Abstract
We have used differential RNA-seq (dRNA-seq) to characterise the transcriptomic architecture of S. Typhimurium SL1344, and its dependence on the bacterial alarmone, guanosine tetraphosphate (ppGpp) during late stationary phase, (LSP). Under LSP conditions we were able to identify the transcriptional start sites (TSSs) for 53% of the S. Typhimurium open reading frames (ORFs) and discovered 282 candidate non-coding RNAs (ncRNAs). The mapping of LSP TSSs enabled a detailed comparison with a previous dRNA-seq study of the early stationary phase (ESP) transcriptional architecture of S. Typhimurium SL1344 and its dependence on ppGpp. For the purposes of this study, LSP was defined as an aerobic LB culture grown to a later optical density reading (OD600 = 3.6) compared to ESP (OD600 = 2.3). The precise nucleotide positions of the majority of S. Typhimurium TSSs at LSP agreed closely with those identified at ESP. However, the identification of TSSs at different positions, or where additional or fewer TSSs were found at LSP compared to ESP enabled the genome-wide categorisation of growth phase dependent changes in promoter structure, the first time such an analysis has been done on this scale. Comparison of the ppGpp-dependency LSP and ESP TSSs for mRNAs and ncRNAs revealed a similar breadth of ppGpp-activation and repression. However, we note several ncRNAs previously shown to be involved in virulence were highly ppGpp-dependent at LSP. Finally, although SPI1 was expressed at ESP, we found SPI1 was not as highly expressed at LSP, instead we observed elevated expression of SPI2 encoded genes. We therefore also report an analysis of SPI2 transcriptional architecture at LSP resulting in localisation of SsrB binding sites and identification of a previously unreported SPI2 TSS. We also show that ppGpp is required for nearly all of SPI2 expression at LSP as well as for expression of SPI1 at ESP.
Highlights
Infections caused by non-typhoidal Salmonella are one of the most frequent causes of food-borne illness worldwide
The mapped reads from this study and from the early stationary phase (ESP) dataset [19] can be viewed on JBrowse by following the link provided at www.ifr.ac.uk/Safety/ MolMicro/
Typhimurium at later stationary phase resulted in the prediction of transcriptional start sites (TSSs) for 53% of open reading frames (ORFs) which were found to closely correlate with the TSS positions for these ORFs previously defined at ESP [19]
Summary
Infections caused by non-typhoidal Salmonella are one of the most frequent causes of food-borne illness worldwide. Typhimurium is able to penetrate intestinal epithelium cells, a process that is dependent on the expression of a type 3 secretion system (T3SS) encoded by a horizontally acquired set of virulence genes encoded within Salmonella Pathogenicity Island 1 (SPI1). The reorganization of the nucleoid is accompanied by an overall reprogramming of gene expression, much of it mediated by the bacterial alarmone ppGpp and the stationary phase sigma factor RpoS [6,7]. The adaptation to stationary phase starvation and stress involves the expression of virulence factors including genes encoded within SPI1 and SPI2, several of which have previously been shown to be ppGpp-dependently activated [23,24,26]
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