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J Bacteriol. Feb 2011; 193(4): 952–962.
Published online Dec 17, 2010. doi:  10.1128/JB.01161-10
PMCID: PMC3028676

Pyruvate Formate Lyase Acts as a Formate Supplier for Metabolic Processes during Anaerobiosis in Staphylococcus aureus[down-pointing small open triangle]

Abstract

Previous studies demonstrated an upregulation of pyruvate formate lyase (Pfl) and NAD-dependent formate dehydrogenase (Fdh) in Staphylococcus aureus biofilms. To investigate their physiological role, we constructed fdh and pfl deletion mutants (Δfdh and Δpfl). Although formate dehydrogenase activity in the fdh mutant was lost, it showed little phenotypic alterations under oxygen-limited conditions. In contrast, the pfl mutant displayed pleiotropic effects and revealed the importance of formate production for anabolic metabolism. In the pfl mutant, no formate was produced, glucose consumption was delayed, and ethanol production was decreased, whereas acetate and lactate production were unaffected. All metabolic alterations could be restored by addition of formate or complementation of the Δpfl mutant. In compensation reactions, serine and threonine were consumed better by the Δpfl mutant than by the wild type, suggesting that their catabolism contributes to the refilling of formyl-tetrahydrofolate, which acts as a donor of formyl groups in, e.g., purine and protein biosynthesis. This notion was supported by reduced production of formylated peptides by the Δpfl mutant compared to that of the parental strain, as demonstrated by weaker formyl-peptide receptor 1 (FPR1)-mediated activation of leukocytes with the mutant. FPR1 stimulation could also be restored either by addition of formate or by complementation of the mutation. Furthermore, arginine consumption and arc operon transcription were increased in the Δpfl mutant. Unlike what occurred with the investigated anaerobic conditions, a biofilm is distinguished by nutrient, oxygen, and pH gradients, and we thus assume that Pfl plays a significant role in the anaerobic layer of a biofilm. Fdh might be critical in (micro)aerobic layers, as formate oxidation is correlated with the generation of NADH/H+, whose regeneration requires respiration.

Staphylococcus aureus, a facultative anaerobic bacterium, is multifaceted during colonization and infection, ranging from an asymptomatic nasal colonizer (17, 47) to an aggressive pathogen that leads to acute infection (18, 49). Many strains may also cause chronic or biofilm-associated infections (23), which are particularly difficult to treat because of increasing multiple antibiotic resistances and high tolerance to the immune defense in a biofilm (24). The tolerance of biofilm-embedded cells to various antibiotics is unclear but can probably be attributed not solely to the exopolysaccharide polysaccharide intercellular adhesin (PIA) (11, 25), which protects the cells; rather, resistance to antibiotics probably results from the altered physiology of biofilm cells. Within a biofilm, cells are exposed to various gradients of nutrients, oxygen, or pH; all of these parameters decrease with the depth of the layers. In comparative expression studies of planktonic and biofilm-grown S. aureus cells, it was shown that pfl/Pfl (pyruvate formate lyase), fdh/Fdh (NAD+-dependent formate dehydrogenase), and fhs (formyltetrahydrofolate synthetase) were upregulated at the transcriptional and proteome levels in biofilm (41, 42). It has also been reported that pfl is upregulated under anaerobic conditions (20).

The pfl gene is found in many bacteria, like Haemophilus influenzae, Clostridium spp. (27, 48), Lactobacillus spp. (1), and Streptococcus species (2, 13). However, the enzyme Pfl has been best studied in Escherichia coli, where it is involved in mixed-acid fermentation (30, 43, 44). Pfl catalyzes the reversible conversion of pyruvate to formate, thereby producing acetyl coenzyme A (acetyl-CoA) (4), which is important for energy supply when pyruvate is available. The PflB enzyme has to be activated in E. coli and related bacteria by a special Pfl activase (PflA) under strict anaerobic conditions (19). This is achieved by forming a glycyl-radical where S-adenosylmethionine and reduced flavodoxin serve as cofactors (8).

Soluble Fdh (EC 1.2.1.2) has been reported to occur in methylotrophic bacteria especially (40). In other microorganisms, however, the function has mostly remained unclear thus far. The Fdh enzyme catalyzes the oxidation of formate to carbon dioxide, thereby producing NADH.

Formyl groups, transferred by 10-formyl-tetrahydrofolate (formyl-THF), are important building blocks for a number of key biosynthetic bacterial processes. Bacteria start protein biosynthesis with a formylmethionyl (fMet) start tRNA, with the consequence that all newly synthesized proteins bear an N-terminally formylated methionine. Of note, innate mammalian immune systems can detect formylated peptides very efficiently with their formyl-peptide receptor 1 (FPR1), and the formylated peptides are used as hallmarks for invasive infections, since eukaryotic cytoplasmic ribosomes produce only unformylated peptides. FPR1-dependent detection of formylated peptides has major proinflammatory consequences, such as neutrophil influx and degranulation. The source of the formyl groups for the production of formylated proteins and peptides in anaerobically grown bacterial cells has hardly been studied before.

