九州大学 研究者情報
論文一覧
小椋 義俊(おぐら よしとし) データ更新日:2019.06.19

准教授 /  医学研究院 基礎医学部門


原著論文
1. Yoshitoshi Ogura, Y. Imai, N. Ogasawara, S. Moriya, Autoregulation of the dnaA-dnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis, Journal of bacteriology, 10.1128/JB.183.13.3833-3841.2001, 183, 13, 3833-3841, 2001.07, [URL], In Escherichia coli, the DnaA protein level appears to play a pivotal role in determining the timing of replication initiation. To examine the effects on replication initiation in B. subtilis, we constructed a strain in which a copy of the dnaA gene was integrated at the purA locus on the chromosome under the control of an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter. However, increasing the DnaA level resulted in cell elongation and inhibition of cell growth by induction of the SOS response. Transcription of the native dnaA-dnaN operon was greatly reduced at high DnaA levels, but it was increased in a dnaA-null mutant, indicating autoregulation of the operon by DnaA. When a copy of the dnaN gene was added downstream of the additional dnaA gene at purA, the cells grew at high DnaA levels, suggesting that depletion of DnaN (β subunit of DNA polymerase III) within the cell by repression of the native dnaA-dnaN operon at high DnaA levels was the cause of the SOS induction. Flow cytometry of the cells revealed that the cell mass at initiation of replication increased at a lower DnaA level and decreased at DnaA levels higher than those of the wild type. Proper timing of replication initiation was observed at DnaA levels nearly comparable to the wild-type level. These results suggest that if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in B. subtilis..
2. Makoto Ohnishi, Jun Terajima, Ken Kurokawa, Keisuke Nakayama, Takahiro Murata, Kazumichi Tamura, Yoshitoshi Ogura, Haruo Watanabe, Tetsuya Hayashi, Genomic diversity of enterohemorrhagic Escherichia coli 0157 revealed by whole genome PCR scanning, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.262441699, 99, 26, 17043-17048, 2002.12, [URL], Enterohemorrhagic Escherichia coli O157 is one of the leading worldwide public health concerns, causing large outbreaks of hemorrhagic colitis as well as numerous small outbreaks and sporadic cases. The variability of restriction enzyme-digestion patterns of O157 genomes, which is widely used to distinguish strains in the molecular epidemiology of O157 infections, suggests the presence of some genomic diversity among the strains. Based on the complete genome sequence of O157 Sakai, we analyzed the whole genome structures of eight O157 strains displaying diverse Xbal-digestion patterns by a systematic PCR analysis that we have named whole genome PCR scanning. This analysis identified not only the O157-specific sequences that are highly conserved among the strains, but also revealed an unexpectedly high degree of genomic diversity. In particular, prophages, including Shiga toxin-transducing phages, exhibited extensive structural and positional diversity, implying that variation of bacteriophages is a major factor in generating genomic diversity among the O157 lineage..
3. Yoshitoshi Ogura, Naotake Ogasawara, Elizabeth J. Harry, Shigeki Moriya, Increasing the Ratio of Soj to Spo0J Promotes Replication Initiation in Bacillus subtilis, Journal of bacteriology, 10.1128/JB.185.21.6316-6324.2003, 185, 21, 6316-6324, 2003.11, [URL], The ParA and ParB protein families are well conserved in bacteria. However, their functions are still unclear. In Bacillus subtilis, Soj and Spo0J are members of these two protein families, respectively. A previous report revealed that replication initiated early and asynchronously in spo0J null mutant cells, as determined by flow cytometry. In this study, we examined the cause of this promotion of replication initiation. Deletion of both the soj and spo0J genes restored the frequency of replication initiation to almost the wild-type level, suggesting that production of Soj in the absence of Spo0J leads to early and asynchronous initiation of replication. Consistent with this suggestion, overproduction of Soj in wild-type cells had the same effect on replication initiation as in the spo0J null mutant, and overproduction of both Soj and Spo0J did not. These results indicate that when the ratio of Soj to Spo0J increases, Soj interferes with tight control of replication initiation and causes early and asynchronous initiation. Whereas replication initiation also occurred significantly earlier in the two spo0J mutants, spo0J14 and spo0J17, it occurred only slightly early in the sojK16Q mutant and was delayed in the sojG12V mutant. Although Soj localized to nucleoids in the spo0J mutants, the two Soj mutant proteins were distributed throughout the cell or localized to cell poles. Thus, interestingly, the promotion of replication initiation seems to correlate with localization of Soj to nucleoids. This may suggest that Soj inhibits transcription of some cell cycle genes and leads to early and asynchronous initiation of replication. In wild-type cells Spo0J counteracts this Soj function..
4. Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Genome analysis of enterohaemorrhagic E. coli O157 to the epidemiological study of O157 infections, Rinshō Biseibutsu Jinsoku Shindan Kenkyūkai shi = JARMAM : Journal of the Association for Rapid Method and Automation in Microbiology, 16, 2, 179-181, 2005.01.
5. Miho Hayashi, Yoshitoshi Ogura, Elizabeth J. Harry, Naotake Ogasawara, Shigeki Moriya, Bacillus subtilis YabA is involved in determining the timing and synchrony of replication initiation, FEMS microbiology letters, 10.1016/j.femsle.2005.04.028, 247, 1, 73-79, 2005.06, [URL], It is shown here by flow cytometry that Bacillus subtilis YabA negatively regulates the timing of replication initiation. When the level of YabA was reduced, replication began at a decreased cell mass and when the level was increased, initiation was delayed. Synchrony of replication initiation was also disrupted at low levels of YabA. Yfp-YabA localized as foci in cells. Since YabA was reported to interact with DnaN (β subunit of DNA polymerase III), co-localization of Yfp-YabA with the polymerase was examined using a Cfp fusion with DnaX (τ subunit of DNA polymerase III). It is reported that YabA appears to localize at the replication forks only at a late stage of DNA replication..
6. Yoshitoshi Ogura, Ken Kurokawa, Tadasuke Ooka, Kosuke Tashiro, Toru Tobe, Makoto Ohnishi, Keisuke Nakayama, Takuya Morimoto, Jun Terajima, Haruo Watanabe, Satoru Kuhara, Tetsuya Hayashi, Complexity of the genomic diversity in enterohemorrhagic Escherichia coli O157 revealed by the combinational use of the O157 Sakai OligoDNA microarray and the whole genome PCR scanning, DNA Research, 10.1093/dnares/dsi026, 13, 1, 3-14, 2006.06, [URL], Escherichia coli O157, an etiological agent of hemorrhagic colitis and hemolytic uremic syndrome, is one of the leading worldwide public health threats. Genome sequencing of two O157 strains have revealed that the chromosome is comprised of a 4.1 Mb backbone shared by K-12 and a total of 1.4 Mb O157-specific sequences. Most of the large O157-specific sequences are prophages and prophage-like elements, which have carried many virulence genes into the O157 genome. This suggests that bacteriophages have played the key roles in the emergence of O157. The Whole Genome PCR Scanning (WGPScanning) analysis of O157 strains, on the other hand, revealed a high level of genomic diversity in O157. Variation of prophages has also been suggested as a major factor generating such diversity. In this study, we analyzed the gene content of O157 strains, by an oligoDNA microarray, using the same set of strains as examined by the WGPScanning method. Although most of the strains were typical O157 : H7, they differed remarkably in gene composition, particularly in those on prophages, and we identified more than 400 'variably absent or present' genes which included virulence-related genes. This confirms the role of prophages in generating the genomic diversity, and raises a possibility that some level of variation in potential virulence is present among O157 strains. Fine comparison of the two datasets obtained by microarray and WGPScanning provided much further details on the O157 genome diversity than illustrated by each method alone, indicating the usefulness of this combinational approach in the genomic comparison of closely related strains..
7. Noriko Nakanishi, Hiroyuki Abe, Yoshitoshi Ogura, Tetsuya Hayashi, Kosuke Tashiro, Satoru Kuhara, Nakaba Sugimoto, Toru Tobe, ppGpp with DksA controls gene expression in the locus of enterocyte effacement (LEE) pathogenicity island of enterohaemorrhagic Escherichia coli through activation of two virulence regulatory genes, Molecular Microbiology, 10.1111/j.1365-2958.2006.05217.x, 61, 1, 194-205, 2006.07, [URL], For a new pathogen to emerge, it must acquire both virulence genes and a system for responding to changes in environmental conditions. Starvation of nutrients or growth arrest induces the stringent response in Escherichia coli, via increased ppGpp. We found the adherence capacity of enterohaemorrhagic E. coli (EHEC) and gene expression in the locus of enterocyte effacement (LEE) were enhanced by a downshift in nutrients or by entry into the stationary growth phase, both of which increase the ppGpp concentration. The activation was dependent on relA and spoT, which encode enzymes for the synthesis and degradation of ppGpp, and on dksA, which encodes an RNA polymerase accessory protein required for the stringent response. Upon induction of RelA expression, LEE gene transcription was activated within 20 min, even without starvation. The expression of two LEE transcriptional regulators, Ler and Pch, was activated by ppGpp and essential for the enhancement of LEE gene expression. In addition, the ler and pch promoters were directly activated by ppGpp in an in vitro transcription system. These findings suggest that the regulation of virulence genes in EHEC is integrated with E. coli's stringent response system, through the regulation of virulence regulatory genes..
8. Yoshitoshi Ogura, Tadasuke Ooka, Andrew Whale, Junkal Garmendia, Lothar Beutin, Sharon Tennant, Gladys Krause, Stefano Morabito, Isabel Chinen, Toru Tobe, Hiroyuki Abe, Rosangela Tozzoli, Alfredo Caprioli, Marta Rivas, Roy Robins-Browne, Tetsuya Hayashi, Gad Frankel, TccP2 of O157:H7 and non-O157 enterohemorrhagic Escherichia coli (EHEC)
Challenging the dogma of EHEC-induced actin polymerization, Infection and Immunity, 10.1128/IAI.01491-06, 75, 2, 604-612, 2007.02, [URL], Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, TirEHEC O157 is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspFU, which mimics the function of Nck tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, β-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 ΔtccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway..
9. Andrew D. Whale, Rodrigo T. Hernandes, Tadasuke Ooka, Lothar Beutin, Stephanie Schüller, Junkal Garmendia, Lynette Crowther, Mônica A.M. Vieira, Yoshitoshi Ogura, Gladys Krause, Alan D. Phillips, Tania A.T. Gomes, Tetsuya Hayashi, Gad Frankel, TccP2-mediated subversion of actin dynamics by EPEC 2 - A distinct evolutionary lineage of enteropathogenic Escherichia coli, Microbiology, 10.1099/mic.0.2006/004325-0, 153, 6, 1743-1755, 2007.06, [URL], Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in developing countries. While colonizing the gut mucosa, EPEC triggers extensive actin-polymerization activity at the site of intimate bacterial attachment, which is mediated by avid interaction between the outer-membrane adhesin intimin and the type III secretion system (T3SS) effector Tir. The prevailing dogma is that actin polymerization by EPEC is achieved following tyrosine phosphorylation of Tir, recruitment of Nck and activation of neuronal Wiskott-Aldrich syndrome protein (N-WASP). In closely related enterohaemorrhagic E. coli (EHEC) O157: H7, actin polymerization is triggered following recruitment of the T3SS effector TccP/EspFu (instead of Nck) and local activation of N-WASP. In addition to tccP, typical EHEC O157 : H7 harbour a pseudogene (tccP2). However, it has recently been found that atypical, sorbitol-fermenting EHEC O157 carries functional tccP and tccP2 alleles. Interestingly, intact tccP2 has been identified in the incomplete genome sequence of the prototype EPEC strain B171 (serotype O111 : H-), but it is missing from another prototype EPEC strain E2348/69 (O127 : H7). E2348/69 and B171 belong to two distinct evolutionary lineages of EPEC, termed EPEC 1 and EPEC 2, respectively. Here, it is reported that while both EPEC 1 and EPEC 2 triggered actin polymerization via the Nck pathway, tccP2 was found in 26 of 27 (96.2%) strains belonging to EPEC 2, and in none of the 34 strains belonging to EPEC 1. It was shown that TccP2 was: (i) translocated by the locus of enterocyte effacement-encoded T3SS; (ii) localized at the tip of the EPEC 2-induced actin-rich pedestals in infected HeLa cells and human intestinal in vitro organ cultures ex vivo; and (iii) essential for actin polymerization in infected Nck-/- cells. Therefore, unlike strains belonging to EPEC 1, strains belonging to EPEC 2 can trigger actin polymerization using both Nck and TccP2 actin-polymerization signalling cascades..
10. Tadasuke Ooka, Mônica A.M. Vieira, Yoshitoshi Ogura, Lothar Beutin, Roberto La Ragione, Pauline M. Van Diemen, Mark P. Stevens, Ilknur Aktan, Shaun Cawthraw, Angus Best, Rodrigo T. Hernandes, Gladys Krause, Tania A.T. Gomes, Tetsuya Hayashi, Gad Frankel, Characterization of tccP2 carried by atypical enteropathogenic Escherichia coli, FEMS microbiology letters, 10.1111/j.1574-6968.2007.00707.x, 271, 1, 126-135, 2007.06, [URL], Atypical enteropathogenic Escherichia coli (EPEC) comprise an important group of paediatric pathogens. Atypical EPEC have reservoirs in farm and domestic animals where they can be either commensal or pathogenic; serogroup O26 is dominant in humans and animals. Central to intestinal colonization by EPEC is the translocation of the type III secretion system effector Tir into enterocytes, which following phosphorylation (Tir-Yp) recruits Nck to activate the N-WASP actin signalling cascade. The authors have recently shown that typical EPEC strains, belonging to the EPEC-2 lineage, carry a tir gene encoding Tir-Yp and can also use the alternative TccP2 actin-signalling cascade. The aim of this study was to determine if tccP2 is found in atypical EPEC isolated from human and farm animals. tccP2 was found at a frequency of 41% in non-O26 EPEC isolates and in 82.3% of the O26 strains. TccP2 of human and animal strains show high level of sequence identity. It is shown that most strains carry a tir gene encoding Tir-Yp. In addition the authors identified two new variants of tir genes in EPEC O104:H12 and NT:H19 strains..
11. Yoshitoshi Ogura, Tadasuke Ooka, Asadulghani, Jun Terajima, Jean Philippe Nougayrède, Ken Kurokawa, Kosuke Tashiro, Toru Tobe, Keisuke Nakayama, Satoru Kuhara, Eric Oswald, Haruo Watanabe, Tetsuya Hayashi, Extensive genomic diversity and selective conservation of virulence-determinants in enterohemorrhagic Escherichia coli strains of O157 and non-O157 serotypes, Genome biology, 10.1186/gb-2007-8-7-r138, 8, 7, 2007.07, [URL], Background: Enterohemorrhagic Escherichia coli (EHEC) O157 causes severe food-borne illness in humans. The chromosome of O157 consists of 4.1 Mb backbone sequences shared by benign E. coli K-12, and 1.4 Mb O157-specific sequences encoding many virulence determinants, such as Shiga toxin genes (stx genes) and the locus of enterocyte effacement (LEE). Non-O157 EHECs belonging to distinct clonal lineages from O157 also cause similar illness in humans. According to the 'parallel' evolution model, they have independently acquired the major virulence determinants, the stx genes and LEE. However, the genomic differences between O157 and non-O157 EHECs have not yet been systematically analyzed. Results: Using microarray and whole genome PCR scanning analyses, we performed a whole genome comparison of 20 EHEC strains of O26, O111, and O103 serotypes with O157. In non-O157 EHEC strains, although genome sizes were similar with or rather larger than O157 and the backbone regions were well conserved, O157-specific regions were very poorly conserved. Around only 20% of the O157-specific genes were fully conserved in each non-O157 serotype. However, the non-O157 EHECs contained a significant number of virulence genes that are found on prophages and plasmids in O157, and also multiple prophages similar to, but significantly divergent from, those in O157. Conclusion: Although O157 and non-O157 EHECs have independently acquired a huge amount of serotype- or strain-specific genes by lateral gene transfer, they share an unexpectedly large number of virulence genes. Independent infections of similar but distinct bacteriophages carrying these virulence determinants are deeply involved in the evolution of O157 and non-O157 EHECs..
12. Shu Ishikawa, Yoshitoshi Ogura, Mika Yoshimura, Hajime Okumura, Eunha Cho, Yoshikazu Kawai, Ken Kurokawa, Taku Oshima, Naotake Ogasawara, Distribution of stable DnaA-binding sites on the bacillus subtilis genome detected using a modified ChIP-chip method, DNA Research, 10.1093/dnares/dsm017, 14, 4, 155-168, 2007.09, [URL], We developed a modified ChIP-chip method, designated ChAP-chip (Chromatin Affinity Precipitation coupled with tiling chip). The binding sites of Bacillus subtilis Spo0J determined using this technique were consistent with previous findings. A DNA replication initiator protein, DnaA, formed stable complexes at eight intergenic regions on the B. subtilis genome. Characterization of the binding sequences suggested that two factors - the local density of DnaA boxes and their affinities for DnaA - are critical for stable binding. We further showed that in addition to autoregulation, DnaA directly modulate the expression of sda in a positive, and ywlC and yydA in a negative manner. Examination of possible stable DnaA-binding sequences in other Bacillus species suggested that DnaA-dependent regulation of those genes is maintained in most bacteria examined, supporting their biological significance. In addition, a possible stable DnaA-binding site downstream of gcp is also suggested to be conserved. Furthermore, potential DnaA-binding sequences specific for each bacterium have been identified, generally in close proximity to oriC. These findings suggest that DnaA plays several additional roles, such as control of the level of effective initiator, ATP-DnaA, and/or stabilization of the domain structure of the genome around oriC for the proper initiation of chromosome replication..
13. Ken Kurokawa, Takehiko Itoh, Tomomi Kuwahara, Kenshiro Oshima, Hidehiro Toh, Atsushi Toyoda, Hideto Takami, Hidetoshi Morita, Vineet K. Sharma, Tulika P. Srivastava, Todd D. Taylor, Hideki Noguchi, Hiroshi Mori, Yoshitoshi Ogura, Dusko S. Ehrlich, Kikuji Itoh, Toshihisa Takagi, Yoshiyuki Sakaki, Tetsuya Hayashi, Masahira Hattori, Comparative metagenomics revealed commonly enriched gene sets in human gut microbiomes, DNA Research, 10.1093/dnares/dsm018, 14, 4, 169-181, 2007.09, [URL], Numerous microbes inhabit the human intestine, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects human physiology. To identify the genomic features common to all human gut microbiomes as well as those variable among them, we performed a large-scale comparative metagenomic analysis of fecal samples from 13 healthy individuals of various ages, including unweaned infants. We found that, while the gut microbiota from unweaned infants were simple and showed a high inter-individual variation in taxonomic and gene composition, those from adults and weaned children were more complex but showed a high functional uniformity regardless of age or sex. In searching for the genes over-represented in gut microbiomes, we identified 237 gene families commonly enriched in adult-type and 136 families in infant-type microbiomes, with a small overlap. An analysis of their predicted functions revealed various strategies employed by each type of microbiota to adapt to its intestinal environment, suggesting that these gene sets encode the core functions of adult and infant-type gut microbiota. By analysing the orphan genes, 647 new gene families were identified to be exclusively present in human intestinal microbiomes. In addition, we discovered a conjugative transposon family explosively amplified in human gut microbiomes, which strongly suggests that the intestine is a 'hot spot' for horizontal gene transfer between microbes..
14. Estelle Loukiadis, Rika Nobe, Sylvia Herold, Clara Tramuta, Yoshitoshi Ogura, Tadasuke Ooka, Stefano Morabito, Monique Kérourédan, Hubert Brugère, Herbert Schmidt, Tetsuya Hayashi, Eric Oswald, Distribution, functional expression, and genetic organization of Cif, a phage-encoded type III-secreted effector from enteropathogenic and enterohemorrhagic Escherichia coli, Journal of Bacteriology, 10.1128/JB.00844-07, 190, 1, 275-285, 2008.01, [URL], Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049 E. coli strains of human, animal, and environmental origins. A total of 115 E. coli isolates from diverse origins and geographic locations carried cif. The presence of cif was tightly associated with the LEE, since all the cif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population of E. coli but positively selected within the population of LEE-positive strains. Nonetheless, 66% of cif-positive E. coli strains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in the cif gene. The passenger region of the prophages carrying cif was highly variable and showed various combinations of IS elements and genes coding for other effectors such as nleB, nleC, nleH, nleG, espJ, and nleA/espI (some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism..
15. Atsushi Iguchi, Tadasuke Ooka, Yoshitoshi Ogura, Asadulghani, Keisuke Nakayama, Gad Frankel, Tetsuya Hayashi, Genomic comparison of the O-antigen biosynthesis gene clusters of Escherichia coli O55 strains belonging to three distinct lineages, Microbiology, 10.1099/mic.0.2007/013334-0, 154, 2, 559-570, 2008.02, [URL], Typical enteropathogenic Escherichia coli (EPEC) O55:H7 is regarded as the closest relative of enterohaemorrhagic E. coli (EHEC) O157:H7. Both serotypes usually express the γ1 intimin subclass and trigger actin polymerization by the Tir-TccP pathway. However, atypical O55:H7 strains capable of triggering actin polymerization via the Tir-Nck pathway have recently been identified. In this study, we investigated the genotypic differences and phylogenetic relationships between typical and atypical O55:H7 strains. We show that the atypical O55:H7 strains, which express the θ intimin subclass and lack both tccP and tccP2, belong to an E. coli lineage distinct from the typical O55:H7 and from the EPEC O55:H6, which also uses the Tir-Nck actin polymerization pathway. We conducted genomic comparisons of the chromosomal regions covering the O-antigen gene cluster and its flanking regions between the three O55 lineages by RFLP analysis of PCR products and DNA sequencing analysis of about 65 kb chromosomal regions. This unexpectedly revealed that horizontal transfer of large fragments (≥40 kb) encoding the O55-antigen gene cluster and part of the neighbouring colanic acid gene cluster was involved in the emergence of the three O55 E. coli lineages. The data provide new insights into the mechanisms involved in the generation of a wide variety of O-serotypes in Gram-negative bacteria..
16. Yoshitoshi Ogura, Hiroyuki Abe, Keisuke Katsura, Ken Kurokawa, Md Asadulghani, Atsushi Iguchi, Tadasuke Ooka, Keisuke Nakayama, Atsushi Yamashita, Masahira Hattori, Toru Tobe, Tetsuya Hayashi, Systematic identification and sequence analysis of the genomic islands of the enteropathogenic Escherichia coli strain B171-8 by the combined use of whole-genome PCR scanning and fosmid mapping, Journal of bacteriology, 10.1128/JB.00625-08, 190, 21, 6948-6960, 2008.11, [URL], Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are diarrheagenic pathogens that colonize the intestinal tract through the formation of attaching and effacing lesions, induced by effectors translocated via a type III secretion system (T3SS) encoded on the locus of enterocyte effacement (LEE). In EHEC O157, numerous virulence factors, including around 40 T3SS effectors, have been identified. Most of them are encoded on genomic islands (GEIs) such as prophages and integrative elements. For EPEC, however, no systematic search of GEIs and virulence-related genes carried therein has been done, and only a limited number of virulence factors have been identified so far. In this study, we performed a systemic and genome-wide survey of the GEIs in strain B171-8, one of the prototype strains of EPEC, by the combined use of whole-genome PCR scanning and fosmid mapping and identified 22 large GEIs, including nine lambda-like prophages, three P2-like prophages, the LEE, and three additional integrative elements. On these prophages and integrative elements, we found genes for a set of T3SS proteins, a total of 33 T3SS effectors or effector homologues, and 12 other virulence factors which include five nonfimbrial adhesins. Most of the T3SS effector families identified are also present in EHEC O157, but B171-8 possesses a significantly smaller number of effectors. Not only the presence or absence of Shiga toxin genes but also the difference in the T3SS effector repertoire should be considered in analyzing the pathogenicity of EPEC and EHEC strains..
