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Functional genomics of PycR, a LysR family transcriptional regulator essential for maintenance of Pseudomonas aeruginosa in the rat lung

¹ Centre de Recherche sur la Fonction, Structure et Ingénierie des Protéines, Biologie Médicale, Faculté de Médecine, Université Laval, QC G1K 7P4, Canada
² Department of Microbiology and Immunology, University of Oklahoma, Health Sciences Center, Oklahoma City, OK 73104, USA
³ Centre d'étude de la forêt, Pavillon Charles-Eugène Marchand, Université Laval, QC G1K 7P4, Canada

Correspondence
Roger C. Levesque
rclevesq{at}rsvs.ulaval.ca

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.

A supplementary table listing the PCR primers used and a supplementary figure showing a transcriptome analysis of genes differentially regulated by P. aeruginosa PycR are available with the online version of this paper.

The microarray data for this paper have been deposited in ArrayExpress (http://www.ebi.ac.uk/arrayexpress) with accession number E-MEXP-1591.