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Journal of Bacteriology, June 2007, p. 4449-4455, Vol. 189, No. 12
0021-9193/07/$08.00+0     doi:10.1128/JB.00162-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Membrane-Bound Nitrate Reductase Is Required for Anaerobic Growth in Cystic Fibrosis Sputum ^,

Kelli L. Palmer, Stacie A. Brown, and Marvin Whiteley^*

Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, Texas 78712

Received 31 January 2007/ Accepted 22 March 2007

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The autosomal recessive disorder cystic fibrosis (CF) affects approximately 70,000 people worldwide and is characterized by chronic bacterial lung infections with the opportunistic pathogen Pseudomonas aeruginosa. To form a chronic CF lung infection, P. aeruginosa must grow and proliferate within the CF lung, and the highly viscous sputum within the CF lung provides a likely growth substrate. Recent evidence indicates that anaerobic microenvironments may be present in the CF lung sputum layer. Since anaerobic growth significantly enhances P. aeruginosa biofilm formation and antibiotic resistance, it is important to examine P. aeruginosa physiology and metabolism in anaerobic environments. Measurement of nitrate levels revealed that CF sputum contains sufficient nitrate to support significant P. aeruginosa growth anaerobically, and mutational analysis revealed that the membrane-bound nitrate reductase is essential for P. aeruginosa anaerobic growth in an in vitro CF sputum medium. In addition, expression of genes coding for the membrane-bound nitrate reductase complex is responsive to CF sputum nitrate levels. These findings suggest that the membrane-bound nitrate reductase is critical for P. aeruginosa anaerobic growth with nitrate in the CF lung.

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Cystic fibrosis (CF) is a heritable disease characterized by accumulation of large volumes of mucus (sputum) within the lungs and persistent colonization with opportunistic pathogens. While numerous bacterial species transiently colonize CF sputum or persist at low cell densities, the opportunistic pathogen Pseudomonas aeruginosa establishes a chronic infection and can attain densities of >10⁹ cells/ml of sputum (19). Chronic P. aeruginosa infection is the leading cause of morbidity and mortality in CF patients (20). Once established, P. aeruginosa CF lung infections are highly refractory to antibiotic treatments, and even aggressive therapies fail to fully eradicate the bacterium from the lung (20). Several aspects of P. aeruginosa physiology are believed to contribute to its high antibiotic resistance in vivo, including biofilm formation and overproduction of the viscous exopolysaccharide alginate (23).

P. aeruginosa CF lung infections are frequently modeled under aerobic laboratory conditions; however, direct oxygen measurements within CF lung sputum in situ indicate that sputum contains hypoxic, and potentially anaerobic, regions (38). In addition, strict anaerobic bacteria have been detected in sputum samples from multiple CF patients (21, 29), suggesting that environments capable of sustaining these species are present in vivo. There are several factors that could potentially reduce oxygen levels within CF sputum: cultured CF lung epithelial cells consume oxygen at a higher rate than non-CF lung epithelial cells, oxygen diffusion through sputum is restricted, and oxygen is consumed by resident sputum microorganisms, including P. aeruginosa (38). Collectively these factors may effectively reduce oxygen to levels insufficient for aerobic respiration. Interestingly, antibodies to components of two putative anaerobic nitrate reductases of P. aeruginosa, NapA and NarG, have been detected in sera from CF patients (2), suggesting that P. aeruginosa produces these respiratory enzymes in vivo.

It is clear from several published reports that P. aeruginosa physiology is remarkably different during anaerobic growth and aerobic growth. For example, it has been proposed that P. aeruginosa "prefers" to reside in antibiotic-resistant biofilms under anaerobic conditions (40) and coincidently enhances production of the alginate capsule surrounding the biofilm under these conditions (5, 38). The structure and function of the outer membrane are also altered during anaerobic growth. Specifically, P. aeruginosa modifies the structure of its lipopolysaccharide from a highly electronegative surface to a neutral surface during anaerobic growth (30). This change provides enhanced resistance to cationic antimicrobials, such as aminoglycoside antibiotics (28), and potentially to naturally produced cationic peptides due to inhibition of charge-mediated uptake (17). Although the mechanism is unclear, the bactericidal effects of the noncationic antibiotics meropenem, ceftazidime, and colistin are also diminished under anaerobic growth conditions for P. aeruginosa CF lung isolates (18). These antibiotics are used in combination therapies for treatment of chronic P. aeruginosa lung infection (15). Taken together, these observations suggest that, if anaerobic microenvironments are present in the CF lung sputum layer, growth in these microenvironments may significantly impact P. aeruginosa CF lung infection.

