||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, 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..
||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, 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..
||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, 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..
||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, 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..
||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, 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..
||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, 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..
||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, 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..
||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, 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..
||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, 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..