The toxR gene of Vibrio cholerae encodes a transmembrane, DNA-binding protein that activates transcription of the cholera toxin operon and a gene (tcpA) for the major subunit of a pilus colonization factor. We constructed site-directed insertion mutations in the toxR gene by a novel method employing the chromosomal integration of a mobilizable suicide plasmid containing a portion of the toxR coding sequence. Mutants containing these new toxR alleles had an altered outer membrane protein profile, suggesting that two major outer membrane proteins (OmpT and OmpU) might be under the control of toxR. Physiological studies indicated that varying the concentration of the amino acids asparagine, arginine, glutamate, and serine caused coordinate changes in the expression of cholera toxin, TcpA, OmpT, and OmpU. Changes in the osmolarity of a tryptone-based medium also produced coordinate changes in the expression of these proteins. Other environmental signals (temperature and pH) had a more pronounced effect on the expression of cholera toxin and TcpA than they did on the outer membrane proteins. These results suggest that certain environmental signals (i.e., osmolarity and the presence of amino acids) are tightly coupled to the expression of toxR-regulated proteins and therefore may be signals that are directly sensed by the ToxR protein.Little is known about the in vivo environmental signals that control expression of bacterial virulence determinants. The effects of nutritional and physical parameters on the production of virulence factors in laboratory media reflect the existence of regulatory mechanisms that may help the microbe determine when it is appropriate to express these rather specialized properties. This regulation presumably allows the organism to avoid the metabolic drain of producing toxins, colonization factors, capsules, and other virulence-enhancing proteins in environments where their action is not needed. A wide spectrum of compounds and growth conditions have been implicated in the regulation of virulence properties, including iron (2, 23), divalent cations (24,25,29,33), atmospheric gases (26), temperature (14), and even complex organic molecules like nicotinic acid (33) and phenolic compounds (28). Understanding the signals and mechanisms that are involved in the control of virulence gene expression might someday lead to applications in vaccine development and chemotherapy of bacterial infections.Vibrio cholerae is a gram-negative bacterium that causes a severe diarrheal disease by colonizing the upper intestine of humans and elaborating a protein exotoxin (1). Cholera toxin is a multimeric protein composed of two types of subunits, A and B, that are encoded by the genes ctxA and ctxB, respectively (18). The ctxA and ctxB genes form an operon that is positively regulated at the transcriptional level by the product of the toxR gene (20)(21)(22). Recently, we have shown that the toxR gene regulates not only the ctx operon but also the gene (tcpA) for the major subunit of a toxin-coregulated pilus col...

The transposon TnphoA was used to generate fusions between phoA, the gene for alkaline phosphatase (PhoA), and genes encoding proteins that are secreted by Vibrio cholerae. One of the PhoA' mutants isolated showed a dramatic reduction in its ability to colonize the intestines of suckling mice. This mutant no longer produced a 20.5-kDa protein (TcpA) that we show is the major subunit of a V. cholerae pilus. Amino-terminal sequence analysis of the TcpA pilus subunit showed that it shares amino acid homology with the pilins produced by several other pathogenic bacteria. The TcpA pilus was coordinately expressed with cholera toxin under various culture conditions, and this effect appeared to be dependent on the transcriptional activator encoded by the toxR gene. We conclude that the toxR gene plays a central role in the transcriptional regulation of multiple virulence genes of V. cholerae.

Virulent strains of Yersinia enterocolitica cause disease syndromes ranging from mild gastroenteritis to lymphadenitis and septicemia. The ability of these bacteria to invade intestinal epithelial cells to gain access to the reticuloendothelial system is thought to be an important aspect of their virulence. We report here on the cloning of two Y. enterocolitica chromosomal loci, inv and ail, ehch of which confers an invasive phenotype on Escherichia coli HB101. The inv locus allows a uniformly high level of invasion in several tissue culture lines and is homologous to the inv gene of Yersinia pseudotuberculosis. The second locus, ail, shows more host specificity than inv in that it allows invasion to a variable degree of some cell lines (e.g., HEp-2, HECiB, and CHO cells) but allows no invasion of others (e.g., Madin-Darby canine kidney cells).

The ability to invade the intestinal epithelium of mammals is an essential virulence determinant of Yersinia enterocolitica. The chromosomally encoded Y. enterocolitica 8081v invasion gene, inv, was disrupted to assess its role in pathogenesis. The inv mutant (JP273v) was -80-fold less invasive than wild type for cultured epithelial cells. When mice were infected intragastrically, up to 107 fewer JP273v were recovered from Peyer's patches early (6-18 hr) after infection compared with wild

We have cloned a positive regulatory gene (toxR) from Vibrio cholerae that controls cholera toxin transcription. This was done by first constructing a genetic fusion consisting of the lacZ gene fused to the promoter of the cholera toxin operon ctxAB. This operon fusion was used to screen a V. cholerae genomic library for genes that could activate the ctx promoter in Escherichia coli. This method allowed the identification of a gene, toxR, that increases ctx expression by more than 100-fold. Complementation analysis indicated that certain hypotoxinogenic mutants of V. cholerae 569B probably have mutations in the toxR gene. Southern blot analysis suggests that all V. chokrae, including nontoxinogenic strains, have the toxR gene. Moreover, nontoxinogenic strains not only lack the structural genes for cholera toxin but also sequences associated with the larger 7-kilobase ctx genetic element.The diarrheal disease Asiatic cholera is induced by cholera toxin, a heat-labile protein produced by toxinogenic strains of Vibrio cholerae (1). Each toxin molecule is composed of two types of subunits, A (27 kilodaltons) and B (11.7 kilodaltons), encoded by the genes ctxA and ctxB, respectively (2, 3). The ctxAB operon shows genetic linkage to the nal region of the V. cholerae chromosome in strains of both the classical and the El Tor biotypes (4). All V. cholerae strains of the classical biotype thus far examined carry a structurally similar duplication of the ctxAB operon (5). Although most strains of the El Tor biotype possess only a single toxin operon copy, there are also El Tor strains that have two or more ctxAB copies (5), as well as other V. cholerae strains that have no detectable toxin gene sequences (6). This variation in the toxin operon copy number, together with recent physical data on the structure of ctx duplications (3,5), suggests that the ctx genes reside on a larger genetic element that may undergo duplication, amplification, and transposition events. Thus, the study of cholera toxin regulation offers a system in which to analyze regulatory systems controlling the expression of virulence genes located on accessory genetic elements (e.g., transposons, phages, and plasmids).Previous studies focusing on toxin regulation in V. cholerae have identified at least two different genetic loci that control toxin production in the classical strain 569B (7,8). Mutations in a locus called tox cause a decrease in toxin production by a factor of 1000. Since strain 569B carries a duplication of the ctx operon (2, 5), this result suggests that the tox gene product is required for high expression of both ctx copies of this strain.The two ctx operon copies from strain 569B have been cloned in Escherichia coli, but synthesize 100-fold more B subunit in V. cholerae than in E. coli (3). While it is possible that the ctx transcriptional and translational signals are not recognized efficiently by the E. coli synthetic machinery, an alternative explanation for this difference, consistent with known V. cholerae regulatory muta...