The LysR-type transcriptional regulator BsrA (PA2121) is engaged in the control of vital metabolic pathways in Pseudomonas aeruginosa (ChIP-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). Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis was performed on P. aeruginosa PAO1161 ?bsrA / pMEB99 (lacIQ-tacp-bsrA-flag) (hereafter referred to as BsrA-F) as well as PAO1161 /pABB28.1 (lacIQ-tacp-flag), empty vector control (hereafter called EV-F) strains. Cells were grown under selection in L broth with 0.05 mM IPTG untillOD600 = 0.5). For each biological replicate, two independent cultures of each strain were pooled together. The immunoprecipitation was performed with commercially available polyclonal anti-FLAG antibodies (DYKDDDDK Tag polyclonal antibodies; PA1-985B; Invitrogen).