Functional characterization of TetR-like transcriptional regulator PA3973 from Pseudomonas aeruginosa [ChIP-seq]

Pseudomonas aeruginosa PA3973 encodes a putative TetR family transcriptional regulator, with a helix-turn-helix motif involved in DNA binding. We applied phenotype analyses, as well as transcriptome profiling (RNA-seq), and genome-wide identification of binding sites using ChIP-seq to unravel the biological role of PA3973. The ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome, among them 139 were located in intergenic regions. The 13 bp sequence was identified as the preferential binding site of PA3973. The PA3973 regulon encompasses genes involved in stress response, including the putative PA3973-PA3970 operon. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3973 showed changes in the mRNA level of 648 genes; among them, 374 were down-regulated. Concomitantly, ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome, among them 139 were located in intergenic regions. The 13 bp sequence was identified as the preferential binding site of PA3973. 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 ΔPA3973/pMEB1 (lacIQ-tacp-PA3973-flag) (hereafter referred to as PA3973-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).