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Nitrate serves as a terminal electron acceptor under anaerobic conditions in Pseudomonas aeruginosa. Reduction of nitrate to nitrite generates a transmembrane proton motive force allowing ATP synthesis and anaerobic growth. Inner membrane-bound nitrate reductase NarGHI is encoded within the narK1K2GHJI operon and the periplasmic nitrate reductase NapAB is encoded within the napEFDABC operon. The role of the two dissimilatory nitrate reductases in anaerobic growth, and the regulation of their expression were examined by using a set of deletion mutants in P. aeruginosa PAO1. NarGHI mutants were unable to grow anaerobically, but plate cultures remained viable up to 120 hr. In contrast, nitrate sensor-response regulator mutant ΔnarXL displayed growth arrest initially, but resumed growth after 72 hr and reached early stationary phase in liquid culture after 120 hr.
Genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the NarXL mutant was the result of NapAB periplasmic nitrate reductase expression. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild type P. aeruginosa PAO1, nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase.
Pseudomonas aeruginosa growth in biofilms is characterized by its ability to carry out respiration in oxygen-limited environments. Important for anaerobic respiration is the dissimilatory enzyme pathway that catalyzes the reduction of nitrate (NO3−) to nitrogen gas (N2). Reduction of either nitrate or nitrite provides energy for P. aeruginosa growth in the absence of oxygen, with nitrate reduction to nitrite via nitrate reductase contributing more significantly to proton motive force and, hence, energy production, than nitrite reduction or arginine fermentation (Berks et al., 1995; Zumft, 1997).
Respiratory nitrate reduction to nitrite can be mediated by two different nitrate reductase complexes in P. aeruginosa (Berks et al., 1995; Zumft, 1997): an inner membrane-bound nitrate reductase complex encoded by the narK1K2GHJI operon and a periplasmic nitrate reductase encoded by the napEFDABC operon. NarGHI forms the core membrane-bound nitrate reductase. The γ-subunit NarI, anchored in the inner membrane, accepts electrons from the quinol pool and transfers them via β-hemes to the β-subunit NarG. The α- and β-subunits NarGH extend into the cytoplasm and associate with a molybdopterin cofactor (Philippot & Hojberg, 1999) and the Snr-1 protein (Kerschen et al., 2001). NarJ is a chaperone involved in NarGHI assembly (Liu & DeMoss, 1997), and NarK1K2 are thought to be involved in nitrate and nitrite transport (Goddard et al., 2008; Sharma et al., 2006; Wood et al., 2002). NapAB comprises the periplasmic nitrate reductase, with NapC, a cytochrome c-type protein, likely involved in electron transfer to NapAB. NapD is essential for NapAB activity and is postulated to be a chaperone involved in NapA maturation prior to export into the periplasm (Potter, 2001). NapF appears to be a non-heme, iron-sulfur protein (Nilavongse et al., 2006; Olmo-Mira et al., 2004) implicated in an energy-conserving role coupled to the oxidation of ubiquinol (Brondijk et al., 2002), and may also play a role in the post-translational maturation of NapA (Nilavongse et al., 2006; Olmo-Mira et al., 2004). The physiological role of NapE has not yet been defined.
Adjacent to the 5’ - region of the narK1K2GHJI operon, and divergently transcribed in P. aeruginosa, is the narXL operon encoding the two-component nitrate sensor-response regulator proteins, NarX and NarL (Hartig et al., 1999; Stewart et al., 1989). NarXL is a classic two-component bacterial regulatory system in which a histidyl-aspartyl phosphorelay controls gene expression in response to specific environmental stimuli (Varughese, 2002). The periplasmic domain of the sensor protein, NarX, contains a highly conserved nitrate recognition region termed the P-box. Nitrate recognition by the P-box results in autophosphorylation of a conserved histidyl residue in the NarX cytoplasmic transmitter domain. Transfer of phosphate to the conserved aspartyl residue in the receiver domain of the response regulator NarL results in transcriptional regulation of target operons (Stewart, 2003). NarL, in conjunction with oxygen regulator Anr and N-oxide regulator Dnr, induce narK1K2GHJI operon expression in P. aeruginosa (Schreiber et al., 2007). The regulation of napEFDABC expression in P. aeruginosa is less well understood.
Using a set of P. aeruginosa deletion mutants, we corroborate previous studies (Schreiber et al., 2007) that NarXL activates the narK1K2GHJI operon, and we further show that a deletion in narGH resulted in anaerobic growth arrest. In contrast, a deletion in narXL resulted in subsequent growth recovery, which was correlated with the expression of the napEFDABC operon. A novel transcriptional start site for napEFDABC expression was identified in the NarXL mutant grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding site monomer upstream of the novel transcriptional start site also restored anaerobic growth recovery in the NarXL mutant. The data suggest that during anaerobic growth of wild type P. aeruginosa PAO1, nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase, while inducing maximal expression of membrane nitrate reductase.
P. aeruginosa PAO1 was used throughout this study. Deletion mutants were generated in P. aeruginosa PAO1 as described in (Van Alst et al., 2007), using techniques described by Schweizer and colleagues (Hoang et al., 1998). All strains, corresponding mutants and plasmids used in this study are listed in Supplemental Table 1.
