Construction of a physical map of the chromosome of Shigella flexneri 2a and the direct assignment of nine virulence-associated loci identified by Tn5 insertions

To establish the molecular basis of the chromosomal virulence genes of Shigella flexneri 2a (YSH6000), a Notl restriction map of the chromosome was constructed by exploiting Notl-linking clones, partial Notl digestion and DNA probes from various genes of Escherichia coli K-12. The map revealed at least three local differences in the placements of genes between YSH6000 and E. coli K-12. Using the additional Notl sites introduced by Tn5 insertion, nine virulence loci identified previously by random Tn5 insertions were physically mapped on the chromosome. To demonstrate the versatility of the Notl map in direct assignment of the virulence loci tagged by Tn5 to a known genetic region in E. coli K-12, the major class of avirulent mutants defective in the core structure of lipopolysaccharide (LPS) was examined for the sites of Tn5 insertions. The two Notl segments created by the Tn5 insertion in the Notl fragment were analysed by Southern blotting with two DNA probes for the 5' and 3' flanking regions of the rfa region, and shown to hybridize separately with each of them, confirming the sites of Tn5 in the rfa locus. This approach will facilitate direct comparison genetically mapped Tn5 insertion mutations of S. flexneri with genes physically determined in E. coli K-12.     

Resistance to mecillinam produced by the co-operative action of mutations affecting iipopolysaccharide, spoT, and cya or crp genes of Salmonella typhimurium

Lipopolysaccharide (LPS), spoT, and cya or crp mutations individually do not affect the minimum inhibitory concentration of mecillinam on Salmonella typhimurium. However, when mutations of two of these types were combined in the same strain, high-level resistance appeared, and increased even further when all three types of mutations were present. Most mutations affecting LPS (rfa, rfb, rfc) showed this behaviour, although to different degrees. The highest resistance to mecillinam was caused by galE and rfc mutations whereas almost no effect was noticed with rfaB or rfaK mutations. This phenomenon appears to be specific for mecillinam since none of several other antibiotics elicited it. Reduction of guanosine tetraphosphate (ppGpp) levels by introduction of a relA mutation did not significantly affect the MIC of mecillinam on strains carrying different combinations of SpoT, galE, and cya or crp mutations. All the strains produced spherical cells in medium with a low concentration (0.05 μg ml^?1) of the antibiotic. These results suggest that the antibacterial action of mecillinam on S. typhimurium is somehow dependent on the interaction of LPS, cyclic AMP/cyclic AMP receptor protein (cAMP/CRP), and SpoT. The reported resistance to mecillinam of cya and crp mutants of Escherichia coli K-12 is probably due to the natural LPS defectiveness of this strain.     

Molecular cloning and genetic analysis of the rfb region from Shigella flexneri type 6 in Escherichia coli K-12

The rfb gene cluster which determines the biosynthesis of the Shigella flexneri serotype 6 O-antigen specificity has been cloned in pHC79, generating plasmids pPM3115 and pPM3116. These plasmids mediate expression, in Escherichia coli K-12, of lipopolysaccharides (LPS) immunologically similar to the S. flexneri type 6 LPS as judged by SDS-PAGE and Western-immunoblot analysis using S. flexneri type 6 specific antisera. Thus, unlike other S. flexneri serotypes, no additional loci are required for serotype specificity. This expression is independent of E. coli K-12 rfb genes. Southern-hybridization analysis using the 16.2-kb BglII probe from S. flexneri type 6 rfb region detected very little sequence homology in S. flexneri serotypes 1-5, however, some homology was detected with E. coli O2 and O18, but not in E. coli 0101 strains, Salmonella and Vibrio cholerae.     

Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1

A plasmid that included both an 8.9 kb chromosomal DNA insert containing genes from the rfb cluster of Shigella dysenteriae 1 and a smaller insert containing the rfp gene from a S. dysenteriae 1 multicopy plasmid resulted in efficient expression of O antigen in an rfb-deleted strain of Escherichia coli K-12. Eight genes were identified in the rfb fragment: the rfbB-CAD cluster which encodes dTDP-rhamnose synthesis, rfbX which encodes a hydrophobic protein involved in assembly of the O antigen, rfc which encodes the O antigen polymerase, and two sugar transferase genes. The production of an O antigen also required the E. coli K-12 rfe gene, which is known to encode a transferase which adds N-acetyl-glucosamine phosphate to the carrier lipid unde-caprenol phosphate. Thus Rfe protein appears to function as an analogue of the Salmonella RfbP protein to provide the first sugar of the O unit. Functional analysis of the other genes was facilitated by the fact that partial O units of one, two or three sugars were efficiently transferred to the lipopolysaccharide core. This analysis indicated that the plasmid-encoded Rfp protein is the transferase that adds the second sugar of the O unit while the two rfb transferases add the distal sugars to make an O antigen whose structure is (Rha–Rha–Gal–GlcNAc)n. The use of the rfe gene product as the transferase that adds the first sugar of an O unit is a novel mechanism which may be used for the synthesis of other enteric O antigens.     

Molecular cloning and characterization of the rfc gene of Pseudomonas aeruginosa (serotype O5)

Previous work from our laboratory has shown that cosmid clone pFVl00, containing a 26 kb insert, is able to restore O-antigen synthesis in serotype O5 rough mutants of Pseudomonas aeruginosa. Mobilization of pFV100 into two P. aeruginosa semi-rough (SR) mutants, AK14O1 and rd7513, resulted in O-antigen expression, indicating that pFV100 may contain an O-polymerase (rfc) gene. pFV.TK6, a subclone of pFVl00 that contains a 5.6 kb chromosomal insert, was able to complement O-antigen expression in these SR mutants. Mutagenesis of pFV.TK6 using Tn1000 exposed a 1.5 kb region that was essential for complementing O-antigen expression in AK14O1. A 2.0 kb Xhol-HindIII fragment, containing this region, was cloned into vector pUCP26 and the resulting plasmid called pFV.TK8. In Southern analysis of the 20 P aeruginosa serotypes using a probe generated from the 1.5 kb Xhol fragment of pFV.TK8, the rfc probe hybridized to a common fragment of the cross-reactive O2-O5-O16-O18-O20 serogroup, suggesting that these serotypes may share a common O-polymerase gene. In functional studies of the rfc gene, the PAOl (serotype O5) chromosomal rfc was mutated using a gene-replacement strategy. These knockout mutants expressed the SR lipopolysaccharide (LPS) phenotype, which indicated that they were no longer producing a functional O-polymerase enzyme. Nucleotide sequence analysis of the insert DNA of pFV.TK8 revealed one open reading frame (ORF), designated ORF48.9, which could code for a 48.9 kDa protein. In comparisons of the P. aeruginosa rfc nucleotide and amino acid sequences with DNA and protein databases, no significant homology was found. However, the deduced structure of the P. aeruginosa Rfc protein indicated that it is very hydrophobic and contains 11 putative membrane-spanning domains. Therefore, the predicted structure is similar to that of other reported Rfc proteins. Furthermore, comparison of the amino acid composition and codon usage of the P. aeruginosa Rfc with other Rfc proteins revealed significant similarity between them.     

Effect of mutations in Shigella flexneri chromosomal and plasmid-encoded lipopolysaccharide genes on invasion and serum resistance