To generate energy for processes such as biofilm formation and alginate production in vivo, P. aeruginosa must obtain carbon and energy from its CF sputum growth environment. P. aeruginosa is capable of utilizing a wide range of carbon sources in CF sputum (26) and possesses numerous putative and confirmed oxidases for respiratory growth (7). P. aeruginosa is a denitrifier and can utilize the nitrogenous oxides nitrate, nitrite, and nitrous oxide as electron acceptors for respiratory growth in the absence of oxygen (39). P. aeruginosa can also anaerobically metabolize arginine in a substrate level phosphorylation pathway known as arginine deimination, as well as ferment pyruvate; however, these pathways are likely not important for growth in vivo, since they support little or no bacterial growth (>40-h doubling time for arginine deimination and no net growth for pyruvate fermentation). Instead, these metabolic pathways are likely used for anaerobic cellular maintenance in the absence of nitrogenous electron acceptors (9, 34). Given the evidence that P. aeruginosa potentially encounters anaerobic microenvironments during the course of chronic CF lung infection and the impact of anaerobic growth on P. aeruginosa persistence and antibiotic resistance, we sought to determine the P. aeruginosa metabolic components critical for anaerobic growth in CF sputum. In this study, we identified and characterized a component of P. aeruginosa metabolism, the membrane-bound nitrate reductase, as a required determinant for P. aeruginosa anaerobic growth and nitrate reduction in an in vitro CF sputum medium.

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Bacterial strains and growth media. P. aeruginosa strain PA14 and the isogenic narG and napA transposon insertion mutants were obtained from the MGH-Parabiosys:NHLBI Program for Genomic Applications (http://pga.mgh.harvard.edu/cgi-bin/pa14/mutants/retrieve.cgi). P. aeruginosa mPAO1 and the isogenic narG transposon insertion mutant were obtained from the University of Washington Genome Center (http://genome.washington.edu). Transposon insertion sites in PA14 and PAO1 mutants were confirmed using PCR. P. aeruginosa was routinely cultured on tryptic soy agar. Escherichia coli DH5 was used for routine cloning and was commonly grown in LB Miller broth (EMD Chemicals).

For P. aeruginosa anaerobic growth, a MOPS (morpholinepropanesulfonic acid)-buffered medium (50 mM MOPS, pH 7.2, 93 mM NH₄Cl, 43 mM NaCl, 3.7 mM KH₂PO₄, 1 mM MgSO₄, 3.5 µM FeSO₄·7H₂O, 10 mM glycerol, 10 mM sodium succinate, and 0.001% yeast extract) or brain heart infusion (BHI) broth supplemented with NaNO₂ or KNO₃ was used. Anaerobic media were aliquoted into Balch tubes, capped, sealed, and heated for 30 min at 100°C. The heated tubes were boiled under vacuum to release residual oxygen from the media, and the headspace was replaced with nitrogen gas three times before the tubes were autoclaved. All incubations were performed at 37°C with shaking at 250 rpm. Where applicable, antibiotics were added at the following concentrations: kanamycin, 200 µg/ml; ampicillin, 50 µg/ml; and carbenicillin, 300 µg/ml.

Complementation of the P. aeruginosa narG mutant. The narGHJI genes were amplified from P. aeruginosa PA14 chromosomal DNA using the primers narGHJI-for (5'-GCTCTAGACAACCTCTGATTAGCGTTGTAAC-3') and narGHJI-rev (5'-CCCAAGCTTGATCGATCACCTCGGTTCGGC-3') (underlining indicates XbaI and HindIFF sites, respectively), using the Expand Long Template kit (Roche). The resulting 6,878-bp fragment containing narGHJI was digested with XbaI/HindIII and cloned into pUCP18 (29) to create pKP201. In this plasmid, narGHJI expression is under control of the constitutive lac promoter.

Anaerobic-growth experiments. Aerobic stationary-phase P. aeruginosa cells were inoculated to a final optical density at 600 nm (OD₆₀₀) of 0.001 in anaerobic BHI broth supplemented with various amounts of KNO₃. Growth was measured by OD₆₀₀, and samples were removed for nitrate analysis at 24 h postinoculation. For some experiments, anaerobic MOPS medium with or without 1 mM NaNO₂ was inoculated to 10³ cells/ml with aerobic exponential-phase P. aeruginosa PA14. Samples were removed at 24 h postinoculation for viable-cell counts on tryptic soy agar and nitrite analysis.