Strains were stored at −80°C in 10% skim milk. Aliquots were plated onto either PTSB (50 g/L peptone, 2.5 g/L tryptic soy broth) agar plates or LB (5 g/L yeast extract, 10 g/L NaCl, and 10 g/L tryptone). Growth studies were performed using either reduced LB or reduced NY (25 g/L nutrient broth and 5 g/L yeast extract) (Difco, Detroit, MI) broth supplemented with 100 mM KNO3, 10 mM KNO2 (VWR International, West Chester, PA) or 40 mM arginine (Sigma-Aldrich, St. Louis, MO) as indicated. Reduced liquid medium was prepared by boiling the water for 10 minutes, bubbling N2 gas through the prepared broth for 10 minutes, and sterilization by autoclaving. All antibiotics were obtained from Sigma-Aldrich. Carbenicillin was used at 400 µg/ml.
Aerobic overnight cultures were used to inoculate 200 ml of medium in a 500 ml baffled Erlenmeyer flask to an initial optical density at 660 nm (OD660) of 0.05. Cultures were incubated in a shaking water bath at 250 rpm at 37°C. Anaerobic growth studies were performed in a sealed tent with a gas mix of 85% (v/v) nitrogen, 10% (v/v) hydrogen and 5% (v/v) carbon dioxide. Aerobic overnight cultures were used to inoculate 200 ml of reduced medium in 250 ml bottles at OD660 of 0.05. Anaerobic cultures were grown at 37°C with mixing using a magnetic stir plate. For all growth studies, no more than 10% (v/v) of the starting culture volume was removed over the course of the experiment to monitor growth as measured by OD660 (Wagner et al., 2003).
Preliminary studies demonstrated that wild type P. aeruginosa PAO1 required at least 50 mM nitrate for optimal anaerobic growth, and 100 mM nitrate did not appear to exert a toxic effect during aerobic growth. Identical results were observed using NY medium instead of LB (data not shown), suggesting medium components in LB and NY other than nitrate did not influence anaerobic growth in these strains of P. aeruginosa. For all subsequent experiments, media were supplemented with 100 mM nitrate.
Plasmid and cosmid DNA was isolated using the QIAprep spin kit (QIAGEN, Valencia, CA). Restriction endonucleases, T4 DNA polymerase, calf intestinal phosphatase, and T4 DNA ligase were used as specified by the supplier (New England Biolabs, Beverley, MA). DNA fragments were isolated from agarose gels using Freeze and Squeeze Kit (Bio-Rad, Hercules, CA). PCR was performed using the iProof polymerase kit (Bio-Rad) in an ABI model 2700 thermal cycler (Foster City, CA). Oligonucleotide primers (Sigma Genosys) used in this study are listed in Supplemental Table 2.
Complementation of PAO1 nar and nap mutants was performed in single copy by integration into the chromosome. Integration was performed as described previously (Choi et al., 2005) by mini-Tn7 insertion at the att Tn7 site located downstream of glmS using mini-Tn7 elements that carry the respective chromosomal DNA fragments. Complementing genes were expressed from their native promoters.
Both the intact narXL and ΔnarXL constructs contain narK1 and narK2, as well as the first 49 nucleotides of narG, with the putative narL box upstream of narK1. Transcriptional fusions Φ(narXL-narG-lacZ):pLP170 and Φ(ΔnarXL-narG-lacZ):pLP170 were created by subcloning into the EcoRI site of plasmid pLP170 upstream of a promoterless lacZ (Preston et al., 1997).
A napD transcriptional fusion was created using primers napoperon 1 (sense) and napoperon 2 (antisense) which amplified region upnapE containing oprH5’, napE, napF, and napD5’ (Supplemental Figure 4A). upnapE was subcloned into pLP170 to generate Φ(napD-lacZ):pLP170. The construct was electroporated into either the wild type PAO1, PAO1:ΔnarXL, and PAO1:ΔnarGH backgrounds to evaluate nap operon expression via β-galactosidase activity. As a negative control, pLP170 alone was also electroporated into all three strains.
β-galactosidase assays (Preston et al., 1997) were performed on organisms grown under either anaerobic or aerobic conditions over a 120 hr period. All solutions were freshly prepared on the day of the assay. Activity is expressed in arbitrary (Miller) units based on the formula 100 × [A420/(T × V × OD660)] where T = time in minutes, V = volume of reaction in ml, and OD660 corresponds to the optical density of the culture used (Miller, 1977). Assays were performed in triplicate in two separate experiments.
200 ml cultures were grown anaerobically in NY supplemented with 100 mM KNO3. Both PAO1 and ΔnarXL were harvested at midlog phase (OD660=0.6) at 8 and 96 hr, respectively; ΔnarGH was harvested at 96 hr (OD660=0.12). Total RNA was isolated using RNA Whiz (Ambion; Austin, TX) per manufacturer’s instructions. Contaminating genomic DNA was removed using Turbo DNA-free kit (Ambion) per the manufacturer’s protocol.
RT-PCR was performed using Ambion RETROscript along with Ambion SuperTaq Plus. Removal of contaminating DNA was confirmed by PCR using GroEL oligonucleotides, GroEL F1 and GroEL R1, and omitting reverse transcriptase from the RT-PCR reaction. RNA integrity was confirmed by RT-PCR using GroEL primers, GroEL F1 and GroEL R1, which yield a 550 bp amplicon. Oligonucleotides used to detect narH transcripts were narH S nested 2 and narH AS nested 2, which yield a 390 bp amplicon. Oligonucleotides used to detect napA transcripts were napA RT-PCR S and napA RT-PCR AS, which yield a 677 bp amplicon.
For transcriptional mapping, 5’ RACE was performed as per manufacturer’s protocol (Invitrogen; Carlsbad, CA). Oligonucleotides used to identify the start of transcription upstream of napEFDABC were nap GSP-3, nap GSP-4, and nap GSP-5 which yielded two amplicons in PAO1:ΔnarXL (400 bp and 150 bp) and one in wild type PAO1(150 bp) grown under anaerobic conditions.
