Pseudomonas 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 Mg2+ and Ca2+ 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 a metabolically diverse opportunistic pathogen capable of causing both acute and chronic infections in multiple hosts. Pseudomonas aeruginosa is the third leading cause of nosocomial infections and is the predominant pathogen associated with morbidity and mortality of cystic fibrosis (CF) patients (Govan and Deretic, 1996; Rajan and Saiman, 2002). Outside of its role as an opportunistic pathogen, P. aeruginosa is found in various environments, such as soil, lakes, rivers and the open ocean (Pellett et al., 1983; Khan et al., 2007). The diversity of ecological niches P. aeruginosa can occupy may be due to its large genome and extensive capability to sense, respond and grow in different environments. The large genome size of P. aeruginosa (6.3 Mb) enables the expression of a wide range of biological systems that are appropriate for growth in various different environments (Stover et al., 2000). The number of regulatory systems identified comprises as much as 8–10% of the P. aeruginosa genome (Stover et al., 2000).
The development of P. aeruginosa infections is dependent on the ability of the organism to colonize and persist within the host environment. In addition to human infections, P. aeruginosa can cause infections in experimental models of Drosophila melanogaster (fruit fly), Dictyostelium discoideum, Caenorhabditis elegans (nematode), plants and Galleria mellonella (wax moth) (Hultmark, 1993; Mahajan-Miklos et al., 1999; Tan et al., 1999; Jander et al., 2000; Hendrickson et al., 2001; Unal and Steinert, 2006). While survival in the host is dependent on overcoming many factors, such as innate immunity, antimicrobial peptides and competition by other bacteria, competition for nutrients is also a very important factor. Little is known regarding the nutritional requirements and the sources of nutrients available to P. aeruginosa during an infection. Previous work identified amino acids (Palmer et al., 2005) and the lung surfactant component phosphatidylcholine (Son et al., 2007) as sources of carbon that are present in the CF lung and support in vitro growth. The topic of in vivo carbon sources used by multiple pathogens has been recently reviewed (Whiteley et al., 2001). As the incidence of antimicrobial resistance continues to climb, novel mechanisms of combating microbial infections are urgently required. It is necessary to further our understanding of bacterial physiology, metabolism and nutrient requirements during infections for the development of novel antimicrobials to treat disease.
In the natural environment, extracellular or free DNA is found in the soil at concentrations between 0.08 and2 µg g−1 of soil (Nielsen et al., 2007). In aquatic environments, extracellular DNA can be found at concentrations up to 44 µg l−1 (Lorenz and Wackernagel, 1994). In P. aeruginosa infection sites, including the gastrointestinal tract, DNA is also present in abundance (Hoskins, 1978). In the lung environment of CF patients, DNA reaches levels as high as 20 mg ml−1 in the sputum (Ranasinha et al., 1993; Brandt et al., 1995; Ulmer et al., 1996). Extracellular DNA is also present circulating throughout the body in the blood and other fluids, including urine, milk, amniotic fluid and bronchial lavage (Vlassov et al., 2007).
Biofilms are recognized as the predominant mode of bacterial growth in nature and are responsible for the majority of refractory bacterial infections (Costerton et al., 1999). Biofilms are multicellular surface-associated microbial communities encased in an extracellular matrix that consists of extracellular DNA (Whitchurch et al., 2002; Qin et al., 2007), as well as bacterial exopolysaccharides and proteins (Ryder et al., 2007; Moreau-Marquis et al., 2008). DNA is released from lysed cells, but is also contained in outer membrane vesicles and accumulates throughout the biofilm matrix (Kadurugamuwa and Beveridge, 1995; Webb et al., 2003; Schooling and Beveridge, 2006). Extracellular DNA plays an adhesive role in maintaining biofilm structure (Whitchurch et al., 2002). We have recently identified a novel signalling role for extracellular DNA in P. aeruginosa biofilms, where its ability to chelate cations serves to impose a cation limitation on cells and induces the expression of genes essential for antibiotic resistance and immune system evasion (Mulcahy et al., 2008). The centres of large microcolonies can undergo dispersion triggered by a central hollowing that is accompanied by virus or rhamnolipid-mediated lysis (Boles et al., 2005; Kirov et al., 2007) or autolysis (Ma et al., 2009) and the release of extracellular DNA. This may serve to provide DNA as a nutrient to neighbouring cells in the deeper levels of a biofilm, which may have limited access to nutrient diffusion.
