Fig. 4.

WalRK regulon expression in different walK mutant strains during exponential growth and corresponding cellular amounts of WalR~P. Strains were grown statically in BHI broth at 37°C in an atmosphere of 5% CO₂ to OD₆₂₀ ≈ 0.2. RNA samples for QRT-PCR analysis and protein samples for Phos-tag SDS-PAGE were prepared and analyzed as described in Experimental procedures. walK⁺ parent strain (IU1781); ΔwalK (IU1896); walK^ΔPAS (IU2306); walK^H218A (IU3102); walK^T222A (IU5401). (A) Amounts of spd_1874 transcript (a representative gene in the WalRK regulon) were normalized to that of gyrA. Transcript amounts are expressed relative to that of the walK⁺ parent (≡ 1.0) and represent averages from duplicate samples from at least 2 independent experiments. Unpaired two-tailed t tests of relative transcript amounts compared to that of the WalK⁺ parent were performed using GraphPad Prism 5 software. (B) Percentage of WalR~P compared to total WalR detected in extracts of walK⁺ parent and walK mutant strains determined by Phos-tag SDS-PAGE and quantitative Western blotting with anti-WalR_Spn antibody as described in Supplemental Information. The corrected amount of WalR~P in the walK⁺ parent strain is indicated by the dotted line (see text). Averages are from 2 to 5 independent biological samples resolved on 50 and 75 μM Phos-tag acrylamide gels (see Fig. 5 and S12).