As little is known about Pfl and Fdh in staphylococci, the intention of this study was to investigate the roles of these enzymes under anaerobic conditions and their potential benefit in biofilm. We compared pfl and fdh deletion mutants with wild-type (wt) cells under various growth conditions. Our findings suggest that Pfl contributes significantly to the supply of formate, which is used via formyl-THF for protein and purine synthesis under anoxic conditions. Fdh most probably plays a role in the microaerobic area of the biofilm, where it contributes to the detoxification of formate and to NADH/H+ production, which can be used as fuel in the respiratory chain. The upregulation of pfl and fdh thus appears to be an important survival strategy in biofilm.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

Primers, strains, and plasmids used in this study are listed with their main characteristics in Tables Tables11 to to33 . All S. aureus mutant strains exhibit the SA113 (ATCC 35556) background. Basic medium (BM; 1% soy tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% K2HPO4, 0.1% glucose) was used during cloning procedures. If necessary, the medium was supplemented with ampicillin (Am; 100 mg/liter for E. coli), chloramphenicol (Cm; 10 mg/liter for S. aureus), kanamycin (Km; 30 mg/liter for E. coli or 15 mg/liter for S. aureus), erythromycin (Em; 2.5 mg/liter for S. aureus), or spectinomycin (Spc; 150 mg/liter for S. aureus).

TABLE 1.
Oligonucleotides used in this study
TABLE 2.
Bacterial strains used in this study
TABLE 3.
Plasmids used in this study

For growth studies, bacterial suspensions of overnight cultures were diluted to an optical density at 578 nm (OD578) of 0.1. To achieve conditions with decreased oxygen tension, cells were grown in closed Eppendorf tubes (2 ml), with a shaking rate of 150 rpm at 37°C and without addition of antibiotics.

Construction of the S. aureus Δpfl deletion mutant and complementation.

To generate a pfl mutant, the plasmid pKO1 (derivative of pBT2 [6; personal communication from B. Krismer]) was used. The pflB gene and 249 bp of the pflA gene were replaced by the erythromycin resistance cassette ermB, derived from pEC3. An upstream flanking region of the target gene (1.1 kb) was amplified by PCR with the primers pfl _KO_1F and pfl _KO_2R, and a downstream flanking region (1.0 kb) was amplified with the primers pfl _KO_3F and pfl _KO_4F. The upstream fragment was restricted with Acc65I and SalI and the downstream fragment with XbaI and HindIII. Both fragments were ligated along with the SalI- and XbaI-restricted ermB resistance gene (1.5 kb) into pKO1, which was digested with Acc65I and HindIII, respectively. The resulting plasmid, pKO1-pfl, was consecutively transferred into E. coli XL1-Blue, S. aureus strain RN4220, and finally S. aureus strain SA113. Allelic replacement of the wild-type pfl gene by erm in the reverse orientation was carried out as described in the legend of Fig. 1 A. This was confirmed by PCR, restriction digestion, and sequencing.

FIG. 1.
Scheme of the deletion concept of the pyruvate formate lyase (pfl) (A) and formate dehydrogenase (fdh) (B). Chromosomal regions of the wild type (upper panels) and the Δpfl and Δfdh deletion mutants (lower panel) are depicted. Dotted lines ...

The Δpfl mutant was complemented by cloning pflB and pflA under the control of their shared putative promoter into the vector pRB473 (7). The construct was used to consecutively transform E. coli XL1-Blue, S. aureus RN4220, and finally the deletion mutant S. aureus SA113 Δpfl (Fig. (Fig.1A1A).

Construction of the S. aureus Δfdh deletion mutant and complementation.

To replace fdh, we used the recently described pBT-fdh plasmid (45), a derivate of pBT2, as an allelic-replacement vector. Briefly, a fragment (1.1 kb) of the homologous upstream region of fdh was amplified by PCR using the primers fdh _KO_1F and fdh _KO_2R, which introduced the restriction sites EcoRI and BamHI, respectively. To provide a homologous downstream sequence, a PCR product of 1 kb generated with the primers fdh _KO_3F and fdh _KO_4R was restricted with SalI and EcoRV. A spectinomycin resistance cassette (1.3 kb) was obtained from plasmid pIC156 (46) via BamHI/SalI cleavage. The replacement vector was cloned in two steps. First, the downstream fragment was ligated into SalI- and EcoRV-cut pBT2; second, the vector obtained was restricted with SalI and EcoRI, and the upstream and resistant marker fragments were inserted to yield pBT2-fdh. The construct was used to transform S. aureus RN4220 and S. aureus SA113 by electroporation. Allelic replacement of the wild-type fdh gene with spc in the reverse orientation (Fig. (Fig.1B)1B) was carried out as described earlier (6). This was confirmed by PCR, restriction digestion, and sequencing. To complement the fdh deletion mutant, the fdh gene and the upstream gene encoding a hypothetical protein (SA0170) were cloned together under their own putative promoter into the vector pRB473, resulting in pRB-fdh. The construct was used to transform E. coli XL1-Blue, S. aureus RN4220, and finally the S. aureus SA113 Δfdh mutant (Fig. (Fig.1B1B).

Construction of the S. aureus Δfdh Δpfl double mutant.

To generate a pfl and fdh double mutant, the plasmid pBT2-pfl was introduced into the fdh deletion mutant. After allelic replacement, the genes pfl and fdh were replaced by the resistance markers spc and ermB, respectively.

Detection of the specific formate dehydrogenase activity.

