pAM373, has a high frequency of transfer in a liquid medium when induced by a recipient-produced sex pheromone, cAM373. The sex pheromone inhibitor against cAM373, termed iAM373, was isolated from a culture supernatant of E. faecalis harboring pAM377 (=pAM373::Tn917), and its structure was identified as a heptapeptide, H-Ser-Ile-Phe-Thr-Leu-Val-Ala-OH..
202. Jiro Nakayama, Yuki Abe, Yukitsugu Ono, Akira Isogai, Akinori Suzuki, Isolation and Structure of the Enterococcus faecalis Sex Pheromone, cOB1, that Induces Conjugal Transfer of the Hemolysin/Bacteriocin Plasmids, pOB1 and pYI1, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.59.703, 59, 4, 703-705, 1995.01, A bacterial sex pheromone, cOB1, which induces conjugal transfer of the Enterococcus faecalis hemolysin-bacteriocin (Hly/Bac) plasmid, pOB1, was isolated from the culture broth of pOB1-free E. faecalis. Its structure was found to be a hydrophobic octapeptide, H-Val-Ala-Val-Leu-Val-Leu-Gly-Ala-OH. The cOB1 peptide induced the mating response of not only pOB1 but also another incompatibility group Hly/Bac plasmid, pYI1..
203. Jiro Nakayama, K. Yoshida, H. Kobayashi, A. Isogai, D. B. Clewell, A. Suzuki, Cloning and characterization of a region of Enterococcus faecalis plasmid pPD1 encoding pheromone inhibitor (ipd), pheromone sensitivity (traC), and pheromone shutdown (traB) genes, Journal of Bacteriology, 10.1128/jb.177.19.5567-5573.1995, 177, 19, 5567-5573, 1995.01, Bacteriocin plasmid pPD1 in Enterococcus faecalis encodes a mating response to recipient-produced sex pheromone cPD1. Once a recipient acquires pPD1, transconjugants apparently shut off cPD1 activity in broth culture and no longer behave as recipients for pPD1. This event is performed by synthesis of the pheromone inhibitor iPD1 and also by repression of cPD1 production, the so-called 'pheromone shutdown.' A 5.4-kb EcoRV-HincII segment of pPD1, which expressed iPD1 in Escherichia coli, was sequenced and found to be organized as traC-traB-traA-ipd; each open reading frame is analogous to that found in other pheromone plasmids, pAD1 and pCF10, and thus is designated in accordance with the nomenclature in pAD1. The ipd gene encodes a peptide consisting of 21 amino acids, in which the C-terminal eight residues correspond to iPD1. The putative TraC product has a strong similarity to oligopeptide-binding proteins found in other bacterial species, as do pheromone-binding proteins of pCF10 and pPD1. A strain carrying traC- disrupted pPD1 required a concentration of cPD1 fourfold higher than that needed by the wild-type strain for induction of sexual aggregation. These results suggest that the TraC product contributes to pheromone sensitivity as o pheromone-binding protein. A strain transformed with traB-disrupted pPD1 produced a high level of cPD1 similar to that produced by plasmid-free recipients and underwent self-induction. Thus, the TraB product contributes to cPD1 shutdown..
204. Jiro Nakayama, R. E. Ruhfel, G. M. Dunny, A. Isogai, A. Suzuki, The prgQ gene of the Enterococcus faecalis tetracycline resistance plasmid pCF10 encodes a peptide inhibitor, iCF10, Journal of Bacteriology, 10.1128/jb.176.23.7405-7408.1994, 176, 23, 7405-7408, 1994.01, Conjugative transfer of the Enterococcus faecalis tetracycline resistance plasmid pCF10 is stimulated by a peptide pheromone, cCF10. Once a recipient strain acquires pCF10 and thus becomes a pheromone-responsive donor, cCF10 activity is no longer detected in culture filtrates. Here we show that pCF10 encodes a peptide inhibitor, iCF10, secreted by donor cells; this inhibitor antagonizes the cCF10 activity in culture filtrates. In order to detect and quantitate iCF10, we developed a reverse-phase high-performance liquid chromatography assay in which the inhibitor peptide elutes separately from the pheromone; this type of assay enabled us to determine that lack of pheromone activity in donor culture filtrates was due to secretion of a mixture of iCF10 and cCF10, rather than abolition of cCF10 secretion. The gene encoding iCF10, prgQ, is located on the EcoRI-C fragment of pCF10. The open reading frame comprising the prgQ gene encodes a 23-amino-acid precursor that resembles a signal peptide. This precursor is cleaved to the mature heptapeptide iCF10 during the secretion process..