To evaluate anaerobic growth in CF sputum, sputum was resuspended in deionized water as previously described (26). Sputum samples were obtained by expectoration from nonexacerbating adult CF patients with total P. aeruginosa titers of <10⁸ cells/ml. Briefly, lyophilized CF sputum was resuspended in deionized water to a concentration of 20% (vol/vol) sputum, homogenized with a tip sonicator three to five times for 30 s each time, and centrifuged for 5 min at 16,000 x g before sterilization by filtration through a 0.45-µm-pore-size filter. Since CF sputum medium contains only 20% of the native nitrate levels of CF sputum, the CF sputum medium was amended with nitrate to levels commonly found in CF sputum harvested from the lung (400 µM). CF sputum medium with nitrate was incubated overnight in an anaerobic chamber (Coy Laboratories) prior to inoculation. P. aeruginosa strains were grown aerobically overnight on MOPS-buffered medium agar plates and subsequently transferred to an anaerobic chamber (Coy Laboratories). After 1 h of incubation at 37°C, P. aeruginosa cells were resuspended to an OD₆₀₀ of 0.2 in MOPS-buffered medium with no added electron acceptor. After 4 to 8 h of incubation at 37°C in the anaerobic chamber, the cells were diluted to approximately 10³ in anaerobic CF sputum medium with nitrate. Samples were removed for viable-cell counts immediately after inoculation and after 24 h of incubation at 37°C in the anaerobic chamber.

Nitrate and nitrite measurements. Nitrate concentrations in CF sputum were determined by anion-exchange high-performance liquid chromatography (HPLC) using a Dionex ion chromatography system. Sputum samples from seven adult CF patients were lyophilized, reconstituted to 25% (vol/vol) in deionized water, and homogenized and sterilized as described above prior to HPLC analysis. For analyses of nitrate concentrations in P. aeruginosa culture supernatants, bacterial cells were removed from cultures by centrifugation for 5 min at 7,500 x g, and the supernatant was filtered through a 0.45-µm-pore-size syringe filter prior to HPLC analysis. Nitrite concentrations in P. aeruginosa culture supernatants were determined using the Nitric Oxide Assay Kit (Calbiochem) according to the manufacturer's instructions.

RNA transcript analyses by reverse transcription (RT)-PCR. Total RNA was isolated from late-exponential-phase cultures (OD₆₀₀, 0.6) of P. aeruginosa PA14 grown in anaerobic BHI broth with 20 mM KNO₃ using an RNeasy miniprep kit (QIAGEN). DNA contamination was removed by digestion with RQ1 DNase (Promega). DNA contamination was monitored by PCR amplification of the P. aeruginosa rplU gene with the primers rplU-for (5'-CGCAGTGATTGTTACCGGTG-3') and rplU-rev (5'-AGGCCTGAATGCCGGTGATC-3'). RNA integrity was monitored by agarose gel electrophoresis. Synthesis of cDNA with the random primer (NS)₅ was performed with SuperScript II (Invitrogen) according to the manufacturer's instructions with 100 ng RNA template. A standard PCR was performed with the Expand Long Template kit (Roche) with 25 ng chromosomal DNA, 100 ng RNA, or 25 ng cDNA templates with primer sets overlapping putative cotranscribed coding regions (see Fig. 3A). For PCRs using primer set 8 (see Fig. 3A), 50 ng cDNA template was used.

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FIG. 3. The narG gene is cotranscribed with other genes implicated in anaerobic nitrate reduction. (A) Chromosomal organization of genes surrounding narG (http://www.pseudomonas.com). The numbered horizontal bars represent regions amplified by primer sets overlapping coding regions of putative cotranscribed genes (see Table S1 in the supplemental material). (B) RT-PCR analysis of narG transcript structure. P. aeruginosa PA14 was grown anaerobically and harvested for RNA isolation. RNA was used as a template for amplification of cDNA by random priming, and cDNA was subsequently used as a template for standard PCR with primer sets overlapping coding regions as shown in panel A. Appropriate positive (+; P. aeruginosa chromosomal DNA) and negative (–; RNA) controls were included. Product 8, spanning narK1 to narG, is presented separately due to the larger size of the amplicon.

