Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552–PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on β-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation.

Polyamines from the outer surface were isolated and shown to contain putrescine and spermidine by using highperformance liquid chromatography and mass spectrometry. The PA4774::lux mutant did not produce spermidine on the cell surface, but genetic complementation restored surface spermidine production as well as the antibiotic and oxidative stress resistance phenotypes of the membrane. We have identified new functions for spermidine on the cell surface and propose that polyamines are produced under Mg 2؉ -limiting conditions as an organic polycation to bind lipopolysaccharide (LPS) and to stabilize and protect the outer membrane against antibiotic and oxidative damage.

SummaryPseudomonas aeruginosa is an opportunistic pathogen that occupies a wide variety of environmental niches. Extracellular DNA is ubiquitous in various environments and is a rich source of carbon, nitrogen and phosphate. Here we show that P. aeruginosa is capable of using DNA as a nutrient source. Under phosphate-limiting conditions, or when DNA is supplied as a source of phosphate, expression of PA3909 is induced. PA3909 encodes an extracellular deoxyribonuclease (DNase), which is required for degradation of DNA and utilization of DNA as a source of carbon, nitrogen and phosphate. Stabilization of PA3909 by the addition of excess Mg 2+ and Ca 2+ was required for DNase activity in culture supernatants. Extracellular DNase activity was seen in multiple P. aeruginosa strains and isolates from cystic fibrosis patients. The primary Xcp type II secretion system but not the Hxc type II secretion system is required for DNase activity and the ability to use DNA as a source of nutrients. This study identifies an extracellular DNase produced by P. aeruginosa that enables degradation of extracellular DNA into an accessible source of carbon, nitrogen and phosphate. DNase production by P. aeruginosa also has important implications for virulence and biofilm formation.

Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3) demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo.

The ability of Pseudomonas aeruginosa to cause a broad range of infections in humans is due, at least in part, to its adaptability and its capacity to regulate the expression of key virulence genes in response to specific environmental conditions. Multiple two-component response regulators have been shown to facilitate rapid responses to these environmental conditions, including the coordinated expression of specific virulence determinants. RsmA is a posttranscriptional regulatory protein which controls the expression of a number of virulence-related genes with relevance for acute and chronic infections. Many membrane-bound sensors, including RetS, LadS, and GacS, are responsible for the reciprocal regulation of genes associated with acute infection and chronic persistence. In P. aeruginosa this is due to sensors influencing the expression of the regulatory RNA RsmZ, with subsequent effects on the level of free RsmA. While interactions between an rsmA mutant and human airway epithelial cells have been examined in vitro, the role of RsmA during infection in vivo has not been determined yet. Here the function of RsmA in both acute and chronic models of infection was examined. The results demonstrate that RsmA is involved in initial colonization and dissemination in a mouse model of acute pneumonia. Furthermore, while loss of RsmA results in reduced colonization during the initial stages of acute infection, the data show that mutation of rsmA ultimately favors chronic persistence and results in increased inflammation in the lungs of infected mice.Pseudomomas aeruginosa is a metabolically versatile gramnegative bacterium that is capable of causing opportunistic infections in plants, animals, and humans. In humans, P. aeruginosa infections occur in the gastrointestinal tract and respiratory system and in patients with ocular infections, burn wounds, and cystic fibrosis (CF). The characteristics of acute and chronic infections caused by P. aeruginosa are quite distinct and are associated with selected expression of a certain subset of virulence factors. The pathogenesis of acute infections, such as ventilator-associated pneumonia, is thought to require the expression of a functional type III secretion system (T3SS), along with other toxins and proteases (37). These infections typically result in systemic infections and ultimately mortality. On the other hand, individuals with CF are usually colonized with P. aeruginosa early in life (1), and while P. aeruginosa can reach densities of 10 9 CFU/ml of sputum (33), P. aeruginosa infections in the lungs of CF patients are minimally invasive and rarely progress to septicemia. Rather, it is the inflammation and deterioration of pulmonary function resulting from chronic P. aeruginosa infection that is the main cause of mortality in CF patients. Therefore, investigating the mechanism(s) by which P. aeruginosa colonizes and becomes firmly established is critical for understanding the nature of life-long infections in CF patients.The substantial proportion of the P. aeruginosa g...