Wild type PAO1 and ΔnarXL were grown in nitrate supplemented NY medium for 150 hr. Samples were taken and cells were removed by filtration. Nitrite concentration was determined in the filtrate for each strain at each time point against a standard curve of known nitrite concentrations using the Measure – iT High Sensitivity Nitrite Assay Kit (Molecular Probes).
Nitrate reductase activity was determined in whole cell suspensions (MacGregor et al., 1974; Stewart et al., 2002). Cultures were grown anaerobically in NY supplemented with 100 mM KNO3. To inhibit protein synthesis, 1.5 ml of 50 µg/ml chloramphenicol was added to 1.5 ml of culture. To inhibit further nitrate reduction by the membrane nitrate reductase NarGHI, 1.5 µl of 50 mM NaN3 was added as indicated. Cells were centrifuged, washed twice, and re-suspended in an equal volume of 50 mM phosphate buffer, pH 7.2, and OD660 was determined. An 800 µl aliquot of cells was mixed with 100 µl freshly prepared, 0.5 mg/ml methyl viologen solution. Nitrate reduction was initiated by adding 100 µl of a solution containing 4 mg/ml sodium dithionite, 4 mg/ml sodium bicarbonate, and 100 mM KNO3. Control reactions replaced sodium dithionite with water. Reactions were incubated at room temperature for 5 min, and stopped by vortexing until the solution became clear, indicating the electron donor was oxidized. 1 ml of 1% w/v sulfanilic acid in 20% HCl was added immediately to the stopped reaction, and vortexed for 15 sec. 1 ml of 1.3 mg/ml N-(1-naphthyl) ethylenediamine-HCL was added to allow formation of red azo dye, and the suspension was centrifuged to pellet debris. A540 of the supernatant was measured spectrophotometrically to quantitate dye formation, and A420 was measured to account for absorbance due to light scattering by residual cells or cell fragments. Activity is expressed in arbitrary units based on the formula 100 × [A540 – (0.72 × A420)]/(T × V × OD660) (Stewart & Parales, 1988). T = time in minutes, V = volume of reaction used in ml, OD660 corresponds to the optical density of the culture used. Assays were performed in triplicate in three separate experiments.
The plasmid napEFDABC:pUCP18 was used to construct two independent, site-directed mutant NarL binding sites upstream of napEFDABC, narLBS1napEFDABC and narLBS2napEFDABC (Supplemental Table 1). Site directed mutagenesis of the two potential NarL binding sites were accomplished by SOE-PCR. narLBS1napEFDABC was generated by mutating the wild type sequence CAGGGTACTGAA, denoted as BS1 by the solid underline in Figure 7A, to CAGGGTGCATAA using the oligonucleotides napoperon#1, pnarL1 Sense, pnarL1 AS, and napoperon #11. narLBS2napEFDABC was generated by mutating the wild type sequence TACTACAGTA, denoted as BS2 by the dashed underline in Figure 7A, to CATCATAGTA using the oligonucleotides napoperon#1, pnarL2 Sense, pnarL2 AS, and napoperon#11.
Statistical analysis was performed using the paired Student’s t test to determine statistical significance between strains in the β-galactosidase assay and whole cell nitrate reduction assay. In all cases, P values of ≤ 0.05 were considered significant.
Deletion mutants in the nitrate reductase operon narK1K2GHJI and nitrate sensor-response regulator operon narXL in P. aeruginosa strain PAO1 were generated by allelic exchange (Hoang et al., 1998) to examine their growth under anaerobic conditions. These mutants included a deletion spanning the junctions of narG and narH (ΔnarGH), narJ and narI (ΔnarJI), a deletion encompassing the bulk of narXL (ΔnarXL) and a deletion in narL alone (ΔnarL). The positions of the respective deletions in the PAO1 chromosome of the mutants are listed in Supplemental Table 1, and deletions were confirmed by Southern analysis (data not shown).
Over 24 hr of aerobic culture, ΔnarXL and ΔnarGH grew comparably to the wild type PAO1 strain. In contrast, after 24 hr growth under anaerobic conditions, ΔnarXL and ΔnarGH displayed growth arrest compared to parental strain PAO1 (Figure 1). Genetic complementation of the two mutants with the appropriate genomic DNA fragments showed slightly delayed anaerobic growth compared to wild type PAO1, but all three strains reached similar densities after 24 hr. ΔnarGH was not an anaerobically lethal mutant. Replica plates of ΔnarGH cultures were incubated anaerobically up to 120 hr, with a plate removed daily and shifted to aerobic conditions. Following overnight aerobic incubation, 98% (61 out of 62 colonies) of the ΔnarGH colonies displayed growth recovery by increasing colony size after 120 hr incubation (data not shown). The ΔnarJI mutant displayed a comparable phenotype to ΔnarGH in all cases (data not shown), and was not studied further. A possible explanation for the persistence of the ΔnarGH mutant may be due to pyruvate fermentation as suggested in (Eschbach et al., 2004).
In contrast to ΔnarGH and ΔnarJI, ΔnarXL began to recover from growth arrest after approximately 72 hr, and reached the maximum growth levels observed for wild type after 120 hr (Figure 1). Little anaerobic growth was observed by either wild type PAO1 or ΔnarXL in NY medium alone or NY medium supplemented with 40 mM arginine (Supplemental Figure 1), illustrating the need for nitrate supplementation under these conditions. Arginine fermentation in NY medium alone did not support P. aeruginosa PAO1 growth under anaerobic conditions over 5 days. P. aeruginosa is an obligate respirer and, hence, does not have the ability to ferment pyruvate unless provided with a terminal electron donor such as nitrate. Pyruvate does not sustain P. aeruginosa anaerobic growth but permits anaerobic survival up to 18 days (Eschbach et al., 2004).
