Figure 8.
(A) The stringent response triggers SdsR-specific ompD mRNA decay. Salmonella expressing ompD from a chromosomal constitutive PLtetO-1 promoter (JVS-9488) and derivative strains carrying either only sdsR or rybB of the four relevant sRNA genes (strains JVS-9491 or JVS-9655, respectively) were treated with serine hydroxamate (SHX) to induce the stringent response. Total RNA samples withdrawn from cultures prior to and 15 or 30 min after SHX addition were analysed by northern blotting. SdsR but not RybB was strongly induced upon 30 min of growth in the presence of SHX; ompD mRNA levels decreased concomitantly in strains carrying a functional sdsR allele (see left and middle panels) but remained stable in the absence of sdsR (right panel). Bars represent relative ompD mRNA levels as determined from the quantification of northern blots normalized by probing for 5S RNA; error bars indicate the standard deviation from three independent biological replicates. Black or white boxes mark the presence or absence, respectively, of the indicated sRNA genes in the strains used. (B) RybB promotes selective ompD mRNA repression during membrane stress. The envelope stress response was induced by addition of the antimicrobial peptide polymyxin B (PMB) with the same set of Salmonella strains as above. Northern blot analysis of total RNA samples prepared from cells collected prior to and 5 or 10 min after PMB addition revealed strong induction of RybB but not SdsR. Rapid decay of ompD mRNA was observed to the same extent in WT and the strain only expressing RybB (see left and right panel), but down-regulation was markedly reduced in its absence (see middle panel).