Quantification was performed as described earlier (42). Briefly, cells were grown aerobically in BM supplemented with 0.67% glucose in flasks (1:8 culture-to-flask volume ratio) at 37°C and 150 rpm. After 24 h, cells were harvested, resuspended in 5 ml of buffer containing 5 mM MgCl2, 1 mM EDTA, and 0.8 M Tris-HCl (pH 7.6), and treated for 15 min with 50 μl lysostaphin (0.5 mg/ml; Sigma) at 37°C. Cells were disrupted three times by vortexing them for 30 s with 200 μg glass beads (diameter, 0.1 mm; Sartorius); in between, the suspensions were kept on ice. After centrifugation (10 min at 15,000 × g and 4°C), the supernatant was used for the detection of the formate dehydrogenase. Next, 250 μl of crude cell extract was used, and a buffer containing 10 mM NAD+ and 20 mM formate was added to a final volume of 1 ml. Enzyme activity was detected by an increase of the extinction at 340 nm at room temperature by using the extinction coefficient of NADH (epsilon = 6.3 mM−1 cm−1). Specific activity is expressed as units per mg of protein. Protein concentration was determined by the method of Bradford (5).

Preparation of crude protein extracts and SDS-PAGE.

Proteins were analyzed by 12% Laemmli SDS-PAGE (32). Samples were grown under decreased oxygen tension in tryptic soy broth (TSB; Sigma) with 0.5% glucose and harvested after 48 h. Cells were adjusted to the same OD and treated with lysostaphin to gain crude cell extracts. Proteins were visualized by staining cells with Coomassie brilliant blue R-250.

Measurement of glucose, acetate, lactate, and formate in culture supernatants in TSB via enzymatic reactions.

Bacterial suspensions of overnight cultures grown in TSB were diluted and grown under limited-oxygen conditions. After 3 h, 5 h, 8 h, 24 h, and 48 h, samples were taken and the bacteria were spun down. The culture supernatants were treated for 10 min at 80°C, filtered with a sterile filter (0.22 μm), and stored at −20°C until usage. The concentrations of corresponding metabolites were determined with kits purchased from R-Biopharm Inc. according to the manufacturer's instructions, except with the reduction of the reaction batch to 10% to adjust the assays to a microtiter plate scale. All UV assays were based on the increase of NADH or NADPH, which can be monitored spectrophotometrically at a wavelength of 340 nm. Defined dilutions of the metabolites were used to build a calibration curve.

Analysis of the extracellular metabolome in CDM.

Chemically defined medium (CDM [21]) includes, among other ingredients, alanine, arginine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, and valine at a concentration of 1 mM. For growth analysis, overnight cultures were diluted in prewarmed medium and grown under oxygen-limited conditions. The shaking rate was reduced to 100 rpm. Culture supernatants of cells were harvested after 4 h, 6 h, 8 h, 24 h, and 48 h by centrifugation (5 min at 4,500 × g) and heating (10 min, 80°C). Subsequently, the solution was filtered with a sterile filter and stored at −20°C. The extracellular metabolome was analyzed with 1H nuclear magnetic resonance (1H-NMR), which has recently been described (34). The relative difference was based on (theoretically) added amounts of substances and endpoint quantification (signal intensity relative to that of an internal standard).

Analysis of the extracellular metabolome and detection of fMet polypeptides in IMDM.

Bacterial cultures grown overnight in tryptic soy broth were used to inoculate lipopolysaccharide (LPS)-free Iscove's modified Dulbecco's medium (IMDM) without phenol red (Gibco) at an OD578 of 0.1.

Strains were grown under decreased oxygen tension. When required, 2 mM sodium formate (purissimum pro analysi [p.a.], ≥99.0%; Fluka) was added to the growth medium. For the metabolome analysis, culture supernatants were sampled after 8 h, 24 h, and 48 h, as described for CDM, and analyzed under the same conditions as described above.

Differences in the release of N-terminally formylated peptides were assessed by measuring FPR1-dependent calcium fluxes in human promyelocytic leukemia cells (HL60 cells), which were stably FPR1 transfected (10) upon stimulation with diluted bacterial culture supernatants. To prepare culture supernatants for these measurements, bacterial strains were cultivated either with decreased oxygen tension (as described under “Bacterial strains and growth conditions” above) or aerobically in flasks, with a 1:7 culture-to-flask volume ratio. In some samples, 2 mM sodium formate was added before cultivation. After incubation for 8 or 24 h at 37°C and at 150 rpm, all suspensions were adjusted to an OD578 of 1.0. To remove bacteria, suspensions were centrifuged and the culture supernatants were passed through a 0.22-mm-pore-size sterile filter and stored at −20°C. HL60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS; Sigma), 20 mM HEPES (Biochrom), 100 mg/ml streptomycin, 100 U/ml penicillin, and 2 mM GlutaMAX (Gibco) as described previously (35). The growth medium of transfected cells was supplemented with 1 mg/ml G418 (Invitrogen). In order to measure calcium ion fluxes, 106 cells/ml were mixed with the calcium-sensitive dye Fluo-3-AM (Molecular Probes) at a final concentration of 2 mM and incubated for 20 min at room temperature in RPMI 1640 medium (Biochrom) supplemented with 0.05% human serum albumin (RPMIeHSA). Subsequently, cells were mixed with diluted staphylococcal culture supernatants at final concentrations of 25% (vol/vol). The corresponding calcium fluxes were monitored after 15 s of incubation in a FACSCalibur flow cytometer (Becton Dickinson) as described recently (15, 35). Untransfected HL60 cells, IMDM, IMDM with 2 mM sodium formate, RPMI 1640 medium, and supernatants of RN4220 Δfmt (data not shown and reference 35) served as negative controls, whereas the synthetic formylated peptide formylmethionyl-leucyl-phenylalanine (fMLF; 10 nM, final concentration; Sigma) in RPMIeHSA was used as a positive control.