Microarray analyses. P. aeruginosa was inoculated to an OD₆₀₀ of 0.001 in CF sputum medium with 10% (vol/vol) CF sputum as previously described (26). Briefly, lyophilized CF sputum was sterilized by UV treatment for 20 min, resuspended to a final concentration of 10% (vol/vol) in a MOPS-buffered base (50 mM MOPS [pH 7.2], 93 mM NH₄Cl, 43 mM NaCl, 3.7 mM KH₂PO₄, 1 mM MgSO₄, and 3.5 µM FeSO₄·7H₂O) and homogenized by sonication as described above. CF sputum medium was supplemented with 100 mM KNO₃ for aerobic and anaerobic microarray experiments. The microarray experiments were performed in duplicate for each condition. Cells were harvested for RNA isolation at an OD₆₀₀ of 0.1. RNA was isolated and monitored for integrity and DNA contamination as described above. Microarray analysis with Affymetrix P. aeruginosa GeneChips was performed as previously described (26).

Transcriptional regulation of the nar operon. A 688-bp region upstream of narK1 was amplified from P. aeruginosa PA14 chromosomal DNA using the Expand Long Template kit (Roche) with the primers nar prom-for (5'-GGGGTACCGGCTAAACTCTCTGCACGGAC-3') and nar prom-rev (5'-GAAGATCTGCACCGTGCTGAGCAGTTGCG-3') (underlining indicates KpnI and BglII sites, respectively). The resulting fragment was digested with KpnI/BglII and cloned upstream of a promoterless lacZ gene on the reporter plasmid pQF50 (10) to create pKP301. Clones were verified by restriction enzyme digestion and DNA sequencing at the Laboratory for Genomics and Bioinformatics at the University of Oklahoma Health Sciences Center.

To monitor aerobic expression of narG, P. aeruginosa PA14 carrying pQF50 or pKP301 was inoculated to an OD₆₀₀ of 0.01 in 10 ml BHI broth with or without added nitrate (400 µM or 20 mM KNO₃) in a 125-ml Erlenmeyer flask. ß-Galactosidase activity was monitored during exponential (OD₆₀₀, 0.2 and 0.6) and early stationary (OD₆₀₀, 1.6) phases. To monitor anaerobic expression of narG, P. aeruginosa PA14 carrying pQF50 or pKP301 was inoculated to an OD₆₀₀ of 0.01 in 75 ml BHI broth in a 1-liter flask and incubated with shaking at 37°C. At an OD₆₀₀ of 0.2, 3 ml cells was injected into prewarmed anaerobic Balch tubes containing KNO₃ or water so that the final added KNO₃ concentration in cultures was 0 µM, 400 µM, or 20 mM. After 4 h of incubation, cells were harvested for analysis of ß-galactosidase activity as previously described (24).

Microarray data accession number. The microarray data have been deposited in the EMBL-EBI data bank (www.ebi.ac.uk/miamexpress) under experiment accession number E-MEXP-1051.

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P. aeruginosa induces genes encoding two putative anaerobic nitrate reductases during anaerobic growth in an in vitro CF sputum medium. CF sputum serves as an in vivo growth substrate for P. aeruginosa in the CF lung (19, 25), and we recently developed a method for utilizing CF lung sputum as an in vitro growth substrate for P. aeruginosa (26). Transcriptome analyses of CF sputum-grown P. aeruginosa indicated that amino acids are the likely in vivo aerobic growth substrates (26). Since anaerobiosis commonly influences how bacteria acquire necessary carbon and energy, it is important to understand how anaerobic growth in CF sputum might impact P. aeruginosa metabolism. To determine the impact of anaerobic conditions on P. aeruginosa gene expression during growth in CF sputum, we conducted GeneChip microarray analyses of P. aeruginosa grown aerobically and anaerobically in CF sputum medium amended with 100 mM nitrate. This nitrate concentration was chosen to provide P. aeruginosa with ample electron acceptor for growth. Approximately 3% of the genes within the P. aeruginosa genome (167 genes) were differentially regulated over 10-fold when aerobic and anaerobic CF sputum medium-grown bacteria were compared (see the supplemental material). Results from these analyses suggest that, similar to aerobic growth, amino acids likely serve as the carbon substrates for anaerobic growth in CF sputum (see the supplemental material), since expression of previously reported amino acid catabolism genes (26) was unchanged.

Among the genes differentially regulated during anaerobic growth in CF sputum medium were those encoding factors required for anaerobic respiration with nitrate, anaerobic arginine deimination, and pyruvate fermentation. These genes were highly up-regulated during anaerobic growth in CF sputum medium (Table 1). Interestingly, genes encoding two predicted nitrate reductases, one associated with the cytoplasmic membrane (narGHJI) and one located in the periplasm (napAB), were induced during anaerobic growth in CF sputum. These results contrast with a previous transcriptome study of P. aeruginosa PAO1 grown in common laboratory medium, in which the periplasmic nitrate reductase and genes important for nitrate transport (narK2) (31) were down-regulated during anaerobic growth with nitrate, while no differential regulation of the membrane-bound nitrate reductase was observed (12).

