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

Abstract

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 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 NO 3 − and NO 2 − are those encoding Pf1 bacteriophage. With anaerobic NO 2 − , 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 NO 3 − and NO 2 − 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 NO 3 − and NO 2 − respiration. This study represents a major step in unraveling the molecular machinery involved in anaerobic NO 3 − and NO 2 − 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.