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mBio. 2013 Nov-Dec; 4(6): e00696-13.
Published online 2013 November 12. doi:  10.1128/mBio.00696-13
PMCID: PMC3892780

The Staphylococcus aureus SrrAB Two-Component System Promotes Resistance to Nitrosative Stress and Hypoxia

ABSTRACT  

Staphylococcus aureus is both a commensal and a pathogen of the human host. Survival in the host environment requires resistance to host-derived nitric oxide (NO·). However, S. aureus lacks the NO·-sensing transcriptional regulator NsrR that is used by many bacteria to sense and respond to NO·. In this study, we show that S. aureus is able to sense and respond to both NO· and hypoxia by means of the SrrAB two-component system (TCS). Analysis of the S. aureus transcriptome during nitrosative stress demonstrates the expression of SrrAB-dependent genes required for cytochrome biosynthesis and assembly (qoxABCD, cydAB, hemABCX), anaerobic metabolism (pflAB, adhE, nrdDG), iron-sulfur cluster repair (scdA), and NO· detoxification (hmp). Targeted mutations in SrrAB-regulated loci show that hmp and qoxABCD are required for NO· resistance, whereas nrdDG is specifically required for anaerobic growth. We also show that SrrAB is required for survival in static biofilms, most likely due to oxygen limitation. Activation by hypoxia, NO·, or a qoxABCD quinol oxidase mutation suggests that the SrrAB TCS senses impaired electron flow in the electron transport chain rather than directly interacting with NO· in the manner of NsrR. Nevertheless, like NsrR, SrrAB achieves the physiological goals of selectively expressing hmp in the presence of NO· and minimizing the potential for Fenton chemistry. Activation of the SrrAB regulon allows S. aureus to maintain energy production and essential biosynthetic processes, repair damage, and detoxify NO· in diverse host environments.

IMPORTANCE  

The Hmp flavohemoglobin is required for nitric oxide resistance and is widely distributed in bacteria. Hmp expression must be tightly regulated, because expression under aerobic conditions in the absence of nitric oxide can exacerbate oxidative stress. In most organisms, hmp expression is controlled by the Fe-S cluster-containing repressor NsrR, but this transcriptional regulator is absent in the human pathogen Staphylococcus aureus. We show here that S. aureus achieves hmp regulation in response to nitric oxide and oxygen limitation by placing it under the control of the SrrAB two-component system, which senses reduced electron flow through the respiratory chain. This provides a striking example of convergent evolution, in which the common physiological goals of responding to nitrosative stress while minimizing Fenton chemistry are achieved by distinct regulatory mechanisms.

INTRODUCTION

The ubiquitous human pathogen Staphylococcus aureus commonly resides in the nose and on the skin, two sites at which nitric oxide (NO·) is produced in ample quantities by the host (1, 2). NO· is a versatile molecular mediator with broad-spectrum antimicrobial activity resulting primarily from the targeting of heme and nonheme metal centers, protein thiols, and DNA (3). The ability of S. aureus to grow despite the presence of NO· concentrations that are inhibitory for many other bacteria has contributed to its success as a pathogen. The resistance of S. aureus to nitrosative stress is in part dependent on the Hmp flavohemoglobin, an enzyme that detoxifies NO· by converting it to nitrate (4). Flavohemoglobin expression must be tightly regulated, as this enzyme can also exacerbate oxidative stress when produced in the absence of NO·, either by shuttling electrons to the flavin pool and promoting Fenton chemistry or by the direct generation of superoxide (5).

In many bacteria, hmp flavohemoglobin gene expression is regulated by an Rrf2 family transcriptional repressor known as NsrR (5), which is found in most beta- and gammaproteobacteria as well as in many Firmicutes (6). Nitrosylation of an iron-sulfur cluster in NsrR abrogates the protein’s ability to bind DNA, allowing hmp expression to respond to very low NO· concentrations (7). Regulation by NsrR ensures that hmp is expressed only when NO· is present or when iron concentrations are at very low levels. However, S. aureus lacks an NsrR homolog (8). Expression of the S. aureus hmp flavohemoglobin gene is regulated in part by the two-component system SrrAB, homologous to ResDE in Bacillus subtilis. B. subtilis differs from S. aureus in possessing both NsrR and ResDE, which regulate hmp in concert (9). Both the SrrAB regulatory system and the Hmp enzyme are required for S. aureus resistance to NO· and virulence in mice (4). An srrAB mutant remains attenuated for virulence in an inducible nitric oxide synthase gene (iNOS) knockout mouse lacking high-output NO· synthesis, whereas the loss of iNOS restores full virulence to an hmp mutant. This indicates that the role of SrrAB in staphylococcal virulence extends beyond nitrosative stress resistance and that SrrAB-regulated genes in addition to hmp contribute to the pathogenesis of staphylococcal infections.