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TABLE 1. Anaerobic metabolism genes differentially regulated during anaerobic growth in CF sputum

Genes encoding respiratory enzymes that utilize oxygen as the terminal electron acceptor were also differentially regulated during anaerobic growth in CF sputum medium. Transcript levels for genes encoding the oxygen-scavenging cbb₃-2 cytochrome oxidase (PA1555 to -1557) (data not shown), which are positively regulated by the anaerobic transcriptional regulator Anr, were increased four- to eightfold (7). Conversely, expression of genes encoding homologues of cytochrome o ubiquinol oxidase (PA1317 to -1321), which is important for aerobic respiratory energy generation in E. coli under highly aerated conditions (6), were decreased approximately threefold (data not shown).

The narG gene is required for anaerobic growth with multiple nitrate concentrations. Similar to P. aeruginosa, E. coli possesses both cytoplasmic membrane-bound and periplasmic nitrate reductases. In E. coli, nitrate levels dictate the transcription of genes encoding these two nitrate reductases. Genes encoding the periplasmic nitrate reductase are maximally transcribed anaerobically in the presence of micromolar nitrate concentrations, while genes encoding the membrane-bound nitrate reductase are maximally transcribed anaerobically with millimolar nitrate levels (36). Based on these findings, we reasoned that the levels of nitrate in CF sputum are critical to elucidating the roles of P. aeruginosa nitrate reductases in the proposed anaerobic niches of the CF lung. To examine this possibility, we used anion-exchange HPLC to measure nitrate levels within sputum samples collected from multiple CF patients (Table 2). These studies indicate that CF sputum nitrate concentrations fall within an approximate 10-fold range (73 to 792 µM), with an average concentration of 348 µM. Based on these data, we chose 400 µM nitrate as a physiologically relevant nitrate concentration for further experiments. The concentrations of nitrate observed in this study were within a fivefold range of previous studies examining nitrate levels in CF sputum; however, it should be noted that our results were obtained by direct measurement of nitrate in CF sputum, while previous investigations measured nitrate levels by first enzymatically converting nitrate to nitrite (16, 22).

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TABLE 2. Nitrate levels in CF sputum

Based on E. coli physiology and the observation that micromolar levels of nitrate are present in CF sputum, we hypothesized that the P. aeruginosa periplasmic nitrate reductase (NapAB) would be required for anaerobic growth in CF sputum. Conversely, our hypothesis predicted that the membrane-bound nitrate reductase complex (NarGJHI) would be required for anaerobic growth of P. aeruginosa with nitrate concentrations not relevant to CF disease. To test this hypothesis, we compared anaerobic growth of isogenic P. aeruginosa napA and narG mutants to wild-type P. aeruginosa in the presence of various nitrate concentrations (Fig. 1A). A linear relationship was observed between the wild-type P. aeruginosa growth yield and the concentration of nitrate provided. Contrary to our hypothesis, the napA mutant demonstrated no anaerobic growth defect while the narG mutant demonstrated a severe anaerobic growth defect at all nitrate concentrations, including concentrations normally found in CF sputum (Fig. 1A). We confirmed that the transposon insertion in narG was the basis for this growth defect by complementation analysis. Expression of the narGHJI genes, encoding the entire membrane-bound nitrate reductase, in trans restored growth of the narG mutant to near wild-type levels, while the presence of the complementation vector alone had no impact on growth (Fig. 1A). Complementation analysis was performed with the entire membrane-bound nitrate reductase coding region, since the transposon insertion in the narG mutant exerts polar effects on narHJI. Similar anaerobic growth results were obtained when wild-type P. aeruginosa strain PAO1 was compared to the PAO1 narG mutant, indicating that this growth defect is not specific to strain PA14 (data not shown).

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FIG. 1. P. aeruginosa requires the narG gene for anaerobic growth with nitrate. P. aeruginosa PA14, the PA14 isogenic napA and narG mutants, and the PA14 narG mutant harboring pUCP18 or pUCP18 expressing narGHJI in trans (pKP201) were grown anaerobically in BHI broth in the presence of various nitrate concentrations. (A) Growth at 24 h postinoculation as measured by OD600. Error bars representing standard deviations from the mean are too small to be seen. (B) Nitrate levels immediately after inoculation (t0) and 24 h postinoculation as measured by anion-exchange chromatography in anaerobic P. aeruginosa cultures supplemented with 400 µM nitrate. Uninoculated controls were included to evaluate nonbiological reduction of nitrate. The error bars represent standard deviations from the mean.

