FIGURE 3.

Qrrs repress expression of SmcR redundantly in V. vulnificus. A, nucleotide sequences of five Qrrs that potentially base pair with the 5′-untranslated region (UTR) of SmcR. Putative pairing sequences are boxed in the Qrr sequences and are marked with asterisks in the 5′-UTR of SmcR. The initiation codon and ribosome-binding site of SmcR are noted. B, repression of SmcR by Qrrs at log phase in V. vulnificus. Wild type, ΔsmcR, Δqrr1–5, ΔluxO, Δhfq, luxOD47E, and Δqrr1–5 cells were harvested at both log phase (A₆₀₀ 0.6–0.7) and stationary phase (A₆₀₀ >2.0), and 10 μg of lysate was subjected to Western blotting using an antibody against SmcR. The upper panel represents the relative densities of the bands shown in the lower panel. Band intensities were quantified using MultiGauge version 3.0 software (Fujifilm, Tokyo, Japan). Values are averages normalized to the intensity of the Δqrr1–5 (stationary phase) sample from biological experiments done in triplicate. **, p < 0.005; NS, not significant in Student's t test with p > 0.05. C, regulation of SmcR by individual Qrrs. Wild type V. vulnificus cells harboring pRK415, Δqrr1–5 harboring pRK415, and Δqrr1–5 harboring pRK415-qrr1 through pRK415-qrr5 were harvested at log phase (A₆₀₀ ∼0.6). Ten μg of lysate was subjected to Western blotting using an antibody against SmcR. This result is representative of three independent experiments. D, SmcR expression in cell extracts from qrr deletion strains was measured by Western blot hybridization. Wild type V. vulnificus MO6-24/O, Δqrr1, Δqrr2, Δqrr3, Δqrr4, Δqrr5, Δqrr14, Δqrr134, Δqrr1345, and Δqrr1–5 were harvested at log phase (A₆₀₀ ∼0.6). Ten μg of lysate was subjected to Western blotting using an antibody against SmcR. This result is representative of three independent experiments.