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Vibrio cholerae serotype O139: Swapping genes for surface polysaccharide biosynthesis
Abstract
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... Chemical and structural analysis showed that the capsule and O-antigen (lipopolysaccharide, LPS) consist of the same repeating unit [3,6,7]. The sequence of the entire 40-kb fragment responsible for the synthesis, assembly, and transport of these polysaccharides in V. cholerae O139 made it possible to identify 28 open reading frames (ORFs) [8,9], and a function has been assigned to 16 of them [9]. This 40kb fragment acquired by the new strain also contains an insertion sequence (IS), designated IS1358 yIQW [10]. ...
... Chemical and structural analysis showed that the capsule and O-antigen (lipopolysaccharide, LPS) consist of the same repeating unit [3,6,7]. The sequence of the entire 40-kb fragment responsible for the synthesis, assembly, and transport of these polysaccharides in V. cholerae O139 made it possible to identify 28 open reading frames (ORFs) [8,9], and a function has been assigned to 16 of them [9]. This 40kb fragment acquired by the new strain also contains an insertion sequence (IS), designated IS1358 yIQW [10]. ...
... This 40kb fragment acquired by the new strain also contains an insertion sequence (IS), designated IS1358 yIQW [10]. This IS element is homologous with the ISlike H-rpt (Hinc repeat) elements of Escherichia coli K-12 [11,12], and encodes a putative transposase possibly involved in the horizontal transfer of the Oantigen-and capsule-encoding genes [8,9]. Somatic (O) antigen factors cross-reacting with V. cholerae O139 have been found in V. cholerae O22 and O155 [13] and in other bacterial species including Aeromonas trota [14] and Salmonella enterica [15]. ...
The new epidemic strain O139 of Vibrio cholerae, the etiologic agent of cholera, has probably emerged from the pandemic strain O1 E1 Tor through a genetic rearrangement involving the horizontal transfer of exogenous O-antigen- and capsule-encoding genes of unknown origin. In V. cholerae O139, these genes are associated with an insertion sequence designated IS1358O139. In this work, we studied the distribution of seven genes flanking the IS1358O139 element in 13 serovars of V. cholerae strains. All these O139 genes and an IS1358 element designated IS1358O22-1 were only found in V. cholerae O22 with a similar genetic organization. Sequence analysis of a 4.5-kb fragment containing IS1358O22-1 and the adjacent genes revealed that these genes are highly homologous to those of V. cholerae O139. These results suggest that strains of V. cholerae O22 from the environment might have been the source of the exogenous DNA resulting in the emergence of the new epidemic strain O139.
... V. cholerae O139 Bengal, a novel pathogen that emerged in late 1992, has remained endemic in South Asia [8]. There is overwhelming genetic evidence supporting the hypothesis that the O139 serogroup emerged from an O1 El Tor strain by horizontal transfer of the O-antigen gene cluster [9,10]. The evolution of V. cholerae, as a pathogen, is fairly well understood. ...
... The V. cholerae O139-specific ϕJA1 has been further characterized. It was hypothesized that the O139 Oantigen is the phage receptor since mutants lacking the O-antigen were found to be phage resistant [9,19]. Furthermore, a phage-encoded lyase enzyme was shown to degrade the O139 polysaccharide [21,22]. ...
Background
Vibrio cholerae O139 Bengal is the only serogroup other than O1 implicated in cholera epidemics. We describe the isolation and characterization of an O139 serogroup-specific phage, vB_VchP_VchO139-I (ϕVchO139-I) that has similar host range and virion morphology as phage vB_VchP_JA1 (ϕJA1) described previously. We aimed at a complete molecular characterization of both phages and elucidation of their genetic and structural differences and assessment of their genetic relatedness to the N4-like phage group.
Methods
Host-range analysis and plaque morphology screening were done for both ϕJA1 and ϕVchO139-I. Both phage genomes were sequenced by a 454 and Sanger hybrid approach. Genomes were annotated and protein homologies were determined by Blast and HHPred. Restriction profiles, PFGE patterns and data on the physical genome structure were acquired and phylogenetic analyses were performed.
Results
The host specificity of ϕJA1 has been attributed to the unique capsular O-antigen produced by O139 strains. Plaque morphologies of the two phages were different; ϕVchO139-I produced a larger halo around the plaques than ϕJA1. Restriction profiles of ϕJA1 and ϕVchO139-I genomes were also different. The genomes of ϕJA1 and ϕVchO139-I consisted of linear double-stranded DNA of 71,252 and 70,938 base pairs. The presence of direct terminal repeats of around 1974 base pairs was demonstrated. Whole genome comparison revealed single nucleotide polymorphisms, small insertions/deletions and differences in gene content. Both genomes had 79 predicted protein encoding sequences, of which only 59 were identical between the two closely related phages. They also encoded one tRNA-Arg gene, an intein within the large terminase gene, and four homing endonuclease genes. Whole genome phylogenetic analyses of ϕJA1 and ϕVchO139-I against other sequenced N4-like phages delineate three novel subgroups or clades within this phage family.
Conclusions
The closely related phages feature significant genetic differences, in spite of being morphologically identical. The phage morphology, genetic organization, genomic content and large terminase protein based phylogeny support the placement of these two phages in the Podoviridae family, more specifically within the N4-like phage group. The physical genome structure of ϕJA1 could be demonstrated experimentally. Our data pave the way for potential use of ϕJA1 and ϕVchO139-I in Vibrio cholerae typing and control.
... Two hypotheses have been proposed to explain the emergence of V. cholerae O139. The first hypothesis proposes that a transposition event mediated by the IS element IS1358 resulted in the replacement of the O1 wbe genes with the O139 wbf genes (26,(37)(38)(39). The second hypothesis involves a homologous recombination event resulting in the replacement of the O1 wbe region en masse by the O139 wbf region (12,26,40). ...
... The absence of phage att-like sites in the O139 wbf cluster suggests that the mechanism and the mode of horizontal transfer may be different from that of PAIs, which are thought to be transferred as phages. Two mechanisms have been proposed for the acquisition of O-antigen clusters: IS-mediated transposition (26,38,39) and homologous recombination (11,26,38). It has been proposed that two copies of IS1358 are present in the O139 wbf region (a degenerate copy just upstream of IS1358) and that this region was transferred as a transposable element (38). ...
