Establishment Of A Gene Screen Method Of Vibrio Cholerae Involved In Pumping Bile Salts And Research On Aerobic Growth Associated Proteins SCO1and C-Signal

Cholera is an ancient, prevalent and fulminating intestinal diseases caused by the gram-negative bacterial Vibrio cholerae. Extensive studies show that the ability of V. cholerae strains to cause severe enteric infection in humans dependents on cholera toxin and toxin-coregulated pilus.Bile salts come from bile, and they play a role in digesting fats.Bile salts are detergents, which can destroy cell membranes so that they could kill cells.To some extent, Vibrio cholerae are resistant to bile salts, which indicates that V.cholerae can pump bile salts out of the cells or decompose them. Transposon mutagenesis was employed to screen a V.cholerae mutant whose resistance to crude bile,cholate and deoxycholate was greatly attenuated.And the gene disrupted by the transposon was sequenced and found to be consistant with previous research.That means transposon mutagenesis is a proper method to screen for genes involed in pumping bile salts out of Vibrio cholera cells.Cytochrome c oxidase is the terminal enzyme of the respiratory chain. Copper is passed to the reduction-oxidation centers of cytochrome c oxidase by Socl encoeded by socl in yeast, Bacillus subtilis and other microbes.vca0038is a putative scol gene in V.cholerae. A scol mutant of V.cholerae was constructed via in-frame deletion. Deletion of the gene does not affect the aerobic growth of the mutant in LB medium or M9medium compared with the wide type strain C6706. The oxidative ability and the resistance to H2O2of the mutant are not attenuated. Thus, vca0038may not affect the enzyme activity of cytochrome c oxidase of V.cholerae.There are three quoram sensing systems in V.cholerae:CqsA/S system, LuxS/PQ system and VarS/VarA-CsrA/BCD system。They play a part in the regulation of genes involved in pathogenicity. And C-signal is a quorum sensing signal, which makes cells of Myxococcus xanthus start to aggregate and sporulate during aerobic growth.Thus, it is possible that vc1815, a putative C-signal gene in V.cholerae, may encode proteins that influence activities involved in V.cholerae quorum sensing systems.vc1815was deleted via in-frame deletion.The biofilm formation, TCP production and colonization in host intestine of the mutant were tested, none of them were attenuated compared with the wide type strain C6706.vc1815may not affect quorum sensing systems of V.cholerae.In this research, screen method for bile salts pumping associated genes was established via transposon mutagenesis. Both the putative scol gene, vca0038, and putative C-signal gene, vc1815, may affect aerobic growth of V.cholerae. Mutants of both genes were constructed via in-frame deletion.Aerobic growth and the the enzyme ability of cytochrome c oxidase were not attenuated in the vca0038mutant. Biofilm formation and other characteristics involved in quorum sensing systems of the vc1815mutant were not affected. Thus, the two genes may not affect the aerobic growth of Vibrio cholerae.