The regulatory network of gene expression in Pseudomonas aeruginosa, an opportunistic human pathogen, is very complex. In the PAO1 reference strain, about 10% of genes encode transcriptional regulators, many of which have undefined regulons and unknown functions. The aim of this study is the characterization of PA2577 protein, a representative of the Lrp/AsnC family of transcriptional regulators. This family encompasses proteins involved in the amino acid metabolism, regulation of transport processes or cell morphogenesis. The transcriptome profiling of P. aeruginosa cells with mild PA2577 overproduction revealed a decreased expression of the PA2576 gene oriented divergently to PA2577 and encoding an EamA-like transporter. A gene expression analysis showed a higher mRNA level of PA2576 in P. aeruginosa ΔPA2577, indicating that PA2577 acts as a repressor. Concomitantly, ChIP-seq and EMSA assays confirmed strong interactions of PA2577 with the PA2577/PA2576 intergenic region. Additionally, phenotype microarray analyses indicated an impaired metabolism of ΔPA2576 and ΔPA2577 mutants in the presence of polymyxin B, which suggests disturbances of membrane functions in these mutants. We show that PA2576 interacts with two proteins, PA5006 and PA3694, with a predicted role in lipopolysaccharide (LPS) and membrane biogenesis. Overall, our results indicate that PA2577 acts as a repressor of the PA2576 gene coding for the EamA-like transporter and may play a role in the modulation of the cellular response to stress conditions, including antimicrobial peptides, e.g., polymyxin B.

Pseudomonas aeruginosa, a human opportunistic pathogen, is a common cause of nosocomial infections. Its ability to survive under different conditions relies on a complex regulatory network engaging transcriptional regulators controlling metabolic pathways and capabilities to efficiently use the available resources. P. aeruginosa PA3973 encodes an uncharacterized TetR family transcriptional regulator. In this study, we applied a transcriptome profiling (RNA-seq), genome-wide identification of binding sites using ChIP-seq, as well as the phenotype analyses to unravel the biological role of PA3973. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3973 showed changes in the mRNA level of 648 genes. Concomitantly, ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome. A 13 bp sequence motif was indicated as the binding site of PA3973. The PA3973 regulon encompasses the PA3972-PA3971 genes encoding a probable acyl-CoA dehydrogenase and a thioesterase. In vitro analysis showed PA3973 binding to PA3973p. Accordingly, the lack of PA3973 triggered increased expression of PA3972 and PA3971. The ∆PA3972-71 PAO1161 strain demonstrated impaired growth in the presence of stress-inducing agents hydroxylamine or hydroxyurea, thus suggesting the role of PA3972-71 in pathogen survival upon stress. Overall our results showed that TetR-type transcriptional regulator PA3973 has multiple binding sites in the P. aeruginosa genome and influences the expression of diverse genes, including PA3972-PA3971, encoding proteins with a proposed role in stress response.

Pseudomonas aeruginosa is a common cause of nosocomial infections, especially in patients with cystic fibrosis and burn wounds. An attenuated PAO1 strain and its derivatives are widely used to study the biology of this bacterium, however recent studies indicated an ongoing evolution of the laboratory strains, highlighted by changes in the genomes of PAO1 sublines and derivatives used in different laboratories worldwide. Here we have sequenced the genome of Pseudomonas aeruginosa PAO1161 strain, a leu -, Rif ^R , restriction-modification defective PAO1 derivative. This strain is described as the host of IncP-8 plasmid FP2, conferring the resistance to mercury and used in the past for preparation of genetic map of the P. aeruginosa chromosome. Comparison of PAO1161 genome sequence with PAO1-UW reference genome revealed an inversion of the large genome segment between rrnA and rrnB rRNA operons and more than 100 nucleotide polymorphisms and short insertions-deletions, many of which were also found within the recently re-sequenced PAO1 sublines. The PAO1161 specific sequence variants include E108K mutation in PA3792 ( leuA ), which we showed is responsible for leucine auxotrophy, H531L in PA4270 ( rpoB ), likely conferring rifampicin resistance, as well as nonsense mutations in PA2735 encoding a DNA methyltransferase as well as a putative OLD family endonuclease PA1939. Additionally, PAO1161 genome poses a 12 kb RPG42 prophage and a 108 kbp insertion within tRNA-Lys, encompassing a putative mercury resistance and showing similarity to PAPI-1 like integrative conjugative elements (ICE). Our data indicate that the ICE can excise from the bacterial chromosome and can be transferred to Pseudomonas putida cells where it integrates in the genome at three specific sites and confers the cells with mercury resistance. Overall this data provide insight into the genome variation between P. aeruginosa laboratory strains. Moreover, this data indicate that the FP2 element for years described as a IncP-8 plasmid is an integrative conjugative element.