The present study used bioinformatic and transcriptomic approaches to define the genes controlled by SrrAB in S. aureus. The contribution of these genes to nitrosative stress resistance and the mechanism of SrrAB activation were subsequently analyzed. The results reveal that S. aureus uses the SrrAB two-component regulatory system to meet common physiological goals met by the transcriptional repressor NsrR in other bacteria.

RESULTS

Characterization of the SrrAB regulon during nitrosative stress or hypoxia.

To understand the role of the SrrAB two-component system in the resistance of S. aureus to nitrosative stress, a microarray analysis was performed to compare gene expression in wild-type (WT) S. aureus COL and an isogenic srrAB mutant following treatment with the NO· donor diethylamine NONOate (DEA/NO). The results showed that the SrrAB regulon includes genes involved in maintaining the electron transport chain by supporting cytochrome and quinol-oxidase assembly (ctaB, cydAB, qoxABCD) and heme biosynthesis (hemACDX), anaerobic metabolism (pflAB, adhE, nrdDG), and NO· detoxification or the repair of nitrosative damage (hmp, scdA) (Table 1). In contrast to some previous studies (10, 11), no change in the expression of classic staphylococcal virulence factors was observed under these experimental conditions. The SrrAB regulon identified in our microarray studies more closely resembled the ResDE regulon of B. subtilis, which controls genes involved in heme biosynthesis, cytochrome biogenesis, and NO· detoxification (12).

TABLE 1
Microarray analysis of gene expression in wild-type and srrAB mutant S. aureus COL during nitrosative stressa

To validate the microarray results, quantitative reverse transcription-PCR (qRT-PCR) analysis of NO·-treated (1 mM DEA/NO) or untreated (DEA alone) wild-type and srrAB mutant S. aureus COL was performed for selected SrrAB-regulated genes (i.e., pflA, hmp, nrdG, cydA, and scdA) along with positive and negative controls for NO· induction (ldh1 and gyrA, respectively) (Fig. 1A). Under nitrosative stress, the qRT-PCR measurements corroborated the microarray results, showing higher fold induction following NO· treatment in wild-type cells than in an srrAB mutant. Comparison of untreated and NO·-treated cells showed that some NO·-induced genes appear to be exclusively regulated by SrrAB (nrdG, cydA, scdA), while others exhibit both SrrAB-dependent and independent NO·-mediated induction (pflB, hmp). The ldh1 locus, previously shown to be regulated by the transcriptional repressor Rex, was induced by NO· and uninfluenced by SrrAB. Expression of the housekeeping gene gyrA was not altered by nitrosative stress in either wild-type or srrAB mutant cells. Additionally, the SrrAB regulon was verified across multiple S. aureus strains, including UAMS-1, a biofilm-forming strain (13), and Newman, a virulent clinical isolate (4, 14). The pattern of SrrAB-dependent gene regulation was highly similar across these varied S. aureus strains (see Table S1 in the supplemental material).

FIG 1
Expression of SrrAB-regulated genes during nitrosative stress and hypoxia. (A) Wild-type S. aureus COL or isogenic srrAB mutant strains were grown in PN medium with 1 mM DEA/NO or the DEA parent compound. (B) Wild-type S. aureus ...

Previous studies have reported that the SrrAB two-component system is necessary for growth under oxygen-limited conditions (10, 15). We observed that SrrAB-dependent genes expressed during nitrosative stress are also induced during hypoxia in an SrrAB-dependent fashion (Fig. 1B), suggesting a common mechanism for SrrAB induction under nitrosative stress and hypoxic conditions.

Importance of the SrrAB regulon for NO· resistance and anaerobic growth.

Our previous observations have shown that srrAB mutant S. aureus is more sensitive to NO· than an hmp mutant, indicating that additional SrrAB-dependent genes contribute to nitrosative stress resistance (4). To investigate further, mutations were constructed in several SrrAB-regulated genes belonging to different functional groups. These included mutations in two terminal oxidases of the S. aureus electron transport chain: cydAB, encoding the bd-type cytochrome, and qoxABCD, encoding the aa3-type quinol oxidase. In addition, mutations were constructed in nrdDG, which encodes a class III ribonucleotide reductase that functions under anaerobic conditions (16), and in scdA, encoding a di-iron-containing protein previously implicated in the repair of NO·-damaged Fe-S clusters (17). An scdA hmp double mutant was also constructed to assess the scdA phenotype in the absence of the NO·-detoxifying activity of Hmp.

