We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.Toxigenic strains of the Gram-negative bacterium Vibrio cholerae cause a life-threatening diarrheal disease that can kill its victims within hours of the onset of symptoms. The disease is endemic in the Indian subcontinent and continues to reemerge elsewhere in Asia, Africa, and the Americas with the incidence estimated to exceed 5 million cases each year (1-3). Although over 180 serogroups of V. cholerae exist, only the O1 and O139 serovars are known to cause epidemic cholera. The O1 serogroup can be divided further into two biotypes, classical and El Tor, based on various biochemical and phenotypic differences (4). Since 1817, seven cholera pandemics have occurred, of which the classical biotype is responsible for at least the fifth (1881-1896) and the sixth . The El Tor biotype is responsible for the seventh and current pandemic (1961-present). Toxigenic strains of O139 serovar appeared in India and Bangladesh in late 1992 as the first non-O1 serovar to cause epidemic cholera (5, 6). These strains are closely related and probably derived from toxigenic El Tor O1 strains (7). The factors that allowed El Tor biotype and O139 serotype to overtake the classical strains and establish pandemics remain a mystery.Work focusing on the pathogenesis of V. cholerae led to the identification of several key virulence factors, including the cholera toxin responsible for the diarrhea and the toxincoregulated pilus essential for colonizing the human small intestine (8, 9). Several groups tried to construct live, attenuated V. cholerae vaccines by deleting the genes for cholera toxin and several other putative accessory toxins (10-16). However, many of these strains remain reactogenic in human subjects, causing diarrhea or other significant symptoms (17).The virulence factors responsible for the residual reactogenicity remain to be identified.Bacterial genomics offers an opportunity to identify undiscov...

SummaryWe evaluated a spontaneous mutant of Vibrio cholerae, which was avirulent in an infant mouse and had reduced expression of cholera toxin and TcpA in response to environmental signals. The toxR, toxS and toxT genes in the mutant were normal, but transcription of toxT was absent. A plasmid expressing wild-type tcpP and tcpH complemented the mutant. The mutation resulted from a frameshift in a string of nine G residues within tcpH; similar slipped-strand mutations in tcpH arose at a frequency of 10 ¹4 during overnight growth and in the majority of colonies by the end of 5 days of growth in ToxR-inducing conditions. Transcription of tcpPH was regulated by temperature and pH independently of ToxR or ToxT. These results suggest that TcpH couples environmental signals (temperature and pH) to expression of the ToxR regulon, and provide a model for phase variation in the co-ordinate expression of cholera virulence factors.

Bacteroides frgigli& CS30 is a clinical isolate resistant to high concentrations of benzylpenicillin and cephaloridine but not to cephamycin or penem antibiotics. fl-Lactam resistance is mediated by a chromosomally encoded cephalosporinase produced at a high level. The gene encoding this 13-lactamase was cloned from genomic libraries constructed in Escherichia coli and then mated with B. fragilis 638 for identification of ampicillin-resistant (Apr) strains. Apr transconjugants contained a nitrocefin-reactive protein with the physical and enzymatic properties of the original CS30 isolate. The (3-lactamase gene (cepA) was localized by deletion analysis and subcloned, and its nucleotide sequence was determined. The 903-bp cepA open reading frame encoded a 300-amino-acid precursor protein (predicted molecular mass, 34,070 Da). A 13-lactamase-deficient mutant strain of B. fiugilis 638 was constructed by insertional inactivation with the cepA gene of CS30, demonstrating strict functional homology between these chromosomal 13-lactamase genes. An extensive comparison of the CepA protein sequence by alignment with other 13-lactamases revealed the strict conservation of at least four elements common to Ambler class A. A further comparison of the CepA protein sequence with protein sequences of (-lactamases from two other Bacteroides species indicated that they constitute their own distinct subgroup of class A 13-lactamases.Bacteroides fragilis is responsible for approximately half of all human anaerobic infections and is the most common anaerobe recovered from clinical specimens (12). Numerous reports of B. fragilis isolates resistant to a variety of P-lactam antibiotics indicate that these organisms are becoming increasingly refractory to treatment with these drugs. The primary mechanism of 1-lactam resistance in Bacteroides species is the production of ,B-lactamase (40). At least four types of P-lactamase have been described for members of the B. fragilis group, but the most common type is a constitutively produced, chromosomally encoded cephalosporinase having no activity against cefoxitin or imipenem.This "endogenous" P-lactamase is present in over 90% of clinical isolates tested (10). Unlike the class C chromosomally encoded P-lactamases of members of the family Enterobacteriaceae, the B. fragilis P-lactamase has an isoelectric point in the acid range and is susceptible to inhibition by clavulanic acid and sulbactam, placing it in group 2e in the Bush classification scheme (8).Regulation of the endogenous B. fragilis j-lactamase has not been extensively studied, but this enzyme may be growth rate regulated, with maximal activity occurring 3 h into the stationary phase (7). With regard to the production of the endogenous cephalosporinase, others have grouped B.fragilis clinical isolates into three expression classes (18). Low-level j-lactamase producers are susceptible to all 3-lactams, the MICs of benzylpenicillin and cephaloridine being <2 and <16 ,ug/ml, respectively. For intermediate-level 3-lactamase producers, th...

