In vivo induced clpB1 gene of Vibrio cholerae is involved in different stress responses and affects in vivo cholera toxin production

Previously in global transcription profile approach one of the cosmid clones of Vibrio cholerae containing the genes pnuC, icmF, and a fragment of clpB2 showed higher expression in V. cholerae grown inside rabbit intestine. In the present report, both the stress responsive clpB genes of V. cholerae 0395 were cloned, clpB1 from chromosome I and clpB2 present in chromosome II. From the Northern blot hybridization it was observed that the level of transcription of clpB2 was very low which could be due to the weak promoter strength of clpB2 its predicted in silico. The deduced amino acid sequence showed that clpB1 possesses features typical of the ClpB ATPase family of stress response proteins. The clpB! gene showed about three times higher expression under in vivo condition than in vitro. Increased expression of clpB1 gene was also observed at high temperature, high salt, and in the condition mimicking human intestine viz., 37 degrees C, pH 8.5, 300 mM NaCl, which is known to be the repressive condition for ToxR, the global transcriptional regulator of virulence in V. cholerae. The clpB1 insertion mutant showed increased sensitivity towards high temperature, oxidative stress, and acid pH. ClpB1 also conferred thermotolerance to V cholerae. These effects could be reversed by complementation. Although clpB1 appeared not to be tinder the control of virulence regulatory cascade of V. cholerae, the CT production was reduced in clpB1 mutant when tested in vivo in ail infant mice model. (c) 2005 Elsevier Inc. All rights reserved.