FIG 5.

cFP enhances the expression of genes encoding subunits of a histone-like protein, vHUα and vHUβ, via LeuO. (A) Expression levels of rpoS as measured by qRT-PCR from wild-type V. vulnificus [Wild type (pBBR1-MCS2)], ΔleuO(pBBR1-MCS2), ΔleuO(pBBR1-leuO), ΔleuOΔvhuAB(pRK415), and ΔleuOΔvhuAB(pRK-vhuAB) cultured in AB broth at early stationary phase (A₆₀₀, ∼0.2). (B) β-Galactosidase activities from wild-type (pBBR1-MCS2), ΔleuO(pBBR1-MCS2), and ΔleuO(pBBR1-leuO) strains harboring pMZtc-vhuA (left) or pMZtc-vhuB (right). Overnight cultures of V. vulnificus were subcultured into fresh LB broth supplemented with each concentration of cFP, and when the cells reached exponential phase (optical density [OD], 0.3 to 0.4), the cells were resubcultured in fresh AB broth. β-Galactosidase activities were measured as described in Materials and Methods. The error bars denote standard deviations of the results of three independent experiments. MU, Miller units. **, P < 0.005; *, P < 0.05; NS, not significant. (C) Binding of recombinant LeuO (rLeuO) to the upstream regions of vhuA (left) and vhuB (right) genes as determined by electrophoretic mobility shift assay. Ten nanograms of radiolabeled probes was incubated with increasing concentrations of LeuO. Lanes 1 to 5, LeuO concentrations of 0 nM, 10 nM, 20 nM, 40 nM, and 80 nM, respectively; lanes 6 to 8, 80 nM rLeuO with unlabeled probes as a competitor at 1 ng, 10 ng, and 100 ng, respectively.