Since nitrate serves as the terminal electron acceptor in these experiments, nitrate loss can be used as a marker for anaerobic nitrate reduction. Based on the previous growth yield experiments, we predicted that the narG mutant would not reduce nitrate while the wild-type and the napA mutant would exhibit similar nitrate reduction profiles. Measurement of nitrate concentrations in cell supernatants of wild-type P. aeruginosa, the napA mutant, and the narG mutant grown anaerobically with 400 µM nitrate indicated that after 24 h of growth, wild-type P. aeruginosa and the napA mutant reduced virtually all the nitrate present (Fig. 1B). As expected, the narG mutant was deficient for anaerobic nitrate reduction, and expression of narGHJI in trans restored this ability. Collectively, these data indicate that P. aeruginosa requires the membrane-bound nitrate reductase for anaerobic growth and nitrate reduction at nitrate concentrations relevant to the CF lung.

The narG mutant does not have a general anaerobic growth defect. Denitrification is the progressive reduction of nitrate to dinitrogen, and nitrate reduction to nitrite is the first step (42). Our data indicate that the P. aeruginosa narG gene is required to carry out this initial reaction. P. aeruginosa can also use the denitrification intermediates nitrite and nitrous oxide as anaerobic electron acceptors (39). Previous studies of NarG in Pseudomonas fluorescens revealed that genetic inactivation of narG also resulted in an inability to reduce nitrate, but it also negatively impacted nitrite reduction (14). Based on these studies, it was possible that inactivation of narG in P. aeruginosa resulted in a generalized anaerobic growth defect not specific to nitrate respiration. To determine if the growth defect of the narG mutant was specific to nitrate, we tested the ability of this mutant to grow anaerobically using 1 mM nitrite as the terminal electron acceptor. The results from these experiments indicate that the narG mutant reaches similar cell densities (averages, 6.6 x 10⁷ CFU/ml for wild-type and 5.0 x 10⁷ CFU/ml for the narG mutant) and reduces nitrite to levels (<10 µM) similar to those for wild-type P. aeruginosa. These data indicate that the narG mutant does not possess a general anaerobic growth defect but instead has a nitrate-specific defect.

Nitrate levels in CF sputum support significant anaerobic P. aeruginosa growth, and the narG gene is required for this growth. Previous work in our laboratory indicated that aerobic growth in CF sputum significantly impacts the physiology of P. aeruginosa (26). Having established that narG was required for anaerobic growth of P. aeruginosa in a common laboratory medium using physiologically relevant concentrations of nitrate, we sought to determine whether growth in CF sputum would influence the anaerobic requirement for narG. For these experiments, wild-type P. aeruginosa and the narG mutant were grown in an in vitro CF sputum medium containing nitrate at levels (400 µM) commonly found in CF sputum. Viable-cell counts were then performed to examine the importance of narG for anaerobic growth in CF sputum. The results from these experiments indicated that wild-type P. aeruginosa grows well anaerobically in CF sputum medium, exhibiting an 3.5-log-unit increase in cell numbers (from 1 x 10³ to 5 x 10⁶ bacteria/ml); however, inactivation of narG eliminated the ability of P. aeruginosa to grow anaerobically in CF sputum (Fig. 2). Collectively, these data indicate that the concentration of nitrate commonly found in CF sputum is sufficient to support significant growth of P. aeruginosa and that the membrane-bound nitrate reductase is required for P. aeruginosa anaerobic growth in CF sputum medium.

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FIG. 2. The narG gene is required for anaerobic growth in CF sputum. P. aeruginosa PA14 and the isogenic narG mutant were grown anaerobically in 25% CF sputum medium containing 400 µM nitrate. Viable-bacterial-cell counts immediately after inoculation (t0) and after 24 h of anaerobic growth are shown. The error bars represent standard deviations from the mean. wt, wild type.