It has been suggested that the periplasmic nitrate reductase encoded by napAB is expressed only under aerobic and microaerobic conditions, and is thought to function in the transition from aerobic to anaerobic metabolism in denitrifying bacteria (Potter, 2001). However, recent work by Stewart, et al. demonstrated that napAB is expressed under anaerobic conditions in E. coli K-12 (Stewart et al., 2002). We hypothesized that if anaerobic growth recovery of ΔnarXL was due to expression of napAB, a deletion in napA in the ΔnarXL background would fail to grow anaerobically. Deletions in napA in both parental PAO1 (ΔnapA) and ΔnarXL (ΔnarXL:ΔnapA) were constructed, and the deletions confirmed by Southern analysis.
Strain ΔnapA grew similarly to wild type PAO1. In contrast, strain ΔnapA:ΔnarXL failed to demonstrate anaerobic growth recovery in NY supplemented with nitrate, with the double mutant displaying growth arrest similar to ΔnarGH (Figure 1). Strains ΔnarXL and ΔnarGH:ΔnarXL displayed full growth recovery after 120 hr (Figure 1). ΔnapA:ΔnarXL complemented in trans with narXLK1K2G5’:pUCP18 also grew similarly to wild type (Supplemental Figure 2). When complemented in trans with napEFDABC:pUCP18, ΔnapA:ΔnarXL reached a growth plateau after 48 hr under anaerobic conditions after a slight initial delay (Supplemental Figure 2). All strains grew comparably aerobically in NY supplemented with nitrate (data not shown). These data provide genetic evidence for the hypothesis that napAB expression is required for anaerobic growth recovery by ΔnarXL. Several approaches were taken to understand the basis of the recovery from growth arrest by ΔnarXL under anaerobic conditions.
To test whether the delayed growth recovery by ΔnarXL was due to a defect in nitrate reduction to nitrite, or within subsequent steps in the reduction of nitrogen oxide species by the denitrification pathway (NO3− →NO2−→ NO˙ → N2O → N2), we compared anaerobic growth of wild type PAO1 and ΔnarXL in NY liquid medium supplemented with either nitrate or nitrite. Wild type PAO1 and ΔnarXL were grown in NY supplemented with nitrate under anaerobic conditions for either 4 hr or 24 hr. Cultures were maintained anaerobically, and diluted to O.D.660 of ~0.05 into fresh anaerobic NY supplemented with either 100 mM KNO3 or 10 mM KNO2. After 4 hr of anaerobic adaptation, PAO1 reached early stationary phase (O.D.660 = 1.4) within 24 hr when subcultured back into NY supplemented with 100 mM KNO3 (Figure 2A). PAO1 subcultured in NY supplemented with 10 mM KNO2, as well as ΔnarXL subcultured into either medium, reached an O.D.660 of ~0.25 after 48 hr. In contrast, after 24 hr of adaptation to anaerobic conditions (when subcultured into either medium) ΔnarXL reached a growth plateau comparable to wild type PAO1 after 48 hr, compared to 96 hr for cells not pre-adapted to anaerobic growth on nitrate. The growth of ΔnarXL was slightly delayed compared to wild type PAO1 grown under nitrate supplementation, but comparable to wild type grown under nitrite supplementation (Figure 2B). These data suggest that wild type PAO1 becomes induced to grow on nitrite between 4 and 24 hr. ΔnarXL displays a growth profile comparable to wild type PAO1 sub-cultured in medium supplemented with 10 mM KNO2 after 24 hr of anaerobic adaptation, suggesting that the remainder of the denitrification pathway downstream of nitrate reduction was still functional in ΔnarXL. Hence, the growth delay seen initially in ΔnarXL grown in nitrate (Figure 1) was likely due to differences in the conversion of nitrate to nitrite.
Next, we examined whether the conversion of nitrate to nitrite was different in wild type PAO1 compared to ΔnarXL by quantitation of the nitrite concentration in culture supernatant when the strains were grown under anaerobic conditions. Wild type PAO1 and ΔnarXL were grown in nitrate supplemented NY medium for 150 hr. Samples were taken and cells were removed by filtration. The nitrite concentration in the supernatant was determined for each strain at each time point. Wild type PAO1 nitrite concentration peaked after 6 hours of incubation under anaerobic conditions (335 µM) (Supplemental Figure 3A) coinciding with wild type PAO1 mid-log growth. In contrast to wild type PAO1, nitrite started accumulating in ΔnarXL around 72 hours (64 µM) and peaked at 111 hr (87 µM) coinciding with the beginning of the delayed growth recovery phenomenon exhibited by ΔnarXL. The area under the wild type growth curve was similar to the area under the ΔnarXL growth curve. The corresponding anaerobic growth curve for the nitrite concentration assay is shown in Supplemental Figure 3B. These data suggest that the delayed growth recovery by ΔnarXL is due to a slow conversion of nitrate to nitrite, and that a minimum amount of nitrite accumulation is required to activate the nitrite reductase and, hence, the rest of the denitrification pathway.
Two approaches were taken to determine whether anaerobic growth recovery of ΔnarXL and ΔnarL was due to the induction of the narK1K2GHJI operon by an alternative transcriptional regulator or whether it was due to an alternative energy metabolism pathway exclusive of narK1K2GHJI.