17. Keisuke Nakayama, Atsushi Yamashita, Ken Kurokawa, Takuya Morimoto, Michihiro Ogawa, Masahiro Fukuhara, Hiroshi Urakami, Makoto Ohnishi, Ikuo Uchiyama, Yoshitoshi Ogura, Tadasuke Ooka, Kenshiro Oshima, Akira Tamura, Masahira Hattori, Tetsuya Hayashi, The whole-genome sequencing of the obligate intracellular bacterium orientia tsutsugamushi revealed massive gene amplification during reductive genome evolution, DNA Research, 10.1093/dnares/dsn011, 15, 4, 185-199, 2008.12, [URL], Scrub typhus ('Tsutsugamushi' disease in Japanese) is a mite-borne infectious disease. The causative agent is Orientia tsutsugamushi, an obligate intracellular bacterium belonging to the family Richettsiaceae of the subdivision alpha-Proteobacteria. In this study, we determined the complete genome sequence of O. tsutsugamushi strain lkeda, which comprises a single chromosome of 2 008 987 bp and contains 1967 protein coding sequences (CDSs). The chromosome is much larger than those of other members of Rickettsiaceae, and 46.7% of the sequence was occupied by repetitive sequences derived from an integrative and conjugative element, 10 types of transposable elements, and seven types of short repeats of unknown origins. The massive amplification and degradation of these elements have generated a huge number of repeated genes (1196 CDSs, categorized into 85 families), many of which are pseudogenes (766 CDSs), and also induced intensive genome shuffling. By comparing the gene content with those of other family members of Rickettsiacea, we identified the core gene set of the family Rickettsiaceae and found that, while much more extensive gene loss has taken place among the housekeeping genes of Orientia than those of Rickettsia, O. tsutsugamushi has acquired a large number of foreign genes. The O. tsutsugamushi genome sequence is thus a prominent example of the high plasticity of bacterial genomes, and provides the genetic basis for a better understanding of the biology of O. tsutsugamushi and the pathogenesis of 'Tsutsugamushi' disease..
18. Hiroyuki Abe, Akira Miyahara, Taku Oshima, Kosuke Tashiro, Yoshitoshi Ogura, Satoru Kuhara, Naotake Ogasawara, Tetsuya Hayashi, Toru Tobe, Global regulation by horizontally transferred regulators establishes the pathogenicity of Escherichia coli, DNA Research, 10.1093/dnares/dsm028, 15, 1, 13-23, 2008.12, [URL], Enterohemorrhagic Escherichia coli is an emerging pathogen that causes diarrhea and hemolytic uremic syndrome. Much of the genomic information that affects virulence is acquired by horizontal transfer. Genes necessary for attaching and effacing lesions are located in the locus for enterocyte effacement (LEE) pathogenicity island. LEE gene transcription is positively regulated by Ler, which is also encoded by the LEE, and by Pch regulators, which are encoded at other loci. Here we identified genes whose transcription profiles were similar to those of the LEE genes, by comparing the effects of altering 1er and pch transcript levels. We assigned these genes into two classes, according to their transcription profiles. By determining the binding profiles for Ler and Pch, we showed that both were involved in regulating one class of genes, but only Pch was involved in regulating the other class. Binding sites were found in the coding region as well as the promoter region of regulated genes, which include genes common to K12 strains as well as 0157-specific genes, suggesting that both act as a global regulator. These results indicate that Ler and Pch orchestrate the transcription of virulence genes, which are captured by horizontal transfer and scattered throughout the chromosome..
19. Hiroyuki Abe, Akira Miyahara, Taku Oshima, Kosuke Tashiro, Yoshitoshi Ogura, Satoru Kuhara, Naotake Ogasawara, Tetsuya Hayashi, Toru Tobe, Global regulation by horizontally transferred regulators establishes the pathogenicity of escherichia coli, DNA Research, 10.1093/dnares/dsm033, 15, 1, 25-38, 2008.12, [URL], Enterohemorrhagic Escherichia coli is an emerging pathogen that causes diarrhea and hemolytic uremic syndrome. Much of the genomic information that affects virulence is acquired by horizontal transfer. Genes necessary for attaching and effacing lesions are located in the locus for enterocyte effacement (LEE) pathogenicity island. LEE gene transcription is positively regulated by Ler, which is also encoded by the LEE, and by Pch regulators, which are encoded at other loci. Here we identified genes whose transcription profiles were similar to those of the LEE genes, by comparing the effects of altering ler and pch transcript levels. We assigned these genes into two classes, according to their transcription profiles. By determining the binding profiles for Ler and Pch, we showed that both were involved in regulating one class of genes, but only Pch was involved in regulating the other class. Binding sites were found in the coding region as well as the promoter region of regulated genes, which include genes common to K12 strains as well as 0157-specific genes, suggesting that both act as a global regulator. These results indicate that Ler and Pch orchestrate the transcription of virulence genes, which are captured by horizontal transfer and scattered throughout the chromosome..
20. Kenshiro Oshima, Hidehiro Toh, Yoshitoshi Ogura, Hiroyuki Sasamoto, Hidetoshi Morita, Sang Hee Park, Tadasuke Ooka, Sunao Iyoda, Todd D. Taylor, Tetsuya Hayashi, Kikuji Itoh, Masahira Hattori, Complete genome sequence and comparative analysis of the wild-type commensal escherichia coli strain se11 isolated from a healthy adult, DNA Research, 10.1093/dnares/dsn026, 15, 6, 375-386, 2008.12, [URL], We sequenced and analyzed the genome of a commensal Escherichia coli (E. coli) strain SE11 (O152:H28) recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B1. SE11 harbored a 4.8 Mb chromosome encoding 4679 protein-coding genes and six plasmids encoding 323 protein-coding genes. None of the SE11 genes had sequence similarity to known genes encoding phage- and plasmid-borne virulence factors found in pathogenic E. coli strains. The comparative genome analysis with the laboratory strain K-12 MG1655 identified 62 poorly conserved genes between these two non-pathogenic strains and 1186 genes absent in MG1655. These genes in SE11 were mostly encoded in large insertion regions on the chromosome or in the plasmids, and were notably abundant in genes of fimbriae and autotransporters, which are cell surface appendages that largely contribute to the adherence ability of bacteria to host cells and bacterial conjugation. These data suggest that SE11 may have evolved to acquire and accumulate the functions advantageous for stable colonization of intestinal cells, and that the adhesion-associated functions are important for the commensality of E. coli in human gut habitat..
21. Atsushi Iguchi, Nicholas R. Thomson, Yoshitoshi Ogura, David Saunders, Tadasuke Ooka, Ian R. Henderson, David Harris, M. Asadulghani, Ken Kurokawa, Paul Dean, Brendan Kenny, Michael A. Quail, Scott Thurston, Gordon Dougan, Tetsuya Hayashi, Julian Parkhill, Gad Frankel, Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69, Journal of Bacteriology, 10.1128/JB.01238-08, 91, 1, 347-354, 2009.01, [URL], Enteropathogenic Escherichia coli (EPEC) was the first pathovar of E. coli to be implicated in human disease; however, no EPEC strain has been fully sequenced until now. Strain E2348/69 (serotype O127:H6 belonging to E. coli phylogroup B2) has been used worldwide as a prototype strain to study EPEC biology, genetics, and virulence. Studies of E2348/69 led to the discovery of the locus of enterocyte effacement-encoded type III secretion system (T3SS) and its cognate effectors, which play a vital role in attaching and effacing lesion formation on gut epithelial cells. In this study, we determined the complete genomic sequence of E2348/69 and performed genomic comparisons with other important E. coli strains. We identified 424 E2348/69-specific genes, most of which are carried on mobile genetic elements, and a number of genetic traits specifically conserved in phylogroup B2 strains irrespective of their pathotypes, including the absence of the ETT2-related T3SS, which is present in E. coli strains belonging to all other phylogroups. The genome analysis revealed the entire gene repertoire related to E2348/69 virulence. Interestingly, E2348/69 contains only 21 intact T3SS effector genes, all of which are carried on prophages and integrative elements, compared to over 50 effector genes in enterohemorrhagic E. coli O157. As E2348/69 is the most-studied pathogenic E. coli strain, this study provides a genomic context for the vast amount of existing experimental data. The unexpected simplicity of the E2348/69 T3SS provides the first opportunity to fully dissect the entire virulence strategy of attaching and effacing pathogens in the genomic context..
22. Md Asadulghani, Yoshitoshi Ogura, Tadasuke Ooka, Takehiko Itoh, Akira Sawaguchi, Atsushi Iguchi, Keisuke Nakayama, Tetsuya Hayashi, The defective prophage pool of Escherichia coli O157
Prophage-prophage interactions potentiate horizontal transfer of virulence determinants, PLoS Pathogens, 10.1371/journal.ppat.1000408, 5, 5, 2009.05, [URL], Bacteriophages are major genetic factors promoting horizontal gene transfer (HGT) between bacteria. Their roles in dynamic bacterial genome evolution have been increasingly highlighted by the fact that many sequenced bacterial genomes contain multiple prophages carrying a wide range of genes. Enterohemorrhagic Escherichia coli O157 is the most striking case. A sequenced strain (O157 Sakai) possesses 18 prophages (Sp1-Sp18) that encode numerous genes related to O157 virulence, including those for two potent cytotoxins, Shiga toxins (Stx) 1 and 2. However, most of these prophages appeared to contain multiple genetic defects. To understand whether these defective prophages have the potential to act as mobile genetic elements to spread virulence determinants, we looked closely at the Sp1-Sp18 sequences, defined the genetic defects of each Sp, and then systematically analyzed all Sps for their biological activities. We show that many of the defective prophages, including the Stx1 phage, are inducible and released from O157 cells as particulate DNA. In fact, some prophages can even be transferred to other E. coli strains. We also show that new Stx1 phages are generated by recombination between the Stx1 and Stx2 phage genomes. The results indicate that these defective prophages are not simply genetic remnants generated in the course of O157 evolution, but rather genetic elements with a high potential for disseminating virulence-related genes and other genetic traits to other bacteria. We speculate that recombination and various other types of inter-prophage interactions in the O157 prophage pool potentiate such activities. Our data provide new insights into the potential activities of the defective prophages embedded in bacterial genomes and lead to the formulation of a novel concept of inter-prophage interactions in defective prophage communities..
23. Shana R. Leopold, Vincent Magrini, Nicholas J. Holt, Nurmohammad Shaikh, Elaine R. Mardis, Joseph Cagno, Yoshitoshi Ogura, Atsushi Iguchi, Tetsuya Hayashi, Alexander Mellmanng, Helge Karch, Thomas E. Besser, Stanley A. Sawyer, Thomas S. Whittam, Phillip I. Tarr, A precise reconstruction of the emergence and constrained radiations of Escherichia coli O157 portrayed by backbone concatenomic analysis, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0812949106, 106, 21, 8713-8718, 2009.05, [URL], Single nucleotide polymorphisms (SNPs) in stable genome regions provide durable measurements of species evolution. We systematically identified each SNP in concatenations of all backbone ORFs in 7 newly or previously sequenced evolutionarily instructive pathogenic Escherichia coli O157:H7, O157:H -, and O55:H7. The 1,113 synonymous SNPs demonstrate emergence of the largest cluster of this pathogen only in the last millennium. Unexpectedly, shared SNPs within circumscribed clusters of organisms suggest severely restricted survival and limited effective population sizes of pathogenic O157:H7, tenuous survival of these organisms in nature, source-sink evolutionary dynamics, or, possibly, a limited number of mutations that confer selective advantage. A single large segment spanning the rfb-gnd gene cluster is the only backbone region convincingly acquired by recombination as O157 emerged from O55. This concatenomic analysis also supports using SNPs to differentiate closely related pathogens for infection control and forensic purposes. However, constrained radiations raise the possibility of making false associations between isolates..
24. Noriyo Yoshii, Yoshitoshi Ogura, Tetsuya Hayashi, Takashi Ajiro, Toshiya Sameshima, Muneo Nakazawa, Masahiro Kusumoto, Taketoshi Iwata, Masato Akiba, Pulsed-field gel electrophoresis profile changes resulting from spontaneous chromosomal deletions in enterohemorrhagic Escherichia coli O157:H7 during passage in cattle, Applied and Environmental Microbiology, 10.1128/AEM.00558-09, 75, 17, 5719-5726, 2009.09, [URL], A total of 905 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were recovered from experimentally infected cattle, in addition to the inoculated strain, were analyzed by pulsed-field gel electrophoresis (PFGE). Twelve PFGE profiles other than that of the inoculated strain were observed. We successfully identified five distinct chromosomal deletions that affected the PFGE profiles using whole-genome PCR scanning and DNA sequencing analysis. The changes in PFGE profiles of EHEC O157:H7 isolates after passage through the intestinal tract of cattle were partially generated by deletion of chromosomal regions..
25. Tadasuke Ooka, Jun Terajima, Masahiro Kusumoto, Atsushi Iguchi, Ken Kurokawa, Yoshitoshi Ogura, Md Asadulghani, Keisuke Nakayama, Kazunori Murase, Makoto Ohnishi, Sunao Iyoda, Haruo Watanabe, Tetsuya Hayashi, Development of a multiplex PCR-based rapid typing method for enterohemorrhagic Escherichia coli O157 strains, Journal of Clinical Microbiology, 10.1128/JCM.00792-09, 47, 9, 2888-2894, 2009.09, [URL], Enterohemorrhagic Escherichia coli O157 (EHEC O157) is a food-borne pathogen that has raised worldwide public health concern. The development of simple and rapid strain-typing methods is crucial for the rapid detection and surveillance of EHEC O157 outbreaks. In the present study, we developed a multiplex PCR-based strain-typing method for EHEC O157, which is based on the variability in genomic location of IS629 among EHEC O157 strains. This method is very simple, in that the procedures are completed within 2 h, the analysis can be performed without the need for special equipment or techniques (requiring only conventional PCR and agarose gel electrophoresis systems), the results can easily be transformed into digital data, and the genes for the major virulence markers of EHEC O157 (the stx1, stx2, and eae genes) can be detected simultaneously. Using this method, 201 EHEC O157 strains showing different XbaI digestion patterns in pulsed-field gel electrophoresis (PFGE) analysis were classified into 127 types, and outbreak-related strains showed identical or highly similar banding patterns. Although this method is less discriminatory than PFGE, it may be useful as a primary screening tool for EHEC O157 outbreaks..
26. Tadasuke Ooka, Yoshitoshi Ogura, Md Asadulghani, Makoto Ohnishi, Keisuke Nakayama, Jun Terajima, Haruo Watanabe, Tetsuya Hayashi, Inference of the impact of insertion sequence (IS) elements on bacterial genome diversification through analysis of small-size structural polymorphisms in Escherichia coli O157 genomes, Genome Research, 10.1101/gr.089615.108, 19, 10, 1809-1816, 2009.10, [URL], Mobile genetic elements play important roles in the evolution and diversification of bacterial genomes. In enterohemorrhagic Escherichia coli O157, a major factor that affects genomic diversity is prophages, which generate most of the large-size structural polymorphisms (LSSPs) observed in O157 genomes. Here, we describe the results of a systematic analysis of numerous small-size structural polymorphisms (SSSPs) that were detected by comparing the genomes of eight clinical isolates with a sequenced strain, O157 Sakai. Most of the SSSPs were generated by genetic events associated with only two insertion sequence (IS) elements, IS629 and ISEc8, and a number of genes that were inactivated or deleted by these events were identified. Simple excisions of IS629 and small deletions (footprints) formed by the excision of IS629, both of which are rarely described in bacteria, were also detected. In addition, the distribution of IS elements was highly biased toward prophages, prophage-like integrative elements, and plasmids. Based on these and our previous results, we conclude that, in addition to prophages, these two IS elements are major contributors to the genomic diversification of O157 strains and that LSSPs have been generated mainly by bacteriophages and SSSPs by IS elements. We also suggest that IS elements possibly play a role in the inactivation and immobilization of incoming phages and plasmids. Taken together, our results reveal the true impact of IS elements on the diversification of bacterial genomes and highlight their novel role in genome evolution..
27. Yoshitoshi Ogura, Tadasuke Ooka, Atsushi Iguchi, Hidehiro Toh, Md Asadulghani, Kenshiro Oshima, Toshio Kodama, Hiroyuki Abe, Keisuke Nakayama, Ken Kurokawa, Toru Tobe, Masahira Hattori, Tetsuya Hayashi, Comparative genomics reveal the mechanism of the parallel evolution of O157 and non-O157 enterohemorrhagic Escherichia coli, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.0903585106, 106, 42, 17939-17944, 2009.10, [URL], Among the various pathogenic Escherichia coli strains, enterohemorrhagic E. coli (EHEC) is the most devastating. Although serotype O157:H7 strains are the most prevalent, strains of different serotypes also possess similar pathogenic potential. Here, we present the results of a genomic comparison between EHECs of serotype O157, O26, O111, and O103, as well as 21 other, fully sequenced E. coli/Shigella strains. All EHECs have much larger genomes (5.5-5.9 Mb) than the other strains and contain surprisingly large numbers of prophages and integrative elements (IEs). The gene contents of the 4 EHECs do not follow the phylogenetic relationships of the strains, and they share virulence genes for Shiga toxins and many other factors. We found many lambdoid phages, IEs, and virulence plasmids that carry the same or similar virulence genes but have distinct evolutionary histories, indicating that independent acquisition of these mobile genetic elements has driven the evolution of each EHEC. Particularly interesting is the evolution of the type III secretion system (T3SS). We found that the T3SS of EHECs is composed of genes that were introduced by 3 different types of genetic elements: an IE referred to as the locus of enterocyte effacement, which encodes a central part of the T3SS; SpLE3-like IEs; and lambdoid phages carrying numerous T3SS effector genes and other T3SS-related genes. Our data demonstrate how E. coli strains of different phylogenies can independently evolve into EHECs, providing unique insights into the mechanisms underlying the parallel evolution of complex virulence systems in bacteria..
28. Hidehiro Toh, Kenshiro Oshima, Atsushi Toyoda, Yoshitoshi Ogura, Tadasuke Ooka, Hiroyuki Sasamoto, Sang Hee Park, Sunao Iyoda, Ken Kurokawa, Hidetoshi Morita, Kikuji Itoh, Todd D. Taylor, Tetsuya Hayashi, Masahira Hattori, Complete genome sequence of the wild-type commensal Escherichia coli strain SE15, belonging to phylogenetic group B2, Journal of bacteriology, 10.1128/JB.01543-09, 192, 4, 1165-1166, 2010.02, [URL], Escherichia coli SE15 (O150:H5) is a human commensal bacterium recently isolated from feces of a healthy adult and classified into E. coli phylogenetic group B2, which includes the majority of extraintestinal pathogenic E. coli. Here, we report the finished and annotated genome sequence of this organism..
29. Keisuke Nakayama, Ken Kurokawa, Masahiro Fukuhara, Hiroshi Urakami, Seigo Yamamoto, Kazuko Yamazaki, Yoshitoshi Ogura, Tadasuke Ooka, Tetsuya Hayashi, Genome comparison and phylogenetic analysis of orientia tsutsugamushi strains, DNA Research, 10.1093/dnares/dsq018, 17, 5, 281-291, 2010.10, [URL], Orientia tsutsugamushi (OT) is an obligate intracellular bacterium belonging to the family Rickettsiaceae and is the causative agent of scrub typhus, or Tsutsugamushi disease. The complete genome sequences of two OT strains (Boryong and Ikeda) have recently been determined. In the present study, we performed a fine genome sequence comparison of these strains. Our results indicate that although the core gene set of the family Rickettsiaceae is highly conserved between the two strains, a common set of repetitive sequences have been explosively amplified in both genomes. These amplified repetitive sequences have induced extensive genome shuffling and duplications and deletions of many genes. On the basis of the results of the genome sequence comparison, we selected 11 housekeeping genes and carried out multilocus sequence analysis of OT strains using the nucleotide sequences of these genes. This analysis revealed for the first time the phylogenetic relationships of representative OT strains. Furthermore, the results suggest the presence of an OT lineage with higher potential for virulence, which may explain the clinical and epidemiological differences between 'classic' and 'new' types of Tsutsugamushi disease in Japan..
30. Yoshitoshi Ogura, [Genomic analyses of mechanisms of virulence evolution in enterohemorrhagic E. coli and enteropathogenic E. coli]., Nihon saikingaku zasshi. Japanese journal of bacteriology, 10.3412/jsb.66.175, 66, 2-3, 175-186, 2011.01, [URL].
31. Masahiro Kusumoto, Tadasuke Ooka, Yoshiaki Nishiya, Yoshitoshi Ogura, Takashi Saito, Yasuhiko Sekine, Taketoshi Iwata, Masato Akiba, Tetsuya Hayashi, Insertion sequence-excision enhancer removes transposable elements from bacterial genomes and induces various genomic deletions, Nature communications, 10.1038/ncomms1152, 2, 1, 2011.01, [URL], Insertion sequences (ISs) are the simplest transposable elements and are widely distributed in bacteria. It has long been thought that IS excision rarely occurs in bacterial cells because most bacteria exhibit no end-joining activity to regenerate donor DNA after IS excision. Recently, however, we found that excision of IS629, an IS3 family member, occurs frequently in Escherichia coli O157. In this paper, we describe a protein IS-excision enhancer (IEE) that promotes IS629 excision from the O157 genome in an IS transposase-dependent manner. Various types of genomic deletions are also generated on IEE-mediated IS excision, and IEE promotes the excision of other IS3 family members and ISs from several other IS families. These data and the phylogeny of IEE homologues found in a broad range of bacteria suggest that IEE proteins have coevolved with IS elements and have pivotal roles in bacterial genome evolution by inducing IS removal and genomic deletion..
32. K. B.M.Saiful Islam, Satoru Fukiya, Masahito Hagio, Nobuyuki Fujii, Satoshi Ishizuka, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota, Bile acid is a host factor that regulates the composition of the cecal microbiota in rats, Gastroenterology, 10.1053/j.gastro.2011.07.046, 141, 5, 1773-1781, 2011.01, [URL], Background & Aims: Alterations in the gastrointestinal microbiota have been associated with metabolic diseases. However, little is known about host factors that induce changes in gastrointestinal bacterial populations. We investigated the role of bile acids in this process because of their strong antimicrobial activities, specifically the effects of cholic acid administration on the composition of the gut microbiota in a rat model. Methods: Rats were fed diets supplemented with different concentrations of cholic acid for 10 days. We used 16S ribosomal RNA gene clone library sequencing and fluorescence in situ hybridization to characterize the composition of the cecal microbiota of the different diet groups. Bile acids in feces, organic acids in cecal contents, and some blood parameters were also analyzed. Results: Administration of cholic acid induced phylum-level alterations in the composition of the gut microbiota; Firmicutes predominated at the expense of Bacteroidetes. Cholic acid feeding simplified the composition of the microbiota, with outgrowth of several bacteria in the classes Clostridia and Erysipelotrichi. Externally administered cholic acid was efficiently transformed into deoxycholic acid by a bacterial 7α-dehydroxylation reaction. Serum levels of adiponectin decreased significantly in rats given the cholic acid diet. Conclusions: Cholic acid regulates the composition of gut microbiota in rats, inducing similar changes to those induced by high-fat diets. These findings improve our understanding of the relationship between metabolic diseases and the composition of the gastrointestinal microbiota..
33. Yohsuke Ogawa, Tadasuke Ooka, Fang Shi, Yoshitoshi Ogura, Keisuke Nakayama, Tetsuya Hayashi, Yoshihiro Shimoji, The genome of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, reveals new insights into the evolution of Firmicutes and the organism's intracellular adaptations, Journal of bacteriology, 10.1128/JB.01500-10, 193, 12, 2959-2971, 2011.06, [URL], Erysipelothrix rhusiopathiae is a Gram-positive bacterium that represents a new class, Erysipelotrichia, in the phylum Firmicutes. The organism is a facultative intracellular pathogen that causes swine erysipelas, as well as a variety of diseases in many animals. Here, we report the first complete genome sequence analysis of a member of the class Erysipelotrichia. The E. rhusiopathiae genome (1,787,941 bp) is one of the smallest genomes in the phylum Firmicutes. Phylogenetic analyses based on the 16S rRNA gene and 31 universal protein families suggest that E. rhusiopathiae is phylogenetically close to Mollicutes, which comprises Mycoplasma species. Genome analyses show that the overall features of the E. rhusiopathiae genome are similar to those of other Gram-positive bacteria; it possesses a complete set of peptidoglycan biosynthesis genes, two-component regulatory systems, and various cell wall-associated virulence factors, including a capsule and adhesins. However, it lacks many orthologous genes for the biosynthesis of wall teichoic acids (WTA) and lipoteichoic acids (LTA) and the dltABCD operon, which is responsible for D-alanine incorporation into WTA and LTA, suggesting that the organism has an atypical cell wall. In addition, like Mollicutes, its genome shows a complete loss of fatty acid biosynthesis pathways and lacks the genes for the biosynthesis of many amino acids, cofactors, and vitamins, indicating reductive genome evolution. The genome encodes nine antioxidant factors and nine phospholipases, which facilitate intracellular survival in phagocytes. Thus, the E. rhusiopathiae genome represents evolutionary traits of both Firmicutes and Mollicutes and provides new insights into its evolutionary adaptations for intracellular survival..