The narG gene is cotranscribed with other genes implicated in anaerobic nitrate reduction. A recent study reported that antibodies to P. aeruginosa NarG are present in sera from CF patients (2), suggesting that narG is expressed by P. aeruginosa in vivo. These data, along with our data demonstrating that narG is required for anaerobic growth in CF sputum medium, underscore the need to identify conditions under which narG is transcribed. Such studies will provide important information regarding the microenvironments of the CF lung where the membrane-bound nitrate reductase may be produced. To examine narG transcription, it was important to first identify the genomic context of the gene. The narG gene is the third gene in a putative operon implicated in anaerobic nitrate reduction (31). Aside from narG, this potential operon includes two homologues of the nitrate/nitrite antiporter gene narK, the molybdopterin cofactor synthesis genes PA3871 (nifM) and moaA1, and narHJI, which encode other components of the membrane-bound nitrate reductase (Fig. 3A) (http://www.pseudomonas.com). In silico analysis supports cotranscription of these eight genes, as the start codon and the stop codon of adjacent genes are localized within 75 bp of each other. To experimentally examine the nar operon structure, RT-PCR was used to examine the narG transcript structure. The results from these experiments demonstrate that a transcript containing narK1, narK2, narG, and likely the five other downstream genes exists (Fig. 3B and C). These results also suggest that the promoter controlling nar transcription lies upstream of narK1.

Expression of narG is induced during anaerobic growth with physiologically relevant nitrate levels. Is nar operon expression induced by anaerobiosis and/or by physiologically relevant nitrate concentrations? To address this question, a 688-bp region upstream of narK1 was amplified and cloned into the ß-galactosidase reporter plasmid pQF50 to generate a nar::lacZ transcriptional fusion. P. aeruginosa carrying nar::lacZ was then used to monitor induction of the nar genes under aerobic and anaerobic conditions with or without added nitrate. Our results indicate that anaerobic growth induces transcription of the nar operon approximately 10-fold, and this induction is enhanced further anaerobically with the addition of nitrate (Fig. 4A). More importantly, this enhanced induction with nitrate was observed using 400 µM nitrate, indicating that nar expression is responsive to nitrate levels commonly found in the CF lung.

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FIG. 4. (A) Expression of narG is induced during anaerobic growth with 400 µM nitrate. The DNA region upstream of narK1 was cloned upstream of a promoterless lacZ gene on pQF50 to generate nar::lacZ. P. aeruginosa PA14 harboring pQF50 or the nar::lacZ fusion was grown aerobically and anaerobically in BHI broth with 0 µM, 400 µM, or 20 mM added nitrate. Assays for ß-galactosidase (24) were performed for anaerobic and aerobic bacteria during exponential growth. The error bars represent standard deviations of the mean. Average background ß-galactosidase activity (33 activity units) mediated by promoterless pQF50 was not significantly altered by the presence or absence of nitrate under aerobic or anaerobic conditions, with the exception of an 2-fold increase in activity in anaerobic cultures in the presence of 20 mM nitrate. (B) In silico analysis of the nar operon promoter reveals nitrate- and oxygen-responsive operators. The DNA sequence upstream of narK1 possesses consensus binding sites for FNR-type transcriptional regulators (TTGAT ... . ATCAA, underlined) and the nitrate-responsive transcriptional regulator NarL (TACYNKT, boxed). The predicted translational start codons of narK1 (ATG) and the divergently transcribed narX (CAT) are shown.

In other bacteria, transcriptional regulation in response to oxygen levels and nitrate is attributed to FNR-type proteins and NarL, respectively (33, 35). Previous studies have shown that two FNR-type regulatory proteins, Anr and Dnr, are required for P. aeruginosa denitrification (1, 41). As expected from the nar promoter results described above, in silico examination of the region upstream of narK1 revealed the presence of consensus binding sites for FNR-type proteins (1) and NarL (33) (Fig. 4B). The narXL genes are divergently transcribed from narK1, suggesting that the putative FNR and NarL consensus binding sites in their intergenic region may serve to regulate either or both sets of genes. As predicted by the operon structure, no consensus binding sites for FNR-type proteins or NarL were found in the intergenic regions within the nar operon (Fig. 4A and data not shown), suggesting that the promoter upstream of narK1 likely regulates nar operon expression.

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This study provides evidence that the membrane-bound nitrate reductase is required for P. aeruginosa anaerobic growth in an in vitro CF sputum medium. Genes encoding this enzyme complex are induced under anaerobic conditions in the presence of nitrate concentrations commonly found in CF sputum (16, 22). Based on these results, we anticipate that P. aeruginosa will express narG in the CF lung when exposed to anaerobic microenvironments. These data also suggest that P. aeruginosa requires the membrane-bound nitrate reductase to generate energy in vivo under anaerobic conditions when nitrate is available. A recent study aimed at identifying transposon mutants in P. aeruginosa PAO1 unable to grow anaerobically with nitrate did not identify the membrane-bound anaerobic nitrate reductase as essential (11). The reason for this apparent inconsistency is not strain differences, as P. aeruginosa PAO1 also requires narG for anaerobic growth with nitrate (data not shown); however, since the previous study did not perform a saturating mutagenesis, it is possible that mutants in the nar operon were not examined.

