Salmonella enterica serovar Typhimurium 4/74 transcriptomics using strand-specific RNA-seq

Bacterial transcription networks typically consist of hundreds of transcription factors and thousands of promoters. However, current attempts to map bacterial promoters have failed to report the true complexity of bacterial transcription. The differential RNA-seq (dRNA-seq) approaches only identified a subset of promoters because they involved few growth conditions. Here, we present a simplified approach for global promoter identification in bacteria, based upon the analysis of RNA-seq data from multiple environmental conditions. RNA was extracted from Salmonella enterica serovar Typhimurium (S. Typhimurium) grown in 22 different environmental conditions, which were devised to reflect the pathogenic lifestyle of S. Typhimurium. Individual RNA samples were combined into two pools for sequencing. In just two runs of strand-specific RNA-seq and dRNA-seq of the pooled sample we identified 3701 promoters (Pool sample). In further experiments, we found that individual in vitro conditions stimulate the expression of about 60% of the S. Typhimurium genome, whereas the suite of 22 conditions induced expression of 87% of S. Typhimurium genes. We discovered environmental conditions that induce many genes within Salmonella pathogenicity islands and identified 78 new sRNAs. In S. Typhimurium there is now experimental evidence for 280 sRNAs, and we classified them in terms of location and Hfq-binding. Transcriptome analysis of S. Typhimurium 4/74 using RNA from 22 different conditions using RNA-seq. Also, RNA from each condition was pooled into one sample (RNA Pool). Differential RNA-seq (dRNA-seq) was performed for 5 of the samples from the 22 environmental conditions.

细菌转录网络通常由数百个转录因子与数千个启动子构成。然而，当前绘制细菌启动子图谱的研究尚未能完整呈现细菌转录过程的真实复杂性。差分RNA测序（dRNA-seq）技术仅能鉴定出部分启动子，因此类方法仅覆盖少量生长条件。本研究提出一种简化的细菌全局启动子鉴定方法，该方法基于多环境条件下RNA测序数据的分析。研究团队从22种模拟鼠伤寒沙门氏菌致病生活周期的不同环境条件下培养的鼠伤寒沙门氏菌（Salmonella enterica serovar Typhimurium, S. Typhimurium）中提取RNA，将各独立RNA样本合并为两个混合池用于测序。仅通过对该混合样本开展两轮链特异性RNA测序与dRNA-seq，我们便鉴定出3701个启动子（混合样本组）。后续实验显示，单一体外培养条件可激活鼠伤寒沙门氏菌约60%基因组的表达，而22种环境条件的组合则可诱导87%的鼠伤寒沙门氏菌基因表达。本研究还发现了可诱导沙门氏菌致病岛上大量基因表达的环境条件，并鉴定出78个新型小RNA（small RNA, sRNA）。目前鼠伤寒沙门氏菌中已有280个经实验验证的sRNA，研究团队依据其基因组定位与Hfq结合特性对这些sRNA进行了分类。此外，本研究还利用22种环境条件下获取的RNA，对鼠伤寒沙门氏菌4/74菌株开展了RNA测序转录组分析；同时将每种环境条件下提取的RNA合并为一个混合样本（RNA Pool）。此外，我们还对22种环境条件中的5个样本开展了dRNA-seq实验。