Figure 2.

(A) Non-redundant alignment of the sdsR gene including the upstream promoter region. All nucleotides are coloured regarding their degree of conservation (red: high conservation; blue: partial conservation; black: little or no conservation). σ⁷⁰ or σ^S-specific promoter consensus motifs (61) are indicated above the alignment (W: A/T; R: A/G; Y: C/T; K: T/G); the putative −10 and the −35 sites of the sdsR promoter are boxed in light grey, σ^S-specific extensions of the −10 element are marked in dark grey and the conserved cytosine residue at position −13 is boxed in light blue. ‘+1’ marks the transcriptional start site, and the transcribed SdsR sequence is given in bold letters. The ρ-independent terminator is indicated by arrows. (B) SdsR sRNA is not detectable in the absence of RpoS. SdsR levels were determined by northern blotting of RNA isolated at indicated times over growth from Salmonella wild-type and rpoS mutant cells (JVS-5487) carrying either a control vector (pRH800) or a plasmid constitutively expressing E. coli RpoS (pRL40.1). RpoS expression was monitored by western blotting, and loading was controlled by probing for ribosomal protein S1. (C) SdsR sRNA and osmY mRNA are rapidly induced under osmotic stress in wild-type but not in rpoS mutant bacteria. Salmonella wild-type, ΔrpoS and ΔsdsR (JVS-8827) were grown in supplemented M9 medium to an OD₆₀₀ of 0.3 when cells were split and osmotic shock was induced in one aliquot by addition of NaCl to a final concentration of 0.3 M while the other half was left untreated. Total RNA samples withdrawn prior to and at selected timepoints after NaCl addition were analysed by northern blotting; SdsR and osmY mRNA were detected using riboprobes, 5S levels were determined as loading control. Expression of RpoS was controlled by western blotting, detection of ribosomal protein S1 was used to control equal loading. (D) Transcriptional activity at sdsR and osmY promoters upon osmotic shock. Promoter activities of Salmonella sdsR-lacZ (JVS-8717; red triangles) and osmY-lacZ (JVS-9145; blue squares) fusions were determined after osmotic stress was induced as in (C) by measuring relative β-galactosidase activities over 30 min in culture samples with (filled symbols) or without NaCl added (open symbols). Error bars represent the standard deviation calculated from three biological replicates.