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J Bacteriol. 2008 December; 190(23): 7847–7858. Published online 2008 September 26. doi: 10.1128/JB.00905-08. | PMCID: PMC2583599 |
Copyright © 2008, American Society for Microbiology Characterization of the Oxygen-Responsive NreABC Regulon of Staphylococcus aureus![[down-pointing small open triangle]](/corehtml/pmc/pmcents/x25BF.gif) † Steffen Schlag,1‡ Stephan Fuchs,2‡ Christiane Nerz,1 Rosmarie Gaupp,1 Susanne Engelmann,2 Manuel Liebeke,3 Michael Lalk,3 Michael Hecker,2 and Friedrich Götz1* Microbial Genetics, University of Tübingen, 72076 Tübingen, Germany,1 Institute for Microbiology, University of Greifswald, 17487 Greifswald, Germany,2 Institute for Pharmaceutical Biology, University of Greifswald, 17487 Greifswald, Germany3 Received July 1, 2008; Accepted September 18, 2008. Here, we investigate the functionality of the oxygen-responsive nitrogen regulation system NreABC in the human pathogen Staphylococcus aureus and evaluate its role in anaerobic gene regulation and virulence factor expression. Deletion of nreABC resulted in severe impairment of dissimilatory nitrate and nitrite reduction and led to a small-colony phenotype in the presence of nitrate during anaerobic growth. For characterization of the NreABC regulon, comparative DNA microarray and proteomic analyses between the wild type and nreABC mutant were performed under anoxic conditions in the absence and presence of nitrate. A reduced expression of virulence factors was not observed in the mutant. However, both the transcription of genes involved in nitrate and nitrite reduction and the accumulation of corresponding proteins were highly decreased in the nreABC mutant, which was unable to utilize nitrate as a respiratory oxidant and, hence, was forced to use fermentative pathways. These data were corroborated by the quantification of the extracellular metabolites lactate and acetate. Using an Escherichia coli-compatible two-plasmid system, the activation of the promoters of the nitrate and nitrite reductase operons and of the putative nitrate/nitrite transporter gene narK by NreBC was confirmed. Overall, our data indicate that NreABC is very likely a specific regulation system that is essential for the transcriptional activation of genes involved in dissimilatory reduction and transport of nitrate and nitrite. The study underscores the importance of NreABC as a fitness factor for S. aureus in anoxic environments. Staphylococcus aureus, which is among the most frequently isolated agents of nosocomial sepsis, has the ability to grow under low-oxygen conditions by fermentation or nitrate respiration ( 5). The global adaptation to anaerobic environments has recently been described and involves an induction of genes related to dissimilatory nitrate and nitrite reduction ( 10). Nitrate (NO 3−) and nitrite (NO 2−) can be used as terminal electron acceptors under anaerobic conditions. In the food-grade species Staphylococcus carnosus, used as a starter culture in the production of raw fermented sausages, the membrane-bound respiratory nitrate reductase, NarGHI, generates nitrite, which is further reduced to ammonia by a cytoplasmic NADH-dependent nitrite reductase encoded by the nirBD genes ( 23, 24, 26). In contrast to nitrate reduction, nitrite dissimilation is not coupled to the generation of a proton motive force and, hence, is not a respiratory pathway. In the published genome sequences of S. aureus strains, the nitrite reductase genes are annotated either as nasDE or nirBD, the latter which are referred to in this report. Oxygen sensing by global regulators is crucial for the transcriptional control of genes facilitating adaptation to anaerobiosis or microaerophilic conditions. In S. aureus, the two-component system SrrAB (for staphylococcal respiratory response AB; synonym, SrhSR), homologous to Bacillus subtilis ResDE ( 37), has been described as one major regulatory system involved in anaerobic gene regulation ( 28, 38, 39). Another direct staphylococcal oxygen sensing system, NreABC (for nitrogen regulation), has been identified and characterized in S. carnosus ( 9). Upon oxygen depletion, autophosphorylation activity of the cytoplasmic histidine kinase NreB increases, which depends on the formation of an oxygen-labile iron-sulfur cluster ( 16). The response regulator NreC is phosphorylated by NreB and specifically binds to the promoters of the nitrate reductase and nitrite reductase operons and of the narT gene to enhance transcription ( 9). NarT shares 75% identity with the S. aureus nitrite extrusion protein NarK and is likely to be involved in nitrate and nitrite transport ( 6, 8). The function of NreA, encoded by the first gene of the three-cistron operon nreABC, is as yet unknown. NreA almost exclusively consists of a GAF domain, which is one of the largest families of cyclic nucleotide-binding regulatory domains ( 18). In S. aureus, genes homologous to S. carnosus nreABC are present ( 16), and the respective proteins share 50%, 57%, and 85% identity (NreA [SA2181], NreB [SA2180], and NreC [SA2179], respectively). Here, we investigate