FIG 4.

Manipulating mtlA expression results in corresponding inverse changes in MtlS levels. V. cholerae strains were grown to mid-log (A, B, C) or late log (D, E) phase in minimal medium supplemented with the indicated carbon source. (A, B) The V. cholerae mtlA promoter region was ablated either by deleting the five CRP-binding sites within the promoter (ΔCRPbs) or by creating two point mutations in the −10 promoter region (−10mut). Total RNAs from these strains were used for qRT-PCR analysis with primers specific to mtlA (A) or mtlS (B). The levels of mtlA and MtlS RNA were normalized to those of an endogenous 4.5S RNA control. Reported are the means and standard deviations from three biological replicates (except for mannitol and glucose, where n = 1). P values are based on a two-tailed unpaired t test comparing the mutant to the wild type (WT). *, P < 0.05; **, P < 0.01. (C) Total RNAs from the V. cholerae wild-type or ΔmtlR strain were used for Northern blot analysis. The relative intensity (RI) of each sample compared to the intensity of the mannose wild type is shown underneath each band. (D, E) Cell lysates from the wild type and the ΔmtlR mutant of V. cholerae strains harboring lacZ transcriptional fusions to the 500 bp upstream of the +1 site of mtlA (D) or mtlS (E) were used for LacZ assays, as described in the legend to Fig. 3. Reported are the means and standard deviation from 4 biological replicates. *, statistical analysis indicates that wild type versus ΔmtlR strain are true discoveries (the false-discovery rate q value was set to 1%); NS, not significant. All results shown are representative of those from at least two independent experiments.