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

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 ϳ6.5. 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.Pseudomonas aeruginosa is a gram-negative bacterium of environmental and clinical importance that is capable of both aerobic and anaerobic respiration, the latter of which requires nitrate (NO 3 Ϫ ), nitrite (NO 2 Ϫ ), or nitrous oxide (N 2 O) as an alternative electron acceptor (24). The organism can also utilize arginine for anaerobic growth via substrate-level phosphorylation, although the final cell yield during this form of growth is abysmally low compared to that observed during anaerobic respiration (55). The most facile means to obtain anaerobic energy, however, is via respiration by NO 3 Ϫ reduction. The process of nitrate reduction can occur by two routes, the first of which is an assimilatory pathway where the nitrogen from NO 3 Ϫ is incorporated into macromolecules via formation of NH 3 . Assimilation can proceed under both aerobic and anaerobic conditions. In contrast, respiratory NO 3 Ϫ reduction (denitrification) occurs only under anaerobic conditions and involves the sequential eight-electron reduction of NO 3 Ϫ to nitrogen gas (N 2 ), with intermediates including NO 2 Ϫ , nitric oxide (NO), and N 2 O. The anaerobic process generates respiratory ...

Pseudomonas aeruginosa PAO1 Pyocin Production Affects Population Dynamics within Mixed-Culture Biofilms

“…S3 and Table ST4 in the supplemental material). This confirms the recent observations made by other authors and shows that anaerobic induction of pyocins is not restricted to the biofilm mode of growth (1,16). Pyocins are known to be activated as part of the response to DNA damage.…”

Heterogeneity in Pseudomonas aeruginosa Biofilms Includes Expression of Ribosome Hibernation Factors in the Antibiotic-Tolerant Subpopulation and Hypoxia-Induced Stress Response in the Metabolically Active Population

“…Other studies have suggested that P. aeruginosa may be exposed to anoxic or hypoxic conditions in the cystic fibrosis (CF) lung environment (26,60,74,77). Several transcriptome studies have characterized gene induction during microaerophilic and anaerobic respiratory conditions for P. aeruginosa (3,53,61,66). Microarray results here show that some of the most abundant mRNA transcripts at the top of these biofilms are for genes that are induced by oxygen limitation stress (Fig.…”

Swarming ofPseudomonas aeruginosaIs Controlled by a Broad Spectrum of Transcriptional Regulators, Including MetR

“…Bacteriophage Pf1 has been suggested to be important in CF infections, and studies have shown that Pf1 genes are upregulated during biofilm development (49,64). An increase in expression as great as 325-fold compared to wild type was noted for the helix-destabilizing protein (PA0720) of the phage.…”