The contribution of the SrrAB-regulated genes in resistance to NO· was tested by growth in a Bioscreen C apparatus with (solid lines) or without (dashed lines) the addition of NO· donor compounds NOC-12 (5 mM, half-life [t1/2] of 100 min) and DEA/NO (1 mM, t1/2 of 2 min) (Fig. 2A). As before, growth of both srrAB and hmp mutant strains was found to be significantly impaired during nitrosative stress (P < 0.0005 and P < 0.005, respectively). Mutations in other individual SrrAB-regulated genes conferred various degrees of enhanced susceptibility to NO·. An nrdDG mutant showed only minimal and nonsignificant impairment in NO· resistance under the aerobic conditions tested, while cydAB or scdA mutants exhibited slightly more pronounced phenotypes that also failed to achieve statistical significance. A qoxABCD mutant was found to be significantly more sensitive to nitrosative stress compared to the wild type (P < 0.05), although this mutant also appeared to exhibit a modest growth defect under aerobic conditions in the absence of NO·. Collectively, these results suggest that the NO· susceptibility phenotype of an srrAB mutant represents additive effects from multiple SrrAB-regulated loci, particularly hmp and qoxABCD. To investigate this possibility, double mutants were constructed containing hmp and either cydAB or qoxABCD mutations. The NO· resistance of a cydAB hmp double mutant was only slightly greater than that of the hmp mutant alone (Fig. 2B). However, growth of a qoxABCD hmp mutant after NO· treatment was comparable to that of an srrAB mutant (P < 0.0001), indicating that collectively these two loci account for most of the contribution of SrrAB to nitrosative stress resistance.

FIG 2
Effects of SrrAB and SrrAB-regulated genes on growth during nitrosative stress and hypoxia. Wild-type S. aureus COL or isogenic srrAB (***), hmp (**), scdA, scdA hmp (*), nrdDG, cydAB, and qoxABCD (*) mutants (A) and wild-type S. aureus ...

Previous studies have also implicated SrrAB in growth under anaerobic conditions (10, 15). To analyze the contribution of individual SrrAB-regulated genes to anaerobic growth, the growth of wild-type and mutant strains was measured with aeration (shaking), under standing (static) conditions, and in an anaerobic jar. Our results confirm previous studies showing that SrrAB is required for maximal growth under anaerobic conditions (P < 0.0001) (Fig. 2C). Of the SrrAB-regulated genes tested, only nrdDG had a measurable impact on anaerobic growth (P < 0.0001). The srrAB and qoxABCD mutant strains were unable to attain maximal final cell density under aerobic conditions (P < 0.005 and P < 0.0005, respectively) but showed no significant difference under either static or anaerobic conditions. Thus, the SrrAB two-component system is activated during nitrosative stress or hypoxia and regulates genes that are important for survival and growth under these conditions.

Importance of SrrAB for NO· detoxification.

One of the principal biological effects of NO· is the rapid and reversible inhibition of respiration due to reversible binding of NO· to cytochrome heme centers (18). Respiration is restored once NO· is detoxified, and hmp expression is a major determinant of the duration of respiratory inhibition following bolus administration of NO·. We have previously shown that the inhibition of respiration following treatment of srrAB mutant S. aureus with an NO· donor is intermediate between that of wild-type and hmp mutant S. aureus, presumably because hmp expression is not completely SrrAB-dependent (Fig. 1) (4). In the present study, respiration was monitored following the treatment of cydAB or qoxABCD single mutant strains along with double hmp mutant strains, and the results were compared with those of wild-type, srrAB mutant, and hmp mutant strains. Both oxygen and NO· consumption were recorded, but for clarity only NO· consumption is shown in Fig. 3A (representative respiratory inhibition curves for each strain are provided in Fig. S2 in the supplemental material). Wild-type, srrAB mutant, and hmp mutant strains yielded expected results based on prior studies, and a cydAB mutant exhibited an NO· consumption rate similar to that of the wild type. This suggests that the bd-type cytochrome is not favored under typical aerobic conditions (19). Unexpectedly, the qoxABCD mutant strain exhibited significantly more rapid NO· consumption (Fig. 3A) and a correspondingly briefer period of respiratory inhibition than the wild type (see Fig. S2E in the supplemental material). The cydAB hmp and qoxABCD hmp double mutants were unable to resume respiration after the addition of NO·, phenocopying the single hmp mutant (data not shown).