the function of the oxygen-sensing system NreABC in S. aureus and evaluate its role in anaerobic gene regulation. We therefore constructed mutants lacking the nreABC genes in two different S. aureus backgrounds (SA113 and COL) and used comparative microarray and proteomic analyses to investigate the impact of the nreABC deletion during fermentation and under nitrate respiration conditions on a global scale. Using a two-plasmid system, we confirmed the positive regulation of selected genes involved in dissimilatory nitrate and nitrite reduction. Bacterial strains and growth conditions. S. aureus strains SA113 (ATCC 35556), a derivative of S. aureus NCTC8325 ( 15), and COL ( 34) were used in this study. All staphylococcal strains were cultured at 37°C in BM broth (1% soy peptone, 0.5% yeast extract, 0.5% NaCl, 0.1% K 2HPO 4, 0.1% glucose). Overnight cultures were diluted in fresh medium to an optical density (OD) of 0.07 at 578 nm for staphylococcal strains and 600 nm for Escherichia coli strains. For cloning and beta-galactosidase assays E. coli XL1-Blue (Stratagene) was used, grown in Luria-Bertani (LB) broth. Conditions of decreased oxygen tension were created by growing the cells in closed Eppendorf tubes (2 ml) with moderate shaking (130 rpm) or, when higher cell yields were required (e.g., for RNA isolation), in Falcon tubes (50 ml) that were completely filled with medium. For microarray and proteomic analyses with S. aureus SA113, anaerobic conditions were created by growing the cells in a BioStatQ fermentor (B. Braun Biotech) with helium gas constantly bubbled into all fermentors and moderate agitation at a stir rate of 100 rpm. For anaerobic shift experiments and for the analysis of protein synthesis of S. aureus COL, cells were first grown under aerobic conditions in 100 ml of synthetic medium ( 11) in 500-ml Erlenmeyer flasks under vigorous agitation at 37°C to an OD at 500 nm (OD 500) of 0.5. Cells were then shifted to anaerobic growth by transferring the culture to screw-top ultracentrifugation tubes (10 ml; VWR, Darmstadt, Germany) or Falcon tubes (50 ml) which were completely filled with medium and incubated under vigorous agitation at 37°C. Where applicable, sodium nitrate (NaNO 3) or sodium nitrite (NaNO 2) was added to the medium at concentrations as mentioned in the text. Colony size was monitored by plating appropriate dilutions of overnight cultures on BM agar plates supplemented with 20 mM nitrate. The plates were incubated either aerobically or under oxygen limitation conditions in an anaerobic jar (Merck Anaerocult A) at 37°C for 42 h. Construction and complementation of S. aureus nreABC deletion mutants. The upstream flanking region (1.1 kbp) of nreA was amplified from S. aureus SA113 chromosomal DNA by PCR using DeepVent DNA polymerase (New England Biolabs). The forward primer (1- narJ-F) introduced a BamHI site, and the reverse primer (2- narI-R) introduced an EcoRI site (for primers used in this study, see Table S1 in the supplemental material). The region downstream of nreC (1.0 kbp) was amplified using the forward primer 3- nreC-F and the reverse primer 4- orfX-R, introducing SalI and XbaI sites, respectively. The erythromycin resistance gene ermB of Tn 551 was removed from plasmid pEC3 by EcoRI/SalI digestion and ligated together with the two PCR-generated fragments into the BamHI/XbaI-digested temperature-sensitive shuttle vector pBT2 ( 3), yielding pBT2-Δ nreABC. Cloning was performed in E. coli XL1-Blue (Stratagene). The plasmid pBT2-Δ nreABC was introduced into S. aureus strains SA113 and COL by electroporation ( 1). Insertional inactivation of nreABC by homologous recombination was achieved as described previously ( 3). The insertional disruption of nreABC was confirmed by PCR and DNA sequence analysis. The complementation plasmid pRB-P nreABC was constructed in E. coli by introducing the PCR-generated nreABC operon of S. aureus SA113 with its putative promoter region (253 bp upstream of the start codon) into the shuttle vector pRB473 ( 4). The primers used were P nreA-F and nreC-R, introducing BamHI and SacI sites, respectively. The plasmid pRB-P nreABC was transformed into the nreABC deletion strains of S. aureus SA113 and COL by electroporation ( 1). RNA isolation. Cells were collected and treated with lysostaphin (Dr. Petry Genmedics) for 15 min. Lysostaphin-treated cells were mechanically disrupted by vortexing three times for 30 s with glass beads. RNA was isolated with the RNeasy Mini Kit (Qiagen), followed by treatment with DNase I (Ambion) at 37°C for 30 min according to the manufacturer's instructions. RNA integrity was confirmed by agarose gel electrophoresis. Semiquantitative RT-PCR. The relative expression levels of narG were determined by semiquantitative reverse transcription-PCR (RT-PCR). As an internal standard, the relative expression levels of the dnaA gene, encoding the chromosomal replication initiation protein, were used. RNA was isolated as described above. DNase I-treated RNA (5 μg) was subjected to RT with 200 U of Moloney murine leukemia virus reverse transcriptase (Peqlab) for 2 h. Reverse primers