INTRODUCTION

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Isoprenoids, which are ubiquitous in nature, comprise a family of over 23,000 products, each composed of repeating five-carbon, isopentenyl diphosphate (IPP) subunits. The principal products of IPP in bacteria include the lipid carrier undecaprenol, which is involved in cell wall biosynthesis (33), menaquinones and ubiquinones involved in electron transport (29), and carotenoids (19). Two pathways for the biosynthesis of IPP have been described, the mevalonate pathway (16) and the glyceraldehyde-3-phosphate (GAP)-pyruvate pathway (36, 37). Analysis of the distribution of the genes encoding enzymes involved in the two pathways revealed that Bacillus subtilis and many gram-negative bacteria, including Escherichia coli, Haemophilus influenzae, and Helicobacter pylori, possess only genes that encode the GAP-pyruvate pathway, while the low-G+C gram-positive cocci and Borrelia burgdorferi possess only genes that encode the mevalonate pathway (41). However, the functionality of the implied polypeptides of the mevalonate pathway in bacteria has not been demonstrated.

Enzymes involved in the mevalonate pathway in a number of organisms, including humans, have been isolated and studied. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) is the best-characterized and rate-limiting enzyme of the pathway (9) and is the target of the statin class of cholesterol-lowering drugs (1). Based on amino acid sequence analysis, Bochar et al. (8) suggested that there are two distinct classes of HMG-CoA reductase. Genes that appear to encode class I HMG-CoA reductases are present in all eukaryotes, in many archaea, and in some streptomycetes. By contrast, genes that encode class II forms of the enzyme are present in some eubacteria and in the archaeon Archaeoglobus fulgidus (41). Previously characterized class I HMG-CoA reductases include those of the archaea Haloferax volcanii (6) and Sulfolobus solfataricus (7, 22), the gram-positive eubacterium Streptomyces sp. strain CL190 (40), and numerous eukaryotes. Characterized class II HMG-CoA reductases include that from A. fulgidus (23) and the biodegradative Pseudomonas mevalonii enzyme (4, 20), whose structure has been determined (24, 39) and which converts mevalonate to HMG-CoA, permitting growth on mevalonate (15). No biosynthetic eubacterial class II HMG-CoA reductase has, however, been characterized.

Biosynthetic HMG-CoA reductases catalyze reaction 1, the reductive deacylation of the thioester group of HMG-CoA to the primary alcohol of mevalonate using 2 mol of NADPH (35). The putative intermediates, mevaldyl-CoA and mevaldehyde, remain bound during the course of the reaction. HMG-CoA reductases also catalyze three additional reactions. Reactions 2 and 3 of free mevaldehyde appear to model the second reductive stage and the reverse of the first reductive stage of reaction 1, respectively. Reaction 4, the oxidative acylation of (R)-mevalonate to (S)-HMG-CoA, is the reverse of reaction 1. 

(1)

(2)

(3)

(4)

where CoASH is reduced coenzyme A.

We report here that the mvaA gene of the gram-positive pathogen Staphylococcus aureus is essential for growth, and we report the cloning, purification, and characterization of the mvaA gene product of S. aureus, the first truly biosynthetic class II HMG-CoA reductase characterized from a eubacterium.

	MATERIALS AND METHODS

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Reagents. Purchased reagents included restriction enzymes, calf intestinal alkaline phosphatase, T4 DNA ligase, protein and DNA molecular weight markers (Gibco BRL Life Technologies), Pfu Turbo DNA polymerase (Stratagene), and lysostaphin (Applied Microbiology, Inc.). Fluvastatin was a gift from Novartis. Unless otherwise specified, all other chemicals were from Sigma.

Plasmid, bacterial strains, and culture media. Expression plasmid pET28 was from Novagen. Bacterial strains used included E. coli BL21(DE3) and DH5 and S. aureus strains RN4220 (31), WCUH29 (NCIMB 40771), and NCTC 8325-4. Luria-Bertani broth and agar (38) served for growth of E. coli, and tryptone soy broth (TSB) and tryptone soy agar (TSA) (Oxoid) served for growth of S. aureus. Where required, the E. coli medium was supplemented with 50 µg of kanamycin per ml and 1% (wt/vol) glucose and the S. aureus medium was supplemented with 0.5 µg of erythromycin and 5 µg of tetracycline per ml.

DNA techniques. Plasmid DNA was isolated using the RPM kit (Bio 101 Inc.) or the Wizard Midiprep DNA purification system (Promega). PCR products were isolated by horizontal agarose gel electrophoresis and purified using the GENECLEAN II kit (Bio 101 Inc.). Chromosomal DNA was isolated from S. aureus using standard procedures (27). Incubation with 0.1 µg of lysostaphin per ml was included during the preparation of plasmid and chromosomal DNA from S. aureus to facilitate cell lysis. Procedures for DNA restriction, dephosphorylation and ligation, agarose gel electrophoresis, PCR, transformation of CaCl₂-competent E. coli, and phage transduction were performed as described by Sambrook et al. (38) or as recommended in the manufacturers' instructions. PCR employed a RoboCycler gradient temperature cycler (Stratagene). Synthetic oligonucleotides were prepared, and automated DNA sequencing was performed in-house at SmithKline Beecham.

Determination of essentiality of the mvaA gene. A DNA construct for allelic replacement of the S. aureus mvaA gene was generated by an overlap three-piece PCR technique. This technique is a gene fusion procedure (42) extended to include two separate long flanking sequences (547 bp