Screen Genes Affected The Activity Of Quorum Sensing Regulator VqmA And Functional Analysis Of LysR And MFS Proteins In V.choleare

The gram-negative and facultative bacterium Vibrio cholerae is the causative agent of cholera which is an acute dehydrating diarrhea still endemic in many parts of the developing countries. Extensive studies have indecated that the ability of V. cholerae strains to cause severe enteric infection in humans is dependent on the expression of cholera toxin (CT) and a pilus colonization factor known as the toxin-coregulated pilus (TCP). The genes involved in CT and TCP production are controlled by a series of signal transduction, and a lot of environmental sigals can affect the expression of virulence genes, such as temperature, pH, bile salt and quorum-sensing signals.The main quorum sensing regulator HapR can decrease CT and TCP production by repressing transcription of aphA directly. The LuxR family protein VqmA can decrease CT and TCP production by derepressing HapR at low cell density. In many bacteria, LuxR family proteins could only regulate downstream genes expression at the presence of some small compound which is so-called co-inducer. In this study, with the method of random transposon insertion, we screened the gene which is necessary for VqmA to induce the target genes and got some interesting mutants. We selected M1(AVC0073) which is a putative SAM-dependent methyl transferase for further research and found that VqmA can not induce its taget gene expression in VC0073 in frame deletion strain and the activity of VqmA can be restored in VC0073 complemental strain. Furthermore, the compound synthesized by VC0073 was not existed in the supernate of the bacteria.LysR family protein is a global transcriptional regulator, and there are more than 40 LysR type proteins in V.choleare. Relying on the method of bioinformatics, we found five kinds of special LysR family proteins, the genes downstream of these LysR family proteins encode five MFS type proteins, we chose one of these gene groups VC1617 (Encoding LysR Protein) and VC1618(Encoding Multidrug Resistance protein) as our research material, and find strains with VC1617 in frame deletion and overexpression can grow very well in LB and M9 media, VC1617 is not the growing necessary gene inLB and M9 media.With the method of constructing functional plasmids, we found that VC1617 can regulate not only itself but also the expression of VC1618. Western blot assay indicate that virulence genes expression was not affected in VC1617 deletion strain. Although the expression of VC1618 decreasing sharply in VC1617 disrupted stain, the mutant owns the same resistant ability to some kind of drugs as the WT strain. Because the capacity to some drugs and the expression of virulence genes of the mutants were the same as WT, VC1617 in frame deletion strain can colonize in the intestine of infant mouse very well.