Given that DNA is an excellent source of carbon, nitrogen and phosphate, coupled with its abundance and availability in the environment or the body, the ability to utilize DNA as a nutrient would be of significant benefit to bacteria. DNA utilization has been studied in estuarine microbial communities (Paul et al., 1988) and it is proposed that the rich extracellular DNA content of deep ocean sediments is a major source of phosphorus for prokaryotic growth in ocean sediments (Dell'Anno and Danovaro, 2005). Escherichia coli (Finkel and Kolter, 2001; Palchevskiy and Finkel, 2006) and more recently Shewanella spp. (Pinchuk et al., 2008) were shown to utilize DNA as a sole nutrient source. However, the ability of P. aeruginosa to use DNA as a nutrient has not been investigated. The objective of this study was to determine if P. aeruginosa is capable of utilizing extracellular DNA as a source of carbon, nitrogen and or phosphate.
Ability of P. aeruginosa to use DNA as a sole source of carbon, nitrogen and phosphate
To determine if DNA can be utilized as a nutrient by P. aeruginosa, growth assays were performed wherein DNA was provided as the sole source of carbon, nitrogen or phosphate, and as the source of all three nutrients together. Chemically defined BM2 medium, lacking each or all of the required nutrients, was supplemented with low-molecular-weight fish sperm DNA at concentrations ranging from 0 to 2.5 mg ml−1. Fish sperm DNA was proteinase K-digested, extracted twice with phenol/chloroform/isoamyl alcohol and twice with chloroform to remove any potential contaminating proteins present in the commercial DNA preparation. The P. aeruginosa cells were diluted 1/1000 from overnight cultures and incubated at 37°C in small-volume (150 µl) cultures grown in microplates. Growth (OD600) was measured every 20 min for a period of 18 h (Fig. 1). No growth was observed in media lacking extracellular DNA. Growth was restored to P. aeruginosa cultures supplemented with DNA as the sole source of carbon, nitrogen and phosphate (Fig. 1), indicating the ability of P. aeruginosa to utilize DNA as a nutrient source. DNA is more readily used as a source of phosphate (Fig. 1C), followed by nitrogen (Fig. 1B), when compared with the utilization of DNA as the sole carbon source (Fig. 1A). When DNA is used as a source of all three nutrients, its ability to utilize DNA as a carbon source appears to be the growth-limiting factor (Fig. 1D).
PA3909 expression is regulated by phosphate limitation and DNA
Analysis of the P. aeruginosa genome for signal peptides that target proteins out of the cytoplasm identified a number of putative deoxyribonucleases (DNases) that may be secreted due to the presence of type I signal peptides (PA3909, PA2749, PA2750) (Lewenza et al., 2005a). One possible function for these secreted enzymes is to degrade extracellular DNA into usable sources of carbon, nitrogen and phosphate, which may then be transported into the cell. Previously, we identified a putative extracellular DNase, PA3909, which was transcriptionally induced under phosphate-limiting conditions (Lewenza et al., 2005b). The observation that P. aeruginosa can use DNA as a source of phosphate prompted us to investigate the role of this DNase in DNA utilization as a nutrient. Consistent with our previous results (Lewenza et al., 2005b), expression of PA3909 is induced by phosphate limitation. Furthermore, expression of PA3909 is regulated in a dose-dependent manner, whereby maximal gene expression was observed at 100 µM phosphate (900-fold induction) and was repressed at phosphate concentrations greater than 400 µM (Fig. 2A). Under the conditions examined, PA3909 expression was induced at the onset of stationary phase, after 6–8 h of growth (Fig. 2A).