specific for S. aureus narG (narG-SA-RT-R) and dnaA (dnaA-SA-RT-R) were used to prime the reaction. An equal amount (5 μl) of each reaction mixture was used as a template for PCR amplification (25 cycles) using primer sets for narG (narG-SA-RT-F and narG-SA-RT-R) and dnaA (dnaA-SA-RT-F and dnaA-SA-RT-R). The resulting PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining. The absence of genomic DNA was verified by PCR using DNase I-treated RNA samples as templates with dnaA-specific primers (minus RT control). RNA templates for semiquantitative RT-PCR and for microarray analyses were from independent experiments. cDNA microarray analysis. For DNA microarray analysis sciTRACER S. aureus N315 full genome microarrays (Scienion) containing PCR products corresponding to 2,334 genes derived from the genome sequence of S. aureus N315 were used. S. aureus SA113 was grown anaerobically in BioStatQ fermentors (B. Braun Biotech) in BM broth with helium gas constantly bubbled into all fermentors and moderate agitation at a stir rate of 100 rpm. Where applicable, sodium nitrate (2 mM) was added to the cultures. Bacteria from two samples of each growth condition were collected. RNA was isolated as described above and pooled. Total DNase I-treated RNA (2 μg) was used for microarray hybridization, and data analysis was done as described previously ( 30). The data represent the medians of four parallel microarray analyses, and the significant threshold was set at a level of greater than 2.3-fold change in expression, with a P value cutoff of 0.05 (one-sample t test with a Benjamini and Hochberg multiple testing correction). ß-Galactosidase assays. For ß-galactosidase assays, E. coli strains were grown in LB medium supplemented with 5 g liter −1 lactose, 100 μg ml −1 ampicillin, 40 μg ml −1 chloramphenicol, and 20 μg ml −1 arabinose, unless otherwise noted. The E. coli expression plasmid pAC7 and the promoter probe plasmid pSB40N were described previously ( 31). Plasmids pAC7- nreBC and pAC7- nreABC containing the S. aureus nreBC or nreABC genes, respectively, under the control of the tightly regulated arabinose-inducible P BAD promoter ( 12), were constructed as follows. The nreBC genes were PCR amplified from S. aureus SA113 chromosomal DNA using the primer set pAC7- nreBC-F and pAC7- nreC-R, introducing an NdeI site to the translation initiation codon and an NdeI site downstream of the stop codon, and cloned into NdeI-digested plasmid pAC7. The nreABC genes were cloned analogously using the primer pair pAC7- nreABC-F and pAC7- nreC-R. The promoter probe plasmid pSB40N contains a promoterless lacZα reporter gene. The putative S. aureus promoter regions of nirR, narG, and narK (0.5- to 0.6-kbp fragments upstream of the Shine-Dalgarno sequence) were cloned upstream of lacZα into BamHI/XhoI-cut pSB40N using primer sets pSB40N-P nirR-F/pSB40N-P nirR-R, pSB40N-P narG-F/pSB40N-P narG-R, and pSB40N-P narK-F/pSB40N-P narK-R, yielding plasmids pSB40N-P nirR, pSB40N-P narG, and pSB40N-P narK, respectively. These plasmids were transformed into E. coli containing pAC7, pAC7- nreBC, and pAC7- nreABC. ß-Galactosidase activity was visualized on LBACX-ara agar plates (LB medium with 5 g liter −1 lactose, 100 μg ml −1 ampicillin, 40 μg ml −1 chloramphenicol, 20 μg ml −1 X-Gal [5-bromo-4-chloro-3-indolyl-β- d-galactopyranoside], and 20 μg ml −1 arabinose), as described previously ( 14). For ß-galactosidase assays according to the method of Miller ( 20), strains were grown in LBAC-ara medium (LBACX-ara medium without X-Gal) either aerobically (1:20 culture-to-flask volume ratio) or under conditions with reduced oxygen tension (see above) for 24 h (at 37°C and 130 rpm). Preparation of cytoplasmic proteins for preparative two-dimensional (2D) gel electrophoresis. Cells of 50-ml cultures of S. aureus SA113 were harvested on ice and centrifuged for 10 min at 7,000 × g and 4°C. Cells were washed twice with ice-cold TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5), resuspended in TE buffer (10 μl/mg wet weight), and disrupted using glass beads and a tissue lyser (Qiagen). In order to remove membrane fragments and insoluble proteins, the cleared lysate was centrifuged for 30 min at 21,000 × g (at 4°C). The protein concentration was determined using Roti Nanoquant (Roth, Germany), and the protein solution was stored at −20°C. Analytical and preparative 2D-PAGE and protein identification by mass spectrometry. 