The bacterial species Vibrio cholerae includes harmless aquatic strains as well as strains capable of causing epidemics and global pandemics of cholera. While investigating the relationship between pathogenic and nonpathogenic strains, we identified a chromosomal pathogenicity island (PAI) that is present in epidemic and pandemic strains but absent from nonpathogenic strains. Initially, two ToxRregulated genes (aldA and tagA) were studied and were found to be associated with epidemic and pandemic strains but absent in nontoxigenic strains. The region containing aldA and tagA comprises 13 kb of previously unidentified DNA and is part of a PAI that contains a regulator of virulence genes (ToxT) and a gene cluster encoding an essential colonization factor and the cholera toxin phage receptor (toxin-coregulated pilus; TCP). The PAI is 39.5 kb in size, has low %G؉C (35%), contains putative integrase and transposase genes, is f lanked by att sites, and inserts near a 10Sa RNA gene (ssrA), suggesting it may be of bacteriophage origin. We found this PAI in two clinical non-O1͞non-O139 cholera toxin-positive strains, suggesting that it can be transferred within V. cholerae. The sequence within this PAI includes an ORF with homology to a gene associated with the type IV pilus gene cluster of enteropathogenic Escherichia coli, a transposase from Vibrio anguillarum, and several ORFs with no known homology. As the PAI contains the CTX⌽ receptor, it may represent the initial genetic factor required for the emergence of epidemic and pandemic cholera. We propose to call this island VPI (V. cholerae pathogenicity island).In the last decade, the life-threatening diarrheal disease cholera has reached a wider distribution than at any other time in the 20th century. Cholera is caused by the bacterium Vibrio cholerae, which can be classified into over 140 serogroups (1). Prior to 1992, it was believed that only V. cholerae of the O1 serogroup were responsible for pandemic cholera and that strains of serogroups other than O1 were avirulent or caused only sporadic illness. However, in 1992 a O139 serogroup strain emerged and caused epidemic disease (2). The factors required for epidemic and pandemic ability are not fully understood, and there have been large outbreaks of cholera caused by toxigenic non-O1͞non-O139 strains that have not resulted in significant epidemic or pandemic disease (3, 4).Epidemic and pandemic strains of V. cholerae secrete cholera toxin (CT), the toxin responsible for the secretory diarrhea that is characteristic of the disease. CT is encoded by the ctxAB genes that are carried on a filamentous bacteriophage designated CTX⌽ (5). The bacterial receptor for this phage is the toxin-coregulated pilus (TCP), an essential colonization factor in human and animal models (6, 7). Expression of CT and TCP are coregulated by the ToxR regulatory system consisting of the proteins ToxR, ToxS, and ToxT (8, 9). Recently, it has been shown that the genes tcpP and tcpH within the TCP cluster also regulate virulence factors...
We have characterized a family of repetitive DNA elements with homology to the MgPa cellular adhesion operon of Mycoplasma genitalium, a
It was previously demonstrated that the intestinal pathogen Vibrio cholerae could undergo an adaptive stress response known as the acid tolerance response (ATR). The ATR is subdivided into two branches, inorganic ATR and organic ATR. The transcriptional regulator ToxR, while not involved in inorganic ATR, is required for organic ATR in a ToxT-independent manner. Herein, we investigate the effect of organic acid stress on global protein synthesis in V. cholerae and show by two-dimensional gel electrophoresis that the stress response alters the expression of more than 100 polypeptide species. The expression of more than 20 polypeptide species is altered in a toxR strain compared to the wild type. Despite this, ectopic expression of the porin OmpU from an inducible promoter is shown to be sufficient to bypass the toxR organic ATR defect. Characterization of the effect of organic acid stress on ompU and ompT transcription reveals that while ompU transcription remains virtually unaffected, ompT transcription is repressed in a ToxR-independent manner. These transcript levels are similarly reflected in the extent of accumulation of OmpU and OmpT. Possible roles for OmpU in organic acid resistance are discussed.Vibrio cholerae is the causative agent of the epidemic diarrheal disease cholera. After ingestion by a human host, passage through the gastric acid barrier, and colonization of the small intestine, this gram-negative bacterium produces cholera toxin and a subsequent profuse secretory diarrhea that is the hallmark of cholera (13). It was recently shown that V. cholerae is able to mount an adaptive stress response known as the acid tolerance response (ATR) (17). In addition, the acid-adapted V. cholerae was shown to be more virulent in a murine model of cholera than V. cholerae grown at neutral pH. These results have interesting implications for the V. cholerae ATR in the fitness of this pathogen in an individual host as well as in rapid epidemic spread.