205. Naoyuka Fukuchi, Akira Isogai, Jiro Nakayama, Seiji Takayama, Shuichi Yamashita, Kazuo Suyama, Jon Y. Takemoto, Akinori Suzuki, Structure and stereochemistry of three phytotoxins, syringomycin, syringotoxin and syringostatin, produced by Pseudomonas syringae pv. syringae, Journal of the Chemical Society, Perkin Transactions 1, 9, 1149-1157, 1992.12, The structures of two phytotoxins, syringomycin and syringotoxin, produced by Pseudomonas syringae pv. syringae, were determined. Several amino acid residues of syringomycin were different from those in the syringostatins. Syringotoxin B proved to be [Gly3]syringostatin A. The three kinds of phytotoxins showed close structural similarity, and the stereochemistry of their components was deduced and compared..
206. Tomohiro Kiyosue, Jiro Nakayama, Shinobu Satoh, Akira Isogai, Akinori Suzuki, Hiroshi Kamada, Hiroshi Harada, Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein, and enhancement of its accumulation by abscisic acid in somatic embryos, Planta, 10.1007/BF00195313, 186, 3, 337-342, 1992.02, ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70-80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227-291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos. © 1992 Springer-Verlag. -6.
207. Naoyuki Fukuchi, Jiro Nakayama, Seiji Takayama, Akira Isogai, Akinori Suzuki, Structural Elucidation of Rotihibin B by Tandem Mass Spectrometry, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.56.1152, 56, 7, 1152-1153, 1992.01.
208. Akira Isogai, Jiro Nakayama, Seiji Takayama, Akinori Suzuki, Akihiko Kusai, Structural Elucidation of Minor Components of Peptidyl Antibiotic P168s (Leucinostatins) by Tandem Mass Spectrometry, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.56.1079, 56, 7, 1079-1085, 1992.01, Tandem mass spectrometry with a four-sector type mass spectrometer was used to elucidate the structures of minor components of the peptidyl antibiotic P168s (leucinostatins). As N-terminal fragments, ions by B-type cleavage were dominant, while V-type cleavages were observed along with X, Y, and Z types as C-terminal ions. The V-type ions were predominant in the cleavages of the amino terminals of leucyl and hydroxyleucyl residues. The structures of several minor components could be deduced from the tandem mass spectra..
209. Naoyuki Fukuchi, Akira Isogai, Jiro Nakayama, Seiji Takayama, Shuichi Yamashita, Kazuo Suyama, Akinori Suzuki, Isolation and structural elucidation of syringostatins, phytotoxins produced by pseudomonas syringae pv. syringae lilac isolate, Journal of the Chemical Society, Perkin Transactions 1, 7, 875-880, 1992.01, A bacterial strain of Pseudomonas syringae pv. syringae isolated from lilac was found to produce a homologous mixture of phytotoxins different from syringomycin and syringotoxin. The toxins were termed syringostatins and the structures of the main components, syringostatins A and B, were determined by 2D-NMR spectroscopy and mass spectrometry. Minor component structures were elucidated from mass/mass spectra..
210. Jiro Nakayama, Hiroshi Watarai, Akira Isogai, Akinori Suzuki, Immunological Characterization of Pheromone-induced Proteins Associated with Sexual Aggregation in Enterococcus faecalis, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.56.264, 56, 2, 264-269, 1992.01, Sexual aggregation involved in conjugative transfer of the Enterococcus faecalis plasmids pPDl and pADl is enhanced by sex pheromones cPDl and cADl, respectively, which are excreted from recipient cells. PD78 (78kDa) and AD74 (74kDa) were detectable on the surface of donors harboring pPDl and pAD1, respectively, at the time of cell aggregation. The proteins PD78 and AD74 were purified and used to raise anti-PD78 and anti-AD74 antibodies. The antibodies blocked the sexual aggregation and the plasmid transfer. Anti-AD74 antibody reacted to both 153 kDa proteins extracted from cPDl and cADl-induced donor cells after lysozyme digestion of cell wall. Pheromone-induced synthesis of PD78 and AD74, when both plasmids were present in the same cell, was independent..
211. Jiro Nakayama, Hiroshi Watarai, Akira Isogai, Akinori Suzuki, C-Terminal Identification of AD74, a Proteolytic Product of Enterococcus faecalis Aggregation Substance
Application of Liquid Chromatography/Mass Spectrometry, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.56.127, 56, 1, 127-131, 1992.01, Sexual aggregation involved in conjugative transfer of Enterococcus faecalis plasmid pADl is enhanced by the sex pheromone cADl, which is excreted from recipient cells. A membrane-anchored 137 kDa protein is a pADl-encoded aggregation substance designated asal, which is responsible for cell-cell contact and leads to the aggregation of cells. An AD74 protein is a proteolytic product corresponding to the N-terminal half of asal. The C-terminal of AD74 was identified as lysine at position 510 (K-510) by liquid chromatography/mass spectrometry (LC/MS): it indicates that asal is cleaved specifically between K-510 and G-511..