This study shows that both length and distribution of lipopolysaccharide (LPS) are important for Shigella flexneri invasion and virulence. Mutants were generated in the chromosomal LPS synthesis genes rfa , rfb , and rol , and in a plasmid-encoded O-antigen chain-length regulator, cld _pHS-2. LPS analysis showed that mutations in rfb genes and in a candidate rfaL gene either eliminated the entire O-antigen side chains or produced chains of greatly reduced length. Mutation in a previously unidentified gene, rfaX , affected the LPS core region and resulted in reduced amounts of O-antigen. Mutants defective in cld _pHS-2 or rol had different distributions of O-antigen chain lengths. The results of tissue-culture cell invasion and plaque assays, the Serény test, and serum-sensitivity assay suggested roles for the different LPS synthesis genes in bacterial survival and virulence; rfaL, rfaX and rfb loci are required for serum resistance and intercellular spread, but not for invasion; cld _pHS-2 is required for resistance to serum killing and for full inflammation in the Serény test, but not for invasion or intercellular spread, while rol is required for normal invasiveness and plaque formation, but not for serum resistance. Thus, O-antigen synthesis and chain-length regulation genes encoded on both the chromosome and the small plasmid pHS-2 play important roles in S. flexneri invasion and virulence.     

Genetic Transfer of Salmonella typhimurium and Escherichia coli Lipopolysaccharide Antigens to Escherichia coli K-12

Escherichia coli K-12 varkappa971 was crossed with a smooth Salmonella typhimurium donor, HfrK6, which transfers early the ilv-linked rfa region determining lipopolysaccharide (LPS) core structure. Two ilv(+) hybrids differing in their response to the LPS-specific phages FO and C21 were then crossed with S. typhimurium HfrK9, which transfers early the rfb gene cluster determining O repeat unit structure. Most recombinants selected for his(+) (near rfb) were agglutinated by Salmonella factor 4 antiserum. Transfer of an F' factor (FS400) carrying the rfb-his region of S. typhimurium to the same two ilv(+) hybrids gave similar results. LPS extracted from two ilv(+),his(+), factor 4-positive hybrids contained abequose, the immunodominant sugar for factor 4 specificity. By contrast, his(+) hybrids obtained from varkappa971 itself by similar HfrK9 and F'FS400 crosses were not agglutinated by factor 4 antiserum, indicating that the parental E. coli varkappa971 does not have the capacity to attach Salmonella O repeat units to its LPS core. It is concluded that the Salmonella rfb genes are expressed only in E. coli varkappa971 hybrids which have also acquired ilv-linked genes (presumably rfa genes affecting core structure or O-translocase ability, or both) from a S. typhimurium donor. When E. coli varkappa971 was crossed with a smooth E. coli donor, Hfr59, of serotype O8, which transfers his early, most his(+) recombinants were agglutinated by E. coli O8 antiserum and lysed by the O8-specific phage, Omega8. This suggests that, although the parental E. coli K-12 strain varkappa971 cannot attach Salmonella-specific repeat units to its LPS core, it does have the capacity to attach E. coli O8-specific repeat units.     

Acetylation of O-specific lipopolysaccharides from Shigella flexneri 3a and 2a occurs in Escherichia coli K-12 carrying cloned S. flexneri 3a and 2a rfb genes.

Most of the Shigella flexneri O-specific serotypes result from O-acetyl and/or glucosyl groups added to a common O-repeating unit of the lipopolysaccharide (LPS) molecule. The genes involved in acetylation and/or glucosylation of S. flexneri LPS are physically located on lysogenic bacteriophages, whereas the rfb cluster contains the biosynthesis genes for the common O-repeating unit (D.A.R. Simmons and E. Romanowska, J. Med. Microbiol. 23:289-302, 1987). Using a cosmid cloning strategy, we have cloned the rfb regions from S. flexneri 3a and 2a. Escherichia coli K-12 containing plasmids pYS1-5 (derived from S. flexneri 3a) and pEY5 (derived from S. flexneri 2a) expressed O-specific LPS which reacted immunologically with S. flexneri polyvalent O antiserum. However, O-specific LPS expressed in E. coli K-12 also reacted with group 6 antiserum, indicating the presence of O-acetyl groups attached to one of the rhamnose components of the O-repeating unit. This was confirmed by measuring the amounts of acetate released from purified LPS samples and also by the chemical removal of O-acetyl groups, which abolished group 6 reactivity. The O-acetylation phenotype was absent in an E. coli strain with an sbcB-his-rfb chromosomal deletion and could be restored upon conjugation of F' 129, which carries sequences corresponding to a portion of the deleted region. Our data demonstrate that E. coli K-12 strains possess a novel locus which directs the O acetylation of LPS and is located in the sbcB-rfb region of the chromosomal map.     

Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a.

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.     

Biological Activities of Lipopolysaccharide Isolated from Vibrio cholerae O139, a New Epidemic Strain for Recent Cholera in Indian Subcontinent

Biological activities of lipopolysaccharide (LPS) isolated from Vibrio cholerae O139, a new causative agent for recent cholera epidemic in Indian subcontinent, were investigated in comparison with those of LPS from O1 V. cholerae. V. cholerae O139 LPS exerted mitogenic activity, lethal toxicity and Shwartzman reaction to the same extent as those observed for O1 V. cholerae LPS, although these activities except for lethal toxicity were obviously lower than those of Salmonella typhimurium LT-2 LPS used as a reference. It was, therefore, suggested that O139 LPS does not contribute to the high infective and pathogenic potentials of the V. cholerae O139 strain as in the case of O1 V. cholerae.     

A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome

Many Salmonella typhimurium genes are required for bacterial entry into host cells. P22 transduction analysis has localized several invasion loci near minute 59 on the S. typhimurium chromosome. To further characterize the 59–60 min chromosomal region, we determined the physical and genetic map of 85 kb of S. typhimurium DNA between srl and cysC. It was previously shown that some of the invasion genes from this region are not present in Escherichia coli K-12. We examined whether other S. typhimurium genes on the 85 kb of DNA were similarly absent from E. coli We found that a contiguous 40 kb fragment of the S. typhimurium chromosome which encodes invasion genes is absent from the corresponding region of the E. coli K-12 chromosome and may represent a pathogenicity island. We speculate that acquisition of the 40 kb region must have significantly advanced the evolution of Salmonella as a pathogen.     

An N-[(R)-(-)-2-Hydroxypropionyl]-α-L-Perosamine Homopolymer Constitutes the O Polysaccharide Chain of the Lipopolysaccharide from Vibrio cholerae O144 Which Has Antigenic Factor(s) in Common with V. cholerae O76

Chemical and serological studies were performed with the lipopolysaccharide (LPS) from Vibrio cholerae O144 (O144). The LPS of O144 contained D -glucose, D -galactose, L -glycero-D -manno-heptose, D -fructose, D -quinovosamine (2-amino-2,6-dideoxy-D -gluco-pyranose) and L -perosamine (4-amino-4,6-dideoxy-L -manno-pyranose). The perosamine, a major component sugar of the LPS from O144, was in an L -configuration, as is also the case in the LPS from V. cholerae O76 (O76), in contrast to the D -configuration of the perosamine in the LPS of V. cholerae O1. A structural analysis revealed that the O polysaccharide chain of the LPS from O144 is an α(1 → 2)-linked homopolymer of (R)-(-)-2-hydroxypropionyl-L -perosamine. The serological cross-reactivity between O144 and O76 was clearly revealed by cross-agglutination and cross-agglutinin absorption tests with whole cells, as well as by passive hemolysis tests with sheep red-blood cells that had been sensitized with the LPS from O144 and O76. In contrast, in passive hemolysis tests, the LPS of O144 did not cross-react serologically with the LPSs from other strains such as V. cholerae O1 (Ogawa and Inaba), V. cholerae O140, Vibrio bio-serogroup 1875 (Original and Variant) and Yersinia enterocolitica O9. The LPSs from these strains consist of O polysaccharide chains composed of α(1 → 2)-linked homopolymers of D -perosamine with various N-acyl groups, and they share the Inaba antigen factor C of V. cholerae O1 in common. The results obtained in this study demonstrate that the absolute configuration of the perosamine residue in homopolymers plays a very important role in the expression of the serological specificity of the Inaba antigen factor C of V. cholerae O1.     