The DNA sequence of the O-antigen biosynthesis cluster (wbf) of a recently emergent pathogen, Vibrio cholerae serogroup O139, has been determined. Here we report the sequence of the genes downstream of the O139 wbfX gene and analysis of the genes flanking the wbf gene cluster in other serogroups. The gene downstream of wbfX, designated rjg (right junction gene), is predicted to be not required for O-antigen biosynthesis but appears to be a hot spot for DNA rearrangements. Several variants of the rjg gene (three different insertions and a deletion) have been found in other serogroups. DNA dot blot analysis of 106 V. cholerae strains showed the presence of the left and right junction genes, gmhD and rjg, respectively, in all strains. Further, these genes mapped to a single I-CeuI fragment in all 21 strains analyzed by pulsed-field gel electrophoresis, indicating a close linkage. The insertion sequence element IS1358, found in both O1 and O139 wb* regions, is present in 61% of the strains tested; interestingly, where present, it is predominantly linked to the wb* region. These results indicated a cassette-like organization of the wb* region, with the conserved genes (gmhD and rjg) flanking the divergent, serogroup-specific wb* genes and IS1358. A similar organization of the wb* region in other serogroups raises the possibility of the emergence of new pathogens by homologous recombination via the junction genes.
... DNA sequence analyses of the wb* clusters of two other serogroups (O22 and O139) revealed a similar organization of this region; i.e., serogroup-specific genes are flanked by gmhD (which encodes D-glycero-D-manno-heptose 1-phosphate guanosyltransferase, involved in lipopolysaccharide core biosynthesis) at the left junction and by rjg (which encodes a conserved hypothetical protein with similarities to mRNA 3Ј end processing factor) at the right junction (9,14,18,33,48,59). These data led to the idea that the V. cholerae O139 Bengal strain originated from an O1 strain by homologous recombination-mediated replacement of the wbe region of an O1 strain with the O139 wbf region (36,47,49). However, the donor or the vehicle for this horizontal transfer event is not yet known. ...
... However, the donor or the vehicle for this horizontal transfer event is not yet known. An O22 serogroup strain has been proposed to be a possible donor since its wb* region shares extensive homology with the O139 wbf region (59), and a generalized transducing phage or a conjugative plasmid is the speculated vector (36,47). ...
The novel epidemic strain Vibrio cholerae O139 Bengal originated from a seventh-pandemic O1 El Tor strain by antigenic shift resulting from homologous recombination-mediated
exchange of O-antigen biosynthesis (wb*) clusters. Conservation of the genetic organization of wb* regions seen in other serogroups raised the possibility of the existence of pathogenic non-O1 and non-O139 V. cholerae strains that emerged by similar events. To test this hypothesis, 300 V. cholerae isolates of non-O1 and non-O139 serogroups were screened for the presence of virulence genes and an epidemic genetic background
by DNA dot blotting, IS1004 fingerprinting, and restriction fragment length polymorphism (RFLP) analysis. We found four non-O1 strains (serogroups O27,
O37, O53, and O65) with an O1 genetic backbone suggesting exchange of wb* clusters. DNA sequence analysis of the O37 wb* region revealed that a novel ∼23.4-kb gene cluster had replaced all but the ∼4.2-kb right junction of the 22-kb O1 wbe region. In sharp contrast to the backbones, the virulence regions of the four strains were quite heterogeneous; the O53 and
O65 strains had the El Tor vibrio pathogenicity island (VPI) cluster, the O37 strain had the classical VPI cluster, and the
O27 strain had a novel VPI cluster. Two of the four strains carried CTXφ; the O27 strain possessed a CTXφ with a recently
reported immune specificity (rstR-4** allele) and a novel ctxB allele, and the O37 strain had an El Tor CTXφ (rstRET allele) and novel ctxAB alleles. Although the O53 and O65 strains lacked the ctxAB genes, they carried a pre-CTXφ (i.e., rstRcla). Identification of non-O1 and non-O139 serogroups with pathogenic potential in epidemic genetic backgrounds means that attention
should be paid to possible future epidemics caused by these serogroups and to the need for new, rapid vaccine development
strategies.
... Once it was shown that certain strains of V. cholerae O37 were closely related to classical V. cholerae O1 (discussed more deeply elsewhere in this review), the similarity of the sequence of DNA flanking the O-antigen locus in V. cholerae O1 and V. cholerae O37 led to the hypothesis that homologous recombination had caused the genes conferring an O1 genotype to have been exchanged with those encoding the O37 serogroup, causing seroconversion [49]. This was of particular relevance because similar recombination events had been suggested to have enabled seroconversion of V. cholerae O1 to O139 [13,49,67,68]. Subsequently, Blokesch and Schoolnik demonstrated that genomic DNA prepared from O37 serogroup strain ATCC 25872 could be used to transform naturally competent V. cholerae O1 and convert them to serogroup O37 [32]. ...
Between 1965 and 1968, outbreaks of cholera in Sudan and former Czechoslovakia provoked considerable public health concern. These still represent important historical events that need to be linked to the growing genomic evidence describing the aetiological agent of cholera, Vibrio cholerae. Whilst O1 serogroup V. cholerae are canonically associated with epidemic and pandemic cholera, these events were caused by a clone of toxigenic V. cholerae O37 that may be more globally distributed than just to Europe and North Africa. Understanding the biology of these non-O1 strains of V. cholerae is key to understanding how diseases like cholera continue to be globally important. In this article, we consolidate epidemiological, molecular and genomic descriptions of the bacteria responsible for these outbreaks. We attempt to resolve discrepancies in order to summarize the history and provenance of as many commonly used serogroup O37 strains as possible. Finally, we highlight the potential for whole-genome sequencing of V. cholerae O37 isolates from strain collections to shed light on the open questions that we identify.
... Two hypotheses have been proposed to explain the emergence of V. cholerae O139. The first proposes that a transposition event, mediated by the IS element IS1358, resulted in the replacement of the O1 wbe genes with the O139 wbf genes (26,31,35,36). The second hypothesis involves a homologous recombination event resulting in the replacement of the entire O1 wbe region by the O139 wbf region (30,34,35). ...
