FIG 5.

Effects of the crp mutation and glucose on rtxA expression. (A and B) Total RNAs and proteins were isolated from the V. vulnificus strains grown to an A₆₀₀ of 0.5 and used to determine the rtxA transcript and RtxA, OmpU, CRP, and DnaK protein levels. (A) The rtxA transcript levels were determined by qRT-PCR analyses, and the rtxA transcript level in the wild type was set at 1. Error bars represent the SD. Statistical significance was determined by the Student’s t test (***, P < 0.0005; ns, not significant). (B) The secreted levels of RtxA and OmpU (as an internal control) and cellular levels of CRP and DnaK (as an internal control) were determined by Western blot analysis. WT (pJH0311), wild type; crp (pJH0311), crp mutant; crp (pJH0311::crp), crp complemented strain with pKK1502. (C) V. vulnificus lacZ mutant harboring the reporter plasmid with promoterless lacZ fused to P_rtxA was grown to an A₆₀₀ of 0.5 (for exponential phase) and 2.0 (for stationary phase) with or without 0.5% glucose. The β-galactosidase activity of the V. vulnificus cells was measured and expressed in Miller units. Error bars represent the SD. Statistical significance was determined by the Student’s t test (**, P < 0.005; *, P < 0.05). (D) The V. vulnificus strains were grown to an A₆₀₀ of 0.5 with or without 0.5% glucose and used to determine RtxA and OmpU protein levels. The secreted levels of RtxA and OmpU were determined by Western blot analysis. WT, wild type; crp, crp mutant. Molecular size markers (Bio-Rad) are shown in kilodaltons in panels B and D.