34. Henryk Urbanczyk, Yoshitoshi Ogura, Tory A. Hendry, Alison L. Gould, Naomi Kiwaki, Joshua T. Atkinson, Tetsuya Hayashi, Paul V. Dunlap, Genome sequence of Photobacterium mandapamensis strain svers.1.1, the bioluminescent symbiont of the cardinal fish Siphamia versicolor, Journal of Bacteriology, 10.1128/JB.00370-11, 193, 12, 3144-3145, 2011.06, [URL], Photobacterium mandapamensis is one of three luminous Photobacterium species able to form species-specific bioluminescent symbioses with marine fishes. Here, we present the draft genome sequence of P. mandapamensis strain svers.1.1, the bioluminescent symbiont of the cardinal fish Siphamia versicolor, the first genome of a symbiotic, luminous Photobacterium species to be sequenced. Analysis of the sequence provides insight into differences between P. mandapamensis and other luminous and symbiotic bacteria in genes involved in quorumsensing regulation of light production and establishment of symbiosis..
35. Mariko Naito, Keiko Sato, Mikio Shoji, Hideharu Yukitake, Yoshitoshi Ogura, Tetsuya Hayashi, Koji Nakayama, Characterization of the porphyromonas gingivalis conjugative transposon CTnPg1
Determination of the integration site and the genes essential for conjugal transfer, Microbiology, 10.1099/mic.0.047803-0, 157, 7, 2022-2032, 2011.07, [URL], In our previous study, extensive genomic rearrangements were found in two strains of the Gramnegative anaerobic bacterium Porphyromonas (Por.) gingivalis, and most of these rearrangements were associated with mobile genetic elements such as insertion sequences and conjugative transposons (CTns). CTnPg1, identified in Por. gingivalis strain ATCC 33277, was the first complete CTn reported for the genus Porphyromonas. In the present study, we found that CTnPg1 can be transferred from strain ATCC 33277 to another Por. gingivalis strain, W83, at a frequency of 10 -7 to 10 -6. The excision of CTnPg1 from the chromosome in a donor cell depends on an integrase (Int; PGN_0094) encoded in CTnPg1, whereas CTnPg1 excision is independent of PGN_0084 (a DNA topoisomerase I homologue; Exc) encoded within CTnPg1 and recA (PGN_1057) on the donor chromosome. Intriguingly, however, the transfer of CTnPg1 between Por. gingivalis strains requires RecA function in the recipient. Sequencing analysis of CTnPg1- integrated sites on the chromosomes of transconjugants revealed that the consensus attachment (att) sequence is a 13 bp sequence, TTTTCNNNNAAAA. We further report that CTnPg1 is able to transfer to two other bacterial species, Bacteroides thetaiotaomicron and Prevotella oralis. In addition, CTnPg1-like CTns are located in the genomes of other oral anaerobic bacteria, Porphyromonas endodontalis, Prevotella buccae and Prevotella intermedia, with the same consensus att sequence. These results suggest that CTns in the CTnPg1 family are widely distributed among oral anaerobic Gram-negative bacteria found in humans and play important roles in horizontal gene transfer among these bacteria..
36. Atsushi Iguchi, Hiroki Shirai, Kazuko Seto, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Kayo Osawa, Ro Osawa, Wide distribution of O157-antigen biosynthesis gene clusters in Escherichia coli, PloS one, 10.1371/journal.pone.0023250, 6, 8, 2011.08, [URL], Most Escherichia coli O157-serogroup strains are classified as enterohemorrhagic E. coli (EHEC), which is known as an important food-borne pathogen for humans. They usually produce Shiga toxin (Stx) 1 and/or Stx2, and express H7-flagella antigen (or nonmotile). However, O157 strains that do not produce Stxs and express H antigens different from H7 are sometimes isolated from clinical and other sources. Multilocus sequence analysis revealed that these 21 O157:non-H7 strains tested in this study belong to multiple evolutionary lineages different from that of EHEC O157:H7 strains, suggesting a wide distribution of the gene set encoding the O157-antigen biosynthesis in multiple lineages. To gain insight into the gene organization and the sequence similarity of the O157-antigen biosynthesis gene clusters, we conducted genomic comparisons of the chromosomal regions (about 59 kb in each strain) covering the O-antigen gene cluster and its flanking regions between six O157:H7/non-H7 strains. Gene organization of the O157-antigen gene cluster was identical among O157:H7/non-H7 strains, but was divided into two distinct types at the nucleotide sequence level. Interestingly, distribution of the two types did not clearly follow the evolutionary lineages of the strains, suggesting that horizontal gene transfer of both types of O157-antigen gene clusters has occurred independently among E. coli strains. Additionally, detailed sequence comparison revealed that some positions of the repetitive extragenic palindromic (REP) sequences in the regions flanking the O-antigen gene clusters were coincident with possible recombination points. From these results, we conclude that the horizontal transfer of the O157-antigen gene clusters induced the emergence of multiple O157 lineages within E. coli and speculate that REP sequences may involve one of the driving forces for exchange and evolution of O-antigen loci..
37. Tomomi Kuwahara, Yoshitoshi Ogura, Kenshiro Oshima, Ken Kurokawa, Tadasuke Ooka, Hideki Hirakawa, Takehiko Itoh, Haruyuki Nakayama-Imaohji, Minoru Ichimura, Kikuji Itoh, Chieko Ishifune, Yoichi Maekawa, Koji Yasutomo, Masahira Hattori, Tetsuya Hayashi, The lifestyle of the segmented filamentous bacterium
A non-culturable gut-associated immunostimulating microbe inferred by whole-genome sequencing, DNA Research, 10.1093/dnares/dsr022, 18, 4, 291-303, 2011.08, [URL], Numerous microbes inhabit the mammalian intestinal track and strongly impact host physiology; however, our understanding of this ecosystem remains limited owing to the high complexity of the microbial community and the presence of numerous non-culturable microbes. Segmented filamentous bacteria (SFBs), which are clostridia-related Gram-positive bacteria, are among such non-culturable populations and are well known for their unique morphology and tight attachment to intestinal epithelial cells. Recent studies have revealed that SFBs play crucial roles in the post-natal maturation of gut immune function, especially the induction of Th17 lymphocytes. Here, we report the complete genome sequence of mouse SFBs. The genome, which comprises a single circular chromosome of 1 620 005 bp, lacks genes for the biosynthesis of almost all amino acids, vitamins/cofactors and nucleotides, but contains a full set of genes for sporulation/germination and, unexpectedly, for chemotaxis/flagella- based motility. These findings suggest a triphasic lifestyle of the SFB, which comprises two types of vegetative (swimming and epicellular parasitic) phases and a dormant (spore) phase. Furthermore, SFBs encode four types of flagellin, three of which are recognized by Toll-like receptor 5 and could elicit the innate immune response. Our results reveal the non-culturability, lifestyle and immunostimulation mechanisms of SFBs and provide a genetic basis for the future development of the SFB cultivation and gene-manipulation techniques..
38. Jacques G. Mainil, M. Bardiau, T. Ooka, Yoshitoshi Ogura, K. Murase, Y. Etoh, S. Ichihara, K. Horikawa, G. Buvens, D. Piérard, T. Itoh, Tetsuya Hayashi, Typing of O26 enterohaemorrhagic and enteropathogenic Escherichia coli isolated from humans and cattle with IS621 multiplex PCR-based fingerprinting, Journal of Applied Microbiology, 10.1111/j.1365-2672.2011.05089.x, 111, 3, 773-786, 2011.09, [URL], Aims: This study evaluated a typing method of O26:H11 enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) based on the variation in genomic location and copy numbers of IS621. Methods and Results: Two multiplex PCRs, targeting either the left (5') or right (3') IS/chromosome junction of 12 IS621 insertion sites and one PCR specific of another truncated copy, were developed. Thirty-eight amplification profiles were observed amongst a collection of 69 human and bovine O26:H11 EHEC and EPEC. Seventy-one per cent of the 45 EHEC and EPEC with identical IS621 fingerprints within groups of two, three or four isolates had >85% pulsed field gel electrophoresis (PFGE) profile similarity, including four groups of epidemiologically related EHEC or EPEC, while most of the groups had <85% similarity between each others. Epidemiologically related EHEC from each of three independent outbreaks in Japan and Belgium also exhibited identical IS621 fingerprints and PFGE profiles. Conclusions: The IS621 fingerprinting and the PFGE are complementary typing assays of EHEC and EPEC; though, the former is less discriminatory. Significance and Impact of the Study: The IS621 printing method represents a rapid (24h) first-line surveillance and typing assay, to compare and trace back O26:H11 EHEC and EPEC during surveys in farms, multiple human cases and outbreaks..
39. Atsushi Yokota, Satoru Fukiya, Tadasuke Ooka, Yoshitoshi Ogura, Tetsuya Hayashi, Satoshi Ishizuka, Is bile acid a determinant of the gut microbiota on a high-fat diet?, Gut Microbes, 10.4161/gmic.21216, 3, 5, 2012.01, [URL], Recently, we discovered that bile acid, a main component of bile, is a host factor that regulates the composition of the cecal microbiota in rats. Because bile secretion increases on a high-fat diet and bile acids generally have strong antimicrobial activity, we speculated that bile acids would be a determinant of the gut microbiota in response to a high-fat diet. The observed changes in the rat cecal microbiota triggered by cholic acid (the most abundant bile acid in human biliary bile) administration resemble those found in animals fed high-fat diets. Here, we discuss the rationale for this hypothesis by evaluating reported diet-induced gut microbiota alterations based on the postulate that bile acids worked as an underlying determinant. The identification of host factors determining the gut microbiota greatly contributes to understanding the causal relationships between changes in the gut microbiota and disease development, which remain to be elucidated..
40. Kazunori Murase, Tadasuke Ooka, Atsushi Iguchi, Yoshitoshi Ogura, Keisuke Nakayama, Md Asadulghani, Md Rakibul Islam, Hirotaka Hiyoshi, Toshio Kodama, Lothar Beutin, Tetsuya Hayashi, Haemolysin E- and enterohaemolysin-derived haemolytic activity of O55/O157 strains and other Escherichia coli lineages, Microbiology, 10.1099/mic.0.054775-0, 158, 3, 746-758, 2012.03, [URL], Among three haemolysins identified thus far in Escherichia coli, alpha-haemolysin (HlyA) is encoded on the pathogenicity islands of extraintestinal pathogenic strains, while enterohaemolysin (EhxA) is encoded on the virulence plasmids of enterohaemorrhagic E. coli (EHEC) strains. In contrast, the gene for haemolysin E (HlyE) is located on the E. coli chromosome backbone and is therefore widely distributed among E. coli strains. However, because hlyE gene expression is repressed by the H-NS protein and because the gene has been disrupted in many strains, its haemolytic activity cannot be detected in wild-type strains by routine screening on blood agar plates. In this study, we found that the HlyE-derived haemolytic activity of enteropathogenic E. coli (EPEC) O55:H7 can be detected after anaerobic cultivation on a washed blood agar plate (EHX plate) that is used to detect the production of EhxA.We also found that the haemolytic activity of EHECO157:H7 observed on EHX plates under aerobic and anaerobic growth conditions is derived from EhxA and HlyE, respectively; this differential expression of the two haemolysins occurs at the transcriptional level. Our analysis of 60 E. coli strains of various pathotypes and phylogenies for their repertoires of haemolysin genes, haemolytic phenotypes and hlyE gene sequences revealed that HlyE activity can generally be detected on EHX plates under anaerobic growth conditions if the gene is intact. Furthermore, our results indicate that hlyE gene inactivation occurred in three of the five E. coli lineages (phylogroups A, B1 and B2), which demonstrates phylogroup-specific gene disruption patterns..
41. Tadasuke Ooka, Kazuko Seto, Kimiko Kawano, Hideki Kobayashi, Yoshiki Etoh, Sachiko Ichihara, Akiko Kaneko, Junko Isobe, Keiji Yamaguchi, Kazumi Horikawa, Tânia A. Gomes, Annick Linden, Marjorie Bardiau, Jacques G. Mainil, Lothar Beutin, Yoshitoshi Ogura, Tetsuya Hayashi, Clinical significance of Escherichia albertii, Emerging Infectious Diseases, 10.3201/eid1803.111401, 18, 3, 488-492, 2012.03, [URL], Discriminating Escherichia albertii from other Enterobacteriaceae is difficult. Systematic analyses showed that E. albertii represents a substantial portion of strains currently identified as eae-positive Escherichia coli and includes Shiga toxin 2f-producing strains. Because E. albertii possesses the eae gene, many strains might have been misidentified as enterohemorrhagic or enteropathogenic E. coli..
42. Md Rakibul Islam, Yoshitoshi Ogura, Md Asadulghani, Tadasuke Ooka, Kazunori Murase, Yasuhiro Gotoh, Tetsuya Hayashi, A sensitive and simple plaque formation method for the Stx2 phage of Escherichia coli O157
H7, which does not form plaques in the standard plating procedure, Plasmid, 10.1016/j.plasmid.2011.12.001, 67, 3, 227-235, 2012.05, [URL], Bacteriophages are fascinating genetic elements that play key roles in the evolution and diversification of bacterial genomes. Shiga toxin (Stx)-transducing phages are important genetic elements that disseminate the stx genes among enterohemorrhagic Escherichia coli (EHEC). They are generally regarded as lambda-like phages, but their biological and genetic properties have not been fully elucidated. This is partly due to a serious obstacle in obtaining visible plaques. Here, we describe a modified double agar overlay method that allows us to easily detect and accurately enumerate plaques of Sp5, the Stx2 phage of the EHEC O157 Sakai strain, which otherwise does not produce plaques in the standard plating procedure. In the modified method, the top agar was supplemented with mitomycin C (MMC) and Ca
2+
(or Mg
2+
). MMC appears to prevent the lysogenization of Sp5 and/or compel Sp5 to follow the lytic cycle by inducing the SOS response in the host cells. The divalent cations significantly improve phage adsorption to the host cells and thus yield a synergistic effect in combination with MMC. We further applied this method to a receptor analysis of Sp5 and obtained findings that suggest that the YaeT (BamA) protein serves as the receptor of Sp5. This method would be a very useful tool in studies of Stx2 phages and studies of other phages from various bacteria, in which researchers often encounter problems with plaque formation..
43. Eiji Kakizaki, Yoshitoshi Ogura, Shuji Kozawa, Sho Nishida, Taketo Uchiyama, Tetsuya Hayashi, Nobuhiro Yukawa, Detection of diverse aquatic microbes in blood and organs of drowning victims
First metagenomic approach using high-throughput 454-pyrosequencing, Forensic Science International, 10.1016/j.forsciint.2012.02.010, 220, 1-3, 135-146, 2012.07, [URL], Current 454-pyrosequencing technology enables massive parallel sequencing. We used this technology to investigate the diversity of aquatic microbes in 14 specimens (blood and organs) of two drowning victims and in two water samples taken from the discovery sites. The 16S ribosomal RNA (rRNA) genes of microbes, which are often used to identify species (or genera), have nine highly variable regions (V1-V9), each of which is surrounded by conserved regions. Some parts within the conserved regions are common over domains of microbes, such as between bacteria and algae (16S rRNA genes on algal chloroplast genomes). We therefore simultaneously amplified the target regions (V7 and V8) of various microbes in the blood and organs of drowning victims using PCR with custom-designed primers that were based on the conserved regions. We then exhaustively analyzed the PCR products by pyrosequencing using the Genome Sequencer FLX Titanium system (Roche-454 Life Sciences). This approach identified a wide array of bacteria including cyanobacteria and algae including Bacillariophyceae (diatom), Cryptophyceae, Dictyochophyceae, Chrysophyceae and Trebouxiophyceae in the blood and organs of the victims and water at discovery sites. Our data further indicated that when conventional diatom testing of lungs yielded insufficient evidence of water aspiration, the detection of various exogenous microbes by 454-pyrosequencing is very useful to support a conclusion of death by drowning. To the best of our knowledge, this is the first attempt to use a new generation sequencer to investigate diverse aquatic microbes in the blood and closed organs of drowning victims..
44. Takatsugu Goto, Yoshitoshi Ogura, Hideki Hirakawa, Junko Tomida, Yuji Morita, Takaaki Akaike, Tetsuya Hayashi, Yoshiaki Kawamura, Complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia, Journal of bacteriology, 10.1128/JB.00645-12, 194, 14, 3744-3745, 2012.07, [URL], We report the complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia. The PAGU611 genome comprises a 2,078,348-bp chromosome and a 23,054-bp plasmid. The chromosome contains a unique genomic island, encoding a type VI secretion system and clustered regularly interspaced short palindromic repeat (CRISPR) loci..
45. Yoshitaka Tateishi, Seigo Kitad, Keisuke Miki, Ryoji Maekura, Yoshitoshi Ogura, Yuriko Ozeki, Yukiko Nishiuchi, Mamiko Niki, Tetsuya Hayashi, Kazuto Hirata, Kazuo Kobayashi, Sohkichi Matsumoto, Whole-Genome Sequence of the Hypervirulent Clinical Strain Mycobacterium intracellulare M.i.198, Journal of bacteriology, 10.1128/JB.01439-12, 194, 22, 2012.11, [URL], We report herein the draft genome sequence of Mycobacterium intracellulare clinical strain M.i.198, which consistently exhibits hypervirulence in human patients, human macrophages in vitro, and immunocompetent mice..
46. Yoshitaka Tateishi, Aki Tamaru, Yoshitoshi Ogura, Mamiko Niki, Takayuki Wada, Taro Yamamoto, Kazuto Hirata, Tetsuya Hayashi, Sohkichi Matsumoto, Whole-genome sequence of the potentially hypertransmissible multidrug-resistant Mycobacterium tuberculosis Beijing strain OM-V02_005, Genome Announcements, 10.1128/genomeA.00608-13, 1, 4, 2013.01, [URL], We report the draft genome sequence of Mycobacterium tuberculosis Beijing strain OM-V02_005, which exhibits possible hypertransmissible characteristics among the population of patients with multidrug-resistant tuberculosis in Osaka Prefecture, the largest urban area in western Japan..
47. Tomoo Sawabe, Yoshitoshi Ogura, Yuta Matsumura, Gao Feng, A. K.M. Rohul Amin, Sayaka Mino, Satoshi Nakagawa, Toko Sawabe, Ramesh Kumar, Yohei Fukui, Masataka Satomi, Ryoji Matsushima, Fabiano L. Thompson, Bruno Gomez-Gil, Richard Christen, Fumito Maruyama, Ken Kurokawa, Tetsuya Hayashi, Updating the Vibrio clades defined by multilocus sequence phylogeny
Proposal of eight new clades, and the description of Vibrio tritonius sp. nov., Frontiers in Microbiology, 10.3389/fmicb.2013.00414, 4, DEC, 2013.01, [URL], To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera; Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on (1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis); (2) clades amended since the 2007 proposal with recently described new species; (3) orphan clades of genomospecies F6 and F10; (4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3); and (5) description of V. tritonius sp. nov., which is a member of the "Porteresiae" clade..
48. Tadasuke Ooka, Eisuke Tokuoka, Masato Furukawa, Tetsuya Nagamura, Yoshitoshi Ogura, Kokichi Arisawa, Seiya Harada, Tetsuya Hayashi, Human gastroenteritis outbreak associated with Escherichia albertii, Japan, Emerging Infectious Diseases, 10.3201/eid1901.120646, 19, 1, 144-146, 2013.01, [URL], Although Escherichia albertii is an emerging intestinal pathogen, it has been associated only with sporadic human infections. In this study, we determined that a human gastroenteritis outbreak at a restaurant in Japan had E. albertii as the major causative agent..
49. Kana Minegishi, Chihiro Aikawa, Asuka Furukawa, Takayasu Watanabe, Tsubasa Nakano, Yoshitoshi Ogura, Yoshiyuki Ohtsubo, Ken Kurokawa, Tetsuya Hayashi, Fumito Maruyama, Ichiro Nakagawa, Yoshinobu Eishi, Complete genome sequence of a Propionibacterium acnes isolate from a sarcoidosis patient, Genome Announcements, 10.1128/genomeA.00016-12, 1, 1, 2013.01, [URL], Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles and is the only microorganism that has been isolated from sarcoid lesions. We report the complete genome sequence of P. acnes, which was isolated from a Japanese patient with sarcoidosis..
50. Eiji Nagayasu, Yoshitoshi Ogura, Takehiko Itoh, Ayako Yoshida, Gunimala Chakraborty, Tetsuya Hayashi, Haruhiko Maruyama, Transcriptomic analysis of four developmental stages of Strongyloides venezuelensis, Parasitology International, 10.1016/j.parint.2012.09.006, 62, 1, 57-65, 2013.02, [URL], Strongyloides venezuelensis is one of some 50 species of genus Strongyloides, obligate gastrointestinal parasites of vertebrates, responsible for strongyloidiasis in humans and other domestic/companion animals. Although S. venezuelensis has been widely used as a model species for studying human/animal strongyloidiasis, the sequence information for this species has been quite limited. To create a more comprehensive catalogue of expressed genes for identification of genes potentially involved in animal parasitism, we conducted a de novo sequencing analysis of the transcriptomes from four developmental stages of S. venezuelensis, using a Roche 454 GS FLX Titanium pyrosequencing platform. A total of 14,573 contigs were produced after de novo assemblies of over 2. million sequencing reads and formed a dataset "Vene454". BLAST homology search of Vene454 against proteome and transcriptome data from other animal-parasitic and non-animal-parasitic nematode species revealed several interesting genes, which may be potentially related to animal parasitism, including nicotinamide phosphoribosyltransferase and ferrochelatase. The Vene454 dataset analysis also enabled us to identify transcripts that are specifically enriched in each developmental stage. This work represents the first large-scale transcriptome analysis of S. venezuelensis and the first study to examine the transcriptome of the lung L3 developmental stage of any Strongyloides species. The results not only will serve as valuable resources for future functional genomics analyses to understand the molecular aspects of animal parasitism, but also will provide essential information for ongoing whole genome sequencing efforts in this species..
51. Henryk Urbanczyk, Yoshitoshi Ogura, Tetsuya Hayashi, Taxonomic revision of Harveyi clade bacteria (family Vibrionaceae) based on analysis of whole genome sequences, International Journal of Systematic and Evolutionary Microbiology, 10.1099/ijs.0.051110-0, 63, PART7, 2742-2751, 2013.07, [URL], Use of inadequate methods for classification of bacteria in the so-called Harveyi clade (family Vibrionaceae, Gammaproteobacteria) has led to incorrect assignment of strains and proliferation of synonymous species. In order to resolve taxonomic ambiguities within the Harveyi clade and to test usefulness of whole genome sequence data for classification of Vibrionaceae, draft genome sequences of 12 strains were determined and analysed. The sequencing included type strains of seven species: Vibrio sagamiensis NBRC 104589T, Vibrio azureus NBRC 104587T, Vibrio harveyi NBRC 15634T, Vibrio rotiferianus LMG 21460T, Vibrio campbellii NBRC 15631T, Vibrio jasicida LMG 25398T, and Vibrio owensii LMG 25443T. Draft genome sequences of strain LMG 25430, previously designated the type strain of [Vibrio communis], and two strains (MWB 21 and 090810c) from the 'beijerinckii' lineage were also determined. Whole genomes of two additional strains (ATCC 25919 and 200612B) that previously could not be assigned to any Harveyi clade species were also sequenced. Analysis of the genome sequence data revealed a clear case of synonymy between V. owensii and [V. communis], confirming an earlier proposal to synonymize both species. Both strains from the 'beijerinckii' lineage were classified as V. jasicida, while the strains ATCC 25919 and 200612B were classified as V. owensii and V. campbellii, respectively. We also found that two strains, AND4 and Ex25, are closely related to Harveyi clade bacteria, but could not be assigned to any species of the family Vibrionaceae. The use of whole genome sequence data for the taxonomic classification of the Harveyi clade bacteria and other members of the family Vibrionaceae is also discussed..