2D polyacrylamide gel electrophoresis (2D-PAGE) and identification of proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry was performed as described earlier ( 10). Gels were stained with Sypro Ruby (Invitrogen), according to the recommendations of the manufacturer. Preparation of pulse-labeled protein extracts. For the analysis of protein synthesis, proteins were pulse labeled with l-[ 35S]methionine as described previously ( 10). Protein quantitation approaches. The 2D gel image analysis was performed with the software Delta2D (Decodon), including the TIGR Multiexperiment Viewer package for analysis of quantification data ( 32). Gels visualizing intracellular protein accumulation of S. aureus SA113 wild type and the nreABC mutant, grown anaerobically with and without nitrate supplement, were imported into the same project. A fusion gel was created based on all gels to ensure a spot matching of 100%. Spot volumes were normalized by using total normalization before ratios were calculated. For Pavlidis template matching (PTM) ( 27), spot volumes were standardized (division by mean and standard deviation of their expression profile) using the TIGR Multiexperiment Viewer package. The template for expression clustering of proteins was defined by a higher accumulation than the mean in the wild type and a lower accumulation than the mean in the mutant, in both the absence and presence of nitrate (data were retrieved with a threshold P value of 0.05). Autoradiograms representing the protein synthesis pattern of S. aureus COL were also analyzed by using Delta2D. Spot volumes were normalized by using total normalization. Quantification of glucose, lactate, acetate, and nitrite. Extracellular metabolites were detected by 1H nuclear magnetic resonance (NMR) as previously described ( 13). Extracellular concentrations of nitrite were determined colorimetrically by the Griess reaction as described earlier ( 25, 35). Phenotypic characterization of the S. aureus nreABC deletion mutants and complementation studies. The nreABC genes of S. aureus strains SA113 and COL were replaced by an erythromycin resistance cassette ( ermB), as depicted in Fig. . The deletion mutants (also referred to as the Δ nreABC strains) were transformed with the complementation plasmid pRB-P nreABC, carrying the nreABC genes of strain SA113 under the control of the putative native promoter. First, the strains were analyzed with respect to growth and nitrate (NO 3−) and nitrite (NO 2−) reduction under conditions of decreased oxygen tension in BM broth. During growth in the presence of 2 mM NO 3−, the wild type and the complemented mutant accumulated nitrite in the growth medium, which subsequently was further reduced, likely to ammonia. Nitrite accumulation could not be observed in the Δ nreABC strain, and a concentration of merely 0.2 mM was detectable after 24 h (Fig. ). The transcript level of narG, the first gene of the nitrate reductase operon, was markedly reduced in the mutant and restored to the wild-type level by complementation, as assessed by RT-PCR (Fig. , inset). No bands were detected in the controls lacking RT (data not shown). Also, nitrite reduction during growth in the presence of 1 mM NO 2− was severely impaired in the mutant (Fig. ). S. aureus SA113 and COL wild-type, mutant, and complemented mutant strains behaved likewise in these experiments. The wild-type phenotype was not restored when the Δ nreABC strains were transformed with the empty vector pRB473 (data not shown). In the presence of 2 mM nitrate or 1 mM nitrite, growth of the Δ nreABC strains was slightly decreased under reduced oxygen tension in BM broth (data not shown). In S. carnosus wild type, the presence of higher nitrate concentrations (25 mM) under anoxic conditions resulted in a higher growth yield ( 23). During anoxic growth on BM agar plates with 20 mM nitrate, S. aureus SA113 wild type and the complemented mutant formed significantly larger colonies than the nreABC mutant, which displayed a small-colony phenotype under these conditions (Fig. ). This lack of a growth-promoting effect of nitrate in the mutant underscores the importance of NreABC as a fitness factor. Under oxic conditions there was no difference in colony size, independently of the presence of nitrate (data not shown). Microarray-based characterization of the NreABC regulon. Comparative whole-genome microarray experiments were performed to define the S. aureus nreABC regulon and to identify genes differentially expressed in the nreABC mutant during anaerobic growth and during nitrate respiration. Therefore, S. aureus SA113 wild type and the Δ nreABC strain were grown anaerobically in a fermentor with helium gas constantly bubbled through the medium either for 5 h (early stationary phase) without nitrate supplementation or until the culture reached an OD of approximately 0.6 (mid-log phase) in the presence of 2 mM nitrate. Microarray analysis using a stringent cutoff (≥2.3-fold change; P ≤ 0.05) revealed 37 and 40 genes, respectively, that showed differential transcript levels in the Δ nreABC strain under the two tested conditions (Tables 1 and 2), compared to the wild type. Of the 37 differentially expressed genes after 5 h of anaerobic growth, 18 were downregulated, and 19 were upregulated in the mutant (Table 1). Of the 40 differentially expressed genes after growth to an OD of 0.6 with 2 mM nitrate, 16 were downregulated, and 24 were upregulated in the mutant (Table 2). Strikingly, the only genes that displayed differential expression in the nreABC mutant under both anaerobic and nitrate respiration conditions (anaerobic with nitrate) were the genes coding for nitrite reductase ( nirB and nirD) and nitrate reductase ( narG and narH), the putative nitrite extrusion protein ( narK), and genes likely to be involved in