First, transcriptional fusions were generated in which the first fifty base pairs of narG coding sequence were fused to lacZ. Two constructs were made, one containing an intact narXL locus, Φ(narXL-narG-lacZ), and the other containing a narXL deletion, Φ(ΔnarXL-narG-lacZ) (Figure 3A). Each construct was cloned into pLP170 (Preston et al., 1997) and used to transform wild type PAO1 and ΔnarXL, respectively. Wild type PAO1 and ΔnarXL were also transformed with vector pLP170 alone to serve as negative controls. Because the narXL locus is intact in Φ(narXL-narG-lacZ), we were able to use this construct as both a plasmid-based reporter fusion for narK1K2GHJI expression, and to complement ΔnarXL. We hypothesized that if narK1K2GHJI expression was induced by an alternative transcriptional regulator, β-galactosidase levels would gradually increase over time in ΔnarXL:Φ(ΔnarXL-narG-lacZ). However, β-galactosidase levels in ΔnarXL:Φ(ΔnarXL-narG-lacZ) were only 19% of wild type (p ≤ 0.01) after 24 hr (Figure 3B), and we did not see an increase in β-galactosidase levels in this strain through 120 hr. A possible explanation why β-galactosidase expression in ΔnarXL:Φ(ΔnarXL-narG-lacZ) was 19% of wild type may be due to either gratuitous transcription or that Anr is responding to anaerobiosis. Strains provided with intact narXL in trans demonstrated high levels of β-galactosidase activity after 24 hr, confirming the function of the reporter fusion (Figure 3B).
Second, if narK1K2GHJI expression in ΔnarXL was regulated by a transcriptional activator other than NarL, we predicted that a ΔnarXL:ΔnarGH double mutant would abate anaerobic growth recovery. However, this double mutant retained anaerobic growth recovery after 72 hr similar to ΔnarXL (Figure 1). The studies using Φ(ΔnarXL-narG-lacZ) in either a wild type or ΔnarXL background demonstrated that NarXL activated narK1K2GHJI expression (Figure 3B). Diminished β-galactosidase expression in ΔnarXL:Φ(ΔnarXL-narG-lacZ), and the growth recovery by ΔnarXL:ΔnarGH, suggests growth recovery by ΔnarXL and ΔnarL seen in Figure 1 is due to an alternative anaerobic respiratory pathway exclusive of the membrane nitrate reductase NarGHI. Therefore, we hypothesized that the expression of the NapAB periplasmic nitrate reductase could be responsible for growth recovery in ΔnarXL and ΔnarL.
To support the genetic evidence that napAB expression was required for delayed growth recovery seen in ΔnarXL under anaerobic conditions, three additional experiments were performed. First, using RT-PCR, we examined napA and narH transcript levels in wild type PAO1, ΔnarXL, and ΔnarGH under anaerobic conditions in NY supplemented with nitrate. The expression of the napA transcript was detected only in ΔnarXL, while the narH transcript was detected only in wild type PAO1 (Figure 4). As a control for RNA integrity, the groEL transcript was detected in all three strains under anaerobic conditions. PCR products from genomic DNA using each primer pair are shown in lanes marked (+) and, reverse transcriptase was omitted from reactions shown in lanes marked (−).
Second, to confirm that nap operon expression was induced in ΔnarXL under anaerobic conditions in the presence of nitrate, Φ(napD-lacZ) transcriptional fusions were constructed (Supplemental Figure 4A) and used to transform wild type PAO1, ΔnarXL, and ΔnarGH. Each strain was also transformed with vector pLP170 alone to serve as a negative control. In wild type PAO1 and ΔnarGH, β-galactosidase levels remained low at 4000 Miller units and 5000 Miller units, respectively, under anaerobic conditions. In contrast, β-galactosidase levels were significantly elevated in ΔnarXL compared to wild type PAO1 and ΔnarGH (p < 0.001) after 24 hr of anaerobic growth (Supplemental Figure 4B), and remained consistently elevated at 30,000 Miller units for the entire 120 hr growth period. Combined with the RT-PCR data, these results indicated that expression of the napEFDABC operon is induced in ΔnarXL, and suggested that napEFDABC operon expression was responsible for growth recovery seen in ΔnarXL under anaerobic conditions.
Third, nitrate reductase activity was measured in permeablized cells using methyl viologen (MV) as an artificial electron donor. To determine which nitrate reductase was functional in a given strain, 50 µM sodium azide was added to selected cultures. NarGHI activity is azide-sensitive, whereas NapAB activity is azide-resistant (Jones & Garland, 1977; MacGregor et al., 1974; Stewart et al., 2002). Using either benzyl viologen (BV) or methyl viologen (MV) as an electron donor, periplasmic nitrate reductase NapAB would be predicted to function in either the presence or absence of azide, whereas membrane nitrate reductase NarGHI would function only in the absence of azide. Organisms grown under anaerobic conditions were collected during mid-log growth at an OD660 of ~0.6 with exception of ΔnarGH and ΔnapA:ΔnarXL, which were collected at an OD660 of ~ 0.12. In either the absence or presence of sodium azide, ΔnarXL utilized MV comparably as an electron donor to reduce nitrate to nitrite (Figure 5), indicating increased periplasmic nitrate activity in this strain. As predicted, ΔnarGH and ΔnapA:ΔnarXL demonstrated no nitrate reductase activity using MV under any conditions tested. In contrast, wild type PAO1 and ΔnapA demonstrated a significantly reduced (p < 0.002) ability to utilize MV as an electron donor in the presence of sodium azide compared to ΔnarXL, suggesting that the only functional nitrate reductase in wild type PAO1 under anaerobic conditions is the membrane nitrate reductase NarGHI. Similar results were obtained when BV was utilized as an electron donor. Increased NapAB periplasmic nitrate reductase activity in ΔnarXL corresponded to the increased levels of napA transcription observed in this mutant.