Construction of the S. aureus Δpfl ΔarcOp deletion mutant.

To generate a mutant in which the pfl gene and the complete arcABDCR operon (arcOp) were deleted, the plasmid pBT2-pfl was introduced into the marker-free arcOp deletion mutant described previously (33). Homologous recombination yielded the erythromycin-resistant Δpfl ΔarcOp double mutant.

Cloning of an additional arcOp.

To overexpress arcOp in S. aureus SA113, the entire native arcABCDR operon and its putative promoter were amplified by PCR using chromosomal wild-type DNA as a template. The PCR product was cleaved with PstI and SacI and ligated with the similarly restricted pRB473 plasmid.

Arginine deiminase activity.

The Api Staph assay (28) was used to monitor the arginine deiminase (ADH) activity.

Growth under an excess of arginine and RNA isolation.

Strains were grown under limited-oxygen conditions in TSB without glucose (1.7% casein peptone [AppliChem], 0.3% soy peptone A3 SC, 0.5% NaCl, 0.25% K2HPO4; pH 7.2 to 7.4) and in TSB without glucose but supplemented with 20 mM arginine-HCl (pH 7.2 to 7.4); the TSB may, however, contain traces of sugar present in the soy peptone. The absence of glucose is necessary to prevent catabolite repression, as described earlier for S. aureus (37).

To reach higher cell yields for isolating RNA, cells were grown in 50-ml reaction tubes completely filled with 50 ml TSB without glucose under a shaking rate of 130 rpm at 37°C. The medium, where denoted, was supplemented with arginine. After 4 or 8 h of cultivation, cells were killed by addition of 25 ml of approximately 4°C cold killing buffer (5 mM MgCl2, 20 mM NaN3, 20 mM Tris-HCl [pH 7.5]), harvested by centrifugation (10 min, 4,700 rpm, 4°C), washed, and stored at −70°C until usage. The isolation of total RNA was performed using an acid phenol method with some modifications (22, 36). Cells were disrupted mechanically with 0.5 ml glass beads (diameter, 0.1 mm; Sartorius) in 400 μl phenol-chloroform-isoamyl alcohol (25:24:1) using TissueLyser (Qiagen) for 1 min at 30 Hz. After centrifugation, the upper phase containing the total RNA was extracted with 600 μl phenol-chloroform-isoamyl alcohol (25:24:1) and two times with 600 μl chloroform-isoamyl alcohol (25:1, vol/vol). The RNA was precipitated with 96% ethanol and 3 M Na-acetate. The RNA was resuspended in deionized water. The integrity of the RNA was confirmed by agarose gel electrophoresis.

Northern blot analysis.

For the Northern blot analysis, an arcA RNA probe was prepared by in vitro transcription with T7 RNA polymerase using a PCR-generated fragment as the template. Afterward, the probe was labeled with digoxigenin (Roche). The PCR was performed using chromosomal DNA of S. aureus SA113 as a template and the primers arcA _probe_F and arcA _probe_R. By the same procedure, a pfl probe was generated using the primers pflB _probe-F and pflB _probe-R. The 5′ end of the reverse primers represent the T7 promoter. Denaturing 1.5% agarose gels (20 mM MOPS [morpholinepropanesulfonic acid], 5 mM sodium acetate, 1 mM EDTA, 1.85% formaldehyde [pH 7.0]) were used to separate 15 μg (arcA mRNA) or 10 μg (pflB mRNA) total RNA per well at 120 V in 1× MOPS buffer (20 mM MOPS, 5 mM sodium acetate, 1 mM EDTA [pH 7.0]). After electrophoresis, gels were incubated (5 min) with denaturing buffer (50 mM NaOH, 10 mM NaCl) and neutralized (5 min) with 0.1 M Tris-HCl (pH 7.4). The RNA was transferred to a positively charged nylon membrane with 20× SSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA [pH 7.4]) using a VacuGene XL machine. Blotting of the RNA was fixed to the membrane via UV cross-linking for 1 min. Successful transfer was verified by staining the membrane with methylene blue solution (10% [wt/vol] methylene blue, 0.4 M sodium acetate [pH 5.2], 2% [vol/vol] acetate) to detect the 16S and 23S rRNA bands as a loading control. The digoxigenin-labeled RNA probes were used for gene-specific hybridization according to the manufacturer's instructions (Roche). The hybridization signals were detected using Lumi-Film (Roche).

Computer sequence analysis.

MacDNASIS Pro v3.5 (Hitachi Software Engineering) as well as Clone Manager 9 (Scientific & Educational Software) were used.

Statistical analysis.

P values were determined using the paired Student t test.

RESULTS

Construction and characterization of the Δpfl mutant.

In S. aureus, pflB (SA0218) and pflA (SA0219) are oriented in tandem and separated by 23 bp, suggesting that they form an operon that ends with a transcription terminator structure (9.2 kcal) 22 bp downstream of the stop codon of pflA (Fig. (Fig.1A).1A). Genome annotation of the two genes suggests that PflB (749 amino acids [aa], 85 kDa) represents the proenzyme of Pfl, which can be activated by the introduction of a radical in the catalytic center by the activase PflA (251 aa, 28 kDa) (19).