52. miki Kawada-Matsuo, Yuichi Oogai, Takeshi Zendo, Junichi Nagao, Yukie Shibata, Yoshihisa Yamashita, Yoshitoshi Ogura, Tetsuya Hayashi, Kenji Sonomoto, Hitoshi Komatsuzawa, Involvement of the novel two-component nsrrs and lcrrs systems in distinct resistance pathways against nisin a and nukacin isk-1 in streptococcus mutans, Applied and Environmental Microbiology, 10.1128/AEM.00780-13, 79, 15, 4751-4755, 2013.08, [URL], The novel two-component systems nsrrs and lcrrs are individually associated with resistance against the distinct lantibiotics nisin a and nukacin isk-1 in streptococcus mutans. nsrrs regulates the expression of nsrx, which is associated with nisin a binding, and lcrrs regulates the expression of the abc transporter lctfeg..
53. Henryk Urbanczyk, Yoshiko Urbanczyk, Tetsuya Hayashi, Yoshitoshi Ogura, Diversification of two lineages of symbiotic photobacterium, PLoS One, 10.1371/journal.pone.0082917, 8, 12, 2013.12, [URL], Understanding of processes driving bacterial speciation requires examination of closely related, recently diversified lineages. To gain an insight into diversification of bacteria, we conducted comparative genomic analysis of two lineages of bioluminescent symbionts, Photobacterium leiognathi and 'P. mandapamensis'. The two lineages are evolutionary and ecologically closely related. Based on the methods used in bacterial taxonomy for classification of new species (DNA-DNA hybridization and ANI), genetic relatedness of the two lineages is at a cut-off point for species delineation. In this study, we obtained the whole genome sequence of a representative P. leiognathi strain lrivu.4.1, and compared it to the whole genome sequence of 'P. mandapamensis' svers.1.1. Results of the comparative genomic analysis suggest that P. leiognathi has a more plastic genome and acquired genes horizontally more frequently than 'P. mandapamensis'. We predict that different rates of recombination and gene acquisition contributed to diversification of the two lineages. Analysis of lineage-specific sequences in 25 strains of P. leiognathi and 'P. mandapamensis' found no evidence that bioluminescent symbioses with specific host animals have played a role in diversification of the two lineages..
54. Rei Kajitani, Kouta Toshimoto, Hideki Noguchi, Atsushi Toyoda, Yoshitoshi Ogura, Miki Okuno, Mitsuru Yabana, Masayuki Harada, Eiji Nagayasu, Haruhiko Maruyama, Yuji Kohara, Asao Fujiyama, Tetsuya Hayashi, Takehiko Itoh, Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads, Genome Research, 10.1101/gr.170720.113, 24, 8, 1384-1395, 2014, [URL], Although many de novo genome assembly projects have recently been conducted using high-throughput sequencers, assembling highly heterozygous diploid genomes is a substantial challenge due to the increased complexity of the de Bruijn graph structure predominantly used. To address the increasing demand for sequencing of nonmodel and/or wildtype samples, in most cases inbred lines or fosmid-based hierarchical sequencing methods are used to overcome such problems. However, these methods are costly and time consuming, forfeiting the advantages of massive parallel sequencing. Here, we describe a novel de novo assembler, Platanus, that can effectively manage high-throughput data from heterozygous samples. Platanus assembles DNA fragments (reads) into contigs by constructing de Bruijn graphs with automatically optimized k-mer sizes followed by the scaffolding of contigs based on paired-end information. The complicated graph structures that result from the heterozygosity are simplified during not only the contig assembly step but also the scaffolding step. We evaluated the assembly results on eukaryotic samples with various levels of heterozygosity. Compared with other assemblers, Platanus yields assembly results that have a larger scaffold NG50 length without any accompanying loss of accuracy in both simulated and real data. In addition, Platanus recorded the largest scaffold NG50 values for two of the three low-heterozygosity species used in the de novo assembly contest, Assemblathon 2. Platanus therefore provides a novel and efficient approach for the assembly of gigabase-sized highly heterozygous genomes and is an attractive alternative to the existing assemblers designed for genomes of lower heterozygosity..
55. Lisa Nonaka, Fumito Maruyama, Yuki Onishi, Takeshi Kobayashi, Yoshitoshi Ogura, Tetsuya Hayashi, Satoru Suzuki, Michiaki Masuda, Various pAQU plasmids possibly contribute to disseminate tetracycline resistance gene tet(M) among marine bacterial community, Frontiers in Microbiology, 10.3389/fmicb.2014.00152, 5, MAY, 2014.01, [URL], Emergence of antibiotic-resistant bacteria in the aquaculture environment is a significant problem for disease control of cultured fish as well as in human public health. Conjugative mobile genetic elements (MGEs) are involved in dissemination of antibiotic resistance genes (ARGs) among marine bacteria. In the present study, we first designed a PCR targeting traI gene encoding essential relaxase for conjugation. By this new PCR, we demonstrated that five of 83 strains isolated from a coastal aquaculture site had traI-positive MGEs. While one of the five strains that belonged to Shewanella sp. was shown to have an integrative conjugative element of the SXT/R391 family (ICEVchMex-like), the MGEs of the other four strains of Vibrio spp. were shown to have the backbone structure similar to that of previously described in pAQU1. The backbone structure shared by the pAQU1-like plasmids in the four strains corresponded to a ~100-kbp highly conserved region required for replication, partition and conjugative transfer, suggesting that these plasmids constituted "pAQU group." The pAQU group plasmids were shown to be capable of conjugative transfer of tet(M) and other ARGs from the Vibrio strains to E. coli. The pAQU group plasmid in one of the examined strains was designated as pAQU2, and its complete nucleotide sequence was determined and compared with that of pAQU1. The results revealed that pAQU2 contained fewer ARGs than pAQU1 did, and most of the ARGs in both of these plasmids were located in the similar region where multiple transposases were found, suggesting that the ARGs were introduced by several events of DNA transposition into an ancestral plasmid followed by drug selection in the aquaculture site. The results of the present study indicate that the "pAQU group" plasmids may play an important role in dissemination of ARGs in the marine environment..
56. , Sunao Iyoda, Shannon D. Manning, Kazuko Seto, Keiko Kimata, Junko Isobe, Yoshiki Etoh, Sachiko Ichihara, Yuji Migita, Kikuyo Ogata, Mikiko Honda, Tsutomu Kubota, Kimiko Kawano, Kazutoshi Matsumoto, Jun Kudaka, Norio Asai, Junko Yabata, Kiyoshi Tominaga, Jun Terajima, Tomoko Morita-Ishihara, Hidemasa Izumiya, Yoshitoshi Ogura, Takehito Saitoh, Atsushi Iguchi, Hideki Kobayashi, Yukiko Hara-Kudo, Makoto Ohnishi, Kazumi Horikawa, Nanami Asoshima, Mitsuhiro Kameyama, Reiko Arai, Masao Kawase, Yukiko Asano, Kazuki Chiba, Ichiro Furukawa, Toshiro Kuroki, Madoka Hamada, Seiya Harada, Takashi Hatakeyama, Takashi Hirochi, Yumiko Sakamoto, Midori Hiroi, Takashi Kanda, Kaori Iwabuchi, Hitomi Kasahara, Shinya Kawanishi, Koji Kikuchi, Hiroyuki Ueno, Tomoko Kitahashi, Yuka Kojima, Noriko Konishi, Phylogenetic clades 6 and 8 of enterohemorrhagic Escherichia coli O157
H7 With particular stx subtypes are more frequently found in isolates from hemolytic uremic syndrome patients than from asymptomatic carriers, Open Forum Infectious Diseases, 10.1093/ofid/ofu061, 1, 2, 2014.01, [URL], Background. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection causes severe diseases such as bloody diarrhea and hemolytic uremic syndrome (HUS). Although EHEC O157:H7 strains have exhibited high genetic variability, their abilities to cause human diseases have not been fully examined. Methods. Clade typing and stx subtyping of EHEC O157:H7 strains, which were isolated in Japan during 1999-2011 from 269 HUS patients and 387 asymptomatic carriers (ACs) and showed distinct pulsed-field gel electrophoresis patterns, were performed to determine relationships between specific lineages and clinical presentation. Results. Clades 6 and 8 strains were more frequently found among the isolates from HUS cases than those from ACs (P = .00062 for clade 6, P < .0001 for clade 8). All clade 6 strains isolated from HUS patients harbored stx2a and/ or stx2c, whereas all clade 8 strains harbored either stx2a or stx2a/stx2c. However, clade 7 strains were predominantly found among the AC isolates but less frequently found among the HUS isolates, suggesting a significant association between clade 7 and AC (P < .0001). Logistic regression analysis revealed that 0-9 year old age is a significant predictor of the association between clade 8 and HUS. We also found an intact norV gene, which encodes for a nitric oxide reductase that inhibits Shiga toxin activity under anaerobic condition, in all clades 1-3 isolates but not in clades 4-8 isolates. Conclusions. Early detection of EHEC O157:H7 strains that belonged to clades 6/8 and harbored specific stx subtypes may be important for defining the risk of disease progression in EHEC-infected 0- to 9-year-old children..
57. Atsushi Iguchi, Yutaka Nagaya, Elizabeth Pradel, Tadasuke Ooka, Yoshitoshi Ogura, Keisuke Katsura, Ken Kurokawa, Kenshiro Oshima, Masahira Hattori, Julian Parkhill, Mohamed Sebaihia, Sarah J. Coulthurst, Naomasa Gotoh, Nicholas R. Thomson, Jonathan J. Ewbank, Tetsuya Hayashi, Genome evolution and plasticity of serratia marcescens, an important multidrug-resistant nosocomial pathogen, Genome Biology and Evolution, 10.1093/gbe/evu160, 6, 8, 2096-2110, 2014.01, [URL], Serratiamarcescens is an important nosocomial pathogen that can cause an array of infections,most notably of the urinary tract and bloodstream. Naturally, it is found inmany environmental niches, and is capable of infecting plants and animals. The emergence and spread of multidrug-resistant strains producing extended-spectrum or metallo beta-lactamases now pose a threat to public health worldwide. Here we report the complete genome sequences of two carefully selected S. marcescens strains, a multidrug-resistant clinical isolate (strainSM39)andaninsect isolate (strain Db11).Our comparative analyses reveal the coregenomeof S. marcescensand define the potential metabolic capacity, virulence, and multidrug resistance of this species. We show a remarkable intraspecies genetic diversity, both at the sequence level and with regardsgenomeflexibility, whichmay reflect the diversity of niches inhabited by members of this species.Abroader analysis with other Serratia species identifies a set of approximately 3,000 genes that characterize the genus.Within this apparent genetic diversity, we identifiedmany genes implicated in the high virulence potential and antibiotic resistance of SM39, including the metallo beta-lactamase and multiple other drug resistance determinants carried on plasmid pSMC1. We further show that pSMC1 ismost closely related to plasmids circulating in Pseudomonas species. Our data will provide a valuable basis for future studies on S. marcescens and new insights into the genetic mechanisms that underlie the emergence of pathogens highly resistant to multiple antimicrobial agents..
58. Yoshihiko Sakaguchi, Koji Hosomi, Jumpei Uchiyama, Yoshitoshi Ogura, Kaoru Umeda, Masakiyo Sakaguchi, Tomoko Kohda, Masafumi Mukamoto, Naoaki Misawa, Shigenobu Matsuzaki, Tetsuya Hayashi, Shunji Kozaki, Draft genome sequence of Clostridium botulinum type B strain Osaka05, isolated from an infant patient with botulism in Japan, Genome Announcements, 10.1128/genomeA.e01010-13, 2, 1, 2014.01, [URL], Clostridium botulinum strain Osaka05, which has been isolated from an infant patient with botulism in Japan, is the first strain producing botulinum neurotoxin subtype B6. Here, we report the draft genome sequence of C. botulinum Osaka05..
59. Tomoo Sawabe, Yoshitoshi Ogura, Yuta Matsumura, Gao Feng, A. K.M. Rohul Amin, Sayaka Mino, Satoshi Nakagawa, Toko Sawabe, Ramesh Kumar, Yohei Fukui, Masataka Satomi, Ryoji Matsushima, Fabiano L. Thompson, Bruno Gomez Gil, Richard Christen, Fumito Maruyama, Ken Kurokawa, Tetsuya Hayashi, Corrigendum to Updating the Vibrio clades defined by multilocus sequence phylogeny
Proposal of eight new clades, and the description of Vibrio tritonius sp. nov. [Front Microbiol., 4, (2013), 414]. DOI: 10.3389/fmicb.2013.00414, Frontiers in Microbiology, 10.3389/fmicb.2014.00583, 5, NOV, 2014.01, [URL].
60. Henryk Urbanczyk, Yoshitoshi Ogura, Tetsuya Hayashi, Contrasting inter-and intraspecies recombination patterns in the "harveyi clade" vibrio collected over large spatial and temporal scales, Genome Biology and Evolution, 10.1093/gbe/evu269, 7, 1, 71-80, 2014.01, [URL], Recombinationplaysanimportant role in thedivergenceofbacteria, but thefrequencyof interspecies andintraspecies recombination events remains poorly understood. We investigated recombination events that occurred within core genomes of 35 Vibrio strains (family Vibrionaceae, Gammaproteobacteria), from six closely related species in the so-called "Harveyi clade." The strains were selected from a collection of strains isolated in the last 90 years, fromvarious environments worldwide.Wefound a close relationship between the number of interspecies recombination events within core genomes of the 35 strains and the overall genomic identity, as inferred fromcalculations of the average nucleotide identity. The relationship between theoverall nucleotide identity andthe number of detected interspecies recombination events was comparable when analyzing strains isolated over 80 years apart, from different hemispheres, or from different ecologies, as well as in strains isolated from the same geographic location within a short time frame. We further applied the same method of detecting recombination events to analyze 11 strains of Vibrio campbellii, and identified disproportionally high number of intraspecies recombination events within the core genomes of some, but not all, strains. The high number of recombination events was detected between V. campbellii strains that have significant temporal (over 18 years) and geographical (over 10,000 km)differences in theirorigins of isolation. Resultsof this study reveal aremarkablestabilityofHarveyi clade species, and give clues about the origins and persistence of species in the clade..
61. Yoshiyuki Ohtsubo, Takuya Sato, Kouhei Kishida, Michro Tabata, Yoshitoshi Ogura, Tetsuya Hayashi, Masataka Tsuda, Yuji Nagata, Complete genome sequence of Pseudomonas aeruginosa MTB-1, isolated from a microbial community enriched by the technical formulation of hexachlorocyclohexane, Genome Announcements, 10.1128/genomeA.e01130-13, 2, 1, 2014.01, [URL], Pseudomonas aeruginosa MTB-1 does not degrade gamma-hexachlorocyclohexane (γ-HCH), but this bacterium persistently coexists with a γ-HCH-degrading strain, Sphingomonas sp. MM-1, in a microbial community enriched by the technical formulation of HCH. Here we report the complete MTB-1 genome sequence, with a 6.6-Mb circular chromosome..
62. Yoshiyuki Ohtsubo, Kouhei Kishida, Takuya Sato, Michiro Tabata, Toru Kawasumi, Yoshitoshi Ogura, Tetsuya Hayashim, Masataka Tsuda, Yuji Nagata, Complete genome sequence of Pseudomonas sp. strain TKP, isolated from a γ-hexachlorocyclohexane-degrading mixed culture, Genome Announcements, 10.1128/genomeA.e01241-13, 2, 1, 2014.01, [URL], Pseudomonas sp. strain TKP does not degrade γ-hexachlorocyclohexane (γ-HCH), but it persistently coexists with the γ-HCHdegrading Sphingobium sp. strain TKS in a mixed culture enriched by γ-HCH. Here, we report the complete genome sequence of strain TKP, which consists of one circular chromosome with a size of 7 Mb..
63. Keisuke Okada, Yoshitoshi Ogura, Tetsuya Hayashi, Akio Abe, Asaomi Kuwae, Yasuhiko Horiguchi, Hiroyuki Abe, Complete genome sequence of Bordetella bronchiseptica S798, an isolate from a pig with atrophic rhinitis, Genome Announcements, 10.1128/genomeA.00436-14, 2, 3, 2014.01, [URL], Bordetella bronchiseptica colonizes the respiratory tracts of a wide variety of mammals and causes a range of diseases, from lethal pneumonia to asymptomatic chronic infection. We report the complete genome sequence of Bordetella bronchiseptica strain S798, isolated from a pig with atrophic rhinitis in Japan..
64. Naoki Sudo, Akiko Soma, Akira Muto, Sunao Iyoda, Mayumi Suh, Nanako Kurihara, Hiroyuki Abe, Toru Tobe, Yoshitoshi Ogura, Tetsuya Hayashi, Ken Kurokawa, Makoto Ohnishi, Yasuhiko Sekine, A novel small regulatory RNA enhances cell motility in enterohemorrhagic Escherichia coli, Journal of General and Applied Microbiology, 10.2323/jgam.60.44, 60, 1, 44-50, 2014.01, [URL], Small regulatory RNAs (sRNAs) are conserved among a wide range of bacteria. They modulate the translational efficiency of target mRNAs through base-pairing with the help of RNA chaperone Hfq. The present study identified a novel sRNA, Esr41 (enterohemorrhagic Escherichia coli O157 small RNA #41), from an intergenic region of an enterohemorrhagic E. coli (EHEC) O157:H7 Sakai-specific sequence that is not present in the nonpathogenic E. coli K-12. Esr41 was detected as an RNA molecule approximately 70 nucleotides long with a 3′ GC-rich palindrome sequence followed by a long poly(U), which is a characteristic of rho-independent terminators and is also a structural feature required for the action of Hfq. EHEC O157 harboring a multicopy plasmid carrying the esr41 gene increased cell motility and the expression of fliC, a gene encoding a major flagellar component. These results indicate that Esr41 stimulates fliC expression in EHEC O157. Furthermore, the increase in cell motility induced by Esr41 was also observed in the E. coli K-12, suggesting that target genes controlled by Esr41 are present in both EHEC O157 and K-12..
65. Masahiro Kusumoto, Dai Fukamizu, Yoshitoshi Ogura, Eiji Yoshida, Fumiko Yamamoto, Taketoshi Iwata, Tadasuke Ooka, Masato Akiba, Tetsuya Hayashi, Lineage-specific distribution of insertion sequence excision enhancer in enterotoxigenic Escherichia coli isolated from swine, Applied and Environmental Microbiology, 10.1128/AEM.03696-13, 80, 4, 1394-1402, 2014.02, [URL], Insertion sequences (ISs) are the simplest transposable elements and are widely distributed in bacteria; however, they also play important roles in genome evolution. We recently identified a protein called IS excision enhancer (IEE) in enterohemorrhagic Escherichia coli (EHEC) O157. IEE promotes the excision of IS elements belonging to the IS3 family, such as IS629, as well as several other families. IEE-mediated IS excision generates various genomic deletions that lead to the diversification of the bacterial genome. IEE has been found in a broad range of bacterial species; however, among sequenced E. coli strains, IEE is primarily found in EHEC isolates. In this study, we investigated non-EHEC pathogenic E. coli strains isolated from domestic animals and found that IEE is distributed in specific lineages of enterotoxigenic E. coli (ETEC) strains of serotypes O139 or O149 isolated from swine. The iee gene is located within integrative elements that are similar to SpLE1 of EHEC O157. All iee-positive ETEC lineages also contained multiple copies of IS629, a preferred substrate of IEE, and their genomic locations varied significantly between strains, as observed in O157. These data suggest that IEE may have been transferred among EHEC and ETEC in swine via SpLE1 or SpLE1-like integrative elements. In addition, IS629 is actively moving in the ETEC O139 and O149 genomes and, as in EHEC O157, is promoting the diversification of these genomes in combination with IEE..
66. Susumu Yoshizawa, Yohei Kumagai, Hana Kim, Yoshitoshi Ogura, Tetsuya Hayashi, Wataru Iwasaki, Edward F. DeLong, Kazuhiro Kogure, Functional characterization of flavobacteria rhodopsins reveals a unique class of light-driven chloride pump in bacteria, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1403051111, 111, 18, 6732-6737, 2014.05, [URL], Light-activated, ion-pumping rhodopsins are broadly distributed among many different bacteria and archaea inhabiting the photic zone of aquatic environments. Bacterial proton- or sodium-translocating rhodopsins can convert light energy into a chemiosmotic force that can be converted into cellular biochemical energy, and thus represent a widespread alternative form of photoheterotrophy. Here we report that the genome of the marine flavobacterium Nonlabens marinus S1-08T encodes three different types of rhodopsins: Nonlabens marinus rhodopsin 1 (NM-R1), Nonlabens marinus rhodopsin 2 (NM-R2), and Nonlabens marinus rhodopsin 3 (NM-R3). Our functional analysis demonstrated that NM-R1 and NM-R2 are light-driven outward-translocating H+ and Na+ pumps, respectively. Functional analyses further revealed that the lightactivated NM-R3 rhodopsin pumps Cl- ions into the cell, representing the first chloride-pumping rhodopsin uncovered in a marine bacterium. Phylogenetic analysis revealed that NM-R3 belongs to a distinct phylogenetic lineage quite distant from archaeal inward Cl--pumping rhodopsins like halorhodopsin, suggesting that different types of chloride-pumping rhodopsins have evolved independently within marine bacterial lineages. Taken together, our data suggest that similar to haloarchaea, a considerable variety of rhodopsin types with different ion specificities have evolved in marine bacteria, with individual marine strains containing as many as three functionally different rhodopsins..
67. Ryusei Tanaka, Akina Hino, Isheng J. Tsai, Juan Emilio Palomares-Rius, Ayako Yoshida, Yoshitoshi Ogura, Tetsuya Hayashi, Haruhiko Maruyama, Taisei Kikuchi, Assessment of helminth biodiversity in wild rats using 18S rDNA based metagenomics, PloS one, 10.1371/journal.pone.0110769, 9, 10, 2014.10, [URL], Parasite diversity has important implications in several research fields including ecology, evolutionary biology and epidemiology. Wide-ranging analysis has been restricted because of the difficult, highly specialised and time-consuming processes involved in parasite identification. In this study, we assessed parasite diversity in wild rats using 18S rDNA-based metagenomics. 18S rDNA PCR products were sequenced using an Illumina MiSeq sequencer and the analysis of the sequences using the QIIME software successfully classified them into several parasite groups. The comparison of the results with those obtained using standard methods including microscopic observation of helminth parasites in the rat intestines and PCR amplification/sequencing of 18S rDNA from isolated single worms suggests that this new technique is reliable and useful to investigate parasite diversity..
68. Yuta Matsumura, Hidayu Al-Saari, Sayaka Mino, Satoshi Nakagawa, Fumito Maruyama, Yoshitoshi Ogura, Tetsuya Hayashi, Ken Kurokawa, Toko Sawabe, Tomoo Sawabe, Identification of a gene cluster responsible for hydrogen evolution in Vibrio tritonius strain AM2 with transcriptional analyses, International Journal of Hydrogen Energy, 10.1016/j.ijhydene.2015.05.137, 40, 30, 9137-9146, 2015.01, [URL], Vibrio tritonius strain AM2 shows high-yield hydrogen production even under saline conditions (1.7 mol hydrogen/mol mannitol). However, the molecular mechanism of efficient hydrogen production has never been studied in the genus Vibrio. The aim of this study is to identify the genes responsible for hydrogen evolution in V. tritonius and the gene expression pattern. Complete genome analysis revealed an existence of a single 24-kb gene cluster containing 21 genes, which are essential for the formation of an energy-conserving formate hydrogen lyase (FHL) complex, to be more specific the vibrio FHL was structurally rather similar to the hyf (hydrogenase four) gene cluster found in Escherichia coli. Moreover, genes responsible to the formate dehydrogenase (FDH-H), fhlA-type transcriptional activator and hydrogenase maturation proteins (hyp) were also located downstream of the vibrio hyf gene cluster to form a "super-gene-set" of the FHL complex gene cluster. The vibrio gene for the large subunit of the FHL complex hyfG possessed typical motifs coordinating the [NiFe] center at the active site, which indicates the V. tritonius hydrogenase was able to be classified as a [NiFe]-hydrogenase. Furthermore, transcriptional analysis revealed that the expression level of the hyfG gene slightly increased upon pH decrease, which correlates to the pH-dependent hydrogen production of V. tritonius. Therefore, we can conclude that the FHL complex of V. tritonius is key enzyme in the hydrogen production under acidic conditions. Moreover hyfABCDEFGHIJ-hycI-hydN-fdhF and hyp genes could be co-transcribed respectively during the efficient hydrogen production state. Details of the gene cluster are discussed here..
