V. cholerae is the causative agent of the diarrheal Selleck SCH727965 disease cholera. To date, there have been seven recorded pandemics of this severely dehydrating diarrheal disease. The ability of V. cholerae to survive the passage through the human gastric acid barrier, to colonize the human intestine with its pili and other outer membrane proteins and polysaccharides, and to secrete the cholera toxin (CT) are all crucial components of the bacterial life cycle [18]. Secretion of proteins is critical for the pathogenicity of the organism and for its find more survival in the natural environment. The genome of V. cholerae El Tor contains the tatABC operon in chromosome I and the tatA2 (tatE) gene in chromosome
II [19]. To analyze the function and the involvement of the Tat system in the survival and virulence of V. cholerae, we constructed chromosomal in-frame deletion mutations in tatABC and tatE. Our findings demonstrate that the V. cholerae tatABC genes function in the translocation of TMAO reductase. Moreover, we found that the mutation affected MLN8237 biofilm formation, attachment to HT-29 cells, and colonization of suckling mouse intestines. The flagellum biosynthesis and motility, outer membrane integrity, and growth rate in
normal cultures of Tat mutants were not affected. We also observed that the mutation impaired the transcription of the toxin gene, as well as CT production, although the ratio of secreted toxin to toxin stored in the cytoplasm was the same in the mutant and in the wild type strain. Overall, the Tat system is associated with the survival, as well as the virulence of V. cholerae. Methods Bacterial strains, media, and growth conditions The bacterial strains and plasmids used in this study are listed in Table 1. Thymidylate synthase The tatABC deletion mutant N169-dtatABC strain was derived from the wild type O1 El Tor strain N16961 (Table 1). Both E. coli and V. cholerae cells were routinely grown at 37°C in Luria-Bertani broth (LB). For plate culture, LB was used with 1.5% agar (LBA). For the detection of CT production,
V. cholerae were first grown under AKI conditions with sodium bicarbonate (1.5% Bacto Peptone, 0.4% yeast extract, 0.5% NaCl) at 37°C for 4 h, and the culture was then incubated overnight while shaking at 37°C [20]. Antibiotics were used at the following concentrations: ampicillin, 100 μg/ml; streptomycin, 100 μg/ml; and chloramphenicol, 30 μg/ml. The growth kinetics of the bacterial culture was measured spectrophotometrically with the optical density (OD) of the culture at 600 nm. Complementarity of the E. coli tat mutants complemented by the V. cholerae tat genes was analyzed by anaerobic growth in M9-TMAO minimal media. The components of the M9-TMAO medium (for a final volume of 1 liter) in this study are listed below: 12.8 g Na2HPO4; 3.0 g KH2PO4; 0.5 g NaCl; 1.0 g NH4Cl; 2 ml 1 M MgSO4; 0.
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