Genes encoding a putative iron ABC transporter membrane-binding protein (SA0217) as well as a putative two-component sensor histidine kinase (SA0216, not depicted) are located upstream of pflBA, separated by 588 nucleotides (nt). Downstream at a distance of 322 bp is a gene encoding a putative glycerophosphoryl diester phosphodiesterase (SA0220).

In the SA113 Δpfl mutant, pflB and part of pflA (the first 249 bp of a total of 752 bp) were replaced with the ermB (erythromycin resistance gene) cassette as depicted in Fig. Fig.1A.1A. According to the results of SDS-PAGE, the two characteristic protein bands for PflB are absent in the Δpfl mutant but are overexpressed in the pRB-pfl-complemented strain (Fig. (Fig.1C).1C). The appearance of the double band was first observed in E. coli (29) but has also been observed in S. aureus (31). When the Pfl of E. coli, and probably of S. aureus, is radical activated by PflA, exposure to oxygen (during the preparation of the samples) leads to an irreversible cleavage of the enzyme. The 85-kDa band represents the complete Pfl; the 82-kDa band represents the oxygen-inactivated and cleaved form. The Δpfl mutant produced no formate, while the wt excreted up to 2.5 mM formate into the culture supernatant (Fig. (Fig.2A).2A). In the pRB-pfl (carrying the entire pflBA operon)-complemented strain, formate production was mostly restored.

FIG. 2.
Formate accumulation in the TSB culture supernatant of anaerobically grown cells. The accumulation of formate was measured in the culture supernatant of SA113, the Δpfl mutant, and the Δpfl(pRB-pfl) strain (A) and in the culture supernatants ...

Construction and characterization of the Δfdh mutant.

The fdh gene (SA0171) is followed by a transcription terminator (12.6 kcal) and is flanked by two genes of unknown functions. To construct an fdh deletion mutant, the gene was replaced by spc (the spectinomycin resistance cassette) (Fig. (Fig.1B).1B). In the complementation plasmid pRB-fdh, the fdh gene was cloned with the upstream SA0170 gene and its promoter-like sequence. The fdh gene is most probably transcribed starting from the SA0170 promoter because complementation of the fdh mutant was possible only when SA0170 and its upstream region were present.

Under aerobic growth conditions, specific Fdh activity was detectable in the cytoplasmic supernatants of the wt and the complemented mutant but not in supernatant of the Δfdh mutant [SA113, 3.5 ± 2.7 nmol/min/mg protein; Δfdh mutant, no Fdh activity; fdh(pRB-fdh) strain, 205.3 ± 59.8 nmol/min/mg protein; values represent the averages of results from at least eight independent experiments ± standard deviations].

We expected elevated formate production in the Δfdh mutant under anaerobic conditions because formate, as a Pfl reaction product, cannot be further oxidized to CO2. However, the Δfdh mutant excreted the same amount of formate as the wt in TSB (Fig. (Fig.2B).2B). This suggests that the Fdh activity of the wt has little impact on further formate oxidation under anaerobiosis. Only when Fdh was overexpressed in the presence of the complementation plasmid pRB-fdh (Fig. (Fig.1C)1C) was the formate content in the supernatant significantly decreased (Fig. (Fig.2B).2B). No formate was produced in the Δpfl Δfdh (Fig. (Fig.2B)2B) and Δpfl ΔarcOp (Fig. 7B) double mutants; as shown later, both the transcription of arcOp (the arginine deiminase operon) and enzyme activity were enhanced in the Δpfl mutant.

In TSB medium (12.5 mM glucose) under oxygen limitation conditions, no growth differences between the wt, the mutants, and the complemented mutants [the Δpfl, Δfdh, Δpfl Δfdh, Δpfl(pRB-pfl), and Δfdh(pRB-fdh) strains] could be observed during 48 h of cultivation; the OD578 was always in the range of 3.0 (data not shown). All strains produced l- and d-lactate as well as acetate in comparable amounts.

Addition of formate can largely complement the Δpfl mutant.

Extracellular fermentation products were also determined in IMDM in metabolome studies. IMDM is a highly enriched LPS-free synthetic medium which includes an excess of glucose (25 mM, approximately 2 times more than in TSB). The most obvious differences in fermentation patterns and glucose consumption were observed between the wt and Δpfl strains. In the Δpfl mutant, growth was retarded at 6 and 8 h (P < 0.001, n = 5) and 10 h (P < 0.01, n = 4) (SA113 versus the Δpfl mutant), glucose consumption and acidification were markedly delayed, ethanol production was decreased, and, as expected, formate was not produced (Table (Table4).4). In the Δpfl(pRB-pfl) complemented strain, the wt phenotype was largely restored. A very interesting observation was that the addition of formate (2 mM) to the Δpfl mutant during cultivation also largely restored the wt phenotype. Apparently, the addition of formate has a positive effect on midexponential growth, glucose consumption, and the increase of ethanol production. As shown later, formate serves as a substrate in the biosynthesis of formylated proteins.

TABLE 4.
Accumulated fermentation products, glucose consumption, pH of culture supernatant, and OD578 after anaerobic growth in IMDMa

Serine, threonine, and arginine were consumed faster in the Δpfl mutant.