69. Sayaka Tsuchida, Maiko Nezuo, Masatoshi Tsukahara, Yoshitoshi Ogura, Tetsuya Hayashi, Kazunari Ushida, Draft genome sequence of Lactobacillus gorillae strain KZ01T, isolated from a western lowland gorilla, Genome Announcements, 10.1128/genomeA.01196-15, 3, 5, 2015.01, [URL], Here, we report the draft genome sequence of Lactobacillus gorillae strain KZ01T isolated from a western lowland gorilla (Gorilla gorilla gorilla). This genome sequence will be helpful for the comparative genomics between human and nonhuman primate-associated Lactobacillus..
70. Kenta Yamauchi, Shinji Kondo, Makiko Hamamoto, Yurika Takahashi, Yoshitoshi Ogura, Tetsuya Hayashi, Hiromi Nishida, Draft genome sequence of the archiascomycetous yeast Saitoella complicata, Genome Announcements, 10.1128/genomeA.00220-15, 3, 3, 2015.01, [URL], The draft genome sequence of the archiasomycetous yeast Saitoella complicata was determined. The assembly of newly and previously sequenced data sets resulted in 104 contigs (total of 14.1 Mb N50, 239 kbp). On the newly assembled genome, a total of 6,933 protein-coding sequences (7,119 transcripts, including alternative splicing forms) were identified..
71. Tadasuke Ooka, Yoshitoshi Ogura, Keisuke Katsura, Kazuko Seto, Hideki Kobayashi, Kimiko Kawano, Eisuke Tokuoka, Masato Furukawa, Seiya Harada, Shuji Yoshino, Junji Seto, Tetsuya Ikeda, Keiji Yamaguchi, Kazunori Murase, Yasuhiro Gotoh, Naoko Imuta, Junichiro Nishi, Tânia A. Gomes, Lothar Beutin, Tetsuya Hayashi, Defining the genome features of Escherichia albertii, an emerging enteropathogen closely related to Escherichia coli, Genome Biology and Evolution, 10.1093/gbe/evv211, 7, 12, 3170-3179, 2015.01, [URL], Escherichia albertii is a recently recognized close relative of Escherichia coli. This emerging enteropathogen possesses a type III secretion system (T3SS) encoded by the locus of enterocyte effacement, similar to enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC). Shiga toxin-producing strains have also been identified. The genomic features of E. albertii, particularly differences from other Escherichia species, have not yet been well clarified.Here,wesequenced the genomeof 29 E. albertii strains (3 complete and 26 draft sequences) isolated frommultiple sources and performed intraspecies and intragenus genomic comparisons. The sizes of the E. albertii genomes range from 4.5 to 5.1Mb, smaller than those of E. coli strains. Intraspecies genomic comparisons identified five phylogroups of E. albertii. Intragenus genomic comparison revealed that the possible core genome of E. albertii comprises 3,250 genes, whereas that of the genus Escherichia comprises 1,345 genes. Our analysis further revealed several unique or notable genetic features of E. albertii, including those responsible for known biochemical features and virulence factors anda possibly active secondT3SSknownas ETT2(E. coliT3SS2) that is inactivated inE. coli.Althoughthisorganismhasbeenobserved to be nonmotile in vitro, genes for flagellar biosynthesis are fully conserved; chemotaxis-related genes have been selectively deleted. Based onthese results,wehave developed a nested polymerase chain reaction system to directly detect E. albertii.Our data define the genomic features of E. albertii and provide a valuable basis for future studies of this important emerging enteropathogen..
72. Yoshiko Urbanczyk, Yoshitoshi Ogura, Tetsuya Hayashi, Henryk Urbanczyk, Corrigendum to "Description of a novel marine bacterium, Vibrio hyugaensis sp. nov., based on genomic and phenotypic characterization" [Syst. Appl. Microbiol. 38 (July (5)) (2015) 300-304] DOI
10.1016/j.syapm.2015.04.001, Systematic and Applied Microbiology, 10.1016/j.syapm.2015.09.001, 38, 7, 2015.01, [URL].
73. Atsushi Iguchi, Sunao Iyoda, Taisei Kikuchi, Yoshitoshi Ogura, Keisuke Katsura, Makoto Ohnishi, Tetsuya Hayashi, Nicholas R. Thomson, A complete view of the genetic diversity of the Escherichia coli O-antigen biosynthesis gene cluster, DNA Research, 10.1093/dnares/dsu043, 22, 1, 101-107, 2015.01, [URL], The O antigen constitutes the outermost part of the lipopolysaccharide layer in Gram-negative bacteria. The chemical composition and structure of the O antigen show high levels of variation even within a single species revealing itself as serological diversity. Here, we present a complete sequence set for the O-antigen biosynthesis gene clusters (O-AGCs) from all 184 recognized Escherichia coli O serogroups. By comparing these sequences, we identified 161 well-defined O-AGCs. Based on the wzx/wzy or wzm/wzt gene sequences, in addition to 145 singletons, 37 serogroups were placed into 16 groups. Furthermore, phylogenetic analysis of all the E. coli O-serogroup reference strains revealed that the nearly one-quarter of the 184 serogroups were found in the ST10 lineage, which may have a unique genetic background allowing a more successful exchange of O-AGCs. Our data provide a complete view of the genetic diversity of O-AGCs in E. coli showing a stronger association between host phylogenetic lineage and O-serogroup diversification than previously recognized. These data will be a valuable basis for developing a systematic molecular O-typing scheme that will allow traditional typing approaches to be linked to genomic exploration of E. coli diversity..
74. Keisuke Okada, Hiroyuki Abe, Fumio Ike, Yoshitoshi Ogura, Tetsuya Hayashi, Aya Fukui-Miyazaki, Keiji Nakamura, Naoaki Shinzawa, Yasuhiko Horiguchi, Polymorphisms influencing expression of dermonecrotic toxin in Bordetella bronchiseptica, PloS one, 10.1371/journal.pone.0116604, 10, 2, 2015.02, [URL], Bordetella bronchiseptica is a pathogenic bacterium causing respiratory infections in a broad range of mammals. Recently, we determined the whole genome sequence of B. bronchiseptica S798 strain isolated from a pig infected with atrophic rhinitis and found four single-nucleotide polymorphisms (SNPs) at positions -129, -72, +22, and +38 in the region upstream of dnt encoding dermonecrotic toxin (DNT), when compared with a rabbit isolate, RB50. DNT is known to be involved in turbinate atrophy observed in atrophic rhinitis. Immunoblotting, quantitative real-time PCR, and β-galactosidase reporter assay revealed that these SNPs resulted in the increased promoter activity of dnt and conferred the increased ability to produce DNT on the bacteria. Similar or identical SNPs were also found in other pig isolates kept in our laboratory, all of which produce a larger amount of DNT than RB50. Our analysis revealed that substitution of at least two of the four bases, at positions -72 and +22, influenced the promoter activity for dnt. These results imply that these SNPs are involved in the pathogenicity of bordetellae specific to pig diseases..
75. Wataru Shimizu, Shizuo Kayama, Shuntaro Kouda, Yoshitoshi Ogura, Kanao Kobayashi, Norifumi Shigemoto, Norimitsu Shimada, Raita Yano, Junzo Hisatsune, Fuminori Kato, Tetsuya Hayashi, Taijiro Sueda, Hiroki Ohge, Motoyuki Sugai, Persistence and epidemic propagation of a Pseudomonas aeruginosa sequence type 235 clone harboring an IS26 composite transposon carrying the blaIMP-1 integron in Hiroshima, Japan, 2005 to 2012, Antimicrobial Agents and Chemotherapy, 10.1128/AAC.04207-14, 59, 5, 2678-2687, 2015.05, [URL], A 9-year surveillance for multidrug-resistant (MDR) Pseudomonas aeruginosa in the Hiroshima region showed that the number of isolates harboring the metallo-β-lactamase gene blaIMP-1 abruptly increased after 2004, recorded the highest peak in 2006, and showed a tendency to decline afterwards, indicating a history of an epidemic. PCR mapping of the variable regions of the integrons showed that this epidemic was caused by the clonal persistence and propagation of an MDR P. aeruginosa strain harboring the blaIMP-1 gene and an aminoglycoside 6′-N-acetyltransferase gene, aac(6′)-Iae in a class I integron (In113), whose integrase gene intl1 was disrupted by an IS26 insertion. Sequence analysis of the representative strain PA058447 resistance element containing the In113-derived gene cassette array showed that the element forms an IS26 transposon embedded in the chromosome. It has a Tn21 backbone and is composed of two segments sandwiched by three IS26s. In Japan, clonal nationwide expansion of an MDR P. aeruginosa NCGM2.S1 harboring chromosomally encoded In113 with intact intl1 is reported. Multilocus sequence typing and genomic comparison strongly suggest that PA058447 and NCGM2.S1 belong to the same clonal lineage. Moreover, the structures of the resistance element in the two strains are very similar, but the sites of insertion into the chromosome are different. Based on tagging information of the IS26 present in both resistance elements, we suggest that the MDR P. aeruginosa clone causing the epidemic in Hiroshima for the past 9 years originated from a common ancestor genome of PA058447 and NCGM2.S1 through an IS26 insertion into intl1 of In113 and through IS26-mediated genomic rearrangements..
76. Hisaya Kojima, Yoshitoshi Ogura, Nozomi Yamamoto, Tomoaki Togashi, Hiroshi Mori, Tomohiro Watanabe, Fumiko Nemoto, Ken Kurokawa, Tetsuya Hayashi, Manabu Fukui, Ecophysiology of Thioploca ingrica as revealed by the complete genome sequence supplemented with proteomic evidence, ISME Journal, 10.1038/ismej.2014.209, 9, 5, 1166-1176, 2015.05, [URL], Large sulfur-oxidizing bacteria, which accumulate a high concentration of nitrate, are important constituents of aquatic sediment ecosystems. No representative of this group has been isolated in pure culture, and only fragmented draft genome sequences are available for these microorganisms. In this study, we successfully reconstituted the genome of Thioploca ingrica from metagenomic sequences, thereby generating the first complete genome sequence from this group. The Thioploca samples for the metagenomic analysis were obtained from a freshwater lake in Japan. A PCR-free paired-end library was constructed from the DNA extracted from the samples and was sequenced on the Illumina MiSeq platform. By closing gaps within and between the scaffolds, we obtained a circular chromosome and a plasmid-like element. The reconstituted chromosome was 4.8 Mbp in length with a 41.2% GC content. A sulfur oxidation pathway identical to that suggested for the closest relatives of Thioploca was deduced from the reconstituted genome. A full set of genes required for respiratory nitrate reduction to dinitrogen gas was also identified. We further performed a proteomic analysis of the Thioploca sample and detected many enzymes/proteins involved in sulfur oxidation, nitrate respiration and inorganic carbon fixation as major components of the protein extracts from the sample, suggesting that these metabolic activities are strongly associated with the physiology of T. ingrica in lake sediment..
77. Yoshiko Urbanczyk, Yoshitoshi Ogura, Tetsuya Hayashi, Henryk Urbanczyk, Description of a novel marine bacterium, Vibrio hyugaensis sp. nov., based on genomic and phenotypic characterization, Systematic and Applied Microbiology, 10.1016/j.syapm.2015.04.001, 38, 5, 300-304, 2015.07, [URL], Three luminous bacteria strains have been isolated from seawater samples collected in the coastal regions of the Miyazaki prefecture in Japan. Analysis of the 16S rRNA gene sequences identified the three strains as members of the genus Vibrio (Vibrionaceae, Gammaproteobacteria), closely related to bacteria in the so-called 'Harveyi clade.' The genomes of the three strains were estimated to be between 5.49Mbp and 5.95Mbp, with average G+C of 43.91%. The genome sequence data was used to estimate relatedness of the three strains to related Vibrio bacteria, including estimation of frequency of recombination events, calculation of average nucleotide identity (ANI), and a phylogenetic analysis based on concatenated alignment of nucleotide sequences of 135 protein coding genes. Results of these analyses in all cases showed the three strains forming a group clearly separate from previously described Vibrio species. A phenotypic analysis revealed that the three strains have character similar to Vibrio bacteria in the 'Harveyi clade', but can be differentiated from previously described species by testing for hydrolysis of esculin. Based on results of genomic, phylogenetic and phenotypic analyses presented in this study, it can be concluded that the three strains represent a novel species, for which the name Vibrio hyugaensis sp. nov. is proposed. The type strain is 090810aT (=LMG 28466T=NBRC 110633T)..
78. Mariko Naito, Yoshitoshi Ogura, Takehiko Itoh, Mikio Shoji, Masaaki Okamoto, Tetsuya Hayashi, Koji Nakayama, The complete genome sequencing of Prevotella intermedia strain OMA14 and a subsequent fine-scale, intra-species genomic comparison reveal an unusual amplification of conjugative and mobile transposons and identify a novel Prevotella-lineage-specific repeat, DNA Research, 10.1093/dnares/dsv032, 23, 1, 11-19, 2015.08, [URL], Prevotella intermedia is a pathogenic bacterium involved in periodontal diseases. Here, we present the complete genome sequence of a clinical strain, OMA14, of this bacterium along with the results of comparative genome analysis with strain 17 of the same species whose genome has also been sequenced, but not fully analysed yet. The genomes of both strains consist of two circular chromosomes: the larger chromosomes are similar in size and exhibit a high overall linearity of gene organizations, whereas the smaller chromosomes show a significant size variation and have undergone remarkable genome rearrangements. Unique features of the Pre. intermedia genomes are the presence of a remarkable number of essential genes on the second chromosomes and the abundance of conjugative and mobilizable transposons (CTns and MTns). The CTns/MTns are particularly abundant in the second chromosomes, involved in its extensive genome rearrangement, and have introduced a number of strain-specific genes into each strain. We also found a novel 188-bp repeat sequence that has been highly amplified in Pre. intermedia and are specifically distributed among the Pre. intermedia-related species. These findings expand our understanding of the genetic features of Pre. intermedia and the roles of CTns and MTns in the evolution of bacteria..
79. Yoshitoshi Ogura, Shakhinur Islam Mondal, Md Rakibul Islam, Toshihiro Mako, Kokichi Arisawa, Keisuke Katsura, Tadasuke Ooka, Yasuhiro Gotoh, Kazunori Murase, Makoto Ohnishi, Tetsuya Hayashi, The Shiga toxin 2 production level in enterohemorrhagic Escherichia coli O157:H7 is correlated with the subtypes of toxin-encoding phage, Scientific reports, 10.1038/srep16663, 5, 2015.11, [URL], Enterohemorrhagic E. coli (EHEC) causes diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome. Their major virulence factor is Shiga toxin (Stx), which is encoded by bacteriophages. Of the two types of Stx, the production of Stx2, particularly that of Stx2a (a subtype of Stx2), is a major risk factor for severe EHEC infections, but the Stx2 production level is highly variable between strains. Here, we define four major and two minor subtypes of Stx2a-encoding phages according to their replication proteins. The subtypes are correlated with Stx2a titers produced by the host O157 strains, suggesting a critical role of the phage subtype in determining the Stx2a production level. We further show that one of the two subclades in the clade 8, a proposed hyper-virulent lineage of O157, carries the Stx2 phage subtype that confers the highest Stx2 production to the host strain. The presence of this subclade may explain the proposed high virulence potential of clade 8. These results provide novel insights into the variation in virulence among O157 strains and highlight the role of phage variation in determining the production level of the virulence factors that phages encode..
80. Yuriko Ozeki, Masayuki Igarashi, Matsumi Doe, Aki Tamaru, Naoko Kinoshita, Yoshitoshi Ogura, Tomotada Iwamoto, Ryuichi Sawa, Maya Umekita, Shymaa Enany, Yukiko Nishiuchi, Mayuko Osada-Oka, Tetsuya Hayashi, Mamiko Niki, Yoshitaka Tateishi, Masaki Hatano, Sohkichi Matsumoto, A new screen for tuberculosis drug candidates utilizing a luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren, PloS one, 10.1371/journal.pone.0141658, 10, 11, 2015.11, [URL], Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multidrug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 ug/ml, 0.5 ug/ml, and 2.0-7.5 ug/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904-1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904-1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug..
81. Atsushi Iguchi, Sunao Iyoda, Kazuko Seto, Hironobu Nishii, Makoto Ohnishi, Hirohisa Mekata, Yoshitoshi Ogura, Tetsuya Hayashi, Six novel O genotypes from Shiga toxin-producing Escherichia coli, Frontiers in Microbiology, 10.3389/fmicb.2016.00765, 7, MAY, 2016.01, [URL], Serotyping is one of the typing techniques used to classify strains within the same species. O-serogroup diversification shows a strong association with the genetic diversity of O-antigen biosynthesis genes. In a previous study, based on the O-antigen biosynthesis gene cluster (O-AGC) sequences of 184 known Escherichia coli O serogroups (from O1 to O187), we developed a comprehensive and practical molecular O serogrouping (O genotyping) platform using a polymerase chain reaction (PCR) method, named E. coli O-genotyping PCR. Although, the validation assay using the PCR system showed that most of the tested strains were successfully classified into one of the O genotypes, it was impossible to classify 6.1% (35/575) of the strains, suggesting the presence of novel O genotypes. In this study, we conducted sequence analysis of O-AGCs from O-genotype untypeable Shiga toxin-producing E. coli (STEC) strains and identified six novel O genotypes; OgN1, OgN8, OgN9, OgN10, OgN12 and OgN31, with unique wzx and/or wzy O-antigen processing gene sequences. Additionally, to identify these novel O-genotypes, we designed specific PCR primers. A screen of O genotypes using O-genotype untypeable strains showed 13 STEC strains were classified into five novel O genotypes. The O genotyping at the molecular level of the O-AGC would aid in the characterization of E. coli isolates and will assist future studies in STEC epidemiology and phylogeny..
82. Yoshitoshi Ogura, Natsuko Nakayama, Tetsuya Hayashi, Shoko Ueki, Mitochondrial genome sequences of four strains of the bloom-forming raphidophyte Heterosigma akashiwo, Genome Announcements, 10.1128/genomeA.01288-16, 4, 6, 2016.01, [URL], We report here the complete mitochondrial genome sequences of four strains of bloom-forming raphidophytes from Heterosigma akashiwo. These 39-kb sequences contain 42 protein-, two rRNA-, and 26 tRNA-coding sequences. Notable sequence variations were observed among these four newly sequenced and three previously characterized strains, suggesting their potential usage as strain-specific markers..
83. Shu Kuan Wong, Susumu Yoshizawa, Yu Nakajima, Yoshitoshi Ogura, Tetsuya Hayashi, Koji Hamasaki, Draft genome sequence of Lewinella sp. strain 4G2 isolated from the coastal sea surface microlayer, Genome Announcements, 10.1128/genomeA.00754-16, 4, 4, 2016.01, [URL], We report here the draft genome of Lewinella sp. strain 4G2, isolated from the sea surface microlayer (SML) of a coastal marine inlet. The genome sequence of strain 4G2 should contribute to understanding the lifestyles of bacteria living in the SML..
84. Ryo Kaneko, Shu Kuan Wong, Yoshitoshi Ogura, Tetsuya Hayashi, Susumu Yoshizawa, Koji Hamasaki, Draft genome sequences of two Fabibacter sp. strains isolated from coastal surface water of Aburatsubo Inlet, Japan, Genome Announcements, 10.1128/genomeA.01360-16, 4, 6, 2016.01, [URL], Here, we report the draft genome sequences of Fabibacter sp. strain 4D4 and F. misakiensis strain SK-8T, isolated from surface seawater of a semienclosed inlet..
85. Kazunari Ushida, Sayaka Tsuchida, Yoshitoshi Ogura, Tetsuya Hayashi, Akiko Sawada, Goro Hanya, Draft genome sequences of Sarcina ventriculi strains isolated from wild Japanese macaques in Yakushima Island, Genome Announcements, 10.1128/genomeA.01694-15, 4, 1, 2016.01, [URL], We report the draft genome sequences of Sarcina ventriculi strains 14 and 17, both isolated from feces of wild Yakushima macaques (Macaca fuscata yakui). These genomic sequences will be helpful for the phylogenetic consideration of the family Clostridiaceae and understanding of the contribution of intestinal microbiota to the survival of Yakushima macaques..
86. Katsuyuki Katahira, Yoshitoshi Ogura, Yasuhiro Gotoh, Tetsuya Hayashi, Draft genome sequences of five rapidly growing Mycobacterium species, M. thermoresistibile, M. fortuitum subsp. acetamidolyticum, M. canariasense, M. brisbanense, and M. novocastrense, Genome Announcements, 10.1128/genomeA.00322-16, 4, 3, 2016.01, [URL], We report here the draft genome sequences of five rapidly growing Mycobacterium (RGM) species potentially pathogenic to humans, M. thermoresistibile, M. fortuitum subsp. acetamidolyticum, M. canariasense, M. brisbanense, and M. novocastrense. As the clinical importance of RGMs is increasingly being recognized worldwide, these sequences would contribute to further advances in RGM research..
87. Tadayuki Iwase, Yoshitoshi Ogura, Tetsuya Hayashi, Yoshimitsu Mizunoe, Complete genome sequence of Klebsiella oxytoca strain JKo3, Genome Announcements, 10.1128/genomeA.01221-16, 4, 6, 2016.01, [URL], Klebsiella oxytoca can be either pathogenic or beneficial, depending on conditions. These opposing characteristics have not been fully elucidated. Here, we report the complete sequence of the K. oxytoca JKo3 genome, consisting of a single circular chromosome of 5,943,791 bp and four plasmids..
88. Yoshitoshi Ogura, Tetsuya Hayashi, Shoko Ueki, Complete genome sequence of a phycodnavirus, Heterosigma akashiwo virus strain 53, Genome Announcements, 10.1128/genomeA.01279-16, 4, 6, 2016.01, [URL], We report the complete genome sequence of Heterosigma akashiwo virus strain 53. The virus is a member of the Phycodnaviridae, one of the families regarded as giant double-stranded DNA viruses. The 274,793-bp genome contained 246 protein-coding and 3 tRNA-coding sequences..
89. Tadayuki Iwase, Yoshitoshi Ogura, Tetsuya Hayashi, Yoshimitsu Mizunoe, Complete genome sequence of Klebsiella pneumoniae YH43, Genome Announcements, 10.1128/genomeA.00242-16, 4, 2, 2016.01, [URL], We report here the complete genome sequence of Klebsiella pneumoniae strain YH43, isolated from sweet potato. The genome consists of a single circular chromosome of 5,520,319 bp in length. It carries 8 copies of rRNA operons, 86 tRNA genes, 5,154 protein-coding genes, and the nif gene cluster for nitrogen fixation..
90. Akio Tani, Yoshitoshi Ogura, Tetsuya Hayashi, Kazuhide Kimbara, Complete genome sequence of Methylobacterium aquaticum strain 22A, isolated from a Racomitrium japonicum moss, Genome Announcements, 10.1128/genomeA.00266-15, 3, 2, 2016.01, [URL], Methylobacterium species colonize plant surfaces and utilize methanol emitted from plants. Methylobacterium aquaticum strain 22A was isolated from a hydroponic culture of a moss, Racomitrium japonicum, and is a potent plant growth promoter. The complete genome sequencing of the strain confirmed the presence of genes related to plant growth promotion and methylotrophy..
91. Mitsunori Yoshida, Kazue Nakanaga, Yoshitoshi Ogura, Atsushi Toyoda, Tadasuke Ooka, Yuko Kazumi, Satoshi Mitarai, Norihisa Ishii, Tetsuya Hayashi, Yoshihiko Hoshino, Complete genome sequence of Mycobacterium ulcerans subsp. shinshuense, Genome Announcements, 10.1128/genomeA.01050-16, 4, 5, 2016.01, [URL], Mycobacterium ulcerans subsp. shinshuense produces mycolactone and causes Buruli ulcer. Here, we report the complete sequence of its genome, which comprises a 5.9-Mb chromosome and a 166-kb plasmid (pShT-P). The sequence will represent the essential data for future phylogenetic and comparative genome studies of mycolactone-producing mycobacteria..
92. Yoshio Kondo, Yoshitoshi Ogura, Keiko Sato, Keigo Imamura, Tomonori Hoshino, Miyuki Nishiguchi, Tomoyuki Hasuwa, Hiroyuki Moriuchi, Tetsuya Hayashi, Taku Fujiwara, Complete genome sequence of Streptococcus sp. strain NPS 308, Genome Announcements, 10.1128/genomeA.01349-16, 4, 6, 2016.01, [URL], Streptococcus sp. strain NPS 308, isolated from an 8-year-old diagnosed with infective endocarditis, likely presents a novel species of Streptococcus. Here, we present a complete genome sequence of this species, which will contribute to better understanding of the pathogenesis of infective endocarditis..