Construction and characterization of isogenic O-antigen variants of Salmonella typhi

A 7.5 kb Kpnl-generated fragment, from within the rfb cluster of Salmonella typhimurium LT2 that encodes abequose synthase (the rfbJ gene) which is necessary for O4 antigen synthesis, and flanking sequences, was inserted into a suicide vector. Using allelic exchange techniques, these rfb sequences of S. typhimurium were integrated into the rfb clusters of wild-type Salmonella typhi Vi-positive strain ISP 1820 (i.e. serotype 09,12; Vi⁺ H-d), S. typhi Vi-negative strain H400 (i.e. serotype 09,12; Vi⁻; H-d), and a double aro mutant of S. typhi ISP 1820, strain CVD 906, resulting in the isolation of strains H325, H404 and CVD 906-O4, respectively. Immunoblot analysis of lipopolysaccharide (LPS) purified from H325, H404 and CVD 906-O4 demonstrated that these 8trains express the 04 antigen (an abequose residue) in place of the O9 antigen (a tyvelose residue) in the LPS molecule. Hence, the serotype of H325 is O4,12; Vi⁺; H-d and the serotype of H404 is O4,12; Vi⁻; H-d. DNA hybridization analysis of chromosomal DNA from H325, H404 and CVD 906-O4 confirmed that a precise recombination event within sequences flanking rfbSE of S. typhi (which encodes the enzymes necessary for cytidine diphosphate-tyvelose synthesis) resulted in replacement of rfbSE with rfbJ (which encodes abequose synthase and is necessary for O4 synthesis) of S. typhimurium in strains H325, H404 and CVD 906-O4. The resistance of each strain to the bactericidal effects of guinea-pig serum (GPC) was assessed. Whereas ISP 1820, H325 and H404 exhibit similar resistance patterns in GPC, strain H400 is sensitive to the bactericidal effects of GPC. The results suggest that the development of the O-antigen serotype diversity of Salmonella is probably the result of both sequence divergence and recombination     

The absence of a surface protease, OmpT, determines the intercellular spreading ability of Shigella: the relationship between the ompT and kcpA loci

A large plasmid-encoded protein, VirG, on the bacterial surface is essential for the spreading of Shigella by eliciting polar deposition of filamentous actin In the cytoplasm of epithelial cells. VirG expression from the large plasmid is diminished greatly when it is introduced into Escherichia coli K-12 from Shigella. In an attempt to identify factors affecting VirG expression, we found that the absence of the ompT gene, encoding outer membrane protease OmpT, restored full production of VirG protein to E. coli K-12. Conversely, upon introduction of the ompT gene of E. coii K-12 into Shigella, spreading ability was completely abolished, probably because of the proteolytic degradation of VirG protein by OmpT. Analysis of the DNA sequence of the ompT region indicated that the absence of the ompT gene occurred in Shigella and enteroinvasive E. coli strains, and that the absent DNA segment corresponded to a remnant lambdoid phage structure found in E. coli K-12, which encompasses a 21 kb DNA segment spanning from argU through to the ompT genes. Since ompT is located near purE in E. coli K-12 and a virulence locus for provoking keratocon-junctivitis in the eyes of guinea-pigs, named kcpA is located near purE in S. fiexnerl, and the two loci are involved in VirG expression, the KcpA～ mutants of S. flexneri 2a constructed were examined for correlation between acquisition of ompT and VirG degradation. Our data suggest that the previous recognition of a kcpA locus in S. flexneri is the result of transfer of the ompr gene from E. coli K-12, giving rise to a KcpA phenotype. These results indicate that the lack of OmpT protease confers upon Shigella the ability to spread into adjacent epithelial cells.     