Under phosphate starvation conditions, where DNA is added as the sole phosphate source, the expression of PA3909 was also induced (Fig. 2B). Gene expression increased in a dose-dependent manner as the concentration of DNA decreased, which mirrors the gene expression pattern in phosphate-limited cells. Maximal expression of PA3909 was seen in 0.625 mg ml−1 DNA, similar to the level of expression observed in low phosphate (100 µM). At these low concentrations of DNA, there is a 1000-fold induction of PA3909 expression relative to 10 mg ml−1 DNA (data not shown), which is similar to media containing high concentrations of phosphate (> 400 µM). PA3909 expression is not induced under carbon or nitrogen starvation when phosphate concentration is high (> 400 µM). However, if cells are grown under carbon- and nitrogen-limiting growth conditions and in the absence of phosphate, expression of PA3909 is induced (data not shown).
PA3909 and DNase activity is important for utilizing extracellular DNA as a nutrient
To determine if the ability to use DNA as a nutrient source is proportional to DNase activity, exogenous DNase was added to cultures and growth was measured as a function of the exogenous DNase concentration in the media. Experiments were performed in BM2 media containing DNA as the sole phosphate source supplemented with 5 or 10 µg ml−1 of DNase enzyme. Addition of 5 or 10 µg ml−1 of DNase enzyme to PAO1 increased the growth rate during the log phase of growth, and the final OD600 values, in a dose-dependent manner (Fig. 3A). Addition of DNase to wild-type PAO1 cultures grown with DNA as a carbon or nitrogen source also caused increased growth proportional to the amount of DNase enzyme added (data not shown).
Mutation of PA3909 (PA3909::lux) did not affect growth in LB or BM2 media where succinate, ammonium nitrate and phosphate buffer were used as the source of carbon, nitrogen and phosphate respectively (data not shown). However, the PA3909 mutant showed a significantly slower growth rate than PAO1 grown under conditions where DNA was used as the sole source of phosphate (Fig. 3B). At a DNA concentration of 0.625 mg ml−1 PAO1 had a doubling time of 9.4 h compared with 12.28 h for PA3909::lux. Addition of exogenous DNase to PA3909::lux restored the growth rate similar to that of PAO1 (Fig. 3B), with the rate of PA3909::lux growth increased relative to the quantity of DNase added (Fig. 3B). PA3909::lux also has a growth defect when grown in media using DNA as a source of nitrogen or carbon and addition of exogenous DNA to PA3909::lux growing under these conditions also caused increased growth proportional to the amount of DNase enzyme added (data not shown). These results suggest that PA3909 is a functional, secreted DNase that can degrade extracellular DNA and liberate accessible sources of phosphate, carbon and nitrogen to promote growth.
DNase activity of PA3909 is phosphate-regulated and requires calcium and magnesium cations
To demonstrate that PA3909 encodes an extracellular DNase, culture supernatants from PAO1 and PA3909::lux strains were assessed for DNase activity. As PA3909::lux is induced under low-phosphate conditions and multiple DNases require Ca2+ and Mg2+ for activity (Hasegawa et al., 2009), strains were grown under high- and low-phosphate conditions, with and without the addition of excess cations to determine the optimal conditions for PA3909 enzyme activity. Culture supernatants were incubated with high-molecular-weight P. aeruginosa genomic DNA and assessed for their ability to degrade this DNA. The DNase activity in PAO1 culture supernatants was detected under low-phosphate conditions but not when high concentrations of phosphate were present and only when the culture media were supplemented with excess Ca2+ and Mg2+ cations (Fig. 4A). Mutation of PA3909 resulted in decreased DNase activity over time relative to PAO1, indicating the importance of this gene in the degradation of DNA (Fig. 4B). There is residual DNA degradation by the supernatants of PA3909::lux, suggesting the presence of additional, secreted DNases.
