title: Functional genomics of PycR, a LysR family transcriptional regulator essential for maintenance of Pseudomonas aeruginosa in the rat lung.
abstract: The human opportunistic pathogen Pseudomonas aeruginosa is the major cause of morbidity and mortality of cystic fibrosis patients and is responsible for a variety of infections in compromised hosts. Using PCR-based signature-tagged mutagenesis, we identified a P. aeruginosa STM5437 mutant with an insertion into the PA5437 gene (called pycR for putative pyruvate carboxylase regulator). PycR inactivation results in 100,000-fold attenuation of virulence in the rat lung in vivo. PycR has the signature of a transcriptional regulator with a predicted helix-turn-helix motif binding to a typical LysR DNA binding site in the PA5436 (pycA)-PA5437 (pycR) intercistronic region. Two pyruvate carboxylase subunits (pycA and pycB) are divergently transcribed upstream of pycR. Transcriptional start sites of pycR and pycA are located at -127 and -88 bp upstream of their initiation codons with Shine-Dalgarno and putative promoter sequences containing -10 and -35 sequences. The DNA binding of PycR was confirmed by DNA mobility shift assay. Genome-wide transcriptional profiling and quantitative real-time PCR (qRT-PCR) indicated that the genes differentially regulated by PycR include two pyruvate carboxylase genes and genes necessary for lipid metabolism, lipolytic activity, anaerobic respiration and biofilm formation. PycR is a regulator with pleiotropic effects on virulence factors, such as lipase and esterase expression and biofilm formation, which are important for maintenance of P. aeruginosa in chronic lung infection.
authors.affiliation: Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Biologie Médicale, Faculté de Médecine, Université Laval, QC G1K 7P4, Canada.
authors.forename: Irena
authors.initials: I
authors.lastname: Kukavica-Ibrulj
authors.affiliation: Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Biologie Médicale, Faculté de Médecine, Université Laval, QC G1K 7P4, Canada.
authors.forename: François
authors.initials: F
authors.lastname: Sanschagrin
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma, Health Sciences Center, Oklahoma City, OK 73104, USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Peterson
authors.affiliation: Department of Microbiology and Immunology, University of Oklahoma, Health Sciences Center, Oklahoma City, OK 73104, USA.
authors.forename: Marvin
authors.initials: M
authors.lastname: Whiteley
authors.affiliation: Centre d'étude de la forêt, Pavillon Charles-Eugène Marchand, Université Laval, QC G1K 7P4, Canada.
authors.forename: Brian
authors.initials: B
authors.lastname: Boyle
authors.affiliation: Centre d'étude de la forêt, Pavillon Charles-Eugène Marchand, Université Laval, QC G1K 7P4, Canada.
authors.forename: John
authors.initials: J
authors.lastname: MacKay
authors.affiliation: Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Biologie Médicale, Faculté de Médecine, Université Laval, QC G1K 7P4, Canada.
authors.forename: Roger C
authors.initials: RC
authors.lastname: Levesque
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018441:Biofilms / Q000254:growth & development; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D005809:Genes, Regulator; D005816:Genetic Complementation Test; D016680:Genome, Bacterial; D023281:Genomics; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D009876:Operon; D011401:Promoter Regions, Genetic; D011552:Pseudomonas Infections / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity; D051381:Rats; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D014157:Transcription Factors