The sRNA IesR-1 is up-regulated in non-growing intracellular <i>S.</i> Typhimurium.

<p>Relative levels of the sRNA IesR-1 were determined by reverse transcription and RT-qPCR. Data are relative to the transcript levels of IesR-1 in bacteria cultured with shaking in LB broth to early-exponential phase (O.D.₆₀₀ ∼0.2). 16S ribosomal RNA was used as a reference gene. Bars indicate the mean ± standard deviation of three independent experiments. (A) IesR-1 expression in axenic cultures. Bacteria were cultured in LB broth and minimal media such as ISM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077939#pone.0077939-Headley1" target="_blank">[35]</a> and PCN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077939#pone.0077939-Lober1" target="_blank">[34]</a> with shaking to either exponential or stationary growth phases. A third condition consisting in growth with no shaking to stationary phase was also included in the analysis. (B) Expression of IesR-1 in non-growing intracellular <i>S.</i> Typhimurium collected at different post-infection times from rat and human fibroblasts (NRK-49F and BJ-5ta, respectively). For comparison, IesR-1 expression was also monitored in wild-type bacteria proliferating inside HeLa epithelial cells and in <i>phoP</i> mutant bacteria overgrowing within NRK-49F fibroblasts. The 0 h post-infection time point corresponds to bacteria grown overnight with no shaking in LB broth that were used to infect the eukaryotic cells. Note the pronounced up-regulation of IesR-1 (∼200–300 fold) in non-growing intracellular bacteria at late post-infection times, which contrasts with the moderate induction values registered in extracellular bacteria. Data are the mean and the standard deviation of three independent experiments. (C) Northern blot analysis showing the production of an sRNA of ∼600 nt in intracellular bacteria compatible with the expected size of IesR-1. A second transcript of ∼275 nt was also detected specifically in intracellular bacteria, which as denoted by the RACE experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077939#pone-0077939-g001" target="_blank">Figure 1</a> D) could correspond to 5′ region of IesR-1. Note the lack of noticeable amount of these two molecules in wild-type bacteria grown extracellularly.