The DNase activity was also observed in the culture supernatants of multiple P. aeruginosa isolates, including PAK, PA14, PA103, as well as isolates from adult CF patients (Fig. 4C). PAK and PA14 show comparable DNAse activity to PAO1, demonstrating total degradation of 5 µg of bacterial genomic DNA over 16 h. Supernatants from PA103 also show degradation of DNA, although the level of degradation is lower compared with PAO1. Three of five CF isolates demonstrate complete degradation while two show partial DNA digestion under the conditions tested.
The Xcp but not the Hxc type II secretion system is involved in PA3909 secretion
Pseudomonas aeruginosa has two main type II secretion systems (T2SS) that are involved in the transport of exoproteins across the Gram-negative bacterial outer membrane (Ball et al., 2002; Durand et al., 2003). These systems include the main T2SS, Xcp, and the more recently identified Hxc secretion system (Ball et al., 2002). The Hxc system has been shown to be required for secretion of a single effector protein, LapA. LapA is an alkaline phosphatase, which is secreted in an Hxc-dependent manner, under conditions of phosphate limitation (Ball et al., 2002). As PA3909 expression and activity are also phosphate-regulated (Figs 2A and 4A), we reasoned that the Hxc system may play a role in secretion of the DNase encoded by PA3909. Mutants in the outer membrane secretin hxcQ, as well as lapA, were examined for DNase activity and their ability to use DNA as a phosphate source. Because phenol/chloroform purified DNA and unpurified DNA had no impact on the ability of PAO1 to use DNA as a phosphate source (Fig. S1), unpurified DNA was used for this experiment. Mutation of either hxcQ or lapA had no effect on growth using DNA (2.5 mg ml−1) as the sole phosphate source compared with their parent strain PAO1f (Filloux laboratory strain) (Fig. 5A). Doubling times were 5.1, 5.0 and 4.9 h for PAO1f, hxcQ or lapA strains respectively. Similarly, mutation of hxcQ or lapA had no effect on the ability of P. aeruginosa to degrade DNA (Fig. 5B). This indicated that the Hxc secretion system was not involved in the secretion of PA3909.
We next tested the role of the Xcp T2SS in secreting the DNase encoded by PA3909, in DNA degradation and in utilizing DNA as a phosphate source. Mini-Tn5-lux mutants previously constructed with transposon insertions in the xcpS and xcpU genes (Lewenza et al., 2005b) were compared with PAO1 for their ability to use DNA as a phosphate source or to degrade genomic DNA. XcpS::lux and xcpU::lux demonstrated significantly slower growth rates compared with PAO1 under conditions where DNA is provided as the sole source of phosphate, with doubling times of 13.4 and 7.8 h respectively, compared with a doubling time of 5.7 h for PAO1 (Fig. 5C). Supernatants from xcpS::lux and xcpY::lux, failed to degrade high-molecular-weight bacteria DNA while supernatant from xcpU::lux showed partial degradation (Fig. 5D). The longer doubling times observed in xcpS::lux and xcpY::lux mutant strains (13.4 and 11.3 h respectively) correlate with the absence of DNase activity observed by DNase assays and agarose gel electrophoresis (Fig. 5D).
We have characterized the ability of P. aeruginosa to grow in chemically defined medium using extracellular DNA as the sole nutrient source of phosphate, nitrogen and carbon. DNA can be efficiently used as a phosphate and nitrogen source, but is more difficult to support growth when used as a carbon source. In order to begin to understand the mechanism of DNA utilization, we have identified an extracellular DNase that is capable of degrading high-molecular-weight bacterial DNA.
The PA3909 gene is in a two-gene operon, where the first gene encodes a putative secreted phosphatase (PA3910) and the second gene encodes a secreted enzyme with DNase activity (PA3909). Under conditions of phosphate limitation, this operon is highly transcriptionally induced. Under phosphate starvation, and in the presence of low concentrations of extracellular DNA, P. aeruginosa behaves as if phosphate-limited and induces expression of the PA3909 DNase. We detected DNase activity in supernatants of cultures grown in limiting phosphate conditions and which were supplemented with excess divalent cations. Cations were required to stabilize the DNase activity, as has been observed for other DNases (Hasegawa et al., 2009). The primary T2SS in P. aeruginosa is the Xcp system, and this was required for secretion of PA3909. Although the Hxc system is required for the secretion of another phosphate-regulated effector, the LapA phosphatase (Ball et al., 2002), it was not required for PA3909 secretion. The LapA phosphatase was also not required for degradation of extracellular DNA and phosphate acquisition.