cofactor synthesis and assembly ( nirR, nasF, and narJ) (Fig. ). These genes were most highly repressed in the mutant in both experiments (up to 38-fold). However, no overlapping expression profiles of induced transcript levels were found (Fig. ). Notably, after 5 h of anaerobic growth most genes of the urease gene cluster ( ureABCEFGD) were downregulated in the mutant (two- to fourfold), which may rely on pH effects. Also, the genes of the recently characterized ATP-binding cassette transporter HrtAB, which is required for protection against heme toxicity ( 36), displayed reduced expression. On the other hand, the genes of the arginine deaminase (ADI) pathway ( arcABDC), which provides an ATP source, were upregulated two- to threefold in the nreABC mutant. Under conditions of nitrate respiration (anaerobic with nitrate), reduced transcript levels were found for genes encoding lysine biosynthetic enzymes ( lysC, asd, dapA, dapB, and dapD), whereas the fermentation-related genes of alcohol-acetaldehyde dehydrogenase ( adhE), l-lactate dehydrogenase ( lctE), and alcohol dehydrogenase I ( adh1) were induced up to fivefold. Also, transcript levels of the glutamine synthetase operon ( glnRA) and the ferritin-like protein gene ftnA were elevated in the mutant under nitrate respiration conditions. | TABLE 1.Genes differentially expressed in the nreABC deletion mutant after 5 h of anaerobic growth |
| TABLE 2.Genes differentially expressed in the nreABC deletion mutant after anaerobic growth to an OD of 0.6 with 2 mM nitrate |
Sequence similarities in the putative operator regions of nirR, narG, and narK. The response regulator NreC of S. carnosus has been shown to bind upstream of the nitrate reductase and nitrite reductase operons and the narT gene, and the consensus sequence TAGGGN 4CCCTA was identified as the binding site ( 9). Inspection of the upstream regions of nirR, narG, and narK of S. aureus revealed conserved sequences with a high degree of similarity to the S. carnosus consensus motif (Fig. ). However, a direct interaction of NreC with these proposed regions has yet to be demonstrated. Confirmation of positive regulation of nirR, narG, and narK by NreBC using a two-plasmid system. We used a two-plasmid system that had previously been optimized to identify S. aureus σ B-dependent promoters and that is based on two E. coli-compatible plasmids ( 14) to confirm positive regulation of nirR, narG, and narK by NreBC. The expression plasmid pAC7- nreBC carried the nreBC genes of S. aureus SA113 under the control of an arabinose-inducible promoter. The second plasmid contained a lacZα reporter gene under the control of the putative promoter regions (0.5 to 0.6 kbp upstream of the translational start sites) of nirR, narG, and narK (pSB40N-P nirR, pSB40N-P narG, and pSB40N-P narK, respectively). As controls, these promoter probe plasmids were transformed in parallel into E. coli harboring the empty expression vector pAC7. Both pAC7- and pAC7- nreBC-containing clones grew comparably (data not shown). On LBACX-ara plates, transformants harboring pAC7 formed uncolored colonies, indicating that the tested S. aureus promoters were not active in the absence of NreBC. However, transformants containing pAC7- nreBC were blue on the selective plates, indicating that the activity of the nirR, narG, and narK promoters was dependent on arabinose-induced heterologous expression of S. aureus nreBC (Fig. ). Moreover, by quantification of β-galactosidase activity, we could show that the tested NreBC-activated promoters responded to the availability of oxygen (Fig. ). The highest rate of β-galactosidase activity could be observed in cells grown under reduced oxygen tension conditions. The narG promoter showed the highest NreBC-dependent relative induction (290-fold), followed by the narK (40-fold) and nirR (6-fold) promoters, the latter of which displayed slight unspecific activity in E. coli. Aerobic cultivation resulted in a strong reduction of β-galactosidase activity (6- to 29-fold). Also, the activity rate was reduced in the absence of inducer (arabinose) and repressed in the presence of glucose (as tested with the narG promoter) (data not shown). Proteomic analyses of the NreABC regulon. S. aureus SA113 wild type and the nreABC mutant were cultivated anaerobically in a fermentor to mid-log phase with and without nitrate supplement (2 mM). The accumulation of intracellular proteins was investigated by 2D-PAGE with cytoplasmic protein extracts. Sypro staining of the gels allowed a quantification of each protein spot in terms of the relative spot volume. Based on two biological replicates, we performed different comparisons between the genotypes (wild type and nreABC mutant) and conditions (with and without nitrate) in order not to miss effects caused by hypoxia and/or nitrate supplementation. Simple genotype comparisons (relative spot volume of the mutant versus the corresponding spot volume of the wild type, both either with or without nitrate) revealed genotype-dependent differences in the protein expression pattern. Additionally, we calculated ratios of spot volumes of the nreABC