212. Tsuyoshi Kawano, Hisato Kuniyoshi, Jiro Nakayama, Hiromichi Nagasawa, Akinori Suzuki, Expression of a Putative Precursor of Pheromone Biosynthesis Activating Neuropeptide-I (PBAN-I) of the Silkmoth, Bombyx mori, and Its Conversion to the Mature Peptide, PBAN-I, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.55.1943, 55, 7, 1943-1945, 1991.01.
213. Don B. Clewell, Linda T. Pontius, Florence Y. An, Yasuyoshi Ike, Akinori Suzuki, Jiro Nakayama, Nucleotide sequence of the sex pheromone inhibitor (iAD1) determinant ofEnterococcus faecalis conjugative plasmid pAD1, Plasmid, 10.1016/0147-619X(90)90019-9, 24, 2, 156-161, 1990.09, The determinant for the peptide sex pheromone inhibitor iAD1 (iad) on the hemolysin/bacteriocin plasmid pAD1 ofEnterococcus faecalis was sequenced. The sequence reveals a 22-amino-acid precursor with the car{ballot box}yl-terminal 8 residues corresponding to iAD1. It appears that iAD1 is a component of its own signal sequence. © 1990 Academic Press, Inc. All rights reserved..
214. Jiro Nakayama, Hiromich Nagasawa, Akira Isogai, Don B. Clewell, Akinori Suzuki, Amino acid sequence of pheromone-inducible surface protein in Enterococcus faecalis, that is encoded on the conjugative plasmid pPD1, FEBS Letters, 10.1016/0014-5793(90)81019-K, 268, 1, 245-248, 1990.07, The major pheromone-inducible protein, PD78, believed to contribute to bacterial conjugation, was purified from Enterococcus (formerly Streptococcus) faecalis cells containing the plasmid pPD1. A cloned EcoRI-Bg1II 3.6-kbp fragment of the plasmid pAM351(pPd1::Tn916) contained an open reading frame corresponding to 467 amino acid residues representing PD78. In a central region of the deduced protein, there is a repeated sequence of X-X-Pro that is repeated 15 times. This is analogous to the Gin-Gin-Pro repeat in the C-tenninal region of TraD product encoded on the R 100 plasmid in Escherichia coli. © 1990..
215. Naoyuki Fukuchi, Akira Isogai, Jiro Nakayama, Akinori Suzuki, Structure of Syringotoxin B, a Phytotoxin Produced by Citrus Isolates of Pseudomonas syringae pv. syringae, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.54.3377, 54, 12, 3377-3379, 1990.01.
216. Akira Isogai, Masahiro Sato, Shohei Sakuda, Jiro Nakayama, Akinori Suzuki, Stucture of Demethylallosamidin as an Insect Chitinase Inhibitor, Agricultural and Biological Chemistry, 10.1080/00021369.1989.10869709, 53, 10, 2825-2826, 1989.10.
217. Seiichi Taguchit, Izumi Kumagai, Jiro Nakayama, Akinori Suzuki, Kin Ichiro Miura, Efficient extracellular expression of a foreign protein in smptomyos using secretory protease inhibitor (SSI) gene fusions, Nature Biotechnology, 10.1038/nbt1089-1063, 7, 10, 1063-1066, 1989.10, Using the gene for a secreted protease inhibitor, Streptomyces subtilisin inhibitor (SSI), we have established a secretory expression system for the products of foreign genes in Streptomyces. A sex pheromone peptide, cADl, of Enterococcus fae- calis was expressed and secreted as a stable complete fusion protein with SSI in amounts comparable to the native SSI in S. lividans 66. Recombinant cADl, isolated from the fusion protein by chemical digestion with BrCN, has the same amino acid composition and sequence, retention time on reverse phase HPLC, molecular mass, and biological activity as authentic cADl. This system should be useful for the efficient expression of other foreign gene products, and as a model for heterologous gene expression in Streptomyces. © 1989 Nature Publishing Group..
218. Akira Isogai, Shouhei Sakuda, Jiro Nakayama, Satoshi Watanabe, Akinori Suzuki, Isolation and Structural Elucidation of a New Cyclitol Derivative, Streptol, as a Plant Growth Regulator, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb1961.51.2277, 51, 8, 2277-2279, 1987.01, A new cyclitol derivative, streptol (1), was isolated from a culture filtrate of an unidentified Streptomyces sp., and the structure of 1 was determined as 1d-(1,2,4/3)-5- hydroxymethyl-5-cyclohexene-l,2,3,4-tetrol. This structure was elucidated with NMR and MS spectrometry, and the absolute configuration was determined from the CD spectrum of the tetrabenzoate of the hydrogenolized compound of 1. Streptol inhibited the root growth of lettuce seedlings at a concentration above 13 ppm..