Fig. 1. A hybrid protein consisting of the α-helical region 2.5 of σ^S and β-galactosidase (α2.5::LacZ) binds to phosphorylated RssB. Direct interaction between RssB (carrying an S-TRX-His₆ tag, present in lanes 1 and 6–9) and either His₆-RpoS (lane 1) or crude cell extracts of strains overexpressing α2.5::LacZ or α2.5^K173E::LacZ (lanes 2–9) was assayed by affinity chromatography on S-protein–agarose (as detailed in Materials and methods). Proteins adsorbed to S-protein–agarose were washed and eluted with sodium citrate (pH 2), separated by SDS–PAGE and visualized by immunoblotting. Numbers at the left indicate molecular masses (kDa) of size standard proteins.