Given that previous studies have found considerably lower levels of nitrite than of nitrate in CF sputum (16, 22), nitrate would likely be the electron acceptor used for anaerobic respiratory growth of P. aeruginosa in vivo. This nitrate is presumably derived from the spontaneous oxidation of reactive nitrogen species produced by components of the innate immune system (8). Since nitrate is present in CF sputum, it could be argued that P. aeruginosa does not utilize endogenous nitrate as a terminal electron acceptor in the CF lung. It should be noted that the samples used in this study contained low levels of P. aeruginosa (<10⁸ cells/ml sputum) and sufficient nutrients to allow substantial bacterial growth. It should also be noted that the CF lung is not a batch growth system (like that used in these studies) but more analogous to a chemostat or fed-batch system. Under these growth conditions, sputum nutritional components may be continually replenished by endogenous nitrate production. In any case, our results indicate that a P. aeruginosa protein produced in individuals with CF is required for anaerobic growth using the preferred anaerobic electron acceptor (nitrate) present in the CF lung. This study also provides the first experimental evidence that CF sputum nitrate levels are sufficient to support significant anaerobic growth of P. aeruginosa.

Antibodies to NapA were also detected in sera from CF patients (2), indicating that the periplasmic nitrate reductase is also produced by P. aeruginosa in vivo. The results of our study indicate that the periplasmic nitrate reductase is not required for anaerobic energy generation, since no growth defect or nitrate reduction defect is observed in a napA mutant. In other microorganisms, the periplasmic nitrate reductase fills a variety of metabolic roles (27). Some bacteria possess and use only the periplasmic nitrate reductase for anaerobic growth with nitrate (3, 4). As mentioned above, E. coli possesses both membrane-bound and periplasmic enzymes but utilizes the periplasmic enzymes only at low substrate concentrations (32, 36). Alternatively, some microorganisms use the periplasmic nitrate reductase to balance cellular oxidation-reduction processes when the concentration of cellular reducing equivalents is high and oxygen concentrations are low (13, 27). It is possible that P. aeruginosa utilizes the periplasmic nitrate reductase for this purpose.

While P. aeruginosa prefers respiratory energy production in anaerobic environments where nitrate is available, this study does not preclude the potential role of arginine deimination and pyruvate fermentation in the CF lung. It is possible that P. aeruginosa could encounter anaerobic low-nitrate environments in vivo. However, the inability of pyruvate fermentation to support P. aeruginosa anaerobic growth (9) and the observation that an arginine deimination mutant shows no anaerobic growth deficiency in CF sputum (data not shown) suggest that these pathways would be primarily used for acquiring cell maintenance energy or balancing oxidation-reduction processes and not bacterial growth in vivo.

P. aeruginosa possesses five putative or confirmed terminal oxidases that could potentially utilize oxygen for respiratory growth (7). Given the apparent respiratory flexibility of P. aeruginosa during aerobic growth, it is surprising that one physiological component is required for anaerobic growth with nitrate in an in vitro CF sputum medium. These results are encouraging and suggest that the membrane-bound nitrate reductase is a potential candidate for the targeted development of therapies specific for P. aeruginosa growing anaerobically. Anaerobic respiration as a drug target has been proposed for other bacteria, including Mycobacterium bovis (37). M. bovis requires the membrane-bound nitrate reductase for chronic lung infection, thus emphasizing the potential importance of this enzyme complex in persistent infections. Since clinically relevant phenotypes, such as mucoidy and biofilm formation, are influenced by the respiratory and metabolic states of P. aeruginosa, investigations such as this examining P. aeruginosa physiology during growth with in vivo substrates and various oxygen concentrations are critical to the development of effective treatment strategies.

 
We thank Pradeep Singh for providing CF sputum samples.

This work was supported by grants from the NIH (1P20RR15564-01 to M.W.), the Oklahoma Center for the Advancement of Science and Technology (HR03-137S to M.W.), and the Cystic Fibrosis Foundation (WHITEL06G0 to M.W.).

 
* Corresponding author. Mailing address: Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, TX 78712. Phone: (512) 471-5493. Fax: (512) 471-7088. E-mail: mwhiteley{at}mail.utexas.edu

Published ahead of print on 30 March 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, June 2007, p. 4449-4455, Vol. 189, No. 12
0021-9193/07/$08.00+0     doi:10.1128/JB.00162-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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