Proteomic, Microarray, and Signature-Tagged Mutagenesis Analyses of AnaerobicPseudomonas aeruginosaat pH 6.5, Likely Representing Chronic, Late-Stage Cystic Fibrosis Airway Conditions

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Abstract
Patients suffering from cystic fibrosis (CF) commonly harbor the important pathogenPseudomonas aeruginosain their airways. During chronic late-stage CF,P. aeruginosais 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” ofP. aeruginosawas 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 ofP. aeruginosawas 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, andnuo), thecysGanddksAgenes 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 inP. aeruginosato limit the growth of this important CF pathogen when it is either limited or completely restricted in its oxygen supply.