93. Vicky L. Hunt, Isheng J. Tsai, Avril Coghlan, Adam J. Reid, Nancy Holroyd, Bernardo J. Foth, Alan Tracey, James A. Cotton, Eleanor J. Stanley, Helen Beasley, Hayley M. Bennett, Karen Brooks, Bhavana Harsha, Rei Kajitani, Arpita Kulkarni, Dorothee Harbecke, Eiji Nagayasu, Sarah Nichol, Yoshitoshi Ogura, Michael A. Quail, Nadine Randle, Dong Xia, Norbert W. Brattig, Hanns Soblik, Diogo M. Ribeiro, Alejandro Sanchez-Flores, Tetsuya Hayashi, Takehiko Itoh, Dee R. Denver, Warwick Grant, Jonathan D. Stoltzfus, James B. Lok, Haruhiko Murayama, Jonathan Wastling, Adrian Streit, Taisei Kikuchi, Mark Viney, Matthew Berriman, The genomic basis of parasitism in the Strongyloides clade of nematodes, Nature genetics, 10.1038/ng.3495, 48, 3, 299-307, 2016.03, [URL], Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families - families encoding astacin-like and SCP/TAPS proteins - is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism..
94. Masahiro Kusumoto, Yuna Hikoda, Yuki Fujii, Misato Murata, Hirotsugu Miyoshi, Yoshitoshi Ogura, Yasuhiro Gotoh, Taketoshi Iwata, Tetsuya Hayashi, Masato Akiba, Emergence of a multidrug-resistant Shiga toxin-producing enterotoxigenic Escherichia coli lineage in diseased swine in Japan, Journal of Clinical Microbiology, 10.1128/JCM.03141-15, 54, 4, 1074-1081, 2016.04, [URL], Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenic E. coli strains belong to a limited range of O serogroups, including O8, O138, O139, O141, O147, O149, and O157, which are the most frequently reported strains worldwide. However, the circumstances of ETEC and STEC infections in Japan remain unknown; there have been few reports on the prevalence or characterization of swine-pathogenic E. coli. In the present study, we determined the O serogroups of 967 E. coli isolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup of Shigella boydii type 9) were the predominant serogroups. We further analyzed these four O serogroups using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and virulence factor profiling. Most of the O139 and O149 strains formed serogroup-specific PFGE clusters (clusters I and II, respectively), whereas the O116 and OSB9 strains were grouped together in the same cluster (cluster III). All of the cluster III strains belonged to a single sequence type (ST88) and carried genes encoding both enterotoxin and Shiga toxin. This PFGE cluster III/ST88 lineage exhibited a high level of multidrug resistance (to a median of 10 antimicrobials). Notably, these bacteria were resistant to fluoroquinolones. Thus, this lineage should be considered a significant risk to animal production due to the toxigenicity and antimicrobial resistance of these bacteria..
95. Bruna G. Garcia, Tadasuke Ooka, Yasuhiro Gotoh, Mônica A.M. Vieira, Denise Yamamoto, Yoshitoshi Ogura, Dennys M. Girão, Suely C.F. Sampaio, Alexis Bonfim Melo, Kinue Irino, Tetsuya Hayashi, Tânia A.T. Gomes, Genetic relatedness and virulence properties of enteropathogenic Escherichia coli strains of serotype O119
H6 expressing localized adherence or localized and aggregative adherence-like patterns on HeLa cells, International Journal of Medical Microbiology, 10.1016/j.ijmm.2016.02.008, 306, 3, 152-164, 2016.05, [URL], Enteropathogenic Escherichia coli (EPEC) induce attaching and effacing (A/E) lesions in enterocytes and produce the bundle-forming pilus (BFP) contributing to the localized adherence (LA) pattern formation on HeLa cells. Enteroaggregative E. coli (EAEC) produce aggregative adherence (AA) on HeLa cells and form prominent biofilms. The ability to produce LA or AA is an important hallmark to classify fecal E. coli isolates as EPEC or EAEC, respectively. E. coli strains of serotype O119:H6 exhibit an LA+ phenotype and have been considered as comprising a clonal group. of EPEC strains. However, we have recently identified O119:H6 EPEC strains that produce LA and an AA-like pattern concurrently (LA/AA-like+). In this study, we evaluated the relatedness of three LA/AA-like+ and three LA+ O119:H6 strains by comparing their virulence and genotypic properties. We first found that the LA/AA-like+ strains induced actin accumulation in HeLa cells (indicative of A/E lesions formation) and formed biofilms on abiotic surfaces more efficiently than the LA+ strains. MLST analysis showed that the six strains all belong to the ST28 complex. All strains carried multiple plasmids, but as plasmid profiles were highly variable, this cannot be used to differentiate LA/AA-like+ and LA+ strains. We further obtained their draft genome sequences and the complete sequences of four plasmids harbored by one LA/AA-like+ strain. Analysis of these sequences and comparison with 37 fully sequenced E. coli genomes revealed that both O119:H6 groups belong to the E. coli phylogroup B2 and are very closely related with only 58-67 SNPs found between LA/AA-like+ and LA+ strains. Search of the draft sequences of the six strains for adhesion-related genes known in EAEC and other E. coli pathotypes detected no genes specifically present in LA/AA-like+ strains. Unexpectedly however, we found that a large plasmid distinct from pEAF is responsible for the AA-like phenotype of the LA/AA-like+ strains. Although we have not identified any plasmid genes specifically present in all LA/AA-like+ strains and absent in the LA+ strains, these results suggest the presence of an unknown mechanism to promote the AA-like pattern production and biofilm formation by the LA/AA-like+ strains. Because their ability to produce A/E lesions and biofilm concomitantly could exacerbate the clinical condition of the patient and lead to persistent diarrhea, the mechanism underlying the enhanced biofilm formation by the LA/AA-like+ O119:H6 strains and their spread and involvement in severe diarrheal diseases should be more intensively investigated..
96. Kazunari Ushida, Sayaka Tsuchida, Yoshitoshi Ogura, Atsushi Toyoda, Fumito Maruyama, Domestication and cereal feeding developed domestic pig-type intestinal microbiota in animals of suidae, Animal science journal = Nihon chikusan Gakkaiho, 10.1111/asj.12492, 87, 6, 835-841, 2016.06, [URL], Intestinal microbiota are characterized by host-specific microorganisms, which have been selected through host-microbe interactions under phylogenetic evolution and transition of feeding behavior by the host. Although many studies have focused on disease-related intestinal microbiota, the origin and evolution of host-specific intestinal microbiota have not been well elucidated. Pig is the ideal mammal model to reveal the origin and evolution of host-specific intestinal microbiota because their direct wild ancestor and close phylogenetic neighbors are available for comparison. The pig has been recognized as a Lactobacillus-type animal. We analyzed the intestinal microbiota of various animals in Suidae: domestic pigs, wild boars and Red river hogs to survey the origin and evolution of Lactobacillus-dominated intestinal microbiota by metagenomic approach and following quantitative PCR confirmation. The metagenomic datasets were separated in two clusters; the wild animal cluster being characterized by a high abundance of Bifidobacterium, whereas the domesticated (or captured) animal cluster by Lactobacillus. In addition, Enterobacteriaceae were harbored as the major family only in domestic Sus scrofa. We conclude that domestication may have induced a larger Enterobacteriaceae population in pigs, and the introduction of modern feeding system further caused the development of Lactobacillus-dominated intestinal microbiota, with genetic and geographical factors possibly having a minor impact..
97. Masahiro Kusumoto, Yoshitoshi Ogura, Yasuhiro Gotoh, Taketoshi Iwata, Tetsuya Hayashi, Masato Akiba, Colistin-resistant mcr-1-positive pathogenic Escherichia coli in Swine, Japan, 2007-2014, Emerging Infectious Diseases, 10.3201/eid2207.160234, 22, 7, 1315-1317, 2016.07, [URL].
98. Naoko Imuta, Tadasuke Ooka, Kazuko Seto, Ryuji Kawahara, Toyoyasu Koriyama, Tsuyoshi Kojyo, Atsushi Iguchi, Koichi Tokuda, Hideki Kawamura, Kiyotaka Yoshiie, Yoshitoshi Ogura, Tetsuya Hayashi, Junichiro Nishi, Phylogenetic analysis of enteroaggregative Escherichia coli (eaec) isolates from Japan reveals emergence of ctx-m-14-producing eaec o25:h4 clones related to sequence type 131, Journal of Clinical Microbiology, 10.1128/JCM.00711-16, 54, 8, 2128-2134, 2016.08, [URL], Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea. The aggR gene is widely used as a marker for typical EAEC. The heterogeneity of EAEC is well known; however, there are few reports on the phylogenetic relationships of EAEC. Recently, CTX-M extended-spectrum +-lactamase (ESBL)-producing EAEC strains have been reported worldwide. To characterize EAEC strains in Japan, we investigated the population structure of EAEC. A total of 167 aggR-positive strains isolated from stool specimens from diarrheal patients in Kagoshima (139 strains) and Osaka (28 strains), Japan, between 1992 and 2010 were examined for the prevalence of EAEC virulence markers, the blaCTX-M gene, and the capacity to form biofilms. Multilocus sequence typing was also conducted. EAEC strains were widely distributed across four major E. coli phylogroups. Strains of O111: H21/clonal group 40 (CG40) (30 strains), O126:H27/CG200 (13 strains), and O86a:H27/CG3570 (11 strains) in phylogroup B1 are the historical EAEC clones in Japan, and they exhibited strong biofilm formation. Twenty-nine strains of EAEC O25:H4/ CG131 were identified in phylogroup B2, 79% of which produced CTX-M-14. This clone has emerged since 2003. The clone harbored plasmid-encoded EAEC virulence genes but not chromosomal virulence genes and had lower biofilm-forming capacity than historical EAEC strains. This clone most likely emerged from a pandemic uropathogenic O25:H4/sequence type 131 clone by acquiring an EAEC virulence plasmid from canonical EAEC. Surveillance of the horizontal transfer of both virulence and ESBL genes among E. coli strains is important for preventing a worldwide increase in antimicrobial drug resistance..
99. Yoshiko Urbanczyk, Yoshitoshi Ogura, Tetsuya Hayashi, Henryk Urbanczyk, Genomic evidence that Vibrio inhibens is a heterotypic synonym of Vibrio jasicida, International Journal of Systematic and Evolutionary Microbiology, 10.1099/ijsem.0.001173, 66, 8, 3214-3218, 2016.08, [URL], Vibrio inhibens is a marine bacterium species of the genus Vibrio (Vibrionaceae, Gammaproteobacteria). The species has been shown to be closely related to members of the genus Vibrio in the so-called Harveyi clade. The clade includes at least 11 closely related species with similar physiological and biochemical properties. Due to these similarities, species of the Harveyi clade are difficult to characterize taxonomically. Previously phenotypic and genotypic properties of the V. inhibens type strain were compared with six species of the Harveyi clade, resulting in the possibility that V. inhibens could be a synonym of a previously described species. In this study, the taxonomic status of V. inhibens was analyzed using genomic approaches. The whole-genome sequence of the type strain of V. inhibens, CECT 7692T, was obtained and analyzed. Calculations of average nucleotide identity with the BLAST algorithm (ANIb) showed that CECT 7692T has an ANIb of 97.5 % or higher to five strains of Vibrio. jasicida, including the type strain, but an ANIb lower than 93.5 % to other members of the Harveyi clade Vibrio. Phylogenetic analysis based on nucleotide sequences of 133 protein-coding genes showed a close evolutionary relationship of CECT 7692T to V. jasicida. Based on these results, Vibrio inhibens is proposed to be a later heterotypic synonym of V. jasicida..
100. Kazunobu Amako, Ken Ichiro Iida, Mitsumasa Saito, Yoshitoshi Ogura, Tetsuya Hayashi, Shin Ichi Yoshida, Non-exponential growth of Mycobacterium leprae Thai-53 strain cultured in vitro, MICROBIOLOGY and IMMUNOLOGY, 10.1111/1348-0421.12454, 60, 12, 817-823, 2016.12, [URL], In this study, attempts were made to culture this bacterium in media supplemented with a variety of biological materials to determine why cultivation of Mycobacterium leprae in vitro has not this far been successful. A slight increase in the number of cells in medium supplemented with human blood plasma and an extract of nude mouse tissue as observed after more than 3 months of cultivation at 30 °C. To ascertain whether this increase was real growth, the growth was analyzed by droplet digital PCR, which showed a slow increase in the copy number of cell-associated DNA and the release of a large amount of DNA into the culture medium from bacterial cells during cultivation. These results were supported by electron microscopic examination of M. leprae in infected mouse tissues, which showed that most of the replicated bacteria had degenerated and only a few cells survived. Based on these results, it was postulated that many of the replicated cells degenerate during M. leprae growth and that only a few cells remain to participate in the next growth stage. This means that, unlike other cultivable bacteria, the growth of M. leprae is not exponential and the number of cells therefore increase extremely slowly. Thus, accurate judging of the success of M. leprae cultivation requires observation of growth over a long period of time and careful measurement of the increase in number of viable cells..
101. Shakhinur Islam Mondal, Md Rakibul Islam, Akira Sawaguchi, Md Asadulghani, Tadasuke Ooka, Yasuhiro Gotoh, Yasuhiro Kasahara, Yoshitoshi Ogura, Tetsuya Hayashi, Genes essential for the morphogenesis of the Shiga toxin 2-transducing phage from Escherichia coli O157:H7, Scientific reports, 10.1038/srep39036, 6, 2016.12, [URL], Shiga toxin 2 (Stx2), one of the most important virulence factors of enterohaemorrhagic Escherichia coli (EHEC), is encoded by phages. These phages (Stx2 phages) are often called lambda-like. However, most Stx2 phages are short-tailed, thus belonging to the family Podoviridae, and the functions of many genes, especially those in the late region, are unknown. In this study, we performed a systematic genetic and morphological analysis of genes with unknown functions in Sp5, the Stx2 phage from EHEC O157:H7 strain Sakai. We identified nine essential genes, which, together with the terminase genes, determine Sp5 morphogenesis. Four of these genes most likely encoded portal, major capsid, scaffolding and tail fiber proteins. Although exact roles/functions of the other five genes are unknown, one was involved in head formation and four were required for tail formation. One of the four tail genes encoded an unusually large protein of 2,793 amino-acid residues. Two genes that are likely required to maintain the lysogenic state were also identified. Because the late regions of Stx2 phages from various origins are highly conserved, the present study provides an important basis for better understanding the biology of this unique and medically important group of bacteriophages..
102. Arzuba Akter, Tadasuke Ooka, Yasuhiro Gotoh, Seigo Yamamoto, Hiromi Fujita, Fumio Terasoma, Kouji Kida, Masakatsu Taira, Fumiko Nakadouzono, Mutsuyo Gokuden, Manabu Hirano, Mamoru Miyashiro, Kouichi Inari, Yukie Shimazu, Kenji Tabara, Atsushi Toyoda, Dai Yoshimura, Takehiko Itoh, Tomokazu Kitano, Mitsuhiko P. Sato, Keisuke Katsura, Shakhinur Islam Mondal, Yoshitoshi Ogura, Shuji Ando, Tetsuya Hayashi, Extremely low genomic diversity of Rickettsia japonica distributed in Japan, Genome Biology and Evolution, 10.1093/gbe/evw304, 9, 1, 124-133, 2017.01, [URL], Rickettsiae are obligate intracellular bacteria that have small genomes as a result of reductive evolution. Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fevers". The life cycle of SFG rickettsiae is closely associated with that of the tick, which is generally thought to act as a bacterial vector and reservoir that maintains the bacterium through transstadial and transovarial transmission. Each SFG member is thought to have adapted to a specific tick species, thus restricting the bacterial distribution to a relatively limited geographic region. These unique features of SFG rickettsiae allow investigation of how the genomes of such biologically and ecologically specialized bacteria evolve after genome reduction and the types of population structures that are generated. Here, we performed a nationwide, high-resolution phylogenetic analysis of Rickettsia japonica, an etiological agent of Japanese spotted fever that is distributed in Japan and Korea. The comparison of complete or nearly complete sequences obtained from 31 R. japonica strains isolated from various sources in Japan over the past 30 years demonstrated an extremely low level of genomic diversity. In particular, only 34 single nucleotide polymorphisms were identified among the 27 strains of themajor lineage containing all clinical isolates and tick isolates from the three tick species. Our data provide novel insights into the biology and genome evolution of R. japonica, including the possibilities of recent clonal expansion and a long generation time in nature due to the long dormant phase associated with tick life cycles..
103. Yu Nakajima, Susumu Yoshizawa, Keiji Nakamura, Yoshitoshi Ogura, Tetsuya Hayashi, Kazuhiro Kogure, Draft genome sequences of Tersicoccus phoenicis DSM 30849T, isolated from a cleanroom for spacecraft assembly, and Tersicoccus sp. strain Bi-70, isolated from a freshwater lake, Genome Announcements, 10.1128/genomeA.00079-17, 5, 13, 2017.01, [URL], Here, we report the draft genome sequences of Tersicoccus phoenicis DSM 30849T, isolated from a spacecraft assembly cleanroom at the National Aeronautics and Space Administration (NASA), and Tersicoccus sp. strain Bi-70, isolated from Lake Biwa, the largest lake in Japan. These genome sequences facilitate our understanding of the adaptation of these closely related strains to different habitats..
104. Masateru Nishiyama, Yoshitoshi Ogura, Tetsuya Hayashi, Yoshihiro Suzuki, Antibiotic resistance profiling and genotyping of vancomycin-resistant enterococci collected from an urban river basin in the Provincial City of Miyazaki, Japan, Water (Switzerland), 10.3390/w9020079, 9, 2, 2017.01, [URL], The distribution characteristics of vancomycin-resistant enterococci (VRE) and the resistance of enterococcus isolates to various antibiotics were investigated in Yae River, which flows through Miyazaki city, Japan. The prevalence of VRE among specimens collected from the urban river basin using mEI agar was 0.9% (2 of 226 enterococcal isolates). In the 333 enterococcal isolates obtained using mEI agar or vancomycin-supplemented mEI agar, the possession of the vancomycin-resistant genes (vanA, vanB, vanC1, and vanC2/C3) was examined using multiplex PCR analysis. Although VRE possessing vanA and vanB were not detected in any isolates, isolates possessing vanC2/C3 were detected at all sampling sites and on all days. All isolates (101 strains) possessing vanC2/C3 that were obtained on vancomycin-supplemented mEI agar were identified as E. casseliflavus and analyzed for genotypes using pulse-field gel electrophoresis (PFGE) analysis. These E. casseliflavus isolates revealed them to be genetically highly divergent strains, suggesting that many contamination sources were present in this study area. Many of the enterococcal isolates obtained were resistant to erythromycin, ciprofloxacin, and tetracycline; enterococci distributed in the studied urban river basin are resistant to universally applicable antibiotics. These results indicate that VRE carrying vanC2/C3 are distributed in Yae River, and the sources of VRE are scattered across the river basin..
105. Nozomi Ishijima, Ken Ichi Lee, Tomomi Kuwahara, Haruyuki Nakayama-Imaohji, Saori Yoneda, Atsushi Iguchi, Yoshitoshi Ogura, Tetsuya Hayashi, Makoto Ohnishi, Sunao Iyoda, Identification of a new virulent clade in enterohemorrhagic escherichia coli O26:H11/H- Sequence Type 29, Scientific reports, 10.1038/srep43136, 7, 2017.02, [URL], Enterohemorrhagic Escherichia coli (EHEC) O26 infections cause severe human diseases such as hemolytic uremic syndrome and encephalopathy, and is the predominant serogroup among non-O157 EHEC in many countries. Shiga toxin (Stx), which consists of two distinct types (Stx1 and Stx2), plays a central role in EHEC pathogenesis. The major stx gene type in EHEC O26 strains is stx1, although isolates with only stx2 have emerged in Japan since 2012 and have been reported in Europe. In this study, we selected 27 EHEC O26 strains isolated in Japan and identified a distinct genetic clade within sequence type (ST) 29, designated ST29C1, that carried only stx2 and had the plasmid gene profile ehxA. We showed that ST29C1 strains produced higher Stx2a levels, and greater virulence in Vero cells and in germ-free mice than other lineages. We also showed that ST29C1 was a distinct phylogenetic clade by SNP analysis using whole genome sequences and clearly differed from the major European EHEC O26 virulent clone, which was designated ST29C2 in this study. The combination of toxin production analysis, virulence analysis in Vero cells and germ-free mice, and phylogenetic analysis identified a newly emerging virulent EHEC clade..
106. I. Fakih, D. Thiry, J. N. Duprez, M. Saulmont, A. Iguchi, D. Piérard, L. Jouant, G. Daube, Yoshitoshi Ogura, Tetsuya Hayashi, B. Taminiau, J. G. Mainil, Identification of Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli in diarrhoeic calves and comparative genomics of O5 bovine and human STEC, Veterinary Microbiology, 10.1016/j.vetmic.2016.02.017, 202, 16-22, 2017.04, [URL], Escherichia coli producing Shiga toxins (Stx) and the attaching-effacing (AE) lesion (AE-STEC) are responsible for (bloody) diarrhoea in humans and calves while the enteropathogenic E. coli (EPEC) producing the AE lesion only cause non-bloody diarrhoea in all mammals. The purpose of this study was (i) to identify the pathotypes of enterohaemolysin-producing E. coli isolated between 2009 and 2013 on EHLY agar from less than 2 month-old diarrhoeic calves with a triplex PCR targeting the stx1, stx2, eae virulence genes; (ii) to serotype the positive isolates with PCR targeting the genes coding for ten most frequent and pathogenic human and calf STEC O serogroups; and (iii) to compare the MLSTypes and virulotypes of calf and human O5 AE-STEC after Whole Genome Sequencing using two server databases (www.genomicepidemiology.org). Of 233 isolates, 206 were triplex PCR-positive: 119 AE-STEC (58%), 78 EPEC (38%) and 9 STEC (4%); and the stx1+eae+ AE-STEC (49.5%) were the most frequent. Of them, 120 isolates (84% of AE-STEC, 23% of EPEC, 22% of STEC) tested positive with one O serogroup PCR: 57 for O26 (47.5%), 36 for O111 (30%), 10 for O103 (8%) and 8 for O5 (7%) serogroups. The analysis of the draft sequences of 15 O5 AE-STEC could not identify any difference correlated to the host. As a conclusion, (i) the AE-STEC associated with diarrhoea in young calves still belong to the same serogroups as previously (O5, O26, O111) but the O103 serogroup may be emerging, (ii) the O5 AE-STEC from calves and humans are genetically similar..
107. Ken Ichi Lee, Tomoko Morita-Ishihara, Sunao Iyoda, Yoshitoshi Ogura, Tetsuya Hayashi, Tsuyoshi Sekizuka, Makoto Kuroda, Makoto Ohnishi, Hiroko Takenuma, Junji Seto, Yu Suzuki, Kyoko Mashiko, Shigenori Matsui, Shinichiro Hirai, Eiji Yokoyama, Noriko Konishi, Hiromi Obata, Akemi Kai, Atsuko Ogawa, Yuko Matsumoto, Ayako Kikuchi, Emiko Kitagawa, Hitomi Kasahara, Maki Sekiguchi, Yuji Tsuchiya, Hiromi Nakamura, Kazuko Seto, Junko Tanabe, Mayumi Tsujimoto, Hisahiro Kawai, Hiroko Dannnoue, Ritsuko Ohata, Hiroshi Nakajima, Hiroko Yamada, Kanako Masuda, Fuyuki Okamoto, Shuji Yoshino, Kazuyoshi Hozumi, Working Group in Japan EHEC Working Group in Japan, A geographically widespread outbreak investigation and development of a rapid screening method using whole genome sequences of enterohemorrhagic Escherichia coli O121, Frontiers in Microbiology, 10.3389/fmicb.2017.00701, 8, APR, 2017.04, [URL], From 2014 to 2015, we investigated a suspected nationwide outbreak of enterohemorrhagic Escherichia coli serogroup O121. However, similar pulsed field gel electrophoresis (PFGE) profiles and the lack of epidemiological links between the isolates made detection of the outbreak difficult. To elucidate a more precise genetic distance among the isolates, whole genome sequence (WGS) analyses were implemented in the investigation. The WGS-based single nucleotide polymorphism (SNP) analysis showed that 23 out of 44 isolates formed a distinct cluster (the number of intra-cluster SNPs was ≤8). Specific genomic regions in the clustered isolates were used to develop a specific PCR analysis. The PCR analysis detected all the clustered isolates and was suitable for rapid screening during the outbreak investigation. Our results showed that WGS analyses were useful for the detection of a geographically widespread outbreak, especially for isolates showing similar PFGE profiles and for the development of a rapid and cost-effective screening method..