mutant with nitrate supplement versus the corresponding spot volumes of the fermenting wild type to figure out if the presence of nitrate had a further influence on the genotype-dependent expression. Nitrate-induced differences in the expression pattern were detected by comparing the same genotypes under different conditions as defined by the presence or the absence of nitrate. In spite of this detailed look at the expression profiles, we could observe only 22 spots undergoing a threefold or greater change in the accumulation level in at least one of the genotype-dependent comparisons (and also in both biological replicates) (Table 3 and Fig. ). | TABLE 3.Protein spots with differential accumulation levels in the S. aureus SA113 nreABC mutant compared to the wild type after anaerobic growth without or with nitrate |
Proteins involved in dissimilatory nitrate and nitrite reduction do not accumulate in the nreABC mutant. We found only 10 protein spots with a clearly lower accumulation level in at least one of the genotype-dependent (i.e., mutant versus wild type) comparisons (Table 3). Five of these spots represented subunits of the nitrate and nitrite reductases (NarG, NarH, NirB, and NirD). Accumulation of these proteins was not detectable in the mutant independently of nitrate (Fig. ), which could be confirmed by PTM analysis (Fig. ). Expression profiles of only six spots matched with the expression template defined by a clearly higher accumulation in the wild type than in the mutant, independently of the presence of nitrate. Five of these spots were subunits of the nitrate and nitrite reductases; one spot remained unidentified. The gamma subunit of pyruvate dehydrogenase (PdhC) was present as at least three spots on the gel (data not shown). The signal intensity of one of these spots could be detected as strongly decreased in the mutant (compared to the wild type; both strains were grown with nitrate) and also in the wild type under fermentation conditions, compared to the wild type performing nitrate respiration (Fig. ). In the mutant the accumulation level of this spot was similar to that in the fermenting wild type and was not influenced by the presence of nitrate. Among other proteins, ArcA and AdhE, both involved in fermentation, show increased accumulation levels in the nreABC mutant. Twelve spots showed a threefold or greater increase in accumulation in at least one of the genotype-dependent comparisons (Table 3). Unfortunately, we could identify only five spots representing four proteins. Seven spots remained unidentified, which may be explained by either low accumulation or by proximity to spots of abundant accumulation (Fig. ). Interestingly, two enzymes involved in fermentation showed similar expression profiles. Accumulation of ADI (ArcA) and alcohol dehydrogenase (AdhE) was increased threefold (ArcA) or even fivefold (AdhE) in the nreABC mutant compared to the wild type but, importantly, only when nitrate was present (Fig. ). Also, accumulation of both enzymes was reduced to similar levels in nitrate-respiring wild-type cells compared to the fermenting wild-type cells. Accumulation of these enzymes was approximately equal in the fermenting wild type and the mutant with or without nitrate. The expression profile of one spot of the ferritin-like protein FtnA (FtnA1) was very similar to that of AdhE. Also, the level of accumulation of RpoC, the beta′ subunit of DNA-directed RNA polymerase, was fourfold greater in the mutant than in the wild type when both were grown without nitrate supplementation. Metabolic impact of NreABC depletion. To shed light on the metabolic impact of an nreABC deletion, we decided to use the S. aureus COL strain because its anaerobic physiology had previously been described very well ( 10). Cells were cultivated in synthetic medium to allow measurement of extracellular metabolites. It was known that the rate of secretion of lactate and acetate is different in fermenting and nitrate-respiring wild-type cells ( 10). Thus, we compared the concentrations of glucose, lactate, and acetate in the growth medium of the wild type and the nreABC mutant, cultivated both aerobically or anaerobically with or without nitrate (Fig. ). Under aerobic conditions, extracellular concentrations of the tested metabolites revealed no differences between the wild type and the mutant and were not influenced by the availability of nitrate. The wild type and mutant also showed no significant differences in glucose consumption under all tested conditions (Fig. ). Under aerobic conditions, glucose was already depleted from the growth medium 8 h after an OD of 0.5 was reached, whereas under anaerobic conditions complete depletion of glucose required 24 h. This difference may result from the lower growth rate under oxygen limitation conditions. Lactate was shown to be the predominant fermentation product of S. aureus COL ( 10). During aerobic growth, lactate levels remained constant as long as glucose was present (Fig. ). After glucose depletion, lactate was taken up for reutilization. Upon oxygen depletion, lactate was produced and secreted into the