FIG 3
NO· consumption by wild-type or mutant S. aureus. (A) NO· consumption was monitored after the addition of 5 µM Proli/NO to wild-type S. aureus COL or isogenic srrAB, hmp, cydAB, and qoxABCD mutants and compared ...

We hypothesized that the increased NO· resistance of a qoxABCD mutant might result from increased hmp expression. This was confirmed by measurement of hmp expression in wild-type and qoxABCD mutant cells by qRT-PCR during late-logarithmic-phase growth (Fig. 3B). Under conditions in which respiration was induced by the addition of glucose or following treatment with 5 µM Proli-NO, expression of hmp was 2- and 3.5-fold higher, respectively, in a qoxABCD mutant than in the wild type (Fig. 3B). NAD+/NADH ratios for wild-type and qoxABCD mutant cells under these assay conditions were unchanged (data not shown). These results suggest that the increased NO· consumption and resistance observed in a qoxABCD mutant strain result from increased hmp expression.

Importance of SrrAB for biofilm formation.

The ability to form and sustain biofilms is considered to be an important virulence-associated phenotype of S. aureus that is likely to be of particular relevance to device-associated infections. S. aureus is also capable of forming biofilms in bone and other tissues. Biofilms may be difficult to treat with antibiotics because of limited penetration and phenotypic resistance of organisms in the biofilm state. As the ability to sense oxygen and grow in oxygen-limited conditions is necessary for biofilm formation, we determined whether the SrrAB two-component system affects S. aureus biofilm formation in vitro. Because S. aureus strain COL does not form biofilms, srrAB and hmp mutations were constructed in the biofilm-forming strain UAMS-1 (13, 20). The UAMS-1 strains were tested and shown to have similar SrrAB-dependent gene regulation and NO· resistance phenotypes (see Table S1 and Fig. S1A in the supplemental material). UAMS-1 forms primarily proteinaceous biofilms, in contrast to the polysaccharide intercellular adhesin (PIA) capsule-associated biofilms of other S. aureus strains (21). Biofilm formation was initially evaluated by a 1- to 3-day static biofilm method in human plasma-coated 96-well plates and quantified with crystal violet staining. Using this biofilm method, we identified that the srrAB mutant has reduced capacity to form biofilms over time compared to the WT. The sarA mutant strain, previously reported to be defective at biofilm formation, was used as a negative control (22). Over a 3-day period, a gradual decrease in biofilm formation by the srrAB mutant in comparison with the WT was observed: 83.3%, 51.9%, and 35.6% on days 1, 2, and 3, respectively (Fig. 4A). The hmp mutant did not show any difference in biofilm levels compared to WT UAMS-1.

FIG 4
Biofilm formation by wild-type and mutant S. aureus. Wild-type S. aureus UAMS-1 or isogenic srrAB, hmp, and sarA mutants were grown in TSBGN medium for 3 to 5 days in either 24-well or 6-well tissue culture plates with sterile ...

After observing that an srrAB mutant exhibited decreased biofilm formation over time compared to that of the wild-type S. aureus under static aeration conditions (Fig. 2B), we hypothesized that SrrAB may be more important during long-term persistence of biofilms as cell density increases and oxygen and nutrients become limiting. We therefore utilized an alternative static biofilm method in which biofilms are grown on a glass coverslip over a 5-day period, to allow visualization via confocal microscopy and staining with BacLight (Invitrogen) (23). Culture medium was changed daily, and biofilms were washed with phosphate-buffered saline (PBS) to remove nonadherent cells. Each day, biofilms were collected from WT S. aureus UAMS-1 and isogenic srrAB and hmp mutant strains for the duration of the experiment. Biofilms were stained with BacLight (Invitrogen), which labels viable cells with SYTO-9 and nonviable cells with propidium iodide (PI), and visualized using confocal laser scanning microscopy (LSM). All three strains were able to initiate biofilm formation with similar thickness (~20 to 25 µM) observed on day 1 (data not shown) and day 2 (Fig. 4B). By day 4, the biofilms formed by the srrAB mutant strain began to deteriorate and were found to contain significantly more dead cells than either the wild-type or hmp mutant biofilms. To quantify dead cells within the biofilm, profile images from at least 3 different regions were examined on 2 different slides per day for each strain. The srrAB mutant biofilms contained ~5% more dead cells than wild-type biofilms on days 1 and 2 but contained ~10% more dead cells on days 3 to 5 (42.5% dead cells in srrAB mutant biofilms versus 32.5% in the wild type) (Fig. 4C). The increase in dead cells corresponded to the loss of structural integrity in the srrAB mutant biofilms and suggests that the SrrAB two-component regulatory system is important for long-term biofilm stability and survival. The hmp mutant displayed higher levels of dead cells on day 1 (P < 0.05), but this number did not increase over time and was not significantly different from the WT on subsequent days.