The emergence of Vibrio cholerae O139 Bengal during 1992-1993 was
associated with large epidemics of cholera in India and Bangladesh and,
initially, with a total displacement of the existing V. cholerae O1
strains. However, the O1 strains reemerged in 1994 and initiated a
series of disappearance and reemergence of either of the two serogroups
that was associated with temporal genetic and phenotypic changes
sustained by the strains. Since the initial emergence of the O139
vibrios, new variants of the pathogen derived from multiple progenitors
have been isolated and characterized. The clinical and epidemiological
characteristics of these strains have been studied. Rapid genetic
reassortment in O139 strains appears to be a response to the changing
epidemiology of V. cholerae O1 and also a strategy for persistence in
competition with strains of the O1 serogroup. The emergence of V.
cholerae O139 has provided a unique opportunity to witness genetic
changes in V. cholerae that may be associated with displacement of an
existing serogroup by a newly emerging one and, thus, provide new
insights into the epidemiology of cholera. The genetic changes and
natural selection involving both environmental and host factors are
likely to influence profoundly the genetics, epidemiology, and evolution
of toxigenic V. cholerae, not only in the Ganges Delta region of India
and Bangladesh, but also in other areas of endemic and epidemic cholera.
... In the first place, primers VCO1F2 and VCO1R2 were used to detect O1 serogroup of V. cholerae (Yamasaki et al., 1999). Secondly, primers VCO139F2 (Rivera et al., 2003) and VCO139R2 (Stroeher and Manning, 1997) were tested in a multiplex PCR. ...
In this work a sequential multiplex PCR system was designed and validated for the detection of most frequent foodborne pathogen Vibrio species in fish and seafood (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginoliticus and Vibrio mimicus). The method proposed functions in a hierarchical way, being composed of an end-point multiplex PCR to detect the presence of DNA belonging to the studied species, followed by multiplex PCR and fragment analysis allowing the viability assessment of the detected strains. The final multiplex PCR step of the method may be applied if identification of the serogroup, biotype and/or virulence factor level is necessary. Forty samples of commercial fish and seafood products were used at the method validation stage. Sixty three marine organism samples obtained from various estuarine areas of Spain including shrimps, crabs, bivalve mollusks and fishes were screened for presence of Vibrio species and 2 mussel samples were found positive for V. parahaemolyticus. On the whole, the proposed method is robust and readily adaptable in routine molecular diagnostic laboratories, allowing monitoring and simultaneous detection of all these bacterial pathogens in seafood samples, reducing the expenses and time consumed by other analytical methods.
... Several non-O1 strains, including O139, O27, O37, O53, and O65, have been shown to possess the genetic backbone of O1 strains (determined by the genetic fingerprint, AFLP) and were proposed to have arisen from homologous recombination-mediated exchange of the genes responsible for biosynthesis of O-antigen, wb* gene clusters (i.e., wbf for O139-, and wbe for O1-biosynthesis) (109). Extensive analysis of the nature of the new epidemic O139 serogroup suggests that it originated from a seventh-pandemic O1 El Tor strain that underwent antigenic shift by homologous recombination, replacing the wbe region of the O1 antigen with the O139 wbf region (126,179,180). The donor of the O139 wbf gene cluster has been proposed to be O22 serogroup, because of extensive homology with the O139 wbf region (203). ...
Thesis research directed by: Marine-Estuarine-Environmental Sciences. Title from t.p. of PDF. Thesis (Ph. D.) -- University of Maryland, College Park, 2004. Includes bibliographical references. Text.
... It expresses most of the V. cholerae O1 virulence factors (1), and further genetic analyses have shown that it probably arose from the pandemic strain of V. cholerae O1 biotype El Tor (4,18,23,41). However, in contrast to serovar O1 strains, and like most non-O1 strains, this strain was capsulated and the chemical composition of its lipopolysaccharide (LPS) was different from that of O1 strains (5,6,11,12,23,40,43). Genetic analysis of the region involved in O-antigen biosynthesis, formerly designated the rfb locus, has shown that a 22-kb DNA fragment present in O1 strains has been replaced in V. cholerae O139 by a 40-kb DNA fragment constituted by (i) seven genes, wbfA to -F and wzz, some of which are likely involved in the regulation of the O-antigen length (wzz ϭ otnB) and in the capsule transport (wbtF ϭ otnA) (6,30,36); (ii) a putative insertion sequence designated IS1358 (35); and (iii) 21 open reading frames (ORFs) thought to be involved in O-antigen and capsule biosynthesis (6,11,37). ...
The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374-10378, 1995) and designated IS1358O139. We studied the distribution of the IS1358 element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS1358 was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS1358 from V. cholerae O22 and one copy from V. cholerae O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS1358 to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 x 10(-8). Sequence analysis revealed that IS1358 duplicates 10 bp at its insertion site.
... Third, H. pylori R-M genes are one of the major components of the strain-specific genes. The strain-specific genes are believed to be involved in drug resistance (13) and bacterial surface structure (14), as well as restriction-modification (15). A PCR-based subtractive hybridization method was used to investigate genes that are unique to individual H. pylori strains (16,17). ...
Helicobacter pylori is a Gram-negative bacterial pathogen with a small genome of 1.64-1.67 Mb. More than 20 putative DNA restriction-modification (R-M) systems, comprising more than 4% of the total genome, have been identified in the two completely sequenced H. pylori strains, 26695 and J99, based on sequence similarities. In this study, we have investigated the biochemical activities of 14 Type II R-M systems in H. pylori 26695. Less than 30% of the Type II R-M systems in 26695 are fully functional, similar to the results obtained from strain J99. Although nearly 90% of the R-M genes are shared by the two H. pylori strains, different sets of these R-M genes are functionally active in each strain. Interestingly, all strain-specific R-M genes are active, whereas most shared genes are inactive. This agrees with the notion that strain-specific genes have been acquired more recently through horizontal transfer from other bacteria and selected for function. Thus, they are less likely to be impaired by random mutations. Our results also show that H. pylori has extremely diversified R-M systems in different strains, and that the diversity may be maintained by constantly acquiring new R-M systems and by inactivating and deleting the old ones.
