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Pseudomonas aeruginosa LysR PA4203 regulator acts as a repressor of the PA4202 gene encoding a nitronate monooxygenase

ABSTRACT: More than 7% of the Pseudomonas aeruginosa genes are encoding transcriptional regulators, many of which with unknown functions. Among them, those belonging to the LysR family are the most represented. The PA4203 gene lies upstream of the previously characterized ppgL gene (PA4204), which encodes a periplasmic gluconolactonase, which detoxifies gluconolactone by converting it to gluconate. Upstream of PA4203 and in the opposite orientation are the PA4202 gene coding for a nitronate monooxygenase and ddlA (PA4201) encoding a D-alanine alanine ligase. This genetic organization is conserved in all P. aeruginosa genomes, but not in other pseudomonads. The intergenic regions between PA4203 and ppgL, and PA4202 are very short (79 and 107 nucleotides, respectively). PA4203 is a repressor of PA4202 and of its own transcription. A chromatin immunoprecipation analysis confirmed the presence of a single PA4203 binding site between PA4202 and PA4203. Electrophoretic mobility shift assays (EMSAs) with the purified PA4203 protein and in41 gel footprinting with the 1, 10-phenanthroline-copper ion, combined with primer extension analysis to determine transcriptional startpoints allowed the identification of a LysR binding motive in the PA4202 and PA4203 intergenic region. Despite this, a transcriptome analysis revealed more genes to be affected in a PA4203 mutant, likely due to the overexpression of the nitronate monooxygenase (PA4202). Deletion of the PA4202 gene resulted in an increased sensitivity of the cells to 3- nitropropionic acid (3-NPA).

ORGANISM(S): Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1

PROVIDER: GSE59876 | GEO | 2015/01/23

SECONDARY ACCESSION(S): PRJNA256959

REPOSITORIES: GEO

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Project description:Among multiple interconnected pathways for L-Lysine (L-Lys) catabolism in pseudomonads, Pseudomonas aeruginosa PAO1 employed the decarboxylase and the transaminase pathways. However, up till now several genes involved in the operation and regulation of these pathways were still missing. Transcriptome analyses coupled with promoter activity measurements and mutant growth phenotype analysis lead us to identify several new members of the L-Lys and D-Lys catabolic pathways and their regulatory elements, including argR to trigger lysine decarboxylation into cadaverine, PauR for the Î³-glutamylation pathway of polyamine catabolism into 5-aminovalerate, gcdR-gcdHG for glutarate utilization, dpkA, amaR-amaAB and PA2035 for D-Lys catabolism, lysR-lysXE for L-Lys efflux, and lysP for L-Lys uptake. Gene expression microarray, including probe preparation, hybridization, fluidics run and chip scan, was performed by Georgia State University DNA/Protein Core Facility. P. aeruginosa PAO1 was grown aerobically in minimal medium P with 350 rpm shaking at 37C, in the presence of 10mM L-glutamate supplemented with 10 mM L-lysine, cadaverine, 5-amino valerate, glutaric acid, D-lysine or 5mM L-pipecolate. Cells were harvested when the optical density at 600 nm reached 0.5~0.6 by centrifugation for 5 minutes at 4C. Total RNA samples were isolated by RNeasy purification kit following instructions of the manufacturer (Qiagen). Reverse transcription for cDNA synthesis, fragmentation by DNase I treatment, cDNA probe labeling and hybridization were performed according to the instructions of GeneChip manufacturer (Affymetrix). Data were processed by Microarray Suite 5.0 software normalizing the absolute expression signal values of all chips to a target intensity of 500. GeneSpring software (Silicon Genetics) was used for expression pattern analysis and comparison. Data collection was carried out using GCOS 1.4 software (Affymetrix). Probe intensity values were normalized to a target value of 500 with normalization factor equal to 1. Data analysis was performed using GeneSpring GX 11 Software (Aglient, Palo Alto,CA). Related Research Papers: 1. Indurthi SM, Chou HT, Lu CD. Molecular Characterization of lysR-lysXE, gcdR-gcdHG, and amaR-amaAB Operons for Lysine Export and Catabolism: A Comprehensive Lysine Catabolic Network in Pseudomonas aeruginosa PAO1. Microbiology. 2015 Submitted [EMID:04803de65c782] 2. Chou HT, Li J, and Lu CD. Functional Characterization of the agtABCD and agtSR Operons for Î³-Aminobutyrate and Î´-Aminovalerate Uptake and Regulation in Pseudomonas aeruginosa PAO1. Curr. Microbiology. 2014 Jan;68(1):59-63. 3. Chou HT, Li JY, Peng YC, Lu CD. Molecular characterization of PauR and its role in control of putrescine and cadaverine catabolism through the Î³-glutamylation pathway in Pseudomonas aeruginosa PAO1. J Bacteriol. 2013 Sep; 195(17):3906-13. 4. Chou HT, Hegazy M, Lu CD. L-lysine Catabolism is Controlled by Arginine/ArgR in Pseudomonas aeruginosa PAO1. J Bacteriol. 2010 Nov;192(22):5874-80

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Pseudomonas aeruginosa LysR PA4203 regulator acts as a repressor of the PA4202 gene encoding a nitronate monooxygenase

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