108. Mami Tanaka, Shoko Endo, Fumihito Kotake, Nurhidayu Al-Saari, A. K.M.Rohul Amin, Gao Feng, Sayaka Mino, Hidetaka Doi, Yoshitoshi Ogura, Tetsuya Hayashi, Wataru Suda, Masahira Hattori, Isao Yumoto, Toko Sawabe, Tomoo Sawabe, Toshiyoshi Araki, Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed, PloS one, 10.1371/journal.pone.0180053, 12, 6, 2017.06, [URL], A novel strain Vibrio aphrogenes sp. nov. strain CA-1004T isolated from the surface of seaweed collected on the coast of Mie Prefecture in 1994 [1] was characterized using polyphasic taxonomy including multilocus sequence analysis (MLSA) and a genome based comparison. Both phylogenetic analyses on the basis of 16S rRNA gene sequences and MLSA based on eight protein-coding genes (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA) showed the strain could be placed in the Rumoiensis clade in the genus Vibrio. Sequence similarities of the 16S rRNA gene and the multilocus genes against the Rumoiensis clade members, V. rumoiensis, V. algivorus, V. casei, and V. litoralis, were low enough to propose V. aphrogenes sp. nov. strain CA-1004T as a separate species. The experimental DNA-DNA hybridization data also revealed that the strain CA-1004T was separate from four known Rumoiensis clade species. The G+C content of the V. aphrogenes strain was determined as 42.1% based on the genome sequence. Major traits of the strain were nonmotile, halophilic, fermentative, alginolytic, and gas production. A total of 27 traits (motility, growth temperature range, amylase, alginase and lipase productions, and assimilation of 19 carbon compounds) distinguished the strain from the other species in the Rumoiensis clade. The name V. aphrogenes sp. nov. is proposed for this species in the Rumoiensis clade, with CA-1004T as the type strain (JCM 31643T = DSM 103759T)..
109. Yohsuke Ogawa, Kazumasa Shiraiwa, Yoshitoshi Ogura, Tadasuke Ooka, Sayaka Nishikawa, Masahiro Eguchi, Tetsuya Hayashi, Yoshihiro Shimoji, Clonal lineages of Erysipelothrix rhusiopathiae responsible for acute swine erysipelas in Japan identified by using genome-wide single-nucleotide polymorphism analysis, Applied and Environmental Microbiology, 10.1128/AEM.00130-17, 83, 11, 2017.06, [URL], Erysipelothrix rhusiopathiae causes swine erysipelas, an important infectious disease in the swine industry. In Japan, the incidence of acute swine erysipelas due to E. rhusiopathiae serovar 1a has recently increased markedly. To study the genetic relatedness of the strains from the recent cases, we analyzed 34 E. rhusiopathiae serovar 1a swine isolates collected between 1990 and 2011 and further investigated the possible association of the live Koganei 65-0.15 vaccine strain (serovar 1a) with the increase in cases. Pulsed-field gel electrophoresis analysis revealed no marked variation among the isolates; however, sequencing analysis of a hypervariable region in the surface-protective antigen A gene (spaA) revealed that the strains isolated after 2007 exhibited the same spaA genotype and could be differentiated from older strains. Phylogenetic analysis based on genome-wide single-nucleotide polymorphisms (SNPs) revealed that the Japanese strains examined were closely related, showing a relatively small number of SNPs among them. The strains were classified into four major lineages, with Koganei 65-0.15 (lineage III) being phylogenetically separated from the other three lineages. The strains isolated after 2007 and the two older strains constituted one major lineage (lineage IV) with a specific spaA genotype (M203/I257-SpaA), while the recent isolates were further divided into two geographic groups. The remaining older isolates belonged to either lineage I, with the I203/L257-SpaA type, or lineage II, with the I203/ I257-SpaA type. These results indicate that the recent increased incidence of acute swine erysipelas in Japan is associated with two sublineages of lineage IV, which have independently evolved in two different geographic regions..
110. A. K.M.Rohul Amin, Mami Tanaka, Nurhidayu Al-saari, Gao Feng, Sayaka Mino, Yoshitoshi Ogura, Tetsuya Hayashi, Pedro M. Meirelles, Fabiano L. Thompson, Bruno Gomez-Gil, Toko Sawabe, Tomoo Sawabe, Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. within the family Vibrionaceae, isolated from coral reef seawater off Ishigaki Island, Japan, Systematic and Applied Microbiology, 10.1016/j.syapm.2017.04.003, 40, 5, 290-296, 2017.07, [URL], Two phylogenetically distinct Vibrionaceae strains C4II189T and C4V358T isolated from reef seawater off Ishigaki Island, Japan, in 2014 were studied with advanced genome-based taxonomy approaches. All aspects of phylogenetic (16S rRNA phylogeny, MLSA), phenotypic and genetic (ANI, DDH, AAI, and the number of core genes) cohesions between the two identified species were high enough to propose them as members of a new genus within the family Vibrionaceae. Consequently, an eighth genus Thaumasiovibrio gen. nov. is proposed that contains two new species Thaumasiovibrio occultus sp. nov. strain C4II189T (=DSM 101554T = JCM 31629T) (type species) and Thaumasiovibrio subtropicus sp. nov. strain C4V358T (=DSM 101555T = JCM 31630T). Thaumasiovibrio species were phylogenetically distinct from the other Vibrionaceae species based on pyrH gene sequences. The combination of catalase negative, sensitivity to vibriostatic agent O/129, and green colony formation on TCBS for the phylogenetically affiliated strains was the diagnostic features for the current tentative identification of this genus..
111. A. K.M.Rohul Amin, Mami Tanaka, Nurhidayu Al-saari, Gao Feng, Sayaka Mino, Yoshitoshi Ogura, Tetsuya Hayashi, Pedro M. Meirelles, Fabiano L. Thompson, Bruno Gomez-Gil, Toko Sawabe, Tomoo Sawabe, Corrigendum to “Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. within the family Vibrionaceae, isolated from coral reef seawater off Ishigaki Island, Japan” [Syst. Appl. Microbiol. 40 (5) (2017) 290–296](S0723202017300607)(10.1016/j.syapm.2017.04.003)), Systematic and Applied Microbiology, 10.1016/j.syapm.2017.10.001, 41, 1, 62-63, 2017.07, [URL], On page 295, information of type species of Thaumasiovibrio gen. nov. is removed. The abbreviated genus name was spelled out. Corrected description is follows; Description of Thaumasiovibrio gen. nov. (Thau.ma.si.o.vi'bri.o. Gr. adj. thaumasios, unusual, unique; N.L. masc. n. vibrio, a motile bacterium; N.L. masc. n. Thaumasiovibrio, an unusual bacterium): Bacteria are Gram-negative, facultatively anaerobic, motile rods. Sodium ions are essential for growth. No endospores are formed. The single polar flagella is observed when cultivated on solidified and/or liquid media. No swarming is observed. Green colonies are observed on TCBS. Growth in 1%–3% NaCl broth. Negative for lysine decarboxylase, arginine dihydrolase and ornithine decarboxylase. Catalase, acetoin and indole productions are negative. Susceptible to the vibriostatic agent O/129 (150 μg/disc). Isolated from the seawater off Ishigaki coral reef. The DNA G + C content is 47.2–49.4%. The range of estimated genome sizes based on draft genome sequencing is 4.5 Mb–5.4 Mb. The type species is Thaumasiovibrio occultus. The Digital Protologue taxonumber is GA00017. Description of Thaumasiovibrio occultus sp. nov. (oc.cul'tus. L. adj. occultus, hidden; referring to resistant to the discovery of the species in the diverse active coral reef ecosystem): Cells are 1.0–1.2 μm long and 0.7–0.8 μm in width when grown on ZoBell 2216E agar. Colonies on ZoBell 2216E agar are beige, circular, smooth and convex with a diameter of 2 mm after culture for 3 days. Mesophilic and neutrophilic chemo-organotroph that grows at 15–30 °C. No bioluminescence is observed, and there is no requirement for organic growth factors. Oxidase positive. Produces acid from glucose without gas production, and hydrolyses Tween 80, gelatin, DNA; assimilates D-mannose, D-fructose, fumarate, N-acetyl-D-glucosamine, citrate, glycerol, DL-malate, acetate, D-glucosamine, pyruvate, L-glutamate, glycerate, DL-lactate, L-alanine, and L-asparagine. No pigmentation is observed; does not hydrolyze starch, alginate, agar, and κ-carrageenan; does not reduce nitrate. Does not assimilate D-galactose, sucrose, maltose, melibiose, lactose, D-gluconate, succinate, aconitate, meso-erythritol, D-mannitol, γ-aminobutyrate, D-sorbitol, α-ketoglutarate, xylose, trehalose, glucuronate, δ-aminovalate, cellobiose, L-proline, putrescine, propionate, amygdalin, D-galacturonate, D-raffinose, rhamnose, D-ribose, salicin, L-arginine, L-citrulline, glycine, histidine, L-ornithine, and L-serine. The DNA G + C content is 49.4%, and the estimated genome size is 4.5 Mb. The type strain is Thaumasiovibrio occultus DSM 101554T = JCM 31629T = C4II189T. The Digital Protologue taxonumber is TA00038. Description of Thaumasiovibrio subtropicus sp. nov. (sub.tro'pi.cus. N.L. adj. subtropicus, subtropical, referring to the isolation site of the Ishigaki coral reef): Cells are 1.9–2.0 μm long and 0.9–1.0 μm in width when grown on ZoBell 2216E agar. Colonies on ZoBell 2216E agar are beige, circular, smooth and convex, with a diameter of 3 mm after culture for 3 days. Mesophilic and neutrophilic chemo-organotroph that grows at 15–37 °C. No bioluminescence is observed, and there is no requirement for organic growth factors. Produces soluble brown pigments; acid from glucose without gas production, hydrolyses Tween 80, gelatin, DNA; reduces nitrate; assimilates D-mannose, D-fructose, N-acetyl-D-glucosamine, fumarate, citrate, D-mannitol, D-sorbitol, DL-malate, glucuronate, acetate, D-glucosamine, pyruvate, cellobiose, L-proline, L-glutamate, propionate, glycerate, DL-lactate, L-alanine, L-asparagine, histidine, and L-serine. Does not hydrolyze starch, alginate, agar, and κ-carrageenan; capable of reducing nitrate. Oxidase negative. Does not assimilate D-galactose, sucrose, maltose, melibiose, lactose, D-gluconate, succinate, aconitate, meso-erythritol, glycerol, γ-aminobutyrate, α-ketoglutarate, xylose, trehalose, δ-aminovalate, putrescine, amygdalin, D-galacturonate, D-raffinose, rhamnose, D-ribose, salicin, L-arginine, L-citrulline, glycine, and L-ornithine. The DNA G + C content is 47.2%, and the estimated genome size is 5.4 Mb. The type strain is Thaumasiovibrio subtropicus DSM 101555T = JCM 31630T = C4V358T. The Digital Protologue taxonumber is TA00043..
112. Shu Kuan Wong, Sanghwa Park, Jung Sook Lee, Keunchul Lee, Yoshitoshi Ogura, Tetsuya Hayashi, Hiroshi Xavier Chiura, Susumu Yoshizawa, Koji Hamasaki, Algibacter aquaticus sp. Nov., a slightly alkaliphilic marine Flavobacterium isolated from coastal surface water, International Journal of Systematic and Evolutionary Microbiology, 10.1099/ijsem.0.001924, 67, 7, 2199-2204, 2017.07, [URL], A rod-shaped, pale yellow-pigmented, aerobic, Gram-staining-negative strain with gliding motility, designated as strain SK- 16T, was isolated from the coastal surface water of a semi-enclosed coastal inlet in Misaki, Japan. Analysis of 16S rRNA gene sequences revealed that SK-16T represented a member of the family Flavobacteriaceae and was closely related to the genus Algibacter, with sequence similarities ranging from 95.9 to 94.3% to the type strains of species of the genus Algibacter. The major cellular fatty acids were iso-C
15 : 0
, iso-C
17 : 0
3-OH, iso-C
15 : 0
G and iso-C
15 : 0
3-OH. Major polar lipids were phosphatidylethanolamine, an aminophospholipid and an unidentified phospholipid. The DNA G+C content of SK-16
T
was 32.3 mol% and MK-6 was the only predominant isoprenoid quinone. On the basis of the results of phenotypic, genotypic, chemotaxonomic and phylogenetic studies, it was suggested that SK-16
T
represents a novel species within the genus Algibacter, with the newly proposed name Algibacter aquaticus. The type strain is SK-16
T
(=NBRC 110220
T
=KCTC 32974
T
)..
113. Sayaka Tsuchida, Fumito Maruyama, Yoshitoshi Ogura, Atsushi Toyoda, Tetsuya Hayashi, Moriya Okuma, Kazunari Ushida, Genomic characteristics of Bifidobacterium thermacidophilum pig isolates and wild boar isolates reveal the unique presence of a putative mobile genetic element with tetW for pig farm isolates, Frontiers in Microbiology, 10.3389/fmicb.2017.01540, 8, AUG, 2017.08, [URL], Genomic analysis was performed on seven strains of Bifidobacterium thermacidophilum, a Sus-associated Bifidobacterium. Three strains from the feces of domestic pigs (Sus scrofa domesticus) and four strains from the rectal feces of free-range Japanese wild boars (S. s. scrofa) were compared. The phylogenetic position of these isolates suggested by genomic analyses were not concordant with that suggested by 16S rRNA sequence. There was biased distribution of genes for virulence, phage, metabolism of aromatic compounds, iron acquisition, cell division, and DNA metabolism. In particular four wild boar isolates harbored fiber-degrading enzymes, such as endoglucanase, while two of the pig isolates obtained from those grown under an intensive feeding practice with routine use of antimicrobials, particularly tetracycline harbored a tetracycline resistance gene, which was further proved functional by disk diffusion test. The tetW gene is associated with a serine recombinase of an apparently non-bifidobacterial origin. The insertion site of the tetW cassette was precisely defined by analyzing the corresponding genomic regions in the other tetracycline-susceptible isolates. The cassette may have been transferred from some other bacteria in the pig gut..
114. Yu Nakajima, Susumu Yoshizawa, Sanghwa Park, Yohei Kumagai, Shu Kuan Wong, Yoshitoshi Ogura, Tetsuya Hayashi, Kazuhiro Kogure, Draft genome sequence of Rubricoccus marinus SG-29T, a marine bacterium within the family Rhodothermaceae, which contains two different rhodopsin genes, Genome Announcements, 10.1128/genomeA.00990-17, 5, 38, 2017.09, [URL], Here, we report the draft genome sequence of Rubricoccus marinus SG- 29T, a bacterium isolated from the western North Pacific Ocean. R. marinus SG-29T possesses two different types of rhodopsin genes and belongs to the family Rhodothermaceae, with which halophilic, thermophilic, and marine bacteria are associated..
115. Yoshitoshi Ogura, Yasuhiro Gotoh, Takehiko Itoh, Mitsuhiko P. Sato, Kazuko Seto, Shyuji Yoshino, Junko Isobe, Yoshiki Etoh, Mariko Kurogi, Keiko Kimata, Eriko Maeda, Denis Piérard, Masahiro Kusumoto, Masato Akiba, Kiyoshi Tominaga, Yumi Kirino, Yuki Kato, Katsuhiko Shirahige, Tadasuke Ooka, Nozomi Ishijima, Ken Ichi Lee, Sunao Iyoda, Jacques Georges Mainil, Tetsuya Hayashi, Population structure of escherichia coli o26
H11 with recent and repeated stx2 acquisition in multiple lineages, Microbial Genomics, 10.1099/mgen.0.000141, 3, 11, 2017.11, [URL], A key virulence factor of enterohaemorrhagic Escherichia coli (EHEC) is the bacteriophage-encoded Shiga toxin (Stx). Stxs are classified into two types, Stx1 and Stx2, and Stx2-producing strains are thought to cause more severe infections than strains producing only Stx1. Although O26: H11 is the second most prevalent EHEC following O157: H7, the majority of O26: H11 strains produce Stx1 alone. However, Stx2-producing O26 strains have increasingly been detected worldwide. Through a large-scale genome analysis, we present a global phylogenetic overview and evolutionary timescale for E. coli O26: H11. The origin of O26 has been estimated to be 415 years ago. Sequence type 21C1 (ST21C1), one of the two sublineages of ST21, the most predominant O26: H11 lineage worldwide, emerged 213 years ago from one of the three ST29 sublineages (ST29C2). The other ST21 lineage (ST21C2) emerged 95 years ago from ST21C1. Increases in population size occurred in the late 20th century for all of the O26 lineages, but most remarkably for ST21C2. Analysis of the distribution of stx2-positive strains revealed the recent and repeated acquisition of the stx2 gene in multiple lineages of O26, both in ST21 and ST29. Other major EHEC virulence genes, such as type III secretion system effector genes and plasmid-encoded virulence genes, were well conserved in ST21 compared to ST29. In addition, more antimicrobial-resistance genes have accumulated in the ST21C1 lineage. Although current attention is focused on several highly virulent ST29 clones that have acquired the stx2 gene, there is also a considerable risk that the ST21 lineage could yield highly virulent clones..
116. Sarinya Tawthep, Satoru Fukiya, Ja Young Lee, Masahito Hagio, Yoshitoshi Ogura, Tetsuya Hayashi, Atsushi Yokota, Isolation of six novel 7-oxo- or urso-type secondary bile acid-producing bacteria from rat cecal contents, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.06.002, 124, 5, 514-522, 2017.11, [URL], Understanding the dynamics of secondary bile acid (SBA) formation in the gut by SBA-producing bacteria is important for host health, as SBAs have been shown to affect host pathophysiology and gut microbiota composition. However, our knowledge of SBA producers is limited in light of the diversity of gut microbes. Here, we isolated six novel SBA-producing bacteria from rat cecal contents, all of which were members of known species of gut microbes. Anaerostipes caccae D10, Bacteroides nordii C5, Clostridioides difficile D7, and Clostridium cadaveris G11 were capable of oxidizing cholic acid and chenodeoxycholic acid into 7-oxo-derivatives with varying yields. B. nordii C5 and its type strain JCM 12987T had the highest molar yield, ∼90%. Clostridium disporicum F4 and Clostridium subterminale C4 epimerized cholic acid into ursocholic acid with yields of ∼85%; the corresponding type strains lacked epimerization activity. Furthermore, although not novel as an SBA producer, Clostridium scindens G10 that produced deoxycholic acid from cholic acid was isolated for the first time from rodents. These findings will contribute to elucidation of SBA formation in the gut..
117. Yohei Kumagai, Susumu Yoshizawa, Keiji Nakamura, Yoshitoshi Ogura, Tetsuya Hayashi, Kazuhiro Kogure, Complete and draft genome sequences of eight oceanic Pseudomonas aeruginosa strains, Genome Announcements, 10.1128/genomeA.01255-17, 5, 44, 2017.11, [URL], Pseudomonas aeruginosa is one of the most common model bacterial species, and genomes of hundreds of strains of this species have been sequenced to date. However, currently there is only one available genome of an oceanic isolate. Here, we report two complete and six draft genome sequences of P. aeruginosa isolates from the open ocean..
118. Eiko Hayase, Daigo Hashimoto, Kiminori Nakamura, Clara Noizat, Reiki Ogasawara, Shuichiro Takahashi, Hiroyuki Ohigashi, Yuki Yokoi, Rina Sugimoto, Satomi Matsuoka, Takahide Ara, Emi Yokoyama, Tomohiro Yamakawa, Ko Ebata, Takeshi Kondo, Rina Hiramine, Tomoyasu Aizawa, Yoshitoshi Ogura, Tetsuya Hayashi, Hiroshi Mori, Ken Kurokawa, Kazuma Tomizuka, Tokiyoshi Ayabe, Takanori Teshima, R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease, Journal of Experimental Medicine, 10.1084/jem.20170418, 214, 12, 3507-3518, 2017.12, [URL], The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as α-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant α-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spo1 or recombinant α-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host-microbiota cross talk toward therapeutic benefits..
119. Mami Tanaka, Shoko Endo, Fumihito Kotake, Nurhidayu Al-Saari, A. K.M.Rohul Amin, Gao Feng, Sayaka Mino, Hidetaka Doi, Yoshitoshi Ogura, Tetsuya Hayashi, Wataru Suda, Masahira Hattori, Isao Yumoto, Toko Sawabe, Tomoo Sawabe, Toshiyoshi Araki, Correction
Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed (PLoS ONE (2017) 12:6 (e0180053) DOI: 10.1371/journal.pone.0180053), PloS one, 10.1371/journal.pone.0189555, 12, 12, 2017.12, [URL], The strain number is missing in the description of V. aphrogenes in the first and second paragraphs under the subheading “Description of Vibrio aphrogenes sp. nov.” in the Results and discussion section. Please see the corrected paragraph below. Description of Vibrio aphrogenes sp. nov. V. aphrogenes sp. nov. (aph.ro’ge.nes. Gr. n. aphros, foam; Gr. suff. -genes, producing; N.L. adj. aphrogenes, foam-producing, referring to gas formation of the strain). Gram-negative, facultative anaerobic, non-motile rods isolated from surface of seaweed collected in Mie Prefecture in Japan. Colonies on ZoBell 2216E agar medium were cream or transparent white, round, and smooth on the edge. No flagellum was observed. Sodium ion is essential for growth. Growth occurs at NaCl concentrations of 1.0 to 10.0% and at temperatures between 4 and 40C. V. aphrogenes tested positive for production of alginase, lipase and DNase, oxidase, catalase, gas production from D-glucose, arginine dihydrolase, and is able to assimilate D-glucose, D-mannitol, D-mannose, D-galactose, maltose, D-gluconate, fuma-rate, glycerol, acetate, D-glucosamine, pyruvate, L-proline, D-ribose, L-alanine, L-asparagine, and L-serine. The bacteria tested negative for indole production, acetoin production, lysine decarboxylase, ornithine decarboxylase, amylase, agarose, gelatinase and κ-carrageenase productions, and is incapable of assimilating D-fructose, sucrose, melibiose, lactose, N-acetyl-glucosamine, succinate, citrate, aconitate, meso-erythritol, γ-aminobutyrate, L-tyrosine, Dsorbitol, DL-malate, α-ketoglutarate, trehalose, gluconate, δ-aminovalate, cellobiose, L-glutamate, putrescine, propionate, amygdalin, arabinose, D-galacturonate, glycerate, D-raffinose, rhamnose, salicine, DL-lactate, L-arginine, L-citrulline, glycine, histidine, and L-ornithine. The G+C content of DNA is 42.1%. Estimated genome size is 3.4 Mb on the basis of genome sequencing. The type strain is JCM 31643T = DSM 103759T = CA-1004T..
120. Yu Nakajima, Takashi Tsukamoto, Yohei Kumagai, Yoshitoshi Ogura, Tetsuya Hayashi, Jaeho Song, Takashi Kikukawa, Makoto Demura, Kazuhiro Kogure, Yuki Sudo, Susumu Yoshizawa, Presence of a haloarchaeal halorhodopsin-like Cl pump in marine bacteria, Microbes and Environments, 10.1264/jsme2.ME17197, 33, 1, 89-97, 2018.01, [URL], Light-driven ion-pumping rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone of the aquatic environment. H+-pumping rhodopsin (proteorhodopsin: PR), Na+-pumping rhodopsin (NaR), and Cl-pumping rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one of these genes was heterologously expressed in Escherichia coli cells and the rhodopsin protein named Rubricoccus marinus halorhodopsin (RmHR) was identified as a light-driven inward Cl pump. Spectroscopic assays showed that the estimated dissociation constant (Kd,int.) of this rhodopsin was similar to that of haloarchaeal halorhodopsin (HR), while the Cl-transporting photoreaction mechanism of this rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial ClR. This amino acid sequence similarity also suggested that this rhodopsin is similar to haloarchaeal HR and cyanobacterial HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired rhodopsin-related genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl-pumping rhodopsin is present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR..