... understood which accessory elements mediated the horizontal transfer events that led to the current configuration of the O139 antigen gene cluster [2]. Involvement of an unidentified transducing phage in this gene transfer event remains a possibility [25,26]. ...
The identification of accessory genetic elements (plasmids, phages and chromosomal 'pathogenicity islands') encoding virulence-associated genes has facilitated our efforts to understand the origination of pathogenic microorganisms. Toxigenic Vibrio cholerae, the etiologic agent of cholera, represents a paradigm for this process in that this organism evolved from environmental nonpathogenic V. cholerae by acquisition of virulence genes. The major virulence genes in V. cholerae, which are clustered in several chromosomal regions, appear to have been recently acquired from phages or through undefined horizontal gene transfer events. Evidence is accumulating that the interactions of phages with each other can also influence the emergence of pathogenic clones of V. cholerae. Therefore, to track the evolution of pathogens from their nonpathogenic progenitors, it is also crucial to identify and characterize secondary genetic elements that mediate lateral transfer of virulence genes in trans. Understanding the evolutionary events that lead to the emergence of pathogenic clones might provide new approaches to the control of cholera and other infectious diseases.
... Two hypotheses have been proposed to explain the emergence of V. cholerae O139. The first proposes that a transposition event, mediated by the IS element IS1358, resulted in the replacement of the O1 wbe genes with the O139 wbf genes (26,31,35,36). The second hypothesis involves a homologous recombination event resulting in the replacement of the entire O1 wbe region by the O139 wbf region (30,34,35). ...
The emergence of Vibrio cholerae O139 Bengal during 1992-1993 was associated with large epidemics of cholera in India and Bangladesh and, initially, with a total displacement of the existing V. cholerae O1 strains. However, the O1 strains reemerged in 1994 and initiated a series of disappearance and reemergence of either of the two serogroups that was associated with temporal genetic and phenotypic changes sustained by the strains. Since the initial emergence of the O139 vibrios, new variants of the pathogen derived from multiple progenitors have been isolated and characterized. The clinical and epidemiological characteristics of these strains have been studied. Rapid genetic reassortment in O139 strains appears to be a response to the changing epidemiology of V. cholerae O1 and also a strategy for persistence in competition with strains of the O1 serogroup. The emergence of V. cholerae O139 has provided a unique opportunity to witness genetic changes in V. cholerae that may be associated with displacement of an existing serogroup by a newly emerging one and, thus, provide new insights into the epidemiology of cholera. The genetic changes and natural selection involving both environmental and host factors are likely to influence profoundly the genetics, epidemiology, and evolution of toxigenic V. cholerae, not only in the Ganges Delta region of India and Bangladesh, but also in other areas of endemic and epidemic cholera.
... In addition, the O antigen is the bacterium's major protective antigen, and therefore, changes in the O antigen of a preexisting epidemic strain may result in a new pathogen capable of causing disease in populations immune to the original epidemic strain (1,31). For example, the V. cholerae O139 Bengal strain emerged from an O1 epidemic strain by genetic exchange of O-antigen biosynthesis regions (3,26,40), and O139 strains cause disease in persons immune to O1 strains (4,27). ...
Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTXφ), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPIφ.
In order to determine the prevalence of horizontal transfer of VPI and CTXφ among nonepidemic (non-O1 and non-O139 serogroups)
V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster.
Seven of the 11 VPI+ strains have also acquired the CTXφ. Multilocus sequence typing and restriction fragment length polymorphism analyses of
the VPI and CTXφ prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in
lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited
extensive DNA rearrangements. In addition, several CTXφ prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains
identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTXφ independently rather
than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.
... Similarly, Y. bercovieri W601 was not closely related to the Y. enterocolitica strains, including the two strains (8081 and YE 371) with which it shared the O:8 antigen ( Fig. 1 and 2). These observations suggest that Oantigen switching, which has been proposed (54) to have occurred among several other bacteria, may have also occurred between at least some Yersinia species. ...
The intra- and interspecies genetic relationships of 58 strains representing all currently known species of the genus Yersinia were examined by multilocus sequence typing (MLST), using sequence data from 16S RNA, glnA, gyrB, recA, and Y-HSP60 loci. Yersinia aldovae, Y. bercovieri, Y. intermedia, Y. pestis, Y. pseudotuberculosis, Y. rohdei, and Y. ruckeri were genetically more homogeneous than were Y. enterocolitica, Y. frederiksenii, Y. kristensenii, and Y. mollaretii. The MLST data concerning the genetic relatedness within and among various species of Yersinia support the idea that Y. pestis and Y. pseudotuberculosis are two lineages within the same species rather than two distinct species. Y. ruckeri is the genetically most distant species within the genus. There was evidence of O-antigen switching and genetic recombination
within and among various species of Yersinia. The genetic relatedness data obtained by MLST of the four housekeeping genes and 16S RNA agreed in most, but not all, instances.
MLST was better suited for determining genetic relatedness among yersiniae than was 16S RNA analysis. Some strains of Y. frederiksenii and Y. kristensenii are genetically less related to other strains within those species, compared to strains of all other species within the genus.
The taxonomic standing of these strains should be further examined because they may represent currently unrecognized Yersinia species.
... Unlike the O1-encoding region in El Tor strains, the O139-antigen gene cluster was found to specify not only the new O139 side chain, but in addition, a capsular polysaccharide (CPS)131415 that contains the same O139-antigenic determinant. The close genetic relatedness of the ancestral O1 El Tor lineage with the new O139 serogroup16171819, the existence of different O-antigen–encoding cassettes at the same chromosomal locus20212223, and the presence of regions of homology on either side of this site [12,15,24,25] strongly support the hypothesis that some kind of horizontal gene transfer event had occurred, resulting in replacement of the original O1 gene cluster with the O139 gene cluster of a V. cholerae environmental strain [23,2627282930. However, neither the gene transfer mechanism nor the ecological context in which this occurred has been elucidated. ...