DISCUSSION

Survival of Staphylococcus aureus within the human host depends on the microbe’s ability to sense and respond to diverse environments. Nitrosative stress resulting from the enzymatic or nonenzymatic synthesis of NO· is one of the most important environmental conditions to which staphylococci must adapt. The activation of SrrAB signaling by either NO· or hypoxia suggested that these signals converge upon a common regulatory pathway. Our observations are consistent with a model (Fig. 5) in which reduced menaquinone triggers the SrrB-dependent phosphorylation of SrrA, which in turn activates genes required for anaerobic metabolism, cytochrome and heme biosynthesis, and NO· detoxification. In addition to promoting the expression of Hmp, the SrrAB two-component system promotes NO· resistance by increasing the expression of inhibited metabolic pathways (PflBA, HemACDX, QoxABCD, CydAB, CtaB), upregulating alternative pathways (e.g., NrdDG) to bypass targets of NO· inhibition, and activating repair systems (ScdA). These functions appear to have an additive effect in NO· resistance (Fig. 2A). In contrast to earlier studies (10, 11), we did not find evidence that classical virulence genes (e.g., spa, RNAIII gene, ica) are regulated by SrrAB under our experimental conditions. Hmp expression remains inducible by NO· in an srrAB mutant strain (Fig. 1A), and the mechanism responsible for SrrAB-independent activation of hmp is presently unknown.

FIG 5
Model of SrrAB activation by nitrosative stress or hypoxia. Nitric oxide (NO·), a quinol oxidase (qoxABCD) mutation, or oxygen limitation impairs the flow of electrons through the electron transport chain. Sensing of reduced menaquinone by SrrB ...

The branched electron transport chain of S. aureus can terminate in an aa3-type cytochrome encoded by qoxABCD or an alternative bd-type cytochrome encoded by cydAB (19, 24). While most bacteria utilize multiple quinone variants as electron carriers, S. aureus uses menaquinone exclusively. The inhibition of terminal oxidases by NO· or by oxygen limitation limits flow through the electron transport chain, thereby triggering SrrAB activation. A qoxABCD mutation mimics this process (Fig. 3B and 5). By placing hmp under the control of SrrAB in response to flux through the electron transport chain, S. aureus achieves the central aim of limiting Hmp flavohemoglobin expression unless NO· is present and/or oxygen levels are low. We observed a qoxABCD mutation abrogating cytochrome aa3 to have a greater effect on NO· sensitivity than a cydAB mutation in S. aureus. This suggests that QoxABCD is the preferred terminal oxidase in S. aureus during nitrosative stress. A qoxABCD mutant actually exhibited more rapid NO· detoxification due to compensatory SrrAB activation and increased hmp expression. A recent study has suggested that the S. aureus cydAB genes may have been misannotated and might not actually encode a bd-type cytochrome (25). Additionally, heme-spectral analysis of the S. aureus cytochromes indicates the presence of a bo-type and a ba3-type cytochrome (26). The present study shows that an S. aureus cydAB mutant is not more susceptible to NO·, in contrast to Escherichia coli (27), lending further support to the idea that cydAB may not encode a bd-type cytochrome.

A menD mutation, which abrogates menaquinone biosynthesis, has been previously shown to result in the loss of expression of SrrAB-dependent genes and the upregulation of srrAB itself (28). This is consistent with the direct sensing of reduced menaquinone by SrrB, as observed for ArcB in enteric bacteria (29). By monitoring electron transport, SrrAB complements the regulatory function of the repressor Rex, which is responsive to NAD+/NADH balance, in maintaining redox homeostasis (30). Both SrrAB and Rex respond to changes induced during nitrosative stress, leading to the upregulation of diverse regulons necessary for survival under these conditions. Interestingly, Rex appears to bind upstream of srrAB, and SrrA protein levels are increased in a Rex mutant; however, transcript levels do not appear to be altered significantly, suggesting a more complex regulatory mechanism (30).