In contrast to IMDM, the CDM that we used contains only 7.5 mM glucose and the added amino acids are present in higher concentrations (1 mM each). Furthermore, CDM does not contain a high content of organic buffer that interferes with 1H-NMR measurements. Therefore, CDM is better suited for determining extracellular amino acid consumption by metabolome analysis. Among all the added amino acids, only arginine, serine, and threonine (see Materials and Methods) were consumed more quickly by the Δpfl mutant (Fig. (Fig.3).3). For a control, we also tested the previously described ΔarcOp mutant (33), which was unable to consume arginine. Since CDM is very poor in nutrients, growth is limited. The maximum OD578s were reached after 10 h, with wt and Δfdh values of 0.6 (24 h/48 h, 0.5), whereas the Δpfl mutant (OD578 = 0.7; 24 h/48 h, 0.6) grew slightly better and the ΔarcOp mutant (OD578 = 0.4; 24 h/48 h, 0.4) somewhat less than the wt. Regarding arginine, serine, and threonine consumption, no difference was found between the wt and the Δfdh mutant, which also held true for the rate of glucose consumption, as well as the rate of formate production and the degradation profile (Fig. (Fig.44 A and C). Unlike the wt, the Δpfl and Δpfl Δfdh mutants showed no formate accumulation in the culture supernatant (Fig. (Fig.4B).4B). In a rich medium, such as TSB and IMDM, formate accumulated in the wt and the Δfdh mutant and stayed at the same level over at least 48 h (Fig. (Fig.22 and Table Table4).4). However, in defined and poor media, such as CDM, formate accumulated in the first 8 h and was no longer detectable in the next 24 to 48 h (Fig. (Fig.4A).4A). Furthermore, there was no difference with regard to fermentation products due to the low carbon flow through the Pfl (data not shown).

FIG. 3.
Differences in the levels of amino acid consumption after anaerobic growth in CDM detected by 1H-NMR. In the upper section, the conversion of arginine (black) to ornithine (white) is shown in the culture supernatants of SA113 (A), the Δpfl mutant ...
FIG. 4.
Glucose consumption ([filled lozenge]) and formate accumulation (□) in culture supernatants as detected by 1H-NMR. Samples were taken at the indicated times during growth under oxygen limitation in CDM. The measurements with SA113 (A), the Δpfl ...

Growth, pH profile, and arcABDCR transcription under an excess amount of arginine.

As shown above, the Δpfl mutant consumes arginine much faster than the wt. To answer the question of whether enhanced arginine catabolism affects growth, we compared the growth rates and pH courses of the wt, the Δpfl mutant, the ΔarcOp mutant, and the Δpfl ΔarcOp double mutant (Fig. 5A and B). Since arcOp is glucose repressed (37), we used TSB without glucose but enriched with 20 mM arginine. Until 24 h, no difference was seen in levels of growth and pHs. A marked difference was observed in the late stationary growth phase from 24 to 96 h. After 24 h, the Δpfl mutant started to regrow after a lag phase (diauxie) of approximately 16 h; the wt behaved similarly, but the lag phase was much longer. The regrowth of both strains was correlated with an increase of pH in the medium. Regrowth and alkalization after the lag phase can be explained by the onset of the catabolism of arginine catabolism, which generates ATP and NH3. In contrast to the wt and the Δpfl mutant, the ΔarcOp and Δpfl ΔarcOp mutants were unable to regrow or to alkalize the medium.

FIG. 5.
Microaerobic growth in the presence of arginine. Cells were grown in TSB without glucose but enriched with 20 mM arginine. Optical densities (A) as well as the pH values (B) of SA113 and the Δpfl, ΔarcOp, and Δpfl ΔarcOp ...

Northern blots were carried out with all strains, and SA113(pRB-arcOp) was included as an arcOp-overexpressing strain (see Fig. 8A) to test the transcription of arcOp. In the Δpfl mutant, the 5,800-nt transcript for arcOp was enhanced and was very prominent in the arcOp-overexpressing strain. The ΔarcOp mutants showed no transcript, as expected. Northern blotting was carried out with cells harvested after 4 and 8 h, as, in later growth phases, RNA degradation was observed.

We also assayed pfl transcription in this medium by Northern blotting. As expected, pfl was transcribed in the wt and the ΔarcOp mutant. No pflB expression was detected in the Δpfl mutant and the Δpfl ΔarcOp double mutant (Fig. (Fig.66 B). However, pfl transcription was markedly enhanced in the arcOp-overexpressing strain SA113(pRB-arcOp). We assume that ArcR is a positive regulator both of its own operon and, when overexpressed, of the pflBA operon, which is in line with a recent observation that the pfl upstream regulatory region carries a Crp-like consensus binding site (37).

FIG. 6.
Northern blot analysis directed against arcA (A) and pflB (B). Lanes in the upper panels display transcript amounts obtained from SA113, the Δpfl mutant, the ΔarcOp mutant, the Δpfl ΔarcOp double mutant, and, as a positive ...

The Δpfl mutant produces less-formylated polypeptides.

To verify the role of Pfl as a formate supplier for biosynthetic pathways depending on one-carbon-unit synthesis (27), the amounts of formylated peptides produced by the wt and mutant strains were compared. The biosynthesis of formylated peptides, purine bases, and other bacterial molecules is dependent on formyl-THF, which can be generated via the oxidation of methylene-THF (serine or glycine utilization) or by formate-THF synthetase (Fhs), ligating THF with formate during ATP consumption. The amount of formylated peptides released by bacterial cells was assessed by measuring responses such as calcium ion fluxes in FPR1-transfected HL60 cells exposed to bacterial culture supernatants (10, 12).