The environmental reservoirs for Vibrio cholerae are natural aquatic habitats, where it colonizes the chitinous exoskeletons of copepod molts. Growth of V. cholerae on a chitin surface induces competence for natural transformation, a mechanism for intra-species gene exchange. The antigenically diverse O-serogroup determinants of V. cholerae are encoded by a genetically variable biosynthetic cluster of genes that is flanked on either side by chromosomal regions that are conserved between different serogroups. To determine whether this genomic motif and chitin-induced natural transformation might enable the exchange of serogroup-specific gene clusters between different O serogroups of V. cholerae, a strain of V. cholerae O1 El Tor was co-cultured with a strain of V. cholerae O139 Bengal within a biofilm on the same chitin surface immersed in seawater, and O1-to-O139 transformants were obtained. Serogroup conversion of the O1 recipient by the O139 donor was demonstrated by comparative genomic hybridization, biochemical and serological characterization of the O-antigenic determinant, and resistance of O1-to-O139 transformants to bacteriolysis by a virulent O1-specific phage. Serogroup conversion was shown to have occurred as a single-step exchange of large fragments of DNA. Crossovers were localized to regions of homology common to other V. cholerae serogroups that flank serogroup-specific encoding sequences. This result and the successful serogroup conversion of an O1 strain by O37 genomic DNA indicate that chitin-induced natural transformation might be a common mechanism for serogroup conversion in aquatic habitats and for the emergence of V. cholerae variants that are better adapted for survival in environmental niches or more pathogenic for humans.
... An excellent example of the former approach is provided by analyses of Vibrio cholerae O1 and O139 Bengal. Several studies have shown that a V. cholerae O1 progenitor had acquired a new O-antigen gene cluster and emerged as O139 Bengal (Mooi & Bik, 1997; Stroeher & Manning, 1997; Stroeher et al., 1998 ). In this O-antigen shift, a 22 kb region for the O1- antigen gene cluster was replaced by a 35 kb fragment encoding the O139-antigen gene cluster. ...
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 gamma1 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 theta 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 (> or =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.
The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374–10378, 1995) and designated IS 1358 O139 . We studied the distribution of the IS 1358 element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS 1358 was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS 1358 from V. cholerae O22 and one copy from V. cholerae O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS 1358 to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 × 10 ⁻⁸ . Sequence analysis revealed that IS 1358 duplicates 10 bp at its insertion site.
Vibrio cholerae and V. anguillarum are recognized as aquatic-borne human and fish pathogens, respectively. Based upon analyses of several genes and the presence of novel genetic elements it seems that these two species are very closely related. Studies in this laboratory have identified an association of IS1358 with rfb and capsule loci in these two species. The most recent findings suggest that IS1358 is associated with the rfb region in V. cholerae O1 and O139 and in V. anguillarum O1 and O2. In addition, the rfb region in both V. cholerae serogroups and in V. anguillarum O1 is limited at one end by gmhD. These features make it feasible to envisage a mechanism by which the evolution of new rfb genes is taking place involving IS1358 and the region around gmhD. Furthermore, it is possible to envisage that there is or has been an exchange of genetic material between these species leading to new rfb/capsule regions. This review examines the genetics and biosynthesis of the O-antigen and capsule of V. cholerae O1 and O139, as well as the V. anguillarum serogroup O1 and the role of IS1358. Throughout this review we have used the new nomenclature for rfb genes proposed by Reeves et al. (1996).
Vibrio cholerae and V. anguillarum are recognized as aquatic-borne human and fish pathogens, respectively. Based upon analyses of several genes and the presence of novel genetic elements it seems that these two species are very closely related. Studies in this laboratory have identified an association of IS1358 with rfb and capsule loci in these two species. The most recent findings suggest that IS1358 is associated with the rfb region in V. cholerae O1 and O139 and in V. anguillarum O1 and O2. In addition, the rfb region in both V. cholerae serogroups and in V. anguillarum O1 is limited at one end by gmhD. These features make it feasible to envisage a mechanism by which the evolution of new rfb genes is taking place involving IS1358 and the region around gmhD. Furthermore, it is possible to envisage that there is or has been an exchange of genetic material between these species leading to new rfb/capsule regions. This review examines the genetics and biosynthesis of the O-antigen and capsule of V. cholerae O1 and O139, as well as the V. anguillarum serogroup O1 and the role of IS1358. Throughout this review we have used the new nomenclature for rfb genes proposed by Reeves et al. (1996).
Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe
a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated
strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome,
and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level,
seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes.
Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that
these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic
DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into
microbial genetic diversity and evolution.
A second aetiological agent of cholera, Vibrio cholerae O139 Bengal was identified in late 1992 when it caused large outbreaks of diarrhoea in India and Bangladesh. The new strain probably arose as a result of lateral transfer of genes encoding a novel somatic antigen and a capsule from an unknown bacterium to an O1 El Tor strain with the subsequent loss of genes encoding O1 somatic antigen. O139 produces a semi-rough type colony with a truncated lipopolysaccharide which contains a unique sugar, colitose. Otherwise, O139 and O1 are strikingly similar. The diseases produced by the two serogroups are indistinguishable. There is a moderate inflammatory response in cholera, and in spite of the possession of a capsule by O139, it is no more inflammatory than O1. A number of diagnostic tests have been developed for O139 that are modelled after tests for O1 including rapid tests for field use. O139 has spread to countries of south and south-east Asia, China and Russia. Currently, cholera is caused by both these serogroups in these countries with the O1 serogroup predominant. O139 continues to cause occasional local outbreaks in India and Bangladesh. Genotyping of isolates has shown circulation of multiple clones and their derivation from multiple progenitors. Although there have been changes in antibiogram with the resistance genes being carried on a constin, the strains remain susceptible to tetracycline, a preferred antibiotic for treatment. O139 carries a number of filamentous lysogenic and lytic phages and the latter have been utilized to develop a phage typing scheme. A number of environmental variables have been linked to the occurrence of cholera and lytic phages may determine the course of epidemics. Although the capsule confers serum resistance, vibriocidal antibody assay has been developed for O139. However, unlike in O1 cholera, serum vibriocidal antibody does not seem to correlate with protection against O139 infection. Promising O139 vaccines have been developed or are in the pipeline. It is too early to predict whether O139 will become the causative agent of 'the eighth pandemic of cholera'; it will require many years of monitoring and reporting from regions prone to cholera to find the answer.
