Figure 4.

Cis-encoded targets. Shown are expression data and the schematic representation of the sRNAs and their flanking genes. Total RNA was isolated from S. typhimurium cells grown in rich media to logarithmic phase (Log), stationary phase (Sta) and form exponential cells subjected for 30 min to osmotic shock (NaCl; 0.5 M NaCl) oxidative stress (H₂O₂; 1 mM hydrogen peroxide), acid stress (pH 4.9) and cold shock (CS; 15°C) as described in the legend of Figure 2. All RNAs were detected by primer extension except for glpABC and STM0294.1n that were detected by northern analysis. Ordinarily, 30 μg of total RNA were used except for IsrG and IsrD that were detected using 10 μg RNA and 2.5 μg RNA, respectively. (A) 5′-end overlap. IsrA was detected using RNA samples isolated from cultures harboring pGEM3 carrying the intergenic region of isrA (pGEM3-isrA). STM0294.1n (chromosomal) was detected by northern analysis. IsrG and STM2243 were detected using RNA isolated from S. typhimurium cells carrying pGEM-isrG. IsrH-1, IsrH-2 and STM2287 were detected using RNA isolated from cells carrying pGEM-isrH. Two different primers were used to examine expression of IsrH-1 and IsrH-2. The primer of IsrH-2 also detects IsrH-1 since it is contained within IsrH-1. The region of the 230 nt long cDNA of IsrH-1 produced by IsrH-2 primer detecting IsrH-1 was left out. This region was included in the following Figure B. The transcription start sites of isrH-1 and isrH-2 are denoted schematically by vertical lines. (B) 3′-end overlap. IsrC and msgA RNAs were detected using RNA isolated from cells carrying pGEM-isrC or pGEM-msgA with (+) and without (−), in cis, msgA or isrC, respectively. IsrH-1 and IsrH-2 were detected using RNA isolated from cells carrying pGEM-isrH. The region of the 230 nt long cDNA of IsrH-1 produced by IsrH-2 primer is included. glpABC (chromosomal) was detected by northern analysis. Cells were grown with aeration (Aerobic) and without aeration (Anaerobic). STM2765 was detected using RNA isolated from cells carrying pPtac-isrN expressing isrN from an inducible promoter or the vector plasmid pPtac (pKK177-3) and from cells carrying pPisrN expressing isrN from its own promoter or the vector plasmid (pGEM). (C) Promoter overlap. IsrD was detected using RNA isolated from wild type carrying pGEM-isrD with (+) and without (−) STM1263 (in cis).