The VipA-VipB interaction in the reporter strain KDZif1ΔZ leads to β-galactosidase activity, which is influenced by the growth temperature as well as the NaCl concentration of the medium. Shown is the mean β-galactosidase activity ± standard deviation in Miller units produced from two experiments where two independent transformants were tested on each occasion. The temperatures tested were 37°C (High) or 23°C (Low). Data was subjected to a student’s 2-sided t-test to determine whether the β-galactosidase activity produced

at any given condition was significantly different from that produced by KDZif1ΔZ grown under standard assay conditions (85 mM NaCl, 37°C) (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Mutating the VipB-interaction site of VipA leads to unstable DNA Methyltransferas inhibitor VipB and essentially abolishes Hcp secretion Previously, Nutlin-3 manufacturer Bönemann et al. have shown that VipA is essential for secretion of Hcp as well as production of VipB in V. cholerae non-O1 non-0139 strain V52 [9]. The latter was assumed to be a consequence of decreased VipB stability and, thereby, lower

amounts of the VipA/VipB complex. We have recently shown that VipA is required for secretion of Hcp also in V. cholerae O1 strain A1552 [13]. To investigate if any of our vipA deletion or substitution mutants resulted in diminished Hcp secretion and/or VipB production, we expressed them as C-terminal His6 tagged variants from the ptac promoter of pMMB66EH in an A1552 vipA null mutant

background. Importantly, His6-tagged VipA behaved identically to non-tagged VipA in all analyses performed (data not shown). By immunoblot analyses, we could confirm that all of the mutant strains expressed Hcp at levels similar to the parental strain (Figure 4, top panel), but like the vipA null mutant, some did not secrete Hcp into the culture medium. These corresponded to the deletion mutants Δ104-113 and Δ114-123, as well as the multiple substitution mutants V110A/L113A, D104A/V106A, D104A/V106A/V110A and D104A/V106A/V110A/L113A (Figure 4). The same mutants that failed to secrete Hcp also Sorafenib order failed to support stable production of VipB (Figure 4), suggesting that there is a strong correlation between the ability to secrete Hcp and the ability to produce stable VipB in V. cholerae. When expressed together with VipB in E. coli, the same VipA mutants also failed to support stable VipB (compare Figures 2B and 4), demonstrating that the same mechanisms of degradation exist in these closely related species. Figure 4 The influence of vipA mutations on VipB synthesis and Hcp synthesis/secretion. Deletion mutant alleles (lanes c-d), wild-type (lane e) or substitution mutant alleles (lanes f-r) of vipA were expressed from the ptac promoter of pMMB66EH in a vipA null mutant background. Hcp protein contained in the pellet fraction or secreted to the culture medium was separated by SDS-PAGE and identified by immunoblot analysis using antiserum specific for Hcp.