Vibrio cholerae elaborates three types of polysaccharide structures: lipopolysaccharide (LPS), a component of which is the O-polysaccharide or O-antigen, capsular polysaccharide (CPS) or K-antigen, and “rugose” polysaccharide also known as exopolysaccharide (EPS) or Vibrio polysaccharide (VPS). The major protective antigen for V. cholerae is the O-antigen. A strain typing scheme based on the somatic O-antigen has been in use for a number of years. There are 206 serogroups identified so far and of these only O1 and O139 are known to cause epidemic/pandemic cholera, although a handful of non-O1/non-O139 strains are known to possess the major virulence factors. The O-antigen diversity is due to the number and composition of monosaccharide components, linkages, addition of non-sugar moieties, modal length of the polysaccharide chain, and biosynthesis mechanisms. The genetic basis of this diversity is just beginning to be understood with the sequencing of a number of gene clusters that encode O-polysaccharide (OPS)/capsule structures. In this review, we summarize our current knowledge on the biochemical composition and structure of some of the O-polysaccharides, genes involved in their biosynthesis, and touch upon the role of horizontal gene transfer in creating this diversity and possible mechanisms that may be operative in this process. We highlight the fact that the distinction between OPS and CPS seems to be less evident in V. cholerae than in other species since the genes encoding these structures are shared and map in the same region of the genome. We also describe our current understanding of the genetics and regulation of EPS/VPS synthesis and its role in biofilm formation and environmental survival of V. cholerae.
Comparisons of bacterial genomes demonstrate that even strains of one species may strikingly differ in gene set. Strain-specific genes are of considerable interest, as they may be responsible for distinguishing features, such as virulence or drug resistance, of the strain and may be employed as markers in epidemiological or evolutionary studies. Suppression subtractive hybridization (SSH) was shown to be suitable for generating a set of DNA fragments differing between two closely related bacterial strains. More than 95% DNA fragments selected by SSH proved to be specific for Staphylococcus aureus strains ZW compared with strain 29213.
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Pathogenic bacteria produce an elaborate assortment of extracellular and cell-associated bacterial products that enable colonization and establishment of infection within a host. Lipopolysaccharide (LPS) molecules are cell surface factors that are typically known for their protective role against serum-mediated lysis and their endotoxic properties. The most heterogeneous portion of LPS is the O antigen or O polysaccharide, and it is this region which confers serum resistance to the organism. Pseudomonas aeruginosa is capable of concomitantly synthesizing two types of LPS referred to as A band and B band. The A-band LPS contains a conserved O polysaccharide region composed of D-rhamnose (homopolymer), while the B-band O-antigen (heteropolymer) structure varies among the 20 O serotypes of P. aeruginosa. The genes coding for the enzymes that direct the synthesis of these two O antigens are organized into two separate clusters situated at different chromosomal locations. In this review, we summarize the organization of these two gene clusters to discuss how A-band and B-band O antigens are synthesized and assembled by dedicated enzymes. Examples of unique proteins required for both A-band and B-band O-antigen synthesis and for the synthesis of both LPS and alginate are discussed. The recent identification of additional genes within the P. aeruginosa genome that are homologous to those in the A-band and B-band gene clusters are intriguing since some are able to influence O-antigen synthesis. These studies demonstrate that P. aeruginosa represents a unique model system, allowing studies of heteropolymeric and homopolymeric O-antigen synthesis, as well as permitting an examination of the interrelationship of the synthesis of LPS molecules and other virulence determinants.
Vibrio cholerae O139 strains produce a capsule which is associated with complement resistance and is used as a receptor by bacteriophage JA1. Spontaneous JA1-resistant mutants were found to have several phenotypes, with loss of capsule and/or O-antigen from the cell surface. Determination of the residual complement resistance and infant mouse colonization potential of each mutant suggested that production of O-antigen is of much greater significance than the presence of capsular material for both of these properties. Two different in vitro assays of complement resistance were compared and the results of one shown to closely reflect the comparative recoveries of bacteria from the colonization experiments. Preliminary complementation studies implicated two rfb region genes, wzz and wbfP, as being essential for the biosynthesis of capsule but not O-antigen.
Helicobacter pylori is an important pathogen of the gastric system. The clinical outcome of infection is thought to be correlated with some genetic features of the bacterium. However, due to the extreme genetic variability of this organism, it is hard to draw definitive conclusions concerning its virulence factors. Here we describe a novel H. pylori gene which expresses an autolytic enzyme that is also capable of degrading the cell walls of both gram-positive and gram-negative bacteria. We designated this gene lys. We found this gene and observed its expression in a number of unrelated clinical strains, a fact that suggests that it is well conserved in the species. A comparison of the nucleotide sequences of lys and the hypothetical gene HP0339 from H. pylori strain ATCC 26695 revealed almost total identity, except for the presence of an insertion consisting of 24 nucleotides in the lys sequence. The coding sequences of lys and HP0339 show a high degree of homology with the coding sequence of bacteriophage T4 lysozyme. Because of this similarity, it was possible to model the three-dimensional structures of both the lys and HP0339 products.
After repeated passages through embyronated eggs, the Nine Mile strain of Coxiella burnetii exhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS)
structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied
by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol. 132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was
used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions.
The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the
deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes,
arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose
pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for
addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with
reduced G+C contents were found within the deletions.
Comparisons of bacterial genomes demonstrate that even strains of one species may strikingly differ in gene set. Strain-specific genes are of considerable interest, as they may be responsible for distinguishing features, such as virulence or drug resistance, of the strain and may be employed as markers in epidemiological or evolutionary studies. Suppression subtractive hybridization (SSH) was shown to be suitable for generating a set of DNA fragments differing between two closely related bacterial strains. More than 95% DNA fragments selected by SSH proved to be specific for Staphylococcus aureus strains ZW compared with strain 29213.
