Detection of novel island-encoded sRNAs. (A) Detection of small RNAs by northern analysis. Total RNA was isolated from S. typhimurium cells grown in rich media (LB; 1.5, 2.5 and 8 h after dilution at OD₆₀₀ values of 0.3, 1 and 4.5, respectively) or in N minimal medium supplemented with Casamino Acids, glycerol and 10 μM MgCl₂ (L; low magnesium) or 10 mM MgCl₂ (H; high magnesium) and from cells subjected for 30 min to osmotic shock (NaCl; 0.5 M NaCl), oxidative stress (PQ; 0.2 mM paraquat or H₂O₂; 1 mM hydrogen peroxide), low iron conditions (Dp; 0.2 mM 2,2′-dipyridyl), pH stress (pH 4.9 or pH 8.4), cold shock (CS; 15°C) and heat shock (HS; 42°C). For oxygen limitation conditions (OL), single colonies were inoculated in 10 ml LB and grown overnight without agitation in 50 ml Falcon tubes to OD₆₀₀ of 0.9. The RNA lengths (nucleotides) were estimated based on 5′ end mapping by primer extension and the position of the last uridine at the end of the rho-independent transcription terminator. These estimations were confirmed further by the northern blots. There are two IsrB genes each carrying two transcription-start sites (see mapping in B). 5S RNA was used as a control for loading. (B) Detection of small RNAs by primer extension. Total RNA was isolated from S. typhimurium cells harboring pGEM3 carrying the intergenic region of the predicted small RNA. Cells were grown to logarithmic phase (Log), stationary phase (Sta) and subjected to cold shock (CS; 15°C) and acid stress (pH 4.9). IsrD and IsrG were detected using 5 μg RNA and 10 μg RNA, respectively. The RNA lengths (nucleotides) were estimated based on 5′ end mapping by primer extension and the position of the last uridine at the end of the rho-independent transcription terminator.

Schematic representation of three types of rho-independent transcription terminators of putative sRNAs positioned relative to their flanking genes. These three types of transcription terminators are less likely to function as terminators of previously annotated genes. (A) A transcription terminator located in an intergenic region between two divergently oriented genes. (B) A transcription terminator located in an intergenic region on the opposite strand from its two flanking ORFs. (C) A transcription terminator located in an intergenic region, more than 250 bases downstream from the 3′ end of the nearest ORF.

Differential expression of isrE and ryhB. (A) Sequence of isrE and ryhB genes. The start sites observed by primer extension are indicated (+1). Brackets indicate putative −10 and −35 regions of the promoters mapped. The core sequences (nucleotides 37–69 relative to the start sites) are underlined. Three possible Fur binding sites that match Fur consensus (gataatgataatcattatc) at 15, 16 and 13 of 19 bases are indicated by dashed lines above the sequence of IsrE. Fur sites at ryhB, matching 14 of 19 bases of the consensus are indicated under the sequence of ryhB. (B) Detection of IsrE and RyhB in wild type cells. Northern blots as in Figure 2 of RyhB and IsrE using RyhB and IsrE specific primers (1271 and 797, respectively). The conditions used were; rich media (LB; at OD₆₀₀ values of 0.3, 1 and 4.5, respectively), oxygen limitation (OL), osmotic shock (NaCl; 0.5 M NaCl), oxidative stress (PQ; 0.2 mM paraquat or H₂O₂; 1 mM hydrogen peroxide) and low iron conditions (Dp; 0.2 mM 2,2′-dipyridyl). (C) Late stationary phase cultures of wild type and mutants of isrE, ryhB, fur and rpoS were analyzed for isrE and ryhB levels by primer extension (top panel). Exponentially growing cultures were exposed to H₂O₂ (1 mM for 30 min. middle panel) or to 2,2′-dipyridyl (0.2 mM for 30 min. bottom panel). The primer extension reactions (0.5 μg RNA) were carried out using a primer (470) that is complementary to the core sequence of isrE and ryhB. (D) Fur regulation of isrE and ryhB as detected by primer extension. Exponentially growing cells were treated as in C. (E) Growth curves. Overnight cultures of wild type S. typhimurium and isrE and/or ryhB mutants were diluted 1/100 in LB medium and incubated shaking at 37°C. At 30 min after dilution the cultures were treated with 0.2 mM 2,2′-dipyridyl. Optical density was measured at the indicated time intervals.

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).

Expression of island-encoded sRNA genes in J774 macrophages. Activated macrophages and infected with S. typhimurium were lysed at 1 and 8 h post infection. Thereafter, the intracellular bacteria were recovered, from which total RNA was isolated and subjected to assays of Real-Time PCR as described. To quantify changes in the expression of sRNAs within macrophages, the levels of the RNAs obtained 1 and 8 h post infection were compared with the levels of these genes as detected in S. typhimurium grown in the cell culture medium for 1.5 h.

IsrJ is part of the SPI-1 TTSS regulon. (A) isrJ regulation by hilA. RNA samples from cultures of S. typhimurium, wild type, hilA (hilA::kan) and isrJ (ΔisrJ::cm) mutants grown under conditions of low oxygen were subjected to primer extension using an isrJ specific primer. The strains in the right panel carry a multi copy number plasmid expressing IsrJ (pisrJ). IsrJ expression from pisrJ was detected using 1 μg RNA. 5S RNA was used as a control for loading. (B) IsrJ affects invasion of Salmonella into nonphagocytic cells. HeLa cells infected with S. typhimurium strains for 1 h in medium containing gentamicin were washed, lysed and aliquots were plated to determine the number of viable intracellular bacteria. Black bars; wild type, hilA (hilA::kan) and isrJ (ΔisrJ::cm). Grey bars; strains carrying a plasmid expressing IsrJ (pisrJ) or the vector plasmid (pKK). The results are means of three independent experiments, each carried out with four cultures of each strain. (C) IsrJ affects translocation into nonphagocytic cells. HeLa cells preloaded with CCF2 were infected with wild type, hilA and isrJ mutant strains expressing the fusion protein SptP-BlaM. The decrease in CCF2 fluorescence and the increase in the fluorescence emitted by the CCF2 hydrolysis product were recorded at real time.