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ABSTRACTThe dnaKgene of Vibrio choleraewas cloned, sequenced, and used to construct a dnaKinsertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathogen. The central regulator of several virulence genes of V. choleraeis ToxR, a transmembrane DNA binding protein. The V. cholerae dnaKmutant grown in standard laboratory medium exhibited phenotypes characteristic of cells deficient in ToxR activity. Using Northern blot analysis and toxRtranscriptional fusions, we demonstrated a reduction in expression of the toxRgene in the dnaKmutant strain together with a concomitant increase in expression of a htpG-like heat shock gene that is located immediately upstream and is divergently transcribed fromtoxR. This may be due to increased heat shock induction in the dnaKmutant. In vivo, however, although expression from heat shock promoters in the dnaKmutant was similar to that observed in vitro, expression of both toxRandhtpGwas comparable to that by the parental strain. In both strains, in vivo expression of toxRwas significantly higher than that observed in vitro, but no reciprocal decrease inhtpGexpression was observed. These results suggest that the modulation of toxRexpression in vivo may be different from that observed in vitro.