Extracellular DNases are produced by multiple bacteria including many pathogens. All members of the group A Streptococcus have been shown to produce at least one extracellular DNase, and most strains make more than one distinct enzyme (Cunningham, 2000). Although extracellular DNases may perform multiple functions in P. aeruginosa, this is the first study that demonstrates that secretion of an extracellular DNase provides P. aeruginosa with a growth advantage by increasing the pool of available nutrients by DNA degradation.
In other bacteria, extracellular DNase activity also has been hypothesized to be involved in the dissemination of infecting bacteria by lysing pus (Sherry et al., 1948; Tillett et al., 1948) and a number of recent studies demonstrate a role for extracellular DNases in host immune evasion by the degradation of neutrophil extracellular traps (NETs) (Brinkmann et al., 2004; Buchanan et al., 2006). The NETs arise from stimulated neutrophils, which eject a mesh-like lattice of intracellular DNA and antimicrobial proteins that function to trap and kill pathogens (Brinkmann et al., 2004). The NETs are antimicrobial in nature, killing a variety of microorganisms (Brinkmann et al., 2004; Beiter et al., 2006; Buchanan et al., 2006; Urban et al., 2006; Wartha et al., 2007). The antimicrobial property of NETs was previously attributed to DNA-associated histones and other antimicrobial peptides (Brinkmann et al., 2004). However, our previously published results indicate that the DNA component of NETs may also play an important role, as DNA itself is antimicrobial due to cation chelation and membrane destabilization (Mulcahy et al., 2008). Furthermore, the antimicrobial activity of NETs can be inhibited if activated neutrophils are exposed to DNase (Buchanan et al., 2006). Our future work will focus on the role of PA3909 and other putative extracellular DNases in resisting the antimicrobial action of NETs. The DNase activity may also play a role in biofilm dissolution or nutrient acquisition in biofilms, as extracellular DNA accumulates in the matrix of P. aeruginosa biofilms.
PA3909 is induced under phosphate-limiting conditions. A previous study demonstrated that the level of inorganic phosphate is reduced in humans infected with Gram-negative pathogens to a level that is predicted to be suboptimal for bacterial growth (Weinberg, 1974). Recent work on intestinal infections by P. aeruginosa identified local depletion of phosphate as a major environmental cue to which intestinal P. aeruginosa responds by increasing the expression of multiple virulence traits (Long et al., 2008). Other virulence factors that are induced under phosphate limitation include phospholipases, which function to lyse human cells (Shortridge et al., 1992). These observations suggest that responses to phosphate limitation, including the production of a DNase, may be important for virulence of P. aeruginosa infections.
In summary, we have identified the first extracellular DNase, encoded by PA3909, which is secreted by P. aeruginosa. Expression of PA3909 is regulated by phosphate limitation, and functions to degrade DNA and utilize DNA as a nutrient source. Because of the abundance of DNA in the environment, the ability of P. aeruginosa to recycle DNA as a nutrient may provide a competitive advantage in crowded bacterial environments and/or nutrient-limited conditions. Future work aims to enhance our understanding of the role of DNases in P. aeruginosa virulence.
Bacterial strains and growth conditions
Bacterial strains used in this study are described in Table 1. Pseudomonas aeruginosa PAO1, PAK, PA103 and PA14 were used as wild-type strains. PA3909::lux, xcpS::lux, xcpU::lux and xcpY::lux are transposon insertion mutants and transcriptional luxCDABE fusions that were previously constructed (Lewenza et al., 2005b). The lapA mutant (R. Voulhoux, unpublished) and PAO1ΔhxcQ, was constructed as previously described (Ball et al., 2002). Cystic fibrosis isolates were isolated from adult CF patients at different stages of infection with P. aeruginosa at the adult CF clinic, Foothills Hospital, Calgary.
