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J. Bacteriol. doi:10.1128/JB.01683-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Proteomic, Microarray and Signature Tagged Mutagenesis Analysis of Anaerobic Pseudomonas aeruginosa at pH 6.5, Representing Chronic Cystic Fibrosis Airway Conditions

Department of Chemistry, Rensselaer Polytechnic Institute, Troy, NY 12180; Department of Molecular Genetics, Biochemistry and Microbiology, Pulmonary Medicine, and Chemistry, University of Cincinnati College of Medicine, Cincinnati, OH 45267; Department of Microbiology, University of Colorado School of Medicine, Aurora, CO 80045; Department of Biological Sciences, Binghamton University, Binghamton, NY 13902; 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; Department of Molecular Biology and Biochemistry, Simon Fraser University, BC, Canada; Department of Biology, University of Dayton, Dayton, OH 45469; School of Biotechnology and Biomolecular Sciences, and Centre for Marine Biofouling and Bio-Innovation, University of New South Wales, Sydney, New South Wales, Australia; Departments of Chemistry and Pathology, University of Virginia, Charlottesville, VA 22904-4319

^* To whom correspondence should be addressed. Email: Daniel.Hassett{at}UC.Edu.

	Abstract

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 pH 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 in this study. First, two different proteomic approaches were used to identify proteins differentially expressed under anaerobic vs. 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^- were those encoding Pf1 bacteriophage. With anaerobic NO₂^-, the most downregulated genes are those involved post-glycolytically and include many TCA cycle genes and those involved in the electron transport chain, especially those encoding 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, 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 that is either limited or completely restricted in its oxygen supply.