The induction of SrrAB by hypoxia and the reduced survival of an srrAB mutant during anaerobic growth (Fig. 2C) suggested a possible role for SrrAB in biofilms, an oxygen-limited growth condition. Indeed, we found that srrAB mutant S. aureus exhibits grossly deficient biofilm formation with increased levels of cell death (Fig. 4A and C), a finding corroborated by the results of an earlier mutant screen that identified srrA as essential for PIA-independent biofilm formation (31). The defective biofilm phenotype did not appear to be related to NO·, as an isogenic hmp mutant was unimpaired in overall biofilm formation.

In summary, although S. aureus lacks the NO·-sensing transcription factor NsrR, it achieves NO·-responsive expression of the NO·-detoxifying Hmp flavohemoglobin by means of the SrrAB two-component system that senses flux through the electron transport chain. The SrrAB regulon coordinately controls metabolic, detoxification, and repair systems that play an essential role in staphylococcal adaptation to hypoxic and nitrosative stress environments encountered within the infected host. This underscores the intimate relationship between bacterial metabolism and pathogenesis.

MATERIALS AND METHODS

RNA isolation and microarray studies.

Both WT COL and an isogenic srrAB mutant (4) were grown in PN medium (32) to an optical density at 660 nm (OD660) of 0.5, and cells were split into treated or untreated samples. Cells in the treated group were incubated with 1.1 mM of the NO· donor compound DEA/NO (half-life of 2 min) for 20 min, while those in the untreated group were incubated with the parent nucleophile DEA for the same amount of time. After incubation, cells were immediately mixed 1:1 with an ice-cold 1:1 mixture of 100% ethanol and acetone and placed at −80° C until RNA was isolated. RNA was isolated from approximately 5 × 109 CFU and lysed by bead beating for two 40-s pulses separated by 5 min of incubation on ice, followed by pelleting of cell debris and beads. Supernatant was removed from the beads and used for RNA purification by the Qiagen RNeasy Midi protocol. RNA was eluted in diethyl pyrocarbonate (DEPC)-treated H2O and quantified with a Shimazdu spectrophotometer. RNA was tested for induction of hmp expression after NO· treatment to verify the inducing conditions prior to microarray analysis. Microarray studies were performed as previously described (4, 33) using at least two biological replicates of the NO·-treated WT and srrAB mutant samples.

qRT-PCR assay of gene expression.

To verify the microarray results, treated and untreated RNA samples from the microarray studies were analyzed by quantitative real-time RT-PCR. The Qiagen 1-step SYBR mix was used, preceded by an additional cDNA conversion step. Primer sets for the analyzed genes are listed in Table S2 in the supplemental material. qRT-PCR analysis was performed using a Rotor-Gene (Qiagen, Valencia, CA). Data were analyzed by comparing relative steady-state mRNA concentration of the gene of interest to that of the housekeeping rpoD gene, and fold induction during treatment with NO· was calculated.

For measurement of gene expression under hypoxic conditions, WT S. aureus COL and an isogenic srrAB mutant were grown aerobically with shaking in tryptic soy broth (TSB) medium to an OD660 of 0.5. Cells were then transferred either to a 15-ml conical tube, which was filled to the top with the cap fully tightened, or to a 5-ml glass tube and incubated with shaking for 30 min before mixing with an ice-cold (1:1) EtOH acetone mixture. All other RNA was prepared using the Qiagen mini-prep system with lysis via bead beating and analyzed via a 2-step RT-PCR process. RNA was converted to cDNA (QuantiTect, Qiagen) before cDNA was diluted 1:25 for use with the Qiagen FAST-SYBR kit. Data were analyzed as described above.

Strain construction.

