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ABSTRACTEnteroinvasive Escherichia coli(EIEC), a distinctive pathogenic form of E. colicausing dysentery, is similar in many properties to bacteria placed in the four species of Shigella. Shigellahas been separated as a genus but in fact comprises several clones of E. coli. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigellastrains. The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times. Three of the four clusters contain more than one O antigen type. One EIEC strain (an O112ac:H− strain) was found in Shigellacluster 3 but is not identical to the Shigellacluster 3 D2 and B15 strains with the same O antigen. Two forms of the virulence plasmid pINV have been identified in Shigellastrains by using the sequences of ipgDand mxiAgenes, and all but two of our EIEC strains have pINV A. The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with ShigellapINV A strains. The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions. Two strains were found in the pINV B form by using the ipgDsequence, with one strain having an mxiAsequence similar to the divergent sequence of D1. Clearly, EIEC and Shigellaspp. form a pathovar of E. coli.

ABSTRACTAll Shigellaand enteroinvasive Escherichia coli(EIEC) strains carry a 230-kb virulence plasmid (pINV) which is essential for their invasiveness. There are two sequence forms, pINV A and pINV B, of the plasmid (R. Lan, B. Lumb, D. Ryan, and P. R. Reeves, Infect. Immun. 69:6303-6309, 2001), and the recently sequenced pINV plasmid from Shigella flexneriserotype 5 is a pINV B form. In this study we sequenced the majority of the coding region of the pINV A form from S. flexneriserotype 6 other than insertion sequence or related sequences and compared it with the pINV B form. More than half of the genes sequenced appear to be under positive selection based on their low ratio of synonymous to nonsynonymous substitutions. This high proportion of selected differences indicates that the two pINV forms have functional differences, and comparative studies of pathogenicity in different Shigella-EIEC strains could be informative. There are also genes absent in the S. flexneriserotype 6 plasmid, including the sepAgene encoding serine protease, the major secreted protein of S. flexneriserotype 2a, and the stbABgenes, which encode one of the two partition systems found in S. flexneriserotype 5. The incompatibility of the two pINV forms appears to be due to either small differences in the mvpATpostsegregational killing system or the presence of an unknown system in pINVA.

ABSTRACTO antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria and is highly polymorphic. In this study, we obtained sequences of the O-antigen gene clusters for the Yersinia pseudotuberculosisantigens IA, IIA, and IVB. We propose that the IIA gene cluster was derived from the IVB cluster, one of the very few cases in which a parent gene cluster is identified, and that the IA gene cluster could be a hybrid of the IVB and IB gene clusters. All three O antigens contain 6-deoxy-d-mannoheptose, and we identified six genes for the biosynthetic pathway for the precursor of this sugar, GDP-6-deoxy-d-mannoheptose.

ABSTRACTShigellastrains are in reality clones ofEscherichia coliand are believed to have emerged relatively recently (G. M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567–10572, 2000). There are 33 O-antigen forms in these Shigellaclones, of which 12 are identical to O antigens of other E. colistrains. We sequenced O-antigen gene clusters from Shigella boydiiserotypes 4, 5, 6, and 9 and also studied the O53- and O79-antigen gene clusters of E. coli, encoding O antigens identical to those of S. boydiiserotype 4 and S. boydiiserotype 5, respectively. In both cases the S. boydiiand E. coliO-antigen gene clusters have the same genes and organization. The clusters of both S. boydii6 and S. boydii9 O antigens have atypical features, with a functional insertion sequence and a wzxgene located in the orientation opposite to that of all other genes in S. boydiiserotype 9 and an rmlCgene located away from other rmlgenes in S. boydiiserotype 6. Sequences of O-antigen gene clusters from another threeShigellaclones have been published, and two of them also have abnormal structures, with either the entire cluster or one gene being located on a plasmid in Shigella sonneiorShigella dysenteriae, respectively. It appears that a high proportion of clusters coding for O antigens specific toShigellaclones have atypical features, perhaps indicating recent formation of these gene clusters.

ABSTRACTThree genes, ipgD, mxiC, and mxiA,all in the invasion region of the Shigellavirulence plasmid, were sequenced from strains representing a range ofShigellaserotypes and from two enteroinvasiveEscherichia coli(EIEC) isolates. The plasmids can be classified into two relatively homogeneous sequence forms which are quite distinct. pINV A plasmids are found in Shigella flexneristrains F6 and F6A, S. boydiistrains B1, B4, B9, B10, B14, and B15, S. dysenteriaestrains D3, D4, D6, D8, D9, D10, and D13, and the two EIEC strains (M519 and M520). pINV B plasmids are present in S. flexneristrains F1A, F2A, F3A, F3C, F4A, and FY, two S. boydiistrains (B11 and B12), and S. sonnei. The D1 pINV plasmid is a recombinant with ipgDgene more closely related to those of pINV A but with mxiAandmxiCgenes more closely related to those of pINV B. The phylogenetic relationships of the plasmid and those of the chromosomal genes of Shigellastrains are largely consistent. The cluster 1 and cluster 3 strains tested (G.M. Pupo, R. Lan, and P. R. Reeves, Proc. Natl. Acad. Sci. USA 97:10567–10572, 2000) have pINV A and pINV B plasmids, respectively. However, of the three cluster 2 strains (B9, B11, and B15), B9 and B15 have pINV A while B11 has a pINV B plasmid. Those Shigella(D8 and D10 and S. sonnei) and EIEC strains which do not group with the main body of Shigellastrains based on chromosomal genes were found to have plasmids belonging to one or the other of the two types and must have acquired these by lateral transfer.

