Role of DnaK in in vitro and in vivo expression of virulence factors of Vibrio cholerae

The dnaK gene of Vibrio cholerae was cloned, sequenced, and used to constru ct a dnaK insertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathog en. The central regulator of several virulence genes of V. cholerae is ToxR , a transmembrane DNA binding protein. The V. cholerae dnaK mutant grown in standard laboratory medium exhibited phenotypes characteristic of cells de ficient in ToxR activity. Using Northern blot analysis and toxR transcripti onal fusions, we demonstrated a reduction in expression of the toxR gene in the dnaK mutant strain together with a concomitant increase in expression of a htpG-like heat shock gene that is located immediately upstream and is divergently transcribed from toxR. This may be due to increased heat shock induction in the dnaK mutant. In vivo, however, although expression from he at shock promoters in the dnaK mutant was similar to that observed in vitro , expression of both toxR and htpG was comparable to that by the parental s train. In both strains, in vivo expression of toxR was significantly higher than that observed in vitro, but no reciprocal decrease In htpG expression was observed. These results suggest that the modulation of toxR expression in vivo may be different from that observed in vitro.