Comparative analysis of FUR regulons in gamma-proteobacteria

Iron is an essential element for the survival and pathogenesis of bacteria. The strict control of iron homeostasis is mediated by the FUR repressor, which is highly conserved among various bacterial species. Here we apply the comparative genomics approach to analyze candidate Fur-binding sites in the genomes of Escherichia coli (K12 and O157:H7), Salmonella typhi, Yersinia pestis and Vibrio cholerae. We describe a number of new loci encoding siderophore biosynthesis and transport proteins. A new regulator of iron-acquisition systems was found in S.typhi. We predict FUR regulation for several virulence systems. We also predict FUR regulation for the chemotaxis system of V.cholerae that is probably involved in the process of pathogenesis.     

Molecular cloning and expression in Escherichia coli K-12 of the O101 rfb region from E. coli B41 (O101:K99/ F41) and the genetic relationship to other O101 rfb loci

The gene cluster (rfb region) which determines the synthesis of O101 lipopolysaccharide (LPS) O-antigen was cloned from the Escherichia coli O101:K99:F41 reference strain B41 to give plasmid pPM1301. The smallest subclones represented by pPM1305 and pPM1330 expressed O-antigen in E. coli K-12 similar to (but not identical to) B41, as judged by immunogold electron microscopy and silver staining of LPS separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least six proteins were detected by minicell analysis of proteins encoded by pPM1305, which suggests that O-antigen synthesis is genetically complex. Restriction and deletion analysis demonstrated that a minimum of 8.9 kb and a maximum of 11.8 kb are required for O101 O-antigen biosynthesis in E. coli K-12. Examination of LPS banding patterns of other O101 isolates by SDS-PAGE suggested heterogeneity of LPS structure. Southern DNA hybridization analysis using radiolabelled subclones of pPM1305 demonstrated that there was close relationship among the O101 ETEC isolates.     

Genetic analysis of the genes involved in synthesis of the lipopolysaccharide core in Escherichia coli K-12: three operons in the rfa locus.

The region of the Escherichia coli K-12 chromosome encoding the enzymes responsible for the synthesis of responsible for the synthesis of the lipopolysaccharide (LPS) core has been cloned in vivo by using a mini-Mu vector. This region, formerly known as the rfa locus, comprises 18 kb of DNA between the markers tdh and rpmBG. Results of in vitro mutagenesis of this region with MudII1734 indicate the presence of at least 17 open reading frames or genes, a number considerably higher than expected on the basis of genetic and biochemical studies. Specific insertions in different genes have been recombined into the chromosome, and the mutations have been phenotypically characterized. Complementation analysis indicates that these genes are arranged in three different operons transcribed in opposite directions. A detailed physical map of this region has been constructed on the basis of complementation analysis, fusion protein data, and phenotypic characterizations. Additionally, the role of some genes in the synthesis of LPS has been defined by complementation analysis with known Salmonella typhimurium LPS mutants. The genetic organization of this locus seems to be identical in E. coli K-12 and S. typhimurium.     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S. sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey models, vaccinated animals were protected against the challenges of wild S. flexneri 2a strain 2457T and S. sonnei strain S9.     

The complete DNA sequence of the O antigen gene region of Plesiomonas shigelloides serotype O17 which is identical to Shigella sonnei form I antigen

We cloned and determined the sequence of a DNA region of approximately 15-kb containing the cluster of genes required for O17 antigen expression in the Escherichia coli K-12 strain from the chromosome of Plesiomonas shigelloides serotype O17:H2 strain. The sequencing analysis revealed that the minimum essential region of the P. shigelloides O17 antigen gene cluster had a size of approximately 11.5-kb and contained 9 contiguous open reading frames (ORFs), which were almost identical to the corresponding ORFs of Shigella sonnei form I antigen gene region, except for IS630 sequence, at the DNA as well as amino acid levels. The putative function of most of the ORFs could be determined on the basis of amino acid sequence similarities and characteristics. In addition, the G+C content of the P. shigelloides O17 antigen genes was lower than that of the chromosomal DNA of P. shigelloides and S. sonnei, suggesting that both P. shigelloides O17 and S. sonnei form I antigen genes had been derived from the same origin with a low G+C content.