Comparative RNA-seq analysis of the transcriptome of the Pseudomonas aeruginosa PAO1 wild-type strain with that of the ?vreR mutant

Bacterial gene expression is controlled by modifying the promoter affinity of the RNA polymerase (RNAP). This control occurs through the substitution of the RNAP s subunit and by the interaction with transcription factors. The pathogen Pseudomonas aeruginosa contains several s factors, including sVreI. This protein belongs to the extracytoplasmic function group of the s70 family. Expression and activity of sECFs are tightly regulated and only occur in response to specific signals. sVreI is encoded within the vreAIR gene cluster. The expression of this cluster occurs in response to inorganic phosphate (Pi) limitation. sVreI activity is modulated by VreR anti-sigma factor, which keeps sVreI inactive. In response to a still unidentified host signal, VreR is proteolytically degraded and sVreI released and activated. sVreI interacts then with the RNAP and targets expression of the sVreI regulon, which includes virulence genes that increase P. aeruginosa pathogenicity. In this work, we compared the transcriptome of the P. aeruginosa PAO1 wild-type strain with that of the ?vreR mutant upon bacterial growth in Pi starvation. Forty-six transcripts were more abundant in the ?vreR mutant than in the PAO1 wild-type strain, and nine were less abundant, including the vreR transcript. Overall design: P. aeruginosa PAO1 and ?vreR mutant were grown until late exponential phase in low Pi medium and total RNA was isolated. A mixture of the three isolations was used for RNA-seq analyses.

细菌基因表达的调控通过改变RNA聚合酶（RNA polymerase, RNAP）的启动子亲和力实现。此类调控可通过替换RNAP的σ亚基以及与转录因子的相互作用得以完成。致病菌铜绿假单胞菌（Pseudomonas aeruginosa）携带多种σ因子，其中包括σVreI。该蛋白隶属于σ70家族的胞质外功能（extracytoplasmic function, ECF）σ因子组。胞质外功能σ因子的表达与活性受到严格调控，仅在响应特定信号时才会被激活。σVreI由vreAIR基因簇编码，该基因簇的表达会响应无机磷酸（inorganic phosphate, Pi）匮乏条件。σVreI的活性受到VreR抗σ因子的调控，该因子可维持σVreI处于非活性状态。当受到尚未明确的宿主信号刺激时，VreR会被蛋白水解降解，σVreI得以释放并激活。随后σVreI会与RNAP结合，靶向调控σVreI调节子的表达，该调节子包含可增强铜绿假单胞菌致病性的毒力基因。本研究中，我们比较了铜绿假单胞菌PAO1野生型菌株与ΔvreR缺失突变株在Pi饥饿条件下生长时的转录组。ΔvreR突变株中共有46个转录本的丰度高于PAO1野生型菌株，另有9个转录本丰度更低，其中包括vreR转录本。实验整体设计如下：将铜绿假单胞菌PAO1与ΔvreR突变株接种于低Pi培养基中培养至指数生长后期，随后提取总RNA。将三次独立提取的RNA混合后用于RNA测序（RNA-seq）分析。