Multiple environmental cues have been shown to trigger biofilm detachment, the transition from surfaceattached, highly organized communities known as biofilms to the motile lifestyle. The goal of this study was to identify a gene product involved in sensing environmental cues that trigger biofilm dispersion in Pseudomonas aeruginosa. To do so, we focused on novel putative chemotaxis transducer proteins that could potentially be involved in environmental sensing. We identified a locus encoding such a protein that played a role in detachment, as indicated by the observation that an isogenic mutant biofilm could not disperse in response to a variety of environmental cues. The locus was termed bdlA for biofilm dispersion locus. The BdlA protein harbors an MCP (methyl-accepting chemotaxis protein) domain and two PAS (Per-Arnt-Sint) domains that have been shown to be essential for responding to environmental signals in other proteins. The dispersiondeficient phenotype of the bdlA mutant was confirmed by treatment with the biocide H 2 O 2 and by microscopic observations. The dispersion response was independent of motility. bdlA mutant biofilms were found to have increased adherent properties and increased intracellular levels of cyclic di-GMP (c-di-GMP). Our findings suggest that BdlA may be a link between sensing environmental cues, c-di-GMP levels, and detachment. Based on our findings, a possible involvement of BdlA in a signaling cascade resulting in biofilm dispersion is discussed.

Mucoid, mucA mutant Pseudomonas aeruginosa cause chronic lung infections in cystic fibrosis (CF) patients and are refractory to phagocytosis and antibiotics. Here we show that mucoid bacteria perish during anaerobic exposure to 15 mM nitrite (NO2) at pH 6.5, which mimics CF airway mucus. Killing required a pH lower than 7, implicating formation of nitrous acid (HNO2) and NO, that adds NO equivalents to cellular molecules. Eighty-seven percent of CF isolates possessed mucA mutations and were killed by HNO2 (3-log reduction in 4 days). Furthermore, antibiotic-resistant strains determined were also equally sensitive to HNO2. More importantly, HNO2 killed mucoid bacteria (a) in anaerobic biofilms; (b) in vitro in ultrasupernatants of airway secretions derived from explanted CF patient lungs; and (c) in mouse lungs in vivo in a pH-dependent fashion, with no organisms remaining after daily exposure to HNO2 for 16 days. HNO2 at these levels of acidity and NO2 also had no adverse effects on cultured human airway epithelia in vitro. In summary, selective killing by HNO2 may provide novel insights into the important clinical goal of eradicating mucoid P. aeruginosa from the CF airways.

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 ...

SummarySurfactant protein-A (SP-A) is an important antimicrobial protein that opsonizes and permeabilizes membranes of microbial pathogens in mammalian lungs. Previously, we have shown that Pseudomonas aeruginosa flagellum-deficient mutants are preferentially cleared in the lungs of wild-type mice by SP-Amediated membrane permeabilization, and not by opsonization. In this study, we report a flagellummediated mechanism of P. aeruginosa resistance to SP-A. We discovered that flagellum-deficient (DfliC) bacteria are unable to produce adequate amounts of exoproteases to degrade SP-A in vitro and in vivo, leading to its preferential clearance in the lungs of SP-A +/+ mice. In addition, DfliC bacteria failed to degrade another important lung antimicrobial protein lysozyme. Detailed analyses showed that DfliC bacteria are unable to upregulate the transcription of lasI and rhlI genes, impairing the production of homoserine lactones necessary for quorum-sensing, an important virulence process that regulates the production of multiple exoproteases. Thus, reduced ability of DfliC bacteria to quorum-sense attenuates production of exoproteases and limits degradation of SP-A, thereby conferring susceptibility to this major pulmonary host defence protein.