Functional Analysis Of Lysr Family VC2324and Its Target Gene VC2323in V. Cholerae

Vibrio cholerae is a Gram-negative bacterium that causes an acute, severe diarrheal disease by colonizing the upper intestine and it is still epidemic in many developing countries. For V.cholerae in order to cause cholera, two genetic elements, the Vibrio Pathogenicity Island (VPI) and the lysogenic CTX phage, were both required. These virulence factors’expression is controlled by a series transcriptional cascade, and one important activator is AphB, a LysR-type regulator. LysR-type transcriptional regulators (LTTRs) is one of the largest families of regulators in prokaryotes and their protein products can control the expression of their divergently transcribed genes which are involved in a variety of biological processes such as metabolism, quorum sensing, virulence, attachment and secretion. There are at least40LysR-type genes in Vibrio cholera, many of them have not reported their function yet. In this study, we chose four LysR-type genes without reported previously. By constructing their inframe deletions, and using biofilm formation assay, luminescence test and competition assay, we describe here a new member of LysR-type transcriptional regulators, vc2324, and its target gene vc2323, which relates to drug resistance and colonization of Vibrio cholera.Bioinformation analysis shows vc2323is a homologue of E. coli tellurite-resistance protein TehA. The TehA homologues can be found in both eukaryotic and prokaryotic organisms, such as Arabidopsis thaliana SLAC1and Haemophilus influenzae HiTehA, and these TehA homologues have multiple functions such as drug and tellurite resistance, membrane-associated efflux pump, reactive oxygen species (ROS) resistance, ion channel and nitrosative stress. These functions may share same mechanism by which vc2323affect V. cholerae colonization.Through luminescence assay and minimum bactericidal concentration (MBC) test, we found vc2323participate in V. cholera drug resistance. There was no difference on TCP expression and biofilm formation shown in vc2323mutant. Also vc2323has no relationship with ROS resistance, nitrosative stress and resistance to low pH. However, by intestine attachment assay we found tehA mutant showed significantly attenuated for adherence when crude bile existed, while no inhibition of crude bile and bile salt in culture was observed and no difference of adherence ability between vc2323mutant and wild type was shown without crude bile. These results suggest that vc2323may through a new way to play a role in V.cholerae colonization under inner environment containing crude bile. The mechanism still needs further investigation.