Mutations in cydAB, nrdDG, scdA, qoxABCD, and srrB were constructed using the temperature-sensitive plasmid pBT2 (34), with primer pairs listed in Table S2 in the supplemental material and the ΩKm cassette from pUC-4 ΩKm2 (gift from Kevin McIver). All basic cloning was performed in E. coli DH10b, using ampicillin at 100 µg ml−1. The cydAB, nrdDG, scdA, and srrB fragments were amplified using L and R primers containing BamHI and XhoI restriction sites, and the qoxABCD fragment was amplified without any restriction sites (listed in Table S2), before the digested PCR product was cloned into vector pJK392 or a modified version (pTK392) in which the NdeI site was filled in and religated. The resulting constructs were digested using the following conditions: cydAB, EcoRI digested and filled in; nrdDG, HpaI-digested, which deletes an ~300-bp region; scdA and qoxABCD, NdeI digested and filled in; and srrB, HindIII digested and filled in. All were ligated with the SmaI-digested ΩKm fragment from pUC-4 ΩKm2. Each fragment containing cydAB, nrdDG, scdA, qoxABCD, or srrB with ΩKm was removed from pJK392 or pTK392 with KpnI and XbaI, and ends were filled in and then blunt ligated into the EcoRV site of the pBT2 shuttle vector. These plasmids, designated pTK1, pTK2, pTK3, pTK4, or pTK5, respectively, were purified from DH10b, transformed into S. aureus strain RN4220, and maintained using kanamycin at 100 µg ml−1 and chloramphenicol at 10 µg ml−1. From RN4220, plasmids were transduced into S. aureus strain COL (8) using bacteriophage phi-11 (35). Recombination events occurred following passage at the permissive temperature of 30°C and switching to 43°C, followed by serial passage at 30°C without selection to allow for double recombination. A cycloserine enrichment process was used to select Cms recombinants. Mutants were verified by PCR amplification of the flanking region and an internal primer for the ΩKm-cassette (see Table S2).

Double hmp mutants and mutations in UAMS-1 (srrAB and hmp) were constructed using phage transduction (phi-11) from RN4220 containing pTR43 (srrAB) and pTR40 (hmp). Similar passaging at 30°C and 43°C and cycloserine-enrichment protocols were followed as described above. When indicated, erythromycin was added at 5 µg ml−1.

Measurement of NO· susceptibility.

Cells were grown overnight in TSB and then diluted to an OD660 of 1.0 in PN medium. A further 1:30 dilution was made for each strain before being added to the Bioscreen C plate (Growth Curves United States, Piscataway, NJ) for incubation with maximal shaking at 37°C for 40 h with or without the addition of the NO·-donating compounds DEA/NO (1 mM) and NOC-12 (5 mM).

Measurement of hypoxic growth.

Colonies of each strain were isolated from tryptic soy agar (TSA) plates and inoculated into 9 ml of TSB. Subcultures were split into three equivalent 3-ml samples in 13- by 100-mm glass tubes. One tube was placed on a shaker (~250 rpm), one was incubated statically on a shelf, and one was placed inside an anaerobic jar with a BD BBL GasPak Plus envelope at 37°C for 24 h. A 24-h OD660 reading was taken using a Bio-Rad SmartSpec 3000.

Measurement of respiration inhibition by NO·.

To monitor the inhibition of respiration by NO·, both NO· and O2 levels were monitored using specific electrodes. To measure [NO·], an ISO-NO MarkII NO· meter (World Precision Instruments, Sarasota, FL) with an ISO-NOP NO· probe was used. To measure [O2], an MI-730 oxygen probe with the O2-ADPT oxygen adapter (Microelectrodes Inc., Bedford, NH) was used. The output from both probes was recorded using LABCHART software (ADInstruments, Colorado Springs, CO). Cells were grown in TSB to an OD660 of 1.0, upon which they were spun down in 25-ml aliquots, rinsed with PBS, and then repelleted. Cultures were kept on ice until assayed. For assays, cells were resuspended in PBS that had been bubbled with O2 for 30 min at 37°C. Upon resuspension, cells were immediately transferred to a beaker, where the probes were set to begin recording. One minute after transferring the cells, 20% glucose was added to a final concentration of 0.1% to stimulate respiration. When approximately 75% O2 remained, a 10 µM bolus of NO· was added (Proli-NO; half-life of 1.3 s). Recordings were carried out until all oxygen had been consumed or until 12 min after the addition of glucose.

Data were analyzed by determining the maximal point of NO· in the PBS control and calculating the fraction of NO· remaining for each strain tested. This experiment was repeated 3 independent times for each strain.

Biofilm growth and analysis.