The experiments revealed no differences in the stimulating activities of the culture supernatants of the wt, the Δpfl mutant, and the complemented mutant (pRB-pfl) when harvested after 24 h of aerobic growth. This finding is consistent with our expectations, since Pfl is a strictly anaerobic enzyme that is rapidly inactivated when exposed to oxygen. However, after 24 h of anaerobic growth, a 50% decrease in the stimulating capacity of culture supernatants was detected in the Δpfl mutant in comparison to that of wt supernatants (Fig. (Fig.7A).7A). Culture supernatants of the Δpfl ΔarcOp mutant were included as additional controls and showed the same reduction in calcium mobilization as Δpfl mutant supernatants. Stimulation to the wt level could be restored either by complementation of the Δpfl mutant or by supplementation of the growth medium with 2 mM formate (Fig. (Fig.7).7). Externally added formate did not stimulate the FPR1-transfected HL60 cells or enhance the stimulation level of wt supernatants. These results show the importance of Pfl as a formate supplier for the production of formylated peptides under conditions of anaerobic growth.

FIG. 7.
(A) Calcium flux as a response of HL60 cells stably transfected with FPR1 to culture supernatants of SA113, the Δpfl mutant, the Δpfl(pRB-pfl) strain, and, as an additional control, the ΔarcOp Δpfl mutant. HL60 cells were ...

DISCUSSION

In previous studies, it has been shown that under biofilm conditions, genes of formate metabolism, including pflA and pflB (7-fold) as well as fdh (17-fold) and fhs (3-fold), were significantly upregulated compared to their expression under conditions of planktonic growth (42). The enhanced transcriptional expression is also reflected at the protein level, where PflB is enhanced 3-fold under biofilm conditions (41). To investigate the benefits of pfl and fdh in biofilm and in metabolism generally, we generated deletion mutants and compared these mutants with the wild type.

In contrast to the wt, the fdh mutant showed no Fdh activity in crude extract, but the mutation had no apparent effect on growth under aerobic and anaerobic conditions. Formate production profiles differed with the medium used. Under anaerobic conditions in rich medium, such as TSB, formate accumulated up to 2.5 mM and remained more or less at this level in both the wt and the fdh mutant (Fig. 2A and B). In CDM, there was also no difference between the wt and the fdh mutant; however, the accumulated formate was much lower (0.3 mM) and declined after 8 h (Fig. (Fig.4).4). We assume that in nutrient-poor media, the excreted formate is reused for the biosynthesis of formyl-tetrahydrofolate, which is needed for purine and protein synthesis, but that in rich medium, the glucose and amino acid content is much higher, leading to a larger amount of formate. Formate was not produced under aerobic conditions.

First we thought that the pfl mutant would accumulate less acetate but more lactate because no acetyl-CoA can be formed and therefore that fermentation could be shifted to lactate. However, in the pfl mutant, acetate formation was only marginally affected, ethanol was decreased only 3-fold under glucose excess (IMDM), and formate was not produced at all in any of the media tested. No enhanced lactate production was observed, as described for pfl mutants of E. coli (50). Another question is how ethanol or acetate is produced in the pfl mutant. The most likely candidate is pyruvate dehydrogenase (Pdh), but one cannot rule out the involvement of amino acid metabolism and fatty acid degradation. Normally, Pdh should be active only under aerobic conditions, as its activity is inhibited by high NADH levels. On the other hand, in Bacillus subtilis, which lacks pfl in its genome, Pdh provides acetyl-CoA also under anaerobic conditions (38).

Exometabolome studies of CDM-grown S. aureus indicate that the lack of pfl in the mutant can be compensated for by an enhanced utilization of serine, threonine, and arginine. We propose the following assumption, illustrated in Fig. Fig.8A.8A. The catabolic enzymes involved might be the threonine aldolase (SAOUHSC_01307, LtaA [EC 4.1.2.5]) and serine hydroxymethyltransferase (SAOUHSC_02354, GlyA [EC 2.1.2.1]), which are both encoded in the S. aureus genome. GlyA, which appears to be essential in S. aureus (16), catalyzes the conversion of serine to glycine and thereby provides C1 units via methylene-THF. LtaA converts threonine to glycine and acetaldehyde (not indicated). In both cases, glycine can be further converted via the glycine cleavage system (Gcv) to methylene-THF and finally to formyl-THF. The glycine cleavage system involves the glycine dehydrogenase (SAOUHSC_01632-01633, GcvPB), an aminomethyltransferase T (SAOUHSC_01634, GcvS), and a dihydrolipoamide dehydrogenase (SAOUHSC_01043) (not shown). This putative C1-THF synthesis of the pfl mutant is described to occur normally for bacteria under aerobic conditions. The side effects are obvious, since buildup of methylene-THF and formyl-THF from glycine leads to the formation of 1 equivalent of NADH and NADPH. This might be the reason why the wt under anaerobic conditions converts serine to pyruvate via serine-dehydratase (SdaAA). Based on metabolome analysis, the decrease of intracellular serine was correlated with an increase of pyruvate (unpublished data by M. Lalk, University of Greifswald). The formation of NADH causes an adverse NADH/NAD+ ratio, which has been described as being recognized by the Rex regulator, and transcription of the arc operon is derepressed (39). In agreement with this theory, arginine consumption is increased (Fig. (Fig.3B)3B) and arc transcription is also enhanced (Fig. (Fig.6A).6A). ATP is thereby produced and might help even by using traces of formate to form formyl-THF. While the pfl mutant needs to increase serine and threonine catabolism in order to produce sufficient formyl-THF, in the wt the Pfl produces sufficient formate to directly produce formyl-THF via Fhs. The anabolic function of Pfl in building up C1 units for formyl-THF biosynthesis was first described for Clostridium kluyveri (27). In E. coli, Pfl appears to be used preferentially in catabolism, as E. coli does not possess Fhs activity (14).

