The LysR-type transcriptional regulator BsrA (PA2121) is engaged in the control of vital metabolic pathways in Pseudomonas aeruginosa (RNA-seq). The LysR-type transcriptional regulator BsrA (PA2121) is engaged in the control of vital metabolic pathways in Pseudomonas aeruginosa (RNA-seq)

Pseudomonas aeruginosa, a facultative human pathogen causing nosocomial infections, has complex regulatory systems involving many transcriptional regulators. The LTTR family (LysR-Type Transcriptional Regulators) consists of proteins involved in regulation of various processes including stress response, motility, virulence or amino acid metabolism. The aim of this study was characterization of the LysR-type regulator BsrA (PA2121), identified previously as a negative regulator of biofilm formation in P. aeruginosa. To identify the BsrA binding sites in P. aeruginosa the ChIP-seq analysis was performed. It revealed 765 BsrA binding sites in P. aeruginosa PAO1161 genome, among them 367 was localized in the intergenic regions. Parallel transcriptomic analysis identified altered expression of 157 genes in response to BsrA excess, among them 35 had a BsrA binding site in the corresponding promoter regions, indicating direct influence of BsrA on expression of these genes. BsrA-repressed loci encompass genes encoding proteins engaged in key metabolic pathways including the tricarboxylic acid cycle. A group of directly activated genes by BsrA, consists of several loci encoding proteins involved in pili/fimbriae assembly as well as secretion and transport systems. Results also confirmed that BsrA acts as an autorepressor. Presented data uncover the regulon of BsrA protein with its role as transcriptional regulator of genes engaged in vital cellular processes in P. aeruginosa. Overall design: Pseudomonas aeruginosa PAO1161 (leu-, r-, RifR) derivative of PAO1 was used in the experiment (Kawalek et al., 2020; BMC Genomics, 21:14). To determine the impact of an increased BsrA level on the transcriptome, the RNA-seq analysis was performed on RNA isolated from Pseudomonas aeruginosa PAO1161/pMEB63 (lacIQ-tacp-bsrA) cultures grown under selection in L broth with 0.05 mM IPTG (hereafter referred to as BsrA) as well as from Pseudomonas aeruginosa PAO1161 carrying pAMB9.37 (lacIQ-tacp) cells grown under selection in L broth with 0.05 mM IPTG (empty vector control, hereafter called EV). The RNA was isolated from exponentially growing cultures (OD600 0.5). Three independent biological replicates of total RNA were isolated for each strain.

铜绿假单胞菌（Pseudomonas aeruginosa）是一种可引发院内感染的兼性人类致病菌，其具备复杂的调控网络，涉及众多转录调节因子。LysR型转录调节因子家族（LTTR family, LysR-Type Transcriptional Regulators）包含参与调控多种生理过程的蛋白，涵盖应激响应、运动性、毒力及氨基酸代谢等通路。本研究旨在对此前被鉴定为铜绿假单胞菌生物被膜形成负调控因子的LysR型调节蛋白BsrA（PA2121）进行系统表征。为鉴定铜绿假单胞菌中BsrA的结合位点，本研究开展了染色质免疫共沉淀测序（ChIP-seq）分析。该分析在铜绿假单胞菌PAO1161基因组中鉴定得到765个BsrA结合位点，其中367个定位于基因间区。同步进行的转录组分析显示，当BsrA表达量升高时，共有157个基因的表达发生显著改变，其中35个基因的对应启动子区域存在BsrA结合位点，提示BsrA可直接调控这些基因的转录。受BsrA抑制的基因位点涵盖编码参与关键代谢通路（如三羧酸循环）蛋白的基因。由BsrA直接激活的基因集合则包含多个编码菌毛/纤毛组装蛋白以及分泌与转运系统蛋白的基因位点。研究结果同时证实，BsrA可作为自身阻遏蛋白发挥调控功能。本研究揭示了BsrA蛋白的调控子（regulon），明确了其作为铜绿假单胞菌关键细胞过程相关基因转录调节因子的生物学功能。整体实验设计：本实验采用PAO1的衍生菌株铜绿假单胞菌PAO1161（leu-, r-, RifR）（Kawalek等，2020；BMC Genomics, 21:14）。为探究BsrA表达水平升高对转录组的影响，本研究对分别来自两组菌株的总RNA开展RNA测序（RNA-seq）分析：一组为在添加0.05 mM IPTG的LB肉汤培养基中经抗性筛选培养的铜绿假单胞菌PAO1161/pMEB63（lacIQ-tacp-bsrA），以下简称BsrA组；另一组为同样在添加0.05 mM IPTG的LB肉汤培养基中经抗性筛选培养的携带空载体pAMB9.37（lacIQ-tacp）的铜绿假单胞菌PAO1161，以下简称空载体对照组（EV组）。总RNA均提取自指数生长期（OD600为0.5）的培养物，每个菌株设置3次独立生物学重复。