The primary transcriptome of Salmonella enterica Serovar Typhimurium and its dependence on ppGpp during late stationary phase.
The primary transcriptome of Salmonella enterica Serovar Typhimurium and its dependence on ppGpp during late stationary phase.
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DOI：
10.1371/journal.pone.0092690
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Thompson A
中科院分区：
文献类型：
--
作者：
Ramachandran VK;Shearer N;Thompson A
我们使用差异 RNA 序列 (dRNA-seq) 来表征鼠伤寒沙门氏菌 SL1344 的转录组结构，及其在稳定期晚期 (LSP) 期间对细菌报警素、四磷酸鸟苷 (ppGpp) 的依赖性。在 LSP 条件下，我们能够识别 53% 的鼠伤寒沙门氏菌开放阅读框 (ORF) 的转录起始位点 (TSS)，并发现了 282 个候选非编码 RNA (ncRNA)。 LSP TSS 的图谱能够与之前对鼠伤寒沙门氏菌 SL1344 的早期稳定期 (ESP) 转录结构及其对 ppGpp 依赖性的 dRNA-seq 研究进行详细比较。出于本研究的目的，LSP 被定义为与 ESP (OD600 = 2.3) 相比，生长到较晚光密度读数 (OD600 = 3.6) 的需氧 LB 培养物。 LSP 中大多数鼠伤寒沙门氏菌 TSS 的精确核苷酸位置与 ESP 中鉴定的位置非常一致。然而，在不同位置识别 TSS，或者与 ESP 相比，在 LSP 发现更多或更少的 TSS，使得能够对启动子结构的生长阶段依赖性变化进行全基因组分类，这是第一次在这种规模上进行此类分析。 mRNA 和 ncRNA 的 ppGpp 依赖性 LSP 和 ESP TSS 的比较揭示了 ppGpp 激活和抑制的相似广度。然而，我们注意到先前显示与毒力有关的几种 ncRNA 在 LSP 上高度依赖 ppGpp。最后，虽然 SPI1 在 ESP 中表达，但我们发现 SPI1 在 LSP 中的表达并不高，相反，我们观察到 SPI2 编码基因的表达升高。因此，我们还报告了对 LSP 处 SPI2 转录架构的分析，从而定位了 SsrB 结合位点并鉴定了先前未报告的 SPI2 TSS。我们还表明，几乎所有 LSP 上的 SPI2 表达以及 ESP 上的 SPI1 表达都需要 ppGpp。
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.
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