FIG. 8.
Schematic hypothesis based on observed metabolic and calcium flux differences (red) in the Δpfl mutant. (A) Differences in the levels of amino acid catabolism observed in CDM. The consumption of serine, threonine, and arginine was enhanced in ...

We also asked whether formate represents the formyl-group donor for the biosynthesis of fMet polypeptides under anaerobic conditions, which depends on the formylation of the start methionyl tRNA by formyl-THF in all bacteria. As the amounts of formyl-THF can hardly be monitored by common analytical methods, we quantified the end products, formylated peptides, by a sensitive bioassay. This assay is based on the observation that formylated peptides lead to intracellular accumulation of Ca2+ in human leukocytes at picomolar concentrations via the FPR1 receptor. Formate cannot be generated directly from glucose, as in the wild type, but it is most probably generated from amino acids in the pfl mutant. Indeed, we found that stimulatory capacity decreased by 50% in pfl mutant supernatants compared to that in wild-type supernatants (Fig. (Fig.7A7A and and8B),8B), which could be restored by supplementation of the growth medium with formate, indicating the importance of formate as a prerequisite for the production of a normal level of formylated peptides. In this context, it would be interesting to know whether formate also leads to the formylation of Pdh and whether formylation affects Pdh activity. We assume that Pdh activity is positively affected by formylation, because when formate was added to the Δpfl mutant, the ethanol production was also enhanced almost to the wt level (Table 5). Furthermore, in a proteome analysis with Bacillus subtilis, it has been shown that in at least one enzyme of the Pdh, PdhD, the formyl group was retained (3).

The reason to study the physiological roles of fdh and pfl in more detail goes back to the previous observation that both genes were significantly upregulated under biofilm conditions compared to under conditions of planktonic growth. Most of our studies were carried out under planktonic conditions, but we also studied biofilms formed on a dialysis membrane. However, the problems with the latter approach were that the biofilm cells were extremely sticky and hard to separate and that in CDM medium anaerobic growth was very weak and almost no biofilm was formed. Therefore, biofilm-related survival or metabolome studies were technically hardly feasible. However, we anticipate that the results obtained with liquid medium can be transferred to the biofilm situation, as shown in Fig. Fig.9.9. According to this model, upregulation of pfl takes place in the deeper layer of the biofilm, where anoxic conditions are prevalent (Pfl is oxygen sensitive) and nutrients are limited. Under these conditions, Pfl is necessary, as it allows the formation of C1 units (formate) for formyl-THF synthesis and, consequently, for protein and purine biosynthesis. Interestingly, not only fdh and pfl, but also the formyl-THF synthetase gene (fhs), were upregulated under biofilm growth (42). We found no conspicuous phenotype of the fdh mutant under anaerobic or aerobic planktonic conditions (data not shown). Therefore, we suggest that the observed upregulation of fdh is biofilm specific, which is also in line with a 7.5-fold-enhanced Fdh activity in biofilm-grown cells (42). Our hypothesis is that Fdh plays a role in the microaerobic area of the biofilm. Formate is produced in the anaerobic area by Pfl and diffuses to the microaerobic region, where it is oxidized by Fdh to produce CO2 and NADH/H+. This has two advantages: first, formate is detoxified, and second, in the presence of small amounts of oxygen, NADH/H+ can be respired and no longer comprises a burden for S. aureus. The importance of Pfl under anoxic conditions lies in its ability to supply the cells with sufficient formate, which is used via formyl-THF for protein and purine synthesis. The consequence is that in the pfl mutant, much less fMet-polypeptides were produced than in the wt. Because of the mentioned benefits of functional pfl, fdh, and fhs, we conclude that the upregulation of these genes might be an important survival strategy in biofilm.

FIG. 9.
Schematic representation of formate metabolism in S. aureus biofilms. In the anaerobic layers (red) of the mature biofilm, the PFL converts pyruvate to acetyl-CoA and formate. The latter can be used by strictly anaerobically grown cells for the synthesis ...

Acknowledgments

We thank Bernhard Krismer for providing the plasmid pKO1 and discussion, Melanie Kull for advice and discussion, and Ralf Rosenstein for critical reading of the manuscript. For technical assistance, we thank Nele Nikola, Detlinde Futter-Bryniok, and Anne Winter.

This work was supported by the funding networks of the German Research Council (grant SFB-TR34 to F. Götz, A. Peschel, and M. Leibig and grant SFB685 to A. Peschel). M. Liebeke was a recipient of a fellowship from the Alfried Krupp von Bohlen and Halbach-Stiftung (Functional Genomics Approach in Infection Biology).

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 December 2010.

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