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Bacteroides fragilis is an important opportunistic pathogen of humans and is resistant to many drugs commonly used to treat anaerobic infections, including P-lactams. A strain set comprised of B. fragilis isolates producing either low or high levels of the endogenous cephalosporinase activity, CepA, has been described previously (M. B. Rogers, A. C. Parker, and C. J. Smith, Antimicrob. Agents Chemother. 37:2391-2400.Clones containing cepA genes from each of seven representative strains were isolated, and the DNA sequences were determined. Nucleotide sequence comparisons revealed that there were few differences between the cepA coding sequences of the low-and high-activity strains. The cepA coding sequences were cloned into an expression vector, pFD340, and analyzed in a B.fragilis 638 cepA mutant. The results of j-lactamase assays and ampicillin MICs showed that there was no significant difference in the enzymatic activity of structural genes from the high-or low-activity strains. Comparison of sequences upstream of the cepA coding region revealed that 50 bp prior to the translation start codon, the sequence for high-activity strains change dramatically. This region of the high-activity strains shared extensive homology with IS21, suggesting that an insertion was responsible for the increased expression of cepA in these isolates. Northern (RNA) blot analysis of total RNA by using cepA-specific DNA probes supported the idea that differential cepA expression in low-and high-activity strains was controlled at the level of transcription. However, the insertion did not alter the cepA transcription start site, which occurred 27 bp upstream of the ATG translation start codon in both expression classes. Possible mechanisms of cepA activation are discussed.The anaerobe Bacteroides fragilis contains an endogenous, chromosomally encoded P-lactamase which preferentially hydrolyzes cephalosporins and is responsible for the intrinsic resistance to most penicillins and cephalosporins (6, 15). These organisms are generally susceptible to some of the newer ,B-lactams such as cephamycins (cefoxitin) and carbapenems (imipenem), although newly acquired 3-lactamases capable of degrading these antibiotics have been described (18,21,38).The indigenous 3-lactamase is present in between 90 and 99% of B. fragilis strains and at the biochemical level, this P-lactamase has been shown to be species specific (8,16). Recently, the gene for this enzyme, cepA, was cloned and the nucleotide sequence was determined (27). Southern hybridization analyses with a cepA probe showed that there was homology only with other B. fragilis strains, and construction of a cepA mutant provided evidence that this gene did in fact encode for the endogenous ,-lactamase. Comparison of the predicted CepA amino acid sequence with other 3-lactamase sequences indicated that it was not in the Ambler molecular class C like the chromosomal ,-lactamases of most other gram-negative bacteria, but rather CepA belonged to the class A 3-lactamases. The CepA enzyme together with...