Static biofilms were grown using conditions described previously, with WT S. aureus strain UAMS-1 and isogenic sarA, srrAB, and hmp mutants (13, 22). Briefly, for the crystal violet biofilm assays, 24-well tissue culture plates were used, and for confocal microscopy studies, sterile coverslips were placed into 6-well tissue culture dishes. The tissue culture dishes were precoated with 20% human plasma diluted in 0.1 M carbonate buffer (pH 9.6) for 24 h at 4°C. S. aureus UAMS-1 and isogenic mutant strains were grown overnight in TSB supplemented with 0.5% glucose and 3% sodium chloride. The overnight cultures were diluted 1:200 into either 1 or 5 ml of supplemented TSB. Plates were incubated at 37°C for 24 h without shaking. Each day, biofilms were carefully washed once with 1 or 5 ml sterile PBS to wash away nonadherent cells, and fresh medium was added. This process was continued for a total of 3 to 5 days. To quantify biofilm formation, crystal violet stain was eluted from the biofilms with 100% ethanol and diluted 3-fold prior to reading the absorbance at 595 nm on a plate reading spectrophotometer (Molecular Devices OptiMAX microplate reader).

Long-term static biofilms on coverslips were made as described (23). Coverslips were collected on the final day, and the biofilms were washed and stained with BacLight kit dyes SYTO-9 (1.3 µM) and propidium iodide (PI; 4 µM). The coverslips were then flipped onto glass slides and sealed with nail polish.

A Zeiss 510-Meta confocal laser scanning microscope with a 63× oil immersion Plan-Apochromat objective was used to visualize the biofilms. A 488-nm argon laser was used for excitation of SYTO-9 with the emission band-pass filter set at a wavelength of 515 ± 15 nm. For excitation of PI, a 543-nm HeNe laser was used, and band-pass filters for emission were set at 630 ± 15 nm. Images were analyzed using Zeiss Image Examiner and ImageJ software. All image collection was done at the WM Keck Center for Advanced Studies in Neural Signaling (University of Washington).

Statistical analysis.

Statistical analysis of results used Prism (GraphPad Software, La Jolla, CA). All Student’s t tests were unpaired with a two-tailed 95% confidence interval. NO· consumption over time was analyzed using a matched 2-way analysis of variance (ANOVA) with Bonferroni post-tests comparing each mutant strain to the WT over time.

Microarray data accession numbers.

Microarray data have been deposited into the GEO database under the manuscript title.

SUPPLEMENTAL MATERIAL

Figure S1

Effects of SrrAB and SrrAB-regulated genes on growth during nitrosative stress. Wild-type S. aureus UAMS-1 or isogenic srrAB (***) mutant (A) and wild-type S. aureus RN4220 or isogenic srrAB (*) and srrB (*) mutants (B) were grown in PN medium with (solid lines) or without (dotted lines) addition of 5 mM NOC-12 and 1 mM DEA/NO. Data shown are the averages from 4 independent experiments. Statistical significance was determined by Student’s t test comparing Δt (NO· treated minus untreated) to reach an OD600 of 0.3 compared to that of the wild type. Error bars = SEM (*, P < 0.05; and ***, P < 0.0005). Download

Figure S2

NO·-mediated respiratory inhibition in wild-type and mutant S. aureus COL. Representative tracings of oxygen and NO· concentrations from 1 of 3 independent experiments are shown. A 10 μM bolus of NO· was added (Proli-NO; half-life of 1.3 s) when approximately 75% of oxygen was remaining. Download

Figure S3

Biofilm profiles of wild-type or mutant S. aureus. Wild-type S. aureus UAMS-1 or isogenic srrAB or hmp mutants were grown in TSBGN on 20% plasma-coated coverslips in 6-well plates for 5 days. A minimum of 3 different coverslips were analyzed for each strain on each day. Biofilms were stained with SYTO-9 and propidium iodide and imaged with a Zeiss 510-Meta confocal microscope. Representative profiles, generated with LSM software, are shown for day 5. Download

Table S1

SrrAB-regulated gene expression in the wild type and srrAB mutant in various S. aureus strains. Selected SrrAB-regulated gene expression in S. aureus strains COL, UAMS-1, and Newman either under nitrosative or hypoxic stress. Data are presented as the ratio for srrAB/WT and have been normalized to gyrA expression. Results are the averages from 3 independent experiments. *, a different primer pair was used with UAMS-1 due to sequence differences in hmp

Table S2

Strains, plasmids, and primers used in this study.

ACKNOWLEDGMENTS

This work is supported by NIH grant AI39557 to F.C.F. and AI073780 to P.M.D.

We thank Kevin McIver and Mark Smeltzer for the gifts of plasmids and strains.

Footnotes

Citation Kinkel TL, Roux CM, Dunman PM, Fang FC. 2013. The Staphylococcus aureus SrrAB two-component system promotes resistance to nitrosative stress and hypoxia. mBio 4(6):e00696-13. doi:10.1128/mBio.00696-13.

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