growth medium. Twenty-four hours after the shift to anaerobic conditions, secretion of lactate was clearly lower in nitrate-respiring wild-type cells than in the absence of nitrate and lower than in the mutant with or without nitrate. Nitrate-induced reduction of lactate secretion could not be observed in the mutant. Accumulation of lactate was highest in the medium of the mutant grown with nitrate supplement, followed by the mutant and the wild type without nitrate supplement. During aerobic growth, acetate was also taken up from the cells after the depletion of glucose although much more slowly than lactate (Fig. ). Anaerobically, acetate accumulation remained more or less constant with the exception of the nitrate-respiring wild type, which showed additional secretion of acetate into the growth medium. Impact of NreABC depletion on protein synthesis rates during early adaptation to anaerobiosis. In addition, using radioactive pulse labeling of newly synthesized proteins within a 5-min time frame, we investigated whether in S. aureus COL depletion of NreABC had an impact on the de novo synthesis pattern of intracellular proteins in the early phase of adaptation to anaerobiosis. In the first 60 min after an anaerobic shift, the synthesis pattern in wild-type cells of intracellular proteins and in particular of fermentation enzymes has been reported to be very similar in cells adapting to fermentative conditions compared to conditions of nitrate respiration ( 10). Within the first hour of adaptation to anaerobiosis, we found the protein synthesis pattern of the nreABC mutant, grown in the presence of nitrate, to be very similar to that of the wild-type strain (see Fig. S1 in the supplemental material), as previously described by Fuchs et al. Notably, the synthesis rate of proteins of the nitrate and nitrite reductase within the 5-min time frame was so weak that the spots were not detectable in either the wild type ( 10) or the nreABC mutant (this study). In this report we investigated the functionality and the regulon of the direct oxygen-sensing and regulation system NreABC in S. aureus by comparative transcriptome and proteome analyses to evaluate the system's role in anaerobic gene regulation and virulence factor expression. Overall, our data show the following: (i) under two different growth conditions (anaerobic and nitrate respiration) transcription of genes involved in dissimilatory nitrate and nitrite reduction and accumulation of corresponding proteins are markedly decreased in the nreABC mutant; (ii) the promoters upstream of nirR, narG, and narK are under the positive control of NreBC; and (iii) under conditions of nitrate respiration the mutant grows by fermentation. For the microarray-based analyses two different growth conditions (anaerobic growth to early stationary phase and growth under nitrate respiration conditions to mid-log phase) were chosen. However, the genes of the nitrate and nitrite reductase operons and of the nitrite extrusion protein NarK were the only ones highly downregulated in the ΔnreABC strain in both cases. These data correlated well with the proteome analyses. Five of the identified 10 protein spots with severely decreased accumulation in the mutant represented subunits of the nitrate and nitrite reductases. The depletion of these proteins practically resembled a knockout phenotype. Remarkably, the accumulation of the nitrate and nitrite reductase proteins in the wild type was not influenced by the availability of nitrate. In the microarray analyses the downregulation of the urease gene cluster and the upregulation of the ADI pathway in the nreABC mutant under fermentation conditions were somewhat surprising. Both pathways are thought to counteract acidification by ammonia production. However, whereas the urease pathway is part of the acid shock response ( 2), the ADI pathway provides an ATP source under anaerobiosis ( 7). Using the two-plasmid system, the putative promoter of the urease gene cluster was not activated by NreBC (data not shown), suggesting that the downregulation of the urease operon likely is an indirect response, which may rely on pH effects. Under fermentation conditions the mutant should not have to compensate an energetic disadvantage, which could eventually explain the upregulation of the ADI pathway. Thus, the biological significance of this observation remains unclear. However, proteome analyses revealed an accumulation of ADI (ArcA) in the mutant under nitrate respiration conditions and a nitrate-induced decrease in accumulation in the wild type, suggesting that the induction of ArcA in the mutant is likely a missing adaptation to the presence of nitrate and results from the inability to perform nitrate respiration. Upregulation of fermentation-related genes ( adhE, lctE, and adh1) in the mutant, compared to the wild type, occurred during anaerobic growth in the presence of nitrate. These data were in part corroborated by proteomic and metabolic analyses. The induction of AdhE in the mutant can likely be attributed to a lack of adaptation of the energy metabolism to nitrate respiration conditions, and