Our genetic, transcriptional, and biochemical studies demonstrated that anaerobic growth recovery by the nitrate sensor-response regulator mutant ΔnarXL was the result of NapAB periplasmic nitrate reductase expression. These results would predict a regulatory role for NarL in NapAB expression. Under high nitrate concentrations, E. coli NarL competes with orthologous nitrate response regulator NarP for binding consensus regulatory motifs upstream of the periplasmic nitrate reductase operon, causing the repression of periplasmic nitrate reductase expression (Darwin & Stewart, 1995; Overton et al., 2006; Potter et al., 1999; Stewart & Bledsoe, 2003; Stewart et al., 2003). Two different putative NarL binding sites were identified upstream of the P. aeruginosa PAO1 periplasmic nitrate reductase napEFDABC operon. Rather than being organized as the consensus inverted heptamer (TACYNMT) repeats separated by two random base pairs (8, 35, 36), the heptamer pairs overlap slightly (Supplemental Figure 5A). The first heptamer pair BS1 (solid underlines), more distal from the start site of napE translation, contains 6/7 consensus bases in the forward strand and 5/7 consensus bases in the reverse strand. The second, proximal heptamer pair BS2 (dashed underlines) contains 6/7 consensus bases in each strand.
To determine whether either BS1 or BS2 were involved in regulation of napEFDABC expression, site directed mutations in each of the two sites were generated in cloned fragments containing the upstream oprH-napE intergenic region and napEFDABC. The two mutant constructs, along with the wild type fragment, were cloned into pUCP18 and used to transform ΔnapA:ΔnarXL and ΔnapA:ΔnarGH. The six strains were grown under anaerobic conditions in NY supplemented with 100 mM KNO3 (Supplemental Figure 5B). We predicted that a mutation in either BS1 or BS2 important in NarL regulation of napEFDABC would undergo anaerobic growth recovery in a ΔnapA:ΔnarGH background. In the presence of functional NarL and an intact oprH-nap intergenic regulatory region, ΔnarGH is unable to grow anaerobically (Supplemental Figure 5B) and the ΔnapA:ΔnarGH double mutant displays the same phenotype (data not shown). As expected, introduction of the wild type construct into ΔnapA:ΔnarGH did not restore anaerobic growth. Interestingly, mutation of the distal putative NarL binding site BS1, but not the proximal BS2, restored anaerobic growth in ΔnapA:ΔnarGH (Supplemental Figure 5B). When the same plasmid constructs were introduced into a ΔnapA:ΔnarXL background, all strains grew comparably to the ΔnarXL strain under anaerobic conditions (data not shown), demonstrating that the plasmids themselves did not interfere with anaerobic growth, and that growth recovery occurs when napEFDABC is provided in trans in the absence of NarL (data not shown).
Based on these results, the distance of BS1 from the start of napEFDABC translation suggested the possibility of an alternative transcriptional start site during anaerobic growth of ΔnarXL. Wild type PAO1 and ΔnarXL were grown under anaerobic conditions in NY medium supplemented with 100 mM KNO3, and RNA was isolated from cells grown to mid-log phase (OD660 = 0.6). Using a set of napE-specific antisense primers in Rapid Amplification of cDNA Ends-based PCR followed by nucleotide sequencing, we identified a transcriptional start site common to both wild type PAO1 and ΔnarXL 25 bases upstream of the start site of translation. A second transcriptional start site unique to ΔnarXL was identified 118 bases upstream of the start site of translation (Supplemental Figure 5A).
These genetic and transcriptional mapping data support the model (Figure 6) that, in wild type PAO1, phospho-NarL binds to BS1 and represses transcription of napEFDABC and stimulates transcription of narK1K2GHJI. Repression of napEFDABC is lifted either by the absence of NarL or the alteration of BS1, permitting increased transcription of napEFDABC and thereby supporting anaerobic growth over time. We suggest that the transcription of napEFDABC initiated downstream of BS2, and common to both wild type PAO1 and ΔnarXL, represents a constitutive basal level of transcription corresponding to that reported in (Schreiber et al., 2007). However, this level of napEFDABC transcription is insufficient to support robust anaerobic growth of a ΔnarGH mutant.
Several studies using transcriptional profiling have demonstrated the importance of quorum sensing as a global regulator of nitrate metabolism and biofilm formation in P. aeruginosa (Schuster et al., 2003; Wagner et al., 2003; Wagner et al., 2004). In addition to quorum sensing, anaerobic growth via denitrification in P. aeruginosa is regulated by Anr (Ye et al., 1995), an ortholog of Fnr (fumarate and nitrate reductase) in E. coli (Sawers, 1991). A consensus Anr sequence TTGACN4ATCAG (Spiro & Guest, 1990) and the consensus NarL binding sequence TACc/tNa/cT (Tyson et al., 1993) are found in the intergenic region between the narK1K2GHJI membrane-bound nitrate reductase operon and the operon encoding the nitrate sensor-response regulator narXL. NarXL activation of the membrane nitrate reductase narK1K2GHJI operon has been reported in both the denitrifying pseudomonad P. stutzeri (Hartig et al., 1999) and in P. aeruginosa (Schreiber et al., 2007). A third study demonstrated that Anr and NarL act in concert with Integration Host Factor to activate transcription of the hemA gene, which encodes the first enzyme in heme biosynthesis during anaerobic growth in P. aeruginosa (Krieger et al., 2002).