ABSTRACTCapsular polysaccharide (CPS) is a major virulence factor inStreptococcus pneumoniae. CPS gene clusters of S. pneumoniaetypes 4, 6B, 8, and 18C were sequenced and compared with those of CPS types 1, 2, 14, 19F, 19A, 23F, and 33F. All have the same four genes at the 5′ end, encoding proteins thought to be involved in regulation and export. Sequences of these genes can be divided into two classes, and evidence of recombination between them was observed. Next is the gene encoding the transferase for the first step in the synthesis of CPS. The predicted amino acid sequences of these first sugar transferases have multiple transmembrane segments, a feature lacking in other transferases. Sugar pathway genes are located at the 3′ end of the gene cluster. Comparison of the four dTDP-l-rhamnose pathway genes (rmlgenes) of CPS types 1, 2, 6B, 18C, 19F, 19A, and 23F shows that they have the same gene order and are highly conserved. There is a gradient in the nature of the variation of rmlgenes, the average pairwise difference for those close to the central region being higher than that for those close to the end of the gene cluster and, again, recombination sites can be observed in these genes. This is similar to the situation we observed for rmlgenes of O-antigen gene clusters of Salmonella enterica. Our data indicate that the conserved first four genes at the 5′ ends and the relatively conservedrmlgenes at the 3′ ends of the CPS gene clusters were sites for recombination events involved in forming new forms of CPS. We have also identified wzxand wzygenes for all sequenced CPS gene clusters by use of motifs.

ABSTRACTEpidemic Vibrio choleraestrains possess a large cluster of essential virulence genes on the chromosome called theVibriopathogenicity island (VPI). The VPI contains thetcpgene cluster encoding the type IV pilus toxin-coregulated pilus colonization factor which can act as the cholera toxin bacteriophage (CTXΦ) receptor. The VPI also contains genes that regulate virulence factor expression. We have fully sequenced and compared the VPI of the seventh-pandemic (El Tor biotype) strain N16961 and the sixth-pandemic (classical biotype) strain 395 and found that the N16961 VPI is 41,272 bp and encodes 29 predicted proteins, whereas the 395 VPI is 41,290 bp. In addition to various nucleotide and amino acid polymorphisms, there were several proteins whose predicted size differed greatly between the strains as a result of frameshift mutations. We hypothesize that these VPI sequence differences provide preliminary evidence to help explain the differences in virulence factor expression between epidemic strains (i.e., the biotypes) of V. cholerae.

ABSTRACTEscherichia coliSonnei has an O antigen identical to that of Plesiomonas shigelloidesO17, and its O-antigen gene cluster is located on a plasmid. By sequencing the chromosomal O-antigen gene cluster of P. shigelloidesO17 and comparing it with that of Sonnei, we showed that Sonnei gained its O-antigen genes recently.

ABSTRACTStudies of the Vibrio choleraepopulation, using molecular typing techniques, have shown the existence of several pathogenic clones, mainly sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones. However, the relationship of the pathogenic clones to environmental V. choleraeisolates remains unclear. A previous study to determine the phylogeny of V. choleraeby sequencing the asd(aspartate semialdehyde dehydrogenase) gene of V. choleraeshowed that the sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones had very differentasdsequences which fell into separate lineages in theV. choleraepopulation. As gene trees drawn from a single gene may not reflect the true topology of the population, we sequenced the mdh(malate dehydrogenase) and hlyA(hemolysin A) genes from representatives of environmental and clinical isolates of V. choleraeand found that the mdhand hlyAsequences from the three pathogenic clones were identical, except for the previously reported 11-bp deletion inhlyAin the sixth-pandemic clone. Identical sequences were obtained, despite average nucleotide differences in the mdhand hlyAgenes of 1.52 and 3.25%, respectively, among all the isolates, suggesting that the three pathogenic clones are closely related. To extend these observations, segments of the recAand dnaEgenes were sequenced from a selection of the pathogenic isolates, where the sequences were either identical or substantially different between the clones. The results show that the three pathogenic clones are very closely related and that there has been a high level of recombination in their evolution.

ABSTRACTThe O157:H7 clone of Escherichia coli, which causes major, often prolonged outbreaks of gastroenteritis with hemolytic-uremic syndrome (HUS) such as those in Japan, Scotland, and the United States recently, is thought to be resident normally in cattle or other domestic animals. This clone is of major significance for public health and the food industry. We have developed a fast method for sequencing a given O antigen gene cluster and applied it to O157. The O157 O antigen gene cluster is 14 kb in length, comprising 12 genes and a remnant H-repeat unit. Based on sequence similarity, we have identified all the necessary O antigen genes, including five sugar biosynthetic pathway genes, four transferase genes, the O unit flippase gene, and the O antigen polymerase gene. By PCR testing against all 166 E. coliO serogroups and a range of gram-negative bacterial strains, including some that cross-react serologically with E. coliO157 antisera, we have found that certain O antigen genes are highly specific to O157 E. coli. This work provides the basis for a sensitive test for rapid detection of O157 E. coli. This is important both for decisions on patient care, since early treatment may reduce the risk of life-threatening complications, and for detection of sources of contamination. The method for fast sequencing of O antigen gene clusters plus an ability to predict which genes will be O antigen specific will enable PCR tests to be developed as needed for other clones of E. colior, once flanking genes are identified, clones of any gram-negative bacterium.