the nreABC mutant excreted acetate and lactate in amounts similar to those of the fermenting wild type. These observations indicate that a deletion of the nreABC genes abolished the ability to utilize nitrate as a respiratory oxidant and forced the use of fermentative pathways. However, an impact of NreABC depletion on the de novo protein synthesis rates of enzymes involved in fermentation was not apparent during the early phase of adaptation to nitrate respiration conditions, which was also reflected by the metabolic data (referring to the 1-h value after the anaerobic shift). This observation was in agreement with previous findings that, in the early phase of adaptation, the presence of nitrate had no major influence on the synthesis rate of fermentation-related enzymes in S. aureus wild-type cells ( 10). The transcriptional induction of ftnA under nitrate respiration conditions in the mutant was in accordance with the proteomic data (referring to the spot FtnA 1). FtnA belongs to the family of ferritins and is suggested to function as a staphylococcal iron storage protein ( 22). However, the reason for the FtnA induction in the nreABC mutant remains unclear. Since induction of FtnA 1 was similar to the accumulation patterns of ArcA and AdhE, an increase in accumulation as a result of a missing adaptation to nitrate respiration seems likely. Elevated transcript levels of the glutamine synthetase operon ( glnRA), which could be observed in the mutant grown in the presence of nitrate, may be explained by a lack of ammonia in the growth medium as transcriptional induction of the glutamine synthetase operon occurs under ammonia limitation conditions ( 33). One major route for obtaining ammonia as a substrate for the glutamine synthetase reaction is the reduction of nitrate via nitrite to ammonia by nitrate reductase and nitrite reductase ( 21), which is impaired in the mutant. Using an E. coli-compatible two-plasmid system, activation of the nirR, narG, and narK promoters by NreBC was demonstrated. The promoter activity was very low during aerobic growth, while derepression occurred upon oxygen deprivation. This implies that the oxygen-responsive regulation system NreBC of S. aureus is, in principle, functional in E. coli. The tested S. aureus promoters were not unspecifically recognized in E. coli and were activated only upon heterologous expression of NreBC, showing that the response regulator NreC confers selectivity and is essential for transcription initiation. However, direct regulation has yet to be demonstrated. In S. carnosus transcription of the narG promoter was previously found to be strongly induced under aerobic conditions in the presence of nitrate ( 26). Using the two-plasmid system, we could not observe a high level of activity of the S. aureus narG promoter in the presence of oxygen and nitrate (data not shown). This implies either that the S. carnosus narG promoter exhibits diverse regulatory features or that in staphylococci a specific factor involved in nitrate sensing and subsequent gene activation is likely to be present and is not functionally complemented by the nitrate signal transduction systems NarX-NarL and NarQ-NarP of E. coli ( 29). NreA is unlikely to fulfill the role of a staphylococcal nitrate or nitrite sensor, as has previously been speculated ( 9), since we could observe neither nitrate- nor nitrite-induced elevation of narG and narK promoter activity upon heterologous expression of NreABC from plasmid pAC7 in the β-galactosidase assays (data not shown). Hence, the function of NreA remains as yet unknown. In S. carnosus, the nreABC transcript has been reported to be present during aerobic and anaerobic growth, a requirement expected for an oxygen-sensing system ( 9). The putative S. aureus nreABC promoter resembles a typical σ A-dependent promoter (data not shown), which presumably leads to constitutive expression. However, the absence of a transcription terminator between nreABC and the preceding narGHJI operon supports the idea that NreABC expression is likely under positive autoregulatory control by cotranscription of its own genes with narGHJI. Interestingly, the nreABC operon of S. aureus NCTC8325 has previously been found to be downregulated in a mutant lacking the ClpP protease ( 19), revealing a regulatory impact of ClpP-mediated proteolysis on the transcription of this operon. The correlation of microarray data with proteomic and metabolic data certainly proves helpful and allows distinct conclusions to be drawn regarding a regulon. In summary, the data presented in this study indicate that NreABC of S. aureus represents a very specific regulation system, which in response to oxygen depletion activates the transcription of genes involved in dissimilatory reduction and transport of nitrate and nitrite. Oxygen deprivation has been shown to be of crucial importance for virulence factor expression ( 10). In humans the daily nitrate intake is typically a total of 2 to 3 mmol, and the concentrations of nitrate range from up to 0.1 mM in plasma to 1 mM in saliva ( 17). 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