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Proteomic, Microarray, and Signature-Tagged Mutagenesis Analyses of Anaerobic Pseudomonas aeruginosa at pH 6.5, Likely Representing Chronic, Late-Stage Cystic Fibrosis Airway Conditions ^,

Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York 12180,¹ Department of Molecular Genetics, Biochemistry and Microbiology,² Pulmonary Medicine,⁷ Chemistry, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267,⁹ Department of Microbiology, University of Colorado School of Medicine, Aurora, Colorado 80045,³ Department of Biological Sciences, Binghamton University, Binghamton, New York 13902,⁴ Centre de Recherche sur la Fonction, Structure et Ingénierie des Proteines et Faculté de Médecine, Pavillon Charles-Eugène Marchand, Université Laval, Ste-Foy, Québec, Canada G1K 7P4,⁵ Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada,⁶ Department of Biology, University of Dayton, Dayton, Ohio 45469,⁸ School of Biotechnology and Biomolecular Sciences and Centre for Marine Biofouling and Bio-Innovation, University of New South Wales, Sydney, New South Wales, Australia,¹⁰ Departments of Chemistry,¹¹ Pathology, University of Virginia, Charlottesville, Virginia 22904-4319,¹²

Received 18 October 2007/ Accepted 3 January 2008

Patients suffering from cystic fibrosis (CF) commonly harbor the important pathogen Pseudomonas aeruginosa in their airways. During chronic late-stage CF, P. aeruginosa is known to grow under reduced oxygen tension and is even capable of respiring anaerobically within the thickened airway mucus, at a pH of 6.5. Therefore, proteins involved in anaerobic metabolism represent potentially important targets for therapeutic intervention. In this study, the clinically relevant "anaerobiome" or "proteogenome" of P. aeruginosa was assessed. First, two different proteomic approaches were used to identify proteins differentially expressed under anaerobic versus aerobic conditions. Microarray studies were also performed, and in general, the anaerobic transcriptome was in agreement with the proteomic results. However, we found that a major portion of the most upregulated genes in the presence of NO₃^– and NO₂^– are those encoding Pf1 bacteriophage. With anaerobic NO₂^–, the most downregulated genes are those involved postglycolytically and include many tricarboxylic acid cycle genes and those involved in the electron transport chain, especially those encoding the NADH dehydrogenase I complex. Finally, a signature-tagged mutagenesis library of P. aeruginosa was constructed to further screen genes required for both NO₃^– and NO₂^– respiration. In addition to genes anticipated to play important roles in the anaerobiome (anr, dnr, nar, nir, and nuo), the cysG and dksA genes were found to be required for both anaerobic NO₃^– and NO₂^– respiration. This study represents a major step in unraveling the molecular machinery involved in anaerobic NO₃^– and NO₂^– respiration and offers clues as to how we might disrupt such pathways in P. aeruginosa to limit the growth of this important CF pathogen when it is either limited or completely restricted in its oxygen supply.

Published ahead of print on 18 January 2008.

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