In previous studies, it has been demonstrated that outer membrane protein P2 from Haemophilus influenzae type b has porin activity and that antibody directed against P2 is protective in an infant rat bacteraemic model. Outer membrane protein subtyping has been employed to subclassify type b Haemophilus isolates. Strain MinnA has the outer membrane protein subtype 1H and is representative of the dominant clonal group of disease-producing isolates in the United States. In the present study, the P2 gene from strain MinnA was employed to probe EcoRI- and Pvull-digested chromosomal DNA from 24 Haemophilus influenzae type b isolates representative of the common outer membrane protein subtype groups observed throughout the world. Restriction fragment length polymorphisms were identified for the members of the outer membrane protein subtype 3L group, but not for the other subtypes examined. The P2 gene from each of four prototype isolates was then cloned, sequenced and compared to the previously reported sequence of the strain MinnA gene. The P2 gene from each of two isolates with the outer membrane protein subtype 3L was identical to the MinnA P2 sequence. The P2 gene from a subtype 2L isolate differed by a single nucleotide and the gene from a subtype 6U isolate differed by 13 nucleotides. Thus, the P2 protein is highly conserved among type b isolates.
The OmpU outer membrane protein is a member of the ToxR regulon of Vibrio cholerae and has recently been shown to be a potential adherence factor for this species. Using PCR and degenerate oligonucleotide primers based on internal peptide sequences of purified OmpU, we have cloned and sequenced the gene encoding OmpU. The ompU gene is predicted to encode a 36,646-molecular-weight protein which is present in both cholera toxin-positive and-negative V. cholerae O1 and O139 strains.
Haemophilus influenzae type b are heterogeneous antigenically and with respect to apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For determination of the molecular basis for the differences in the P1 proteins, the genes for the P1 proteins from strain 1613, representative of outer membrane protein subtype 3L, and strain 8358, representative of outer membrane protein subtype 6U, were cloned, sequenced, and compared with the previously reported gene for the P1 protein from strain MinnA, a strain with the outer membrane protein subtype 1H. These prototype strains are representatives of the three major clonal families of H. influenzae type b responsible for invasive disease in diverse areas of the world. The nucleotide sequences of the P1 genes from strains 1613 and 8358 were 94 and 90% identical to the MinnA sequence, respectively. The derived amino acid sequences were 91 and 86% identical, respectively. Heterogeneity between the MinnA and 1613 proteins was largely localized to two short variable regions; the protein from strain 8538 contained a third variable region not observed in the other P1 proteins. Thus, the outer membrane protein P1 genes are highly conserved; the variable regions may code for the previously demonstrated strain-specific antigenic determinants.
Origins of replication are known to be highly conserved among widely divergent microbial species, with the gene order in those regions being dnaA-dnaN-recF-gyrB. On the basis of sequence identities to entries in GenBank, the gene order of a 6-kb fragment of Mycoplasma genitalium DNA was determined to be dnaN-orf3ll-gyrB-gyrA-serS, which is structurally similar to the ancestral origin of replication. We have directly linked the dnaN gene to the M. genitlium dnaA gene by PCR amplification. However, we found a novel open reading frame, designated orJ311, in place of an expected sequence encoding recF. Orf311 contains a DnaJ box motif at its N terminus, but it has no overall homology to any other protein or sequence in the database. We are unable to detect any recF homolog in M. genitalium by hybridization or during a random sequencing survey of the genome.Mycoplasmas are significant pathogens of humans, animals, and plants. They are believed to be the smallest free-living organisms and are thought to have evolved from higher gram-positive bacteria through a loss of genetic material (27). We are characterizing Mycoplasma genitalium, which contains a genome of less than 600 kbp, as a model for an organism containing only those genes which are essential for life (25).Since no genetic system for this species exists and clonal selection of individual cells is difficult, a random sequencing approach was undertaken to survey the M. genitalium genome (25). During this study, a collection of 291 independent clones was generated and partially sequenced. This examination of the chromosome allowed the identification of potential metabolic and biosynthetic pathways of M. genitalium and provided the tools necessary to study genome organization.A clone which showed sequence homology to the Escherichia coli gyrA gene was identified during this study. This clone was chosen for further characterization because of the relation of gyrA genes to origins of replication in most bacterial species. On the basis of our own studies showing the linkage of the gyrA and gyrB genes in Mycoplasma pneumoniae (7), we speculated that these genes would also be linked in M. genitalium and that the replication origin of this organism might be similar to those of gram-positive species.Comparison of chromosomal origins of DNA replication among the most common eubacteria (enteric bacteria, pseudomonads, and bacilli) has allowed the determination of several highly conserved structural features of this region (22,30 gene. A comparison of the Bacillus subtilis origin region with that of Pseudomonas putida revealed that the order of more than 12 genes is conserved between the two species (23). The E. coli origin region, the most extensively characterized of all such regions, also fits the pattern if one compensates for an inversion which places its actual point of origin (oriC) away from the defined ancestral origin region (22). In gram-positive species the gyrA gene is linked to the gyrB gene; in gramnegative species the gyrA gene is not usually associated w...
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