Fig. 2. McaS levels are regulated by CRP and Hfq.
A. Effect of Δcrp on McaS and CyaR levels. Wild type MG1655 and the isogenic Δcrp::cat mutant (GSO549), were grown for 20 h at 37°C in LB media. At the indicated OD600 samples were taken and RNA was processed for northern analysis as in Fig. 1.
B. DNase I footprinting of CRP binding at the mcaS promoter. The ~300 nt fragment labeled on the 5′ end of the top and bottom strands were incubated with increasing amounts of CRP+cAMP and then cleaved with DNase I. Sequencing ladders generated with the same labeled oligonucleotides were run along side the footprinting reactions. The positions indicated are relative to the +1 of McaS transcription.
C. Effect of ΔrpoS on McaS levels. Wild type MG1655 and the isogenic ΔrpoS::kan mutant (GSO548), were grown for 20 h in LB media at 37°C. At the indicated OD600 samples were taken and RNA was processed for northern analysis as in Fig. 1.
D. Immunoprecipitation with Hfq. Cells extracts were prepared from wild type MG1655 grown in LB to early stationary phase (OD600 ~1) and subject to immunoprecipitation with α-Hfq or preimmune serum. Northern analysis was carried out on the immunoprecipitated samples (0.5 μg RNA loaded) as well as on total RNA isolated from wild type and the isogenic hfq-1 mutant (GSO550) (5 μg loaded) as in Fig. 1.