An account of our up to date knowledge of the genetics of biosynthesis of Vibrio cholerae lipopolysaccharide (LPS) is presented in this review. While not much information is available in the literature on the genetics of biosynthesis of lipid A of V. cholerae, the available information on the characteristics and proposed functions of the corepolysaccharide (core-PS) biosynthetic genes is discussed. The genetic organizations encoding the O-antigen polysaccharides (O-PS) of V. cholerae of serogroups O1 and O139, the disease causing ones, have been described along with the putative functions of the different constituent genes. The O-PS biosynthetic genes of some non-O1, non-O139 serogroups, particularly the serogroups O37 and O22, and their putative functions have also been discussed briefly. In view of the importance of the serogroup O139, the origination of the O139 strain and the possible donor of the corresponding O-PS gene cluster have been analyzed with a view to having knowledge of (i) the mode of evolution of different serogroups and (ii) the possible emergence of pathogenic strain(s) belonging to non-O1, non-O139 serogroups. The unsolved problems in this area of research and their probable impact on the production of an effective cholera vaccine have been outlined in conclusion.
Understanding of the biology of interaction between pathogens and host is the central question in studying inflammatory disorders. Subtractive DNA cloning is one of the most efficient and comprehensive methods available for identifying eukaryotic genes regulated under specific physiological conditions, including inflammation and host response. Here we explore the utility of subtractive DNA cloning and describe suppression subtractive hybridization (SSH), a polymerase chain reaction (PCR)-based DNA subtraction method that has been developed and evolved in our labs over several years. The SSH method possesses a number of advantages as compared to other subtractive cloning techniques, making it one of the most adventitious methods for cloning differentially expressed genes. Besides isolation of differentially expressed eukaryotic mRNAs, subtractive DNA cloning can be used to identify genes that are differentially expressed between diverse bacterial species. These genes can be of great interest, as some may encode strain-specific traits such as drug resistance, or bacterial surface proteins involved in determining the virulence of a particular strain. Other genes may be useful as markers for epidemiological or evolutionary studies. To demonstrate the potential of the SSH technique, we describe here the comprehensive characterization of 2 SSH subtracted libraries constructed in our laboratories. One library was created using eukaryotic cDNA subtraction and is specific for mRNAs up-regulated in CD25 positive cells from mouse lymph nodes as compared to CD25 negative cells. The second subtracted library is specific for a methicillin-resistant Staphylococcus aureus bacterial strain, but not in a methicillin-sensitive strain. The bacterial genomes of these 2 strains have been completely sequenced and this second library provides an excellent reference for testing the ability of SSH to recover all strain-specific gene content. The analysis of these 2 subtracted libraries serves as the basis for a discussion of the strength and limitations of the SSH technique. We will also compare and contrast subtractive DNA cloning to other current technologies used to isolate differentially expressed genes.
Vibrio cholerae is a free-living bacterium found in water and in association with plankton. V. cholerae non-O1/non-O139 strains are frequently isolated from aquatic ecosystems worldwide. Less frequently isolated are V. cholerae O1 and V. cholerae O139, the aetiological agents of cholera. These strains have two main virulence-associated factors, cholera toxin (CT) and toxin co-regulated pilus (TCP). By extracting total DNA from aquatic samples, the presence of pathogenic strains can be determined quickly and used to improve a microbiological risk assessment for cholera in coastal areas. Some methods suggested for DNA extraction from water samples are not applicable to all water types. We describe here a method for DNA extraction from coastal water and a multiplex polymerase chain reaction (PCR) for O1 and O139 serogroups. DNA extraction was successfully accomplished from 117 sea water samples collected from coastal areas of Perú, Brazil and the USA. DNA concentration in all samples varied from 20 ng to 480 micro g micro l-1. The sensitivity of the DNA extraction method was 100 V. cholerae cells in 250 ml of water. The specificity of multiplex O1/O139 PCR was investigated by analysing 120 strains of V. cholerae, Vibrio and other Bacteria species. All V. cholerae O1 and O139 tested were positive. For cholera surveillance of aquatic environments and ballast water, total DNA extraction, followed by V. cholerae PCR, and O1/O139 serogroup and tcpA/ctxA genes by multiplex PCR offers an efficient system, permitting risk analysis for cholera in coastal areas.
In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDvco139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1-acceptor DNA is probably located downstream of rfbDvco139. The DNA region between rfaDvco139 and rfbQRSvco139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found inV. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.
The gene clusters that determine the biosynthesis of both the Inaba and Ogawa serotypes of the O antigen of the lipopolysaccharide of Vibrio cholerae were cloned and expressed in Escherichia coli K-12. Restriction analysis of the clones demonstrated that about 15 kilobases were common to all clones and a further 5 kilobases were common to the Ogawa clones. The O antigens expressed by E. coli K-12 had the specificity of V. cholerae. Antibodies raised against E. coli K-12 that harbor one of these clones, pPM1001 (Inaba), were as highly protective in the infant mouse model system as were antibodies to V. cholerae itself. Introduction of such clones into suitable carrier strains could be expected to produce a good oral immunogen against cholera.
The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain. Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough. Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA. However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region. This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1. Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1. Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis. We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.
Vibrio cholerae O139 Bengal, although closely related to V. cholerae O1 El Tor, produces a polysaccharide capsule and has a distinct O antigen. We have identified a chromosomal region of at least 11 kb, as defined by three TnphoA mutations, that is required for the expression of both polysaccharides. Electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis show that these TnphoA mutants have lost the abilities both to express capsule and to produce lipopolysaccharide beyond the core oligosaccharide. Reactivity with O139 typing serum and resistance to serum are also lost in the mutants. DNA probes for this region do not hybridize with O1 V. cholerae but do react with other vibrios, implying that the region was recently acquired.
