The Lrp/AsnC-type regulator PA2577 controls the Eam-like transporter gene PA2576 in Pseudomonas aeruginosa (ChIP-seq)

Pseudomonas aeruginosa is a facultative human pathogen especially dangerous for immunocompromised patients. It is known from its high adaptability and complex regulatory systems involving huge repertoire of transcriptional regulators (TRs). The Lrp/AsnC family consists of proteins engaged mostly in regulation of processes such as amino acid metabolism but the function of most of them remains unknown. The aim of this study was characterization of the Lrp/AsnC-type regulator PA2577 of P. aeruginosa. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA2577 revealed changes in mRNA level of 171 genes. Among downregulated loci PA2576 gene, encoding an EamA-like membrane transporter transcribed divergently to PA2577, was identified. ChIP-seq analysis together with EMSA analysis revealed binding site of PA2577 in PA2577/PA2576 intergenic region, indicating direct regulation of PA2576 expression by PA2577. PA2576 was shown to interact with PA5006 and PA3694 proteins predicted to be involved in membrane processes, especially LPS biosynthesis. Defects of growth of PA2577 and PA2576 deficient cells in conditions of polymyxin B supplementation was shown. Overall, presented data uncovered the regulatory properties of PA2577 acting as the repressor of divergently transcribed PA2576 gene, being a part of LPS biogenesis process 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 ?PA2577/pMEB84 (lacIQ-tacp-PA2577-flag) (hereafter referred to as PA2577-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 until OD600 = 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).