The consensus heptamer binding site for gene activation by NarL (Tyson et al., 1993) is organized as an inverted repeat separated by two nucleotides, the 7-2-7 motif. However, some variants of this motif are functional in P. aeruginosa (Krieger et al., 2002). In silico analysis of the P. aeruginosa PAO1 genome using the web-based Regulatory Sequence Analysis-Tools program (RSAT) program (http://rsat.ulb.ac.be/rsat/) demonstrates that, in addition to the regulatory region of the narK1K2GHJI operon, only six sites were identified upstream of a predicted coding region with full 7-2-7 NarL consensus binding motif (maximum of one mismatch). One 7-2-7 site identified is the region upstream of nuoA-N encoding the NADH dehydrogenase complex I, which participates in electron transfer via ubiquinone to the membrane-bound nitrate reductase NarGHI (Berks et al., 1995; Zumft, 1997). NADH dehydrogenase complex I is essential for anaerobic growth in P. aeruginosa (Filiatrault et al., 2006). NADH dehydrogenase complex I and ubiquinone perform a similar function aerobically by coupling to the electron transport chain of the bacterial cytochrome complex (Richardson et al., 2001).
In P. fluorescens, nuo mutants are deficient in plant root colonization (Camacho Carvajal et al., 2002), suggesting that NADH dehydrogenase complex I may be involved in aerobic motility and biofilm formation in addition to involvement in anaerobic growth. Transcriptome analysis of a NarX/NarL mutant in E. coli demonstrated that the nuo operon is part of the NarL regulon (Constantinidou et al., 2006). In contrast, over 10% of intergenic regions in P. aeruginosa PAO1 have at least one consensus NarL half-site (www.pseudomonas.com). The overall importance of full 7-2-7 to half-site recognition in transcription regulation by NarL in P. aeruginosa is unknown. However, in the report cited above (Krieger et al., 2002), mutation of a single NarL heptamer half site in the P. aeruginosa hemA intergenic region was sufficient to abolish hemA expression. Recently, Benkert et al (Benkert et al., 2008) showed that NarL represses the expression of the arginine fermentation pathway arcDABC in P. aeruginosa. Additionally, they identified a heptamer half site for binding by NarL centered at position −56 upstream of arcDABC rather than the traditional 7-2-7 binding site.
Studies in E. coli support our conclusion that NarL plays a role in regulation of P. aeruginosa napEFDABC operon expression. Darwin and Stewart (Darwin & Stewart, 1995) identified a consensus NarP/NarL binding site upstream of the periplasmic nitrate reductase operon and determined that NarL directly repressed E. coli napFDAGHBC operon expression (Potter, 2001; Stewart et al., 2003). Subsequently, Potter and Cole (Potter & Cole, 1999) demonstrated that a strain containing a functional periplasmic nitrate reductase, but lacking a functional membrane nitrate reductase response regulator NarL (napA+narL−), grew better anaerobically than a strain that contained both a functional periplasmic nitrate reductase and NarL (napA+narL+). The failure of the narL+nap+ strain to grow well in batch culture was partly attributed to NarL-dependent repression of nap operon expression by nitrate. Furthermore, the narGHI− napA+ strain grew better anaerobically under nitrate limiting conditions. In contrast, the narGHI+ napA− strain grew better in high nitrate concentrations (Stewart et al., 2002; Wang et al., 1999). In addition, E. coli NarL can bind to sites other than the preferential 7-2-7 site (Darwin & Stewart, 1995; Darwin et al., 1997) to either promote or repress transcription. The sequences TTACAATTG and TAACCACAC, found upstream of the nrfA operon in E. coli at positions −50 and −22, respectively, are single heptamers where NarL can bind and act as a repressor in presence of nitrate (Darwin et al., 1997; Darwin, 1996). Dong et al (Dong et al., 1992) demonstrated in E. coli that single base substitutions in these sequences significantly reduced NarL binding.
Regulation of periplasmic nitrate reductase operon napEFDABC expression by response regulator NarL in P. aeruginosa has not been widely considered because, to date, NarL has only been shown to be important in the activation of expression of the membrane nitrate reductase narK1K2GHJI operon (Schreiber et al., 2007). Unexpectedly, we observed anaerobic growth recovery over time in ΔnarXL. Genetic, transcriptional, and biochemical studies presented here demonstrated that the delayed anaerobic growth recovery in ΔnarXL was attributed to the activity of periplasmic nitrate reductase NapAB. A novel transcriptional start site for napEFDABC expression was identified in ΔnarXL grown anaerobically. Furthermore, mutagenesis of a consensus NarL-binding half-site upstream of the novel transcriptional start site also restored anaerobic growth recovery in ΔnarXL. Taken together, these data support a model (Figure 6) in which the nitrate response regulator NarL directly represses expression of periplasmic nitrate reductase NapAB, while inducing maximal expression of membrane nitrate reductase NarGHI during anaerobic growth of wild type P. aeruginosa PAO1.
Studies of Paracoccus pantotropha and E. coli suggest why P. aeruginosa ΔnarXL displayed a delayed growth phenotype even though NapAB expression was induced very early under anaerobic conditions. P. pantotropha expressing only a functional periplasmic nitrate reductase supported anaerobic respiration and growth on nitrate at levels approximately one-third that of the wild type (Bell et al., 1993). Also, E. coli (Stewart et al., 2002) used periplasmic nitrate reductase and fumarate reductase equally well in the respiratory chain involving glycerol 3-phosphate oxidation. In this case, the fumarate reductase is not a coupling site for generating a proton motive force, suggesting that the periplasmic nitrate reductase is also not a coupling site for generating a proton motive force via quinol oxidation.