Only Vibrio cholerae strains of serotype O1 are known to cause epidemics, while non-O1 strains are associated with sporadic cases of cholera. It was therefore unexpected that the recent cholera epidemic in Asia was caused by a non-O1 strain with the serotype O139. We provide evidence that O139 arose from a strain closely related to the causative agent of the present cholera pandemic, V. cholerae O1 El Tor, by acquisition of novel DNA which was inserted into, and replaced part of, the O antigen gene cluster of the recipient strain. Part of the novel DNA was sequenced and two open reading frames (otnA and otnB) were observed, the products of which showed homology to proteins involved in capsule and O antigen synthesis, respectively. This suggests that the otnAB DNA determines the distinct antigenic properties of the O139 cell surface. The otnAB DNA was not detected in O1 strains, but was present in two non-O1 V. cholerae strains with serotypes O69 and O141. In the O69 and O139 strains the otnAB genes were located proximate to the putative insertion sequence (IS) element rfbQRS, which is associated with O antigen synthesis genes in O1 strains, and may have played a role in the insertion of the otnAB DNA in the recipient chromosome. Our results suggest that the O139 strain arose by horizontal gene transfer between a non-O1 and an O1 strain. The acquired DNA has altered the antigenic properties of the recipient O1 strain, providing a selective advantage in a region where a large part of the population is immune to O1 strains.(ABSTRACT TRUNCATED AT 250 WORDS)
Vibrio cholerae serogroup O139 emerged on the Indian subcontinent in October 1992 to become the first non-O1 V. cholerae serogroup documented to cause epidemic cholera. Although related to V. cholerae El Tor O1 strains, O139 strains have unique surface structures that include a capsular surface layer and lipopolysaccharide (LPS). Immunoblot analysis of either whole-cell lysates or LPS preparations revealed three electrophoretic forms of the O139 antigen: two slowly migrating forms and one rapidly migrating form that appeared identical to O139 LPS. All three forms of the antigen shared an epitope defined by an O139-specific monoclonal antibody. A serum-sensitive nonencapsulated mutant was isolated that lacks only the slow migrating forms. The slow migrating forms did not stain with silver whereas the rapidly migrating form did, suggesting that the former might constitute highly polymerized O-antigen side-chain molecules that were not covalently bound to core polysaccharide and lipid A (an "O-antigen capsule"). A single transposon insertion resulted in the loss of immunoreactivity of both the LPS and the O-antigen capsule, implying that there are genes common to the biosynthesis of both these macromolecules. The O139 LPS and O-antigen capsule were both important for colonization of the small intestine of the newborn mouse and for serum resistance, demonstrating that both of these forms of the O139 serogroup antigen are virulence factors.
Vibrio cholerae is divided into more than 130 O serogroups; however, only organisms of the O1 serogroup have so far been associated with cholera in humans. V. cholerae O1 strains of both biotypes have been further subdivided into three serotypes, designated Inaba, Ogawa, and Hikojima, grouped according to the structure of the O antigens on the lipopolysaccharide (LPS). The LPS of gram-negative bacteria is the most abundant molecule on the cell surface, where it provides a protective barrier to hydrophobic agents and detergents. The most common sugars found in the O polysaccharide are perosamine and quinovosamine. A number of studies designed to correlate the various O-antigen polysaccharides with particular antigenic specificities have been carried out. The genes involved in O-antigen biosynthesis in V. cholerae O1 strains 569B (Inaba, classical) and O17 (Ogawa, EI Tor) have been cloned and expressed in Escherichia coli K-12. The E. coli RfaD is an ADP-L-glycero-D-mannoheptose epimerase and is one of the critical proteins involved in the synthesis of the core oligosaccharide of the LPS in E. coli. Genetic complementation studies have suggested that the determinant responsible for Ogawa specificity lies at the distal end of the rfb region, an area in which no readily detectable differences could be discerned.
We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.
The rfaD gene of Escherichia coli encodes ADP-L-glycero-D-mannoheptose-6- epimerase, an enzyme required for the biosynthesis of the lipopolysaccharide (LPS) precursor ADP-L-glycero-D- mannoheptose, associated with production of the core oligosaccharide. We have identified an rfaD homologue in Vibrio cholerae O1. This gene maps adjacent to the rfb region encoding O-antigen biosynthesis, but is transcribed divergently. The complete nucleotide sequence of rfaD and the flanking DNA has been determined, and rfaD would appear to be the only gene homologous to known LPS core biosynthesis genes in this region. Comparison with the E. coli rfaD shows many similar structural features such as the ADP-binding beta alpha beta fold at the N terminus, as well as a high degree of homology of both the nucleotide and amino-acid sequences. Based on homology, rfaD of V. cholerae may be transcribed using both sigma 70- and sigma 54-dependent promoters.
Vibrio cholerae serogroup O139 Bengal is the first documented serogroup other than O1 to cause epidemic cholera. The O139 Bengal strains are very similar to V. cholerae serogroup O1 biotype El Tor strains. The major differences between the two serogroups are that O139 Bengal contains a distinct O antigen and produces a polysaccharide capsule. We previously described three TnphoA mutants of O139 strain AI1837 which abolish both O antigen and capsule production. These TnphoA insertions were mapped to a 21.5 kb EcoRI fragment of the O139 chromosome. We describe here the cloning and mapping of this 21.5 kb EcoRI fragment and it was shown to complement each of the mutants in trans to produce O antigen and capsule. The EcoRI fragment contains 13 kb of DNA that is specific to O139 and 8.5 kb of DNA that is common to O1 and O139. Sequence analysis of the 13 kb of O139-specific DNA revealed that it contains 11 open reading frames all of which are transcribed in the same direction. Eight of the 11 open reading frames are homologous to sugar biosynthesis genes from other organisms. Using extended polymerase chain reactions, we show that the extent of the DNA region in O139 that is not present in O1 is approximately 35 kb. The site of insertion of this O139-specific DNA in the O1 chromosome was mapped to the rfbO1 region. We also demonstrate that O139 Bengal strain AI1837 contains a deletion of 22 kb that in serogroup O1 strains contains the rfb region. Therefore, O139 Bengal probably arose from an O1 strain that had undergone genetic rearrangements including deletion of the O1 rfb region and acquisition of a 35 kb region of DNA which encodes O139 surface polysaccharide.
Gene nomenclature for bacterial surface polysaccharides is complicated by the large number of structures and genes. We propose a scheme applicable to all species that distinguishes different classes of genes, provides a single name for all genes of a given function and greatly facilitates comparative studies.
The emergence of the novel Vibrio cholerae strain, O139 Bengal, which caused a large epidemic in Southeast Asia, underlines the adaptability of pathogenic microorganisms. Recent studies reveal that horizontal transfer of cell-wall polysaccharide genes played a central role in the emergence of this strain and that its genesis may not be as unique as initially believed.
- Bik E.M.