The Primary Transcriptome of Salmonella enterica Serovar Typhimurium and Its Dependence on ppGpp during Late Stationary Phase

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.本研究采用差异RNA测序（differential RNA-seq, dRNA-seq）对鼠伤寒沙门氏菌（S. Typhimurium）SL1344的转录组架构进行解析，并探究其在稳定期后期（late stationary phase, LSP）中对细菌警报素四磷酸鸟苷（guanosine tetraphosphate, ppGpp）的依赖特性。在LSP条件下，本研究可鉴定出53%的鼠伤寒沙门氏菌开放阅读框（open reading frames, ORFs）的转录起始位点（transcriptional start sites, TSSs），并发现282个候选非编码RNA（non-coding RNAs, ncRNAs）。通过对LSP阶段TSSs的定位分析，可与此前针对鼠伤寒沙门氏菌SL1344稳定期早期（early stationary phase, ESP）转录组架构及其ppGpp依赖特性的dRNA-seq研究开展精细比对。本研究中，LSP被定义为需氧LB培养基培养至光密度值（OD600=3.6）的培养物，相较之下ESP的OD600为2.3。LSP阶段绝大多数沙门氏菌TSSs的精确核苷酸位置，与ESP阶段鉴定得到的结果高度吻合。不过，相较于ESP阶段，LSP阶段存在TSSs位置改变、新增或缺失TSS的情况，借此可实现全基因组范围内生长阶段依赖的启动子结构变化分类——这是此类规模分析的首次报道。对mRNA和ncRNA的LSP与ESP阶段ppGpp依赖型TSSs进行比对后发现，ppGpp介导的激活与抑制作用覆盖范围相似。但本研究同时注意到，此前已报道的多种毒力相关ncRNAs在LSP阶段呈现高度ppGpp依赖性。尽管SPI1在ESP阶段存在表达，但本研究发现其在LSP阶段的表达水平显著降低，取而代之的是致病岛2（SPI2）编码基因的表达上调。据此，本研究同时报道了LSP阶段SPI2的转录组架构分析结果，包括SsrB结合位点的定位，以及此前未被报道的SPI2 TSS的鉴定。本研究还证实，LSP阶段几乎所有SPI2的表达以及ESP阶段SPI1的表达，均依赖于ppGpp。