121. Mami Tanaka, Sayaka Mino, Yoshitoshi Ogura, Tetsuya Hayashi, Tomoo Sawabe, Availability of Nanopore sequences in the genome taxonomy for Vibrionaceae systematics
Rumoiensis clade species as a test case, PeerJ, 10.7717/peerj.5018, 2018, 6, 2018.01, [URL], Whole genome sequence comparisons have become essential for establishing a robust scheme in bacterial taxonomy. To generalize this genome-based taxonomy, fast, reliable, and cost-effective genome sequencing methodologies are required. MinION, the palm-sized sequencer from Oxford Nanopore Technologies, enables rapid sequencing of bacterial genomes using minimal laboratory resources. Here we tested the ability of Nanopore sequences for the genome-based taxonomy of Vibrionaceae and compared Nanopore-only assemblies to complete genomes of five Rumoiensis clade species: Vibrio aphrogenes, V. algivorus, V. casei, V. litoralis, and V. rumoiensis. Comparison of overall genome relatedness indices (OGRI) and multilocus sequence analysis (MLSA) based on Nanopore-only assembly and Illumina or hybrid assemblies revealed that errors in Nanopore-only assembly do not influence average nucleotide identity (ANI), in silico DNA-DNA hybridization (DDH), G+C content, or MLSA tree topology in Vibrionaceae. Our results show that the genome sequences from Nanopore-based approach can be used for rapid species identification based on the OGRI and MLSA..
122. Yuichi Oogai, Yasuhiro Gotoh, Yoshitoshi Ogura, Miki Kawada-Matsuo, Tetsuya Hayashi, Hitoshi Komatsuzawa, Small RNA repertoires and their intraspecies variation in Aggregatibacter actinomycetemcomitans, DNA Research, 10.1093/dnares/dsx050, 25, 2, 207-215, 2018.04, [URL], Aggregatibacter actinomycetemcomitans is a major periodontal pathogen that has several virulence factors such as leukotoxin and cytolethal distending toxin. Although the genes responsible for virulence have been identified, little is known about their regulatory mechanisms. Small RNA (sRNA) has been recognized as an important factor for gene regulation. To identify new regulatory mechanisms via sRNA in A. actinomycetemcomitans HK1651, we performed a systematic search for sRNAs by RNA-seq and identified 90 intergenic region sRNAs and 30 antisense sRNAs. Of the 85 analysable sRNAs, we successfully detected and quantified 70 sRNAs by developing an RT-PCR system, and we identified 17 sRNAs that were differentially expressed during different growth phases. In addition, we found notable intraspecies variation in the sRNA repertoire of A. actinomycetemcomitans, thus suggesting that frequent acquisition or deletion of sRNAs occurred during the evolution of this species. The predicted target genes of the intergenic region sRNAs indicated the possibility of sRNA interaction with several virulence genes including leukotoxin and cytolethal distending toxin. Our results should serve as an important genomic and genetic basis for future studies to fully understand the regulatory network in A. actinomycetemcomitans and provide new insights into the intraspecies variation of the bacterial sRNA repertoire in bacteria..
123. Yohei Kumagai, Susumu Yoshizawa, Yu Nakajima, Mai Watanabe, Tsukasa Fukunaga, Yoshitoshi Ogura, Tetsuya Hayashi, Kenshiro Oshima, Masahira Hattori, Masahiko Ikeuchi, Kazuhiro Kogure, Edward F. Delong, Wataru Iwasaki, Solar-panel and parasol strategies shape the proteorhodopsin distribution pattern in marine Flavobacteriia, ISME Journal, 10.1038/s41396-018-0058-4, 12, 5, 1329-1343, 2018.05, [URL], Proteorhodopsin (PR) is a light-driven proton pump that is found in diverse bacteria and archaea species, and is widespread in marine microbial ecosystems. To date, many studies have suggested the advantage of PR for microorganisms in sunlit environments. The ecophysiological significance of PR is still not fully understood however, including the drivers of PR gene gain, retention, and loss in different marine microbial species. To explore this question we sequenced 21 marine Flavobacteriia genomes of polyphyletic origin, which encompassed both PR-possessing as well as PR-lacking strains. Here, we show that the possession or alternatively the lack of PR genes reflects one of two fundamental adaptive strategies in marine bacteria. Specifically, while PR-possessing bacteria utilize light energy ("solar-panel strategy"), PR-lacking bacteria exclusively possess UV-screening pigment synthesis genes to avoid UV damage and would adapt to microaerobic environment ("parasol strategy"), which also helps explain why PR-possessing bacteria have smaller genomes than those of PR-lacking bacteria. Collectively, our results highlight the different strategies of dealing with light, DNA repair, and oxygen availability that relate to the presence or absence of PR phototrophy..
124. Masumi Hasegawa, Yu Nakajima, Shu Kuan Wong, Keiji Nakamura, Yoshitoshi Ogura, Tetsuya Hayashi, Kazuhiro Kogure, Susumu Yoshizawa, Draft genome sequence of Saccharospirillum sp. strain MSK14-1, isolated from surface seawater collected at Aburatsubo Inlet in Japan, Genome Announcements, 10.1128/genomeA.00469-18, 6, 22, 2018.05, [URL], Here, we report the draft genome sequence of Saccharospirillum sp. strain MSK14-1, isolated from surface seawater collected at Aburatsubo Inlet in Japan. The genome sequence of strain MSK14-1 should contribute to our understanding of the characteristics of the genus Saccharospirillum..
125. Kengo Inoue, Yoshitoshi Ogura, Yoshihiro Kawano, Tetsuya Hayashi, Complete genome sequence of Geobacter sulfurreducens strain YM18, isolated from river sediment in Japan, Genome Announcements, 10.1128/genomeA.00352-18, 6, 19, 2018.05, [URL], Geobacter sulfurreducens is known to be a dominant species in the anode biofilms of microbial fuel cells. Here, we report the complete genome sequence of G. sulfurreducens strain YM18. Strain YM18 was isolated from a biofilm formed on an anode poised at- 400 mV (versus an Ag/AgCl electrode) in a bioelectrochemical system..
126. Mitsunori Yoshida, Hanako Fukano, Yoshitoshi Ogura, Yuko Kazumi, Satoshi Mitarai, Tetsuya Hayashi, Yoshihiko Hoshino, Complete genome sequence of Mycobacterium shigaense, Genome Announcements, 10.1128/genomeA.00552-18, 6, 25, 2018.06, [URL], Mycobacterium shigaense is a slowly growing scotochromogenic species and a member of the Mycobacterium simiae complex group. Here, we report the complete sequence of its genome, comprising a 5.2-Mb chromosome. The sequence will represent the essential data for future phylogenetic and comparative genome studies of the Mycobacterium simiae complex group..
127. Huei Mien Ke, Dang Liu, Yoshitoshi Ogura, Tetsuya Hayashi, Henryk Urbanczyk, Isheng J. Tsai, Tracing genomic divergence of Vibrio bacteria in the Harveyi clade, Journal of bacteriology, 10.1128/JB.00001-18, 200, 15, 2018.08, [URL], The mechanism of bacterial speciation remains a topic of tremendous interest. To understand the ecological and evolutionary mechanisms of speciation in Vibrio bacteria, we analyzed the genomic dissimilarities between three closely related species in the so-called Harveyi clade of the genus Vibrio, V. campbellii, V. jasicida, and V. hyugaensis. The analysis focused on strains isolated from diverse geographic locations over a long period of time. The results of phylogenetic analyses and calculations of average nucleotide identity (ANI) supported the classification of V. jasicida and V. hyugaensis into two species. These analyses also identified two well-supported clades in V. campbellii; however, strains from both clades were classified as members of the same species. Comparative analyses of the complete genome sequences of representative strains from the three species identified higher syntenic coverage between genomes of V. jasicida and V. hyugaensis than that between the genomes from the two V. campbellii clades. The results from comparative analyses of gene content between bacteria from the three species did not support the hypothesis that gene gain and/or loss contributed to their speciation. We also did not find support for the hypothesis that ecological diversification toward associations with marine animals contributed to the speciation of V. jasicida and V. hyugaensis. Overall, based on the results obtained in this study, we propose that speciation in Harveyi clade species is a result of stochastic diversification of local populations, which was influenced by multiple evolutionary processes, followed by extinction events..
128. Hanako Fukano, Mitsunori Yoshida, Yuko Kazumi, Nagatoshi Fujiwara, Kinya Katayama, Yoshitoshi Ogura, Tetsuya Hayashi, Yuji Miyamoto, Noriki Fujimoto, Wang Hongsheng, Chisaki Mizumoto, Yusuke Koizumi, Hiroyoshi Maeda, Osamu Hiranuma, Satoshi Mitarai, Norihisa Ishii, Yoshihiko Hoshino, Mycobacterium shigaense sp. Nov., a slow-growing, scotochromogenic species, is a member of the mycobacterium simiae complex, International Journal of Systematic and Evolutionary Microbiology, 10.1099/ijsem.0.002845, 68, 8, 2437-2442, 2018.08, [URL], Among non-tuberculous mycobacteria (NTM), the Mycobacterium simiae complex is one of the largest groups, consisting of 18 species of slow-growing mycobacteria. In 2009, a case of NTM-associated infectious skin disease was reported in Shiga Prefecture, Japan. The patient presented with scattered nodules on the chest, back and extremities, and an M. simiae-like organism was isolated from skin biopsy specimens obtained from one of these lesions. Based on several assessments, including multiple-gene analyses, biochemical characterization and drug susceptibility testing, we concluded that this isolate represented a novel species of NTM, and proposed the name ‘Mycobacterium shigaense’. Since 2009, five more cases of NTM-associated infectious disease in which there was a suspected involvement of ‘M. shigaense’ have been reported. Interestingly, four of these six cases occurred in Shiga Prefecture. Here we performed multiple-gene phylogenetic analyses, physiological and biochemical characterization tests, drug susceptibility tests, and profiling of proteins, fatty acids and mycolic acids of eight clinical isolates from the six suspected ‘M. shigaense’ cases. The results confirmed that all of the clinical isolates were ‘M. shigaense’, a slow-growing, scotochromogenic species. Here M. shigaense is validly proposed as a new member of the M. simiae complex, with the type strain being UN-152T (=JCM 32072T =DSM 46748T)..
129. Atsushi Hirano, Junji Umeno, Yasuharu Okamoto, Hiroki Shibata, Yoshitoshi Ogura, Moriyama Tomohiko, takehiro torisu, Shin Fujioka, Yuta Fuyuno, Yutaka Kawarabayasi, Takayuki Matsumoto, Takanari Kitazono, Motohiro Esaki, Comparison of the microbial community structure between inflamed and non-inflamed sites in patients with ulcerative colitis, Journal of Gastroenterology and Hepatology (Australia), 10.1111/jgh.14129, 33, 9, 1590-1597, 2018.09, [URL], Background and Aim: The gut microbiota is suggested to play an important role in the pathogenesis of ulcerative colitis (UC). However, interindividual and spatial variations hamper the identification of UC-related changes. We thus investigated paired mucosa-associated microbiota obtained from both inflamed and non-inflamed sites of UC patients and corresponding sites of non-inflammatory bowel disease (IBD) controls. Methods: Mucosal biopsies of both inflamed and non-inflamed sites were obtained from 14 patients with active UC of the left-sided or proctitis type. Paired mucosal biopsies of the corresponding sites were obtained from 14 non-IBD controls. The microbial community structure was investigated using 16S ribosomal RNA gene sequences, followed by data analysis using qiime and LEfSe softwares. Results: Microbial alpha diversity in both inflamed and non-inflamed sites was significantly lower in UC patients compared with non-IBD controls. There were more microbes of the genus Cloacibacterium and the Tissierellaceae family, and there were less microbes of the genus Neisseria at the inflamed site when compared with the non-inflamed site in UC patients. Decreased abundance of the genera Prevotella, Eubacterium, Neisseria, Leptotrichia, Bilophila, Desulfovibrio, and Butyricimonas was evident at the inflamed site of UC patients compared with the corresponding site of non-IBD controls. Among these taxa, the genera Prevotella and Butyricimonas were also less abundant at the non-inflamed site of UC patients compared with the corresponding site in non-IBD controls. Conclusions: Mucosal microbial dysbiosis occurs at both inflamed and non-inflamed sites in UC patients. The taxa showing altered abundance in UC patients might mediate colonic inflammation..
130. Shu Kuan Wong, Susumu Yoshizawa, Yu Nakajima, Marie Johanna Cuadra, Yuichi Nogi, Keiji Nakamura, Hideto Takami, Yoshitoshi Ogura, Tetsuya Hayashi, Hiroshi Xavier Chiura, Koji Hamasaki, Amylibacter kogurei sp. Nov., a novel marine alphaproteobacterium isolated from the coastal sea surface microlayer of a marine inlet, International Journal of Systematic and Evolutionary Microbiology, 10.1099/ijsem.0.002911, 68, 9, 2872-2877, 2018.09, [URL], A novel Gram-negative bacterium, designated 4G11T, was isolated from the sea surface microlayer of a marine inlet. On the basis of 16S rRNA gene sequence analysis, the strain showed the closest similarity to Amylibacter ulvae KCTC 32465T (99.0 %). However, DNA-DNA hybridization values showed low DNA relatedness between strain 4G11Tand its close phylogenetic neighbours, Amylibacter marinus NBRC 110140T (8.0±0.4 %) and Amylibacter ulvae KCTC 32465T(52.9±0.9 %). Strain 4G11T had C18: 1, C16: 0and C18: 2as the major fatty acids. The only isoprenoid quinone detected for strain 4G11T was ubiquinone-10. The major polar lipids were phosphatidylglycerol, phosphatidylcholine, one unidentified polar lipid, one unidentified phospholipid and one unidentified aminolipid. The DNA G+C content of strain 4G11T was 50.0 mol%. Based on phenotypic and chemotaxonomic characteristics and analysis of the 16S rRNA gene sequence, the novel strain should be assigned to a novel species, for which the name Amylibacter kogurei sp. nov. is proposed. The type strain of Amylibacter kogurei is 4G11T(KY463497=KCTC 52506T=NBRC 112428T)..
131. Yoshihiro Suzuki, Kotaro Teranishi, Tomonori Matsuwaki, Kei Nukazawa, Yoshitoshi Ogura, Effects of bacterial pollution caused by a strong typhoon event and the restoration of a recreational beach
Transitions of fecal bacterial counts and bacterial flora in beach sand, Science of the Total Environment, 10.1016/j.scitotenv.2018.05.265, 640-641, 52-61, 2018.11, [URL], To determine the effects of bacteria pollution associated with a strong typhoon event and to assess the restoration of the normal bacterial flora, we used conventional filtration methods and nextgeneration sequencing of 16S rRNA genes to analyze the transition of fecal and total bacterial counts in water and core sand samples collected from a recreational beach. Immediately after the typhoon event, Escherichia coli counts increased to 82 CFU/100 g in the surface beach sand. E. coli was detected through the surface to sand 85-cm deep at the land side point (10-m land side from the high-water line). However, E. coli disappeared within a month from the land side point. The composition of the bacterial flora in the beach sand at the land point was directly influenced by the typhoon event. Pseudomonas was the most prevalent genus throughout the sand layers (0–102-cm deep) during the typhoon event. After 3 months, the population of Pseudomonas significantly decreased, and the predominant genus in the surface layer was Kaistobacter, although Pseudomonas was the major genus in the 17- to 85-cm layer. When the beach conditions stabilized, the number of pollutant Pseudomonas among the 10 most abundant genera decreased to lower than the limit of detection. The bacterial population of the sand was subsequently restored to the most populous pre-event orders at the land point. A land-side beach, where users directly contact the sand, was significantly affected by bacterial pollution caused by a strong typhoon event. We show here that the normal bacterial flora of the surface sand was restored within 1 month..
132. Kazue Nakanaga, Yoshitoshi Ogura, Atsushi Toyoda, Mitsunori Yoshida, Hanako Fukano, Nagatoshi Fujiwara, Yuji Miyamoto, Noboru Nakata, Yuko Kazumi, Shinji Maeda, Tadasuke Ooka, Masamichi Goto, Kazunari Tanigawa, Satoshi Mitarai, Koichi Suzuki, Norihisa Ishii, Manabu Ato, Tetsuya Hayashi, Yoshihiko Hoshino, Naturally occurring a loss of a giant plasmid from Mycobacterium ulcerans subsp. shinshuense makes it non-pathogenic, Scientific reports, 10.1038/s41598-018-26425-1, 8, 1, 2018.12, [URL], Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a WHO-defined neglected tropical disease. All Japanese BU causative isolates have shown distinct differences from the prototype and are categorized as M. ulcerans subspecies shinshuense. During repeated sub-culture, we found that some M. shinshuense colonies were non-pigmented whereas others were pigmented. Whole genome sequence analysis revealed that non-pigmented colonies did not harbor a giant plasmid, which encodes elements needed for mycolactone toxin biosynthesis. Moreover, mycolactone was not detected in sterile filtrates of non-pigmented colonies. Mice inoculated with suspensions of pigmented colonies died within 5 weeks whereas those infected with suspensions of non-pigmented colonies had significantly prolonged survival (>8 weeks). This study suggests that mycolactone is a critical M. shinshuense virulence factor and that the lack of a mycolactone-producing giant plasmid makes the strain non-pathogenic. We made an avirulent mycolactone-deletion mutant strain directly from the virulent original..
133. Yoshitoshi Ogura, Kazuko Seto, Yo Morimoto, Keiji Nakamura, Mitsuhiko P. Sato, Yasuhiro Gotoh, Takehiko Itoh, Atsushi Toyoda, Makoto Ohnishi, Tetsuya Hayashi, Genomic characterization of β-glucuronidase–positive escherichia coli O157:H7 producing Stx2a, Emerging Infectious Diseases, 10.3201/eid2412.180404, 24, 12, 2219-2227, 2018.12, [URL], Among Shiga toxin (Stx)–producing Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a β-glucuronidase–positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/ stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (β-glucuronidase–negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones..
134. Yasuhiro Gotoh, Takako Taniguchi, Dai Yoshimura, Keisuke Katsura, Yuji Saeki, Yasutoshi Hirabara, Mayumi Fukuda, Ichiro Takajo, Junko Tomida, Yoshiaki Kawamura, Yoshitoshi Ogura, Takehiko Itoh, Naoaki Misawa, Akihiko Okayama, Tetsuya Hayashi, Multi-step genomic dissection of a suspected intra-hospital helicobacter cinaedi outbreak, Microbial Genomics, 10.1099/mgen.0.000236, 5, 1, 2019.01, [URL], Helicobacter cinaedi is an emerging pathogen causing bacteraemia and cellulitis. Nosocomial transmission of this microbe has been described, but detailed molecular-epidemiological analyses have not been performed. Here, we describe the results of a multi-step genome-wide phylogenetic analysis of a suspected intra-hospital outbreak of H. cinaedi that occurred in a hospital in Japan. The outbreak was recognized by the infectious control team (ICT) of the hospital as a sudden increase in H. cinaedi bacteraemia. ICT defined this outbreak case based on 16S rRNA sequence data and epidemiological information, but were unable to determine the source and route of the infections. We therefore re-investigated this case using whole-genome sequencing (WGS). We first performed a species-wide analysis using publicly available genome sequences to understand the level of genomic diversity of this under-studied species. The clusters identified were then separately analysed using the genome sequence of a representative strain in each cluster as a reference. These analyses provided a high-level phylogenetic resolution of each cluster, identified a confident set of outbreak isolates, and discriminated them from other closely related but distinct clones, which were locally circulating and invaded the hospital during the same period. By considering the epidemiological data, possible strain transmission chains were inferred, which highlighted the role of asymptomatic carriers or environmental contamination. The emergence of a subclone with increased resistance to fluoroquinolones in the outbreak was also recognized. Our results demonstrate the impact of the use of a closely related genome as a reference to maximize the power of WGS..
135. Yoshihiro Suzuki, Yuki Imafuku, Masateru Nishiyama, Kotaro Teranishi, Atsushi Jikumaru, Kei Nukazawa, Yoshitoshi Ogura, A highly efficient method for concentrating DNA from river water by combined coagulation and foam separation, Separation Science and Technology (Philadelphia), 10.1080/01496395.2019.1565774, 2019.01, [URL], Both Escherichia coli and Enterococci were collected in foam within 7 min from 500 mL of bacteria-spiked water by coagulation and foam separation using ferric chloride and milk casein. These bacterial DNA isolated in the 100 µL of extract from the foam more than 87.5% recovery using the DNeasy PowerWater® Kit. To test this method with water from three natural rivers, 0.67–2.70 µg of DNA were concentrated in 100 µL of extract from 1,000 mL of river water. When the DNA extract was subjected to 16S rRNA gene sequencing analysis, information on the bacterial flora could be obtained..
136. Keiji Nakamura, Noriko Shinoda, Yukihiro Hiramatsu, Shinya Ohnishi, Shigeki Kamitani, Yoshitoshi Ogura, Tetsuya Hayashi, Yasuhiko Horiguchi, BspR/BtrA, an Anti-σ Factor, Regulates the Ability of Bordetella bronchiseptica To Cause Cough in Rats, mSphere, 10.1128/mSphere.00093-19, 4, 2, 2019.04, [URL], Bordetella pertussis, B. parapertussis, and B. bronchiseptica cause respiratory infections, many of which are characterized by coughing of the infected hosts. The pathogenesis of the coughing remains to be analyzed, mainly because there were no convenient infection models of small animals that replicate coughing after Bordetella infection. Here, we present a coughing model of rats infected with B. bronchiseptica Rats, which are one of natural hosts of B. bronchiseptica, were readily infected with the organisms and showed frequent coughing. B. pertussis also caused coughing in rats, which is consistent with previous reports, but the cough response was less apparent than the B. bronchiseptica-induced cough. By using the rat model, we demonstrated that adenylate cyclase toxin, dermonecrotic toxin, and the type III secretion system are not involved in cough production, but BspR/BtrA (different names for the same protein), an anti-σ factor, regulates the production of unknown factor(s) to cause coughing. Rat coughing was observed by inoculation of not only the living bacteria but also the bacterial lysates. Infection with bspR (btrA)-deficient strains caused significantly less frequent coughing than the wild type; however, intranasal inoculation of the lysates from a bspR (btrA)-deficient strain caused coughing similarly to the wild type, suggesting that BspR/BtrA regulates the production of the cough factor(s) only when the bacteria colonize host bodies. Moreover, the cough factor(s) was found to be heat labile and produced by B. bronchiseptica in the Bvg+ phase. We consider that our rat model provides insight into the pathogenesis of cough induced by the Bordetella infection.IMPORTANCE Whooping cough is a contagious respiratory disease caused by Bordetella pertussis This disease is characterized by severe paroxysmal coughing, which becomes a heavy burden for patients and occasionally results in death; however, its pathogenesis remains largely unknown. The major obstacle to analyzing Bordetella-induced coughing is the lack of conventional animal models that replicate coughing. As Bordetella pertussis is highly adapted to humans, infection models in experimental animals are not considered to be well established. In the present study, we examined coughing in rats infected with B. bronchiseptica, which shares many virulence factors with B. pertussis Using this rat model, we demonstrated that some of the major virulence factors of Bordetella are not involved in cough production, but an anti-σ factor, BspR/BtrA, of B. bronchiseptica regulates the production of unknown cough-causing bacterial factor(s). Our results provide important clues to understand the mechanism by which Bordetella induces cough..
137. Naoki Takizawa, Yoshitoshi Ogura, Yoko Fujita, Takeshi Noda, Hideki Shigematsu, Tetsuya Hayashi, Ken Kurokawa, Local structural changes of the influenza A virus ribonucleoprotein complex by single mutations in the specific residues involved in efficient genome packaging, Virology, 10.1016/j.virol.2019.03.004, 531, 126-140, 2019.05, [URL], The influenza A virus genome consists of eight single-stranded negative-sense RNA segments. The noncoding regions located at the 3′- and 5′- ends of each segment are necessary for genome packaging, and the terminal coding regions are required to precisely bundle the eight segments. However, the nucleotide residues important for genome bundling are not defined. Here, we introduced premature termination codons in the hemagglutinin (HA) or matrix protein 2 (M2) gene and constructed virus libraries containing random sequences in the terminal coding regions. Using these virus libraries, we identified nucleotide residues involved in efficient virus propagation. Viral genome packaging was impaired in viruses that contained single mutations at these identified residues. Furthermore, these single mutations altered the local structure of the viral ribonucleoprotein complex. Our results show that specific nucleotide residues in the viral protein coding region are involved in forming the precise structure of the viral ribonucleoprotein complex..

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pure2017年10月2日から、「九州大学研究者情報」を補完するデータベースとして、Elsevier社の「Pure」による研究業績の公開を開始しました。
 
 
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