A Vibrio cholerae arcA mutant was constructed and used to examine the role of the global anaerobiosis response regulator ArcA in the expression of virulence factors in this important human pathogen. In V. cholerae, expression of the major virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) is regulated by the transcriptional activator ToxT. toxT expression, in turn, is controlled by the transmembrane DNA binding proteins ToxR and TcpP. In the V. cholerae arcA mutant, although ToxR and TcpP were unaffected, Northern blot and reverse transcription-PCR analyses indicated that the expression of toxT was significantly decreased with concomitant reduction in the expression of CT and TCP. CT and TCP expression was completely restored in the V. cholerae arcA mutant strain by expressing a cloned toxT gene in the mutant. These results suggest that ArcA functions as a positive regulator of toxT expression under both aerobic and anaerobic conditions, although as expected, the effect was more pronounced during anaerobic growth. This was reflected in a reduction of virulence of the V. cholerae arcA mutant strain in the infant mouse cholera model.

In Vibrio cholerae, the transmembrane DNA binding proteins, ToxR and TcpP, activate expression of the regulatory gene toxT in response to specific environmental signals. The resulting enhanced level of ToxT leads to a coordinated increase in the production of a subset of virulence factors, including cholera toxin (CT) and toxin-coregulated pilus (TCP). The effect of anaerobiosis on expression of the V. cholerae virulence regulatory cascade was examined. The expression of the major regulatory genes, tcpP, toxR, and toxT, in anaerobically grown V. cholerae was comparable to that in cells grown under aerobic conditions, and no significant difference in the ToxT-dependent expression of tcpA was detected when aerobic and anaerobic cultures were compared. However, in spite of the presence of functional ToxT, ctxAB expression was drastically reduced, and practically no CT was detected in cells grown under anaerobic conditions. In a V. cholerae hns mutant, however, high levels of ctxAB expression occurred even under anaerobic conditions. Also, deletion of the H-NS binding site from the ctxAB promoter eliminated anaerobic repression of ctxAB expression. These results suggest that H-NS directly represses ctxAB expression under anaerobic growth conditions. It has been reported that in the first stage of infection of infant mice by V. cholerae, tcpA is expressed but ctxAB expression is shut off (S. H. Lee, D. L. Hava, M. K. Waldor, and A. Camilli, Cell 99: 625-634, 1999). This pattern is similar to the pattern in anaerobic cultures of V. cholerae. Under all other in vitro conditions, ctxAB and tcpA are known to be coordinately expressed.

Bile-mediated repression of virulence gene expression is relieved in a Vibrio cholerae hns mutant. The mutant also exhibited reduced motility due to lower flrA expression, higher in vivo production of the virulence factors, and lower colonization efficiency. The colonization defect of the mutant was due to low FlrA production.Vibrio cholerae, a gram-negative, noninvasive enteric bacterium is the causative agent of the diarrheal disease cholera (13). Cholera continues to be a cause of human mortality, especially in the developing countries, where conditions of poor sanitation, lack of safe drinking water, malnourishment, war, and famine contribute to regular episodes of cholera, estimated to be more than 5 million cases per year.Expression of a subset of virulence factors of V. cholerae is controlled by a regulatory cascade known as the ToxR regulon (14). The transcriptional regulators ToxR and TcpP (8) act synergistically to control the production of a second regulator, ToxT (5), that directly activates the expression of several virulence genes, including ctxAB and tcpA, coding for cholera toxin (CT) and TcpA, the major subunit of the toxin-coregulated pilus, respectively. The toxin-coregulated pilus is thought to be essential for colonization of the intestinal epithelium by the bacterium (10, 27). Expression of the ToxR regulon is strongly influenced by physicochemical parameters that exert their effects at different levels of the regulatory cascade (reviewed in references 17, 20, and 26). Under nonpermissive conditions of temperature and pH, transcription of the tcpP gene is repressed, leading to down regulation of the entire virulence regulon (15). However, under anaerobic or microaerobic conditions, TcpP, ToxR, and ToxT are produced and the tcpA gene is optimally expressed, but expression of ctxAB is drastically reduced (16,18). The presence of bile influences expression of the virulence regulon in yet another manner. Although production of the regulatory factors ToxR, TcpP, and ToxT is unaffected in cells grown in the presence of bile, expression of both ctxAB and tcpA is drastically reduced by an unknown mechanism (7, 24). A role of the nucleoid-associated histone-like nucleoid structuring protein (H-NS) in the silencing of virulence gene expression under nonpermissive conditions of temperature, pH, and oxygen concentration has been demonstrated (22,28). In this study we have shown that H-NS also represses ctxAB and tcpA gene expression in cells grown in the presence of bile and in vivo in rabbit intestine. Furthermore, the motility and colonization efficiency of a V. cholerae hns mutant strain were lower than those of the wild-type strain.A V. cholerae hns mutant produces CT and TcpA in the presence of bile. The V. cholerae O395 hns mutant strain O395H29, previously used to demonstrate the role of H-NS in anaerobic repression of CT production (16), was used in this study. CT in culture supernatants and sonicated cell lysates of V. cholerae strains O395 and O395H29 (hns) grown under inducing conditions (LB...

In the enteric pathogen Vibrio cholerae, expression of the major virulence factors is controlled by the hierarchical expression of several regulatory proteins comprising the ToxR regulon. In this study, we demonstrate that disruption of the fadD gene encoding a long-chain fatty acyl coenzyme A ligase has marked effects on expression of the ToxR virulence regulon, motility, and in vivo lethality of V. cholerae. In the V. cholerae fadD mutant, expression of the major virulence genes ctxAB and tcpA, encoding cholera toxin (CT), and the major subunit of the toxin-coregulated pilus (TCP) was drastically repressed and a growth-phase-dependent reduction in the expression of toxT, encoding the transcriptional activator of ctxAB and tcpA, was observed. Expression of toxT from an inducible promoter completely restored CT to wild-type levels in the V. cholerae fadD mutant, suggesting that FadD probably acts upstream of toxT expression. Expression of toxT is activated by the synergistic effect of two transcriptional regulators, TcpP and ToxR. Reverse transcription-PCR and Western blot analysis indicated that although gene expression and production of both TcpP and ToxR are unaffected in the fadD mutant strain, membrane localization of TcpP, but not ToxR, is severely impaired in the fadD mutant strain from the mid-logarithmic phase of growth. Since the decrease in toxT expression occurred concomitantly with the reduction in membrane localization of TcpP, a direct correlation between the defect in membrane localization of TcpP and reduced toxT expression in the fadD mutant strain is suggested.