When grown under anaerobic conditions, P. aeruginosa favors expression of the membrane nitrate reductase NarGHI (Schreiber et al., 2007) since its activity is coupled to energy production (Berks et al., 1995; Zumft, 1997). The two component sensor-regulator system NarXL is required for the expression of the membrane nitrate reductase as demonstrated in Figure 3B and shown in (Schreiber et al., 2007). Hence, ΔnarXL and ΔnarGH show no growth over the first 72 hours of incubation under anaerobic conditions (Figure 1). However, delayed growth recovery was observed in ΔnarXL but not in ΔnarGH. This phenomenon was due to the expression of the periplasmic nitrate reductase (Figure 5). The delayed onset of growth by ΔnarXL is likely to be a multifaceted process requiring first the de-repression and expression of the periplasmic nitrate reductase. Even though periplasmic nitrate reductase is expressed as early as 3 hr (Supplemental Figure 4B), the onset of the delayed growth recovery by ΔnarXL does not occur until approximately 72 hours under anaerobic conditions suggesting that the expression, quantity, and/or functionality of the periplasmic nitrate reductase may be sub-optimal. This may be due in part to a dosage effect which can be overcome by providing the napEFDABC operon in trans on a multi-copy plasmid as illustrated in Supplemental Figure 5 and Supplemental Figure 2.
Alternatively, the delayed growth recovery observed in ΔnarXL may be due to a threshold of nitrite concentration accumulating that must be met. Because there may be a slow generation of nitrite by ΔnarXL due to the slow conversion of nitrate to nitrite by NapAB, the subsequent induction of the rest of denitrification pathway may be delayed as seen in Supplemental Figure 3. Maximal nitrite accumulation in wild type PAO1 occurred after 6 hr of anaerobic growth, coinciding with mid-log phase (Supplemental Figure 3A). Nitrite accumulation in ΔnarXL began at 72 hr and reached a maximum concentration at 111 hr of anaerobic growth, also coinciding with mid-log phase in this mutant. However, following 24 hr pre-adaptation of ΔnarXL in medium supplemented with nitrate and subculture into medium supplemented with nitrite, both wild type PAO1 and ΔnarXL (Figure 2B) grew comparably, suggesting that the activity of periplasmic nitrate reductase is the rate limiting step for growth, since the rest of the denitrification pathway is not induced unless an adequate concentration of nitrite is generated. Hence, under normal conditions in wild type P. aeruginosa PAO1, the shift from aerobic to anaerobic growth prefers membrane nitrate reductase utilization to drive energy metabolism. However in ΔnarXL, a shift from aerobic to anaerobic growth requires the electron transport system to be remodeled because the periplasmic nitrate reductase is not a direct coupling site. Hence, P. aeruginosa must rely on the remainder of the denitrification pathway and the arginine fermentation pathway for this purpose. (Eschbach et al., 2004; Schreiber et al., 2006; Vander Wauven et al., 1984).
Anaerobic growth curves of wild type PAO1 (solid shapes) and ΔnarXL (open shapes) grown in different media for 168 hr. Diamonds: NY medium alone. Squares: NY + 100 mM KNO3. Triangles: NY + 40 mM arginine.
Anaerobic growth curves of PAO1, ΔnarXL, ΔnapA:ΔnarXL, and ΔnapA:ΔnarXL complemented with either napEFDABC or narXL in trans in NY + 100 mM KNO3.
A. Quantitation of nitrite concentration accumulation (µM) in supertanants of wild type PAO1, ΔnarXL, ΔnapA:ΔnarXL, and ΔnapA:ΔnarXL-C grown for 150 hours in NY + 100 mM KNO3. B. Anaerobic growth curves of PAO1, ΔnarXL, ΔnapA:ΔnarXL, and ΔnapA:ΔnarXL complemented with the napEFDABC upstream of the glmS site in NY + 100 mM KNO3.
A. Diagram of transcriptional fusion Φ(napD-lacZ), containing nap operon regulatory region upstream of napE (Φ(napD-lacZ)). B. β-galactosidase expression of P. aeruginosa PAO1, ΔnarXL, and ΔnarGH transformed with (Φ(napD-lacZ) and grown under anaerobic conditions in NY + 100mM KNO3 for 120 hours. As a negative control for background expression levels of β-galactosidase, each strain was transformed with pLP170 alone.
A. A schematic of the intergenic sequence upstream of the periplasmic nitrate reductase operon napEDFABC. Two putative NarL binding sites were identified that contain overlapping heptamer sequences (TACYNMT). Binding Site 1 (BS1) is denoted by the solid underline and Binding Site 2 (BS2) is denoted by the dashed underline. The bases changed by site-directed mutagenesis are shown in boldface. The start of transcription under anaerobic conditions in ΔnarXL is denoted by +1; the start of transcription for basal constitutive expression of the periplasmic nitrate reductase is denoted by +91. The italicized ATG denotes the start of translation for the napEFDABC operon. B. Growth curves of site-directed mutants in two putative NarL consensus binding sites. narLBS1napEFDABC:pUCP18, narLBS2napEFDABC:pUCP18 and napEFDABC:pUCP18 were used to transform ΔnapA:ΔnarXL. All strains were grown under anaerobic conditions in NY + 100 mM KNO3.
This work was supported by North American Cystic Fibrosis Foundation Program Project Grant IGLEWS03FGO, NIH R37 AI033713 and NIH T32 AI007285. The authors thank Virginia L. Clark for critical reading of the manuscript. The P. aeruginosa PAO1 genomic library was generously provided by Melanie J. Filiatrault.