Pseudomonas aeruginosa is a common bacterium, but also a facultative human pathogen, especially fatal for immunocompromised patients. It is dangerous due to its high adaptability and intrinsic antibiotic resistance mechanisms making treatment of P. aeruginosa infections particularly difficult. Sophisticated, complex, multilevel regulatory systems engaging huge repertoire of transcriptional regulators (TRs) are the main factors that direct cellular processes to keep cellular homeostasis and adapt to changeable conditions. The PA3458 gene encodes a putative MarR-type TR. Most representatives of MarR family are described as repressors involved in response to various environmental signals and control of different processes e.g. adhesion, virulence, antibiotic resistance. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in mRNA level of 133 genes, among them 100 was down-regulated, pointing out the role of PA3458 as a repressor. ChIP-seq analysis identified multiple PA3458 binding sites in P. aeruginosa genome suggesting its role as a global regulator. The preferred sequence motif recognised by PA3458 was identified. Many genes involved in stress response are under control of PA3458. The PA3459-PA3461 operon, divergent to PA3458 gene is regulated by PA3458. It encodes: asparagine synthase, GNAT-family acetyltransferase and glutamyl aminopeptidase, respectively engaged in the production of potent osmoprotectant an N-acetylglutaminylglutamine amide (NAGGN). Overall, the PA3458 is engaged in osmoadaptation control 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 ΔPA3458 / pKKB2.12 (araC araBADp-PA3458-flag) (hereafter referred to as PA3458-F) as well as PAO1161 / pABB28.3 (araC araBADp-flag), empty vector control (hereafter called EV-F) strains. Cells were grown under selection in L broth with 0.02% arabinose 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).
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