FIG 4.

Effects of MBP-ToxT alanine mutagenesis on response to negative effectors bile and linoleic acid. V. cholerae O395 ΔtoxT mutant with plasmid-borne WT or mutant MBP-ToxT was grown under virulence-inducing conditions (LB, pH 6.5, 30°C, shaking) with or without the addition of negative effectors for 3 h. (Top) Light gray bars, V. cholerae grown without bile (A) or with dimethyl sulfoxide (DMSO) alone (B); dark gray bars, V. cholerae grown with 0.05% bile (A) or 32 μM linoleic acid (B) dissolved in DMSO. β-Galactosidase activity produced from chromosomal tcpA::lacZ in classical strain O395 ΔtoxT was measured. Statistical significance of ToxT mutant activation of tcpA::lacZ under each condition compared to WT ToxT activation of tcpA::lacZ under the same condition was calculated using Student's t test. Statistical significance of activation by a ToxT mutant without effector or with is denoted by an asterisk or plus sign, respectively (* or +, P < 0.05). (Bottom) Mean fold decrease in activation of tcpA::lacZ upon the addition of 0.05% bile (A) or 32 μM linoleic acid (B) for each ToxT mutant. Statistical significance of fold change in activity of a MBP-ToxT mutant compared to the activity of WT MBP-ToxT was determined by Student's t test (*, P < 0.05); the dashed line represents fold change of WT ToxT with the addition of effector. ToxT mutants with results above the line had increased sensitivity to effector, while results below the line represent decreased sensitivity to effector. Error bars represent ±SEM.