Table 1. Bacterial strains.
Source or reference
R.E. Hancock, University of British Columbia, Vancouver
S. Lory, Harvard Medical School, Boston
F. Ausubel, Massachusetts General Hospital, Boston
This defined growth medium includes the following components: 0.1 M Hepes pH 7, 7 mM ammonium chloride, 20 mM sodium succinate pH 6.7, 2 mM magnesium sulfate, 10 µM iron sulfate, 1600 µM phosphate buffer pH 7.2, 1.62 µM mangenese sulfate, 2.45 µM calcium chloride, 13.91 µM zinc chloride, 4.69 µM boric acid, 0.67 µM cobalt chloride. Limiting phosphate, carbon and nitrogen conditions contain 400 µM phosphate, 2.5 mM sodium succinate and 35 µM ammonium chloride respectively. DNA was added as a nutrient source at concentrations ranging from 0 to 2.5 mg ml−1. The source of DNA was fish sperm DNA-potassium salt (USB, Cleveland, OH), which was either resuspended directly in BM2 medium or was purified, as follows, to remove any potential contaminating proteins present in the commercial DNA preparation. DNA was digested with proteinase K (20 µg ml−1) for 30 min, extracted twice in phenol/chloroform/isoamyl alcohol and twice in chloroform. Initial experiments were performed to compare purified protein-free DNA to the commercial DNA preparation. However, no significant difference was observed in the growth rate between PAO1 grown using unpurified DNA as a source of phosphate (Fig S1), carbon or nitrogen (data not shown) and proteinase K-digested and phenol/chloroform-extracted DNA (Fig. 1).
Growth kinetics and analysis of gene expression
Growth kinetics of P. aeruginosa was carried out in small-volume (100–150 µl) cultures grown in transparent 96-well plates (Nunc). Gene expression assays were carried out as previously described (Mulcahy et al., 2008). Briefly, overnight cultures were normalized and diluted 1/1000 into BM2 chemically defined medium or BM2 medium lacking certain nutrients and supplemented with DNA, at the concentrations indicated, as sources of nitrogen, carbon or phosphate. Cultures were overlayed with mineral oil to prevent evaporation. OD600 and CPS were monitored every 20 min over time in the Wallac Victor3 luminescence plate reader (Perkin-Elmer).
DNase activity assays
Pseudomonas aeruginosa strains were grown in BM2 medium containing low phosphate (400 µM), supplemented with 10 mM MgSO4 and 10 mM CaCl2. Overnight cultures of P. aeruginosa strains were normalized to an OD600 of 1 and supernatants were collected by centrifugation at 8000 r.p.m. for 10 min. Fifteen microlitres of supernatant was incubated with 5 µg of P. aeruginosa genomic DNA between 1 and 16 h. Pseudomonas aeruginosa genomic DNA was purified using the standard method of extracting lysed cells with phenol/chloroform followed by precipitating with ethanol. DNA degradation was visualized on ethidium bromide stained 1% agarose gels.
Statistical analysis was performed using Graphpad Prism 5 software. Two-way anova was used to calculate significant differences in OD600 values between PAO1 and mutant strains. Doubling time was calculated using non-linear regression analysis for exponential growth. Where indicated, significant differences refer to P-values < 0.05.
This research was supported by the Westaim Corporation and the Alberta Science and Research Authority (ASRA). H.M. is the recipient of a Canadian Cystic Fibrosis Foundation fellowship. S.L. holds the Westaim-ASRA Chair in Biofilm Research. The authors would like to thank M.G. Surette for providing CF isolates, R. Voulhoux for supplying lapA and hxcQ mutant strains, C. Whitchurch for helpful discussions and H. Nguyen for carrying out preliminary experiments.