Figure 3.

DPO inhibits AphA production. (A) V. cholerae wild-type and vqmR mutants carrying the indicated plasmids were cultivated in M9 minimal media supplemented with casamino acids (0.4% final conc.). At the indicated growth phases, total RNA and protein samples were collected. AphA::3XFLAG production was monitored on Western Blots and RNAP served as the loading control. VqmR and aphA::3XFLAG mRNA levels were probed on Northern Blots using 5S rRNA as loading control. (B) Total RNA and protein samples were collected from V. cholerae wild-type, ΔvqmA, ΔvqmR and Δtdh strains at low cell density (OD₆₀₀ = 0.2). Cells were cultivated in M9 minimal media and one set of cultures was supplemented with DPO (100 μM final conc.). AphA::3XFLAG production was determined using Western Blot. Northern Blot was used to probe the expression of aphA-3XFLAG and VqmR. RNAP and 5S rRNA served as loading controls for the Western and Northern Blot analyses, respectively. (C) V. cholerae Δtdh cells were cultivated in M9 medium supplemented with the indicated DPO concentrations (x-axis) and total RNA and protein samples were harvested at low cell densities (OD₆₀₀ = 0.2). AphA production was analyzed on Western Blots (left y-axis), sRNA levels (VqmR and Qrr4) were determined on Northern Blots (right y-axis). Error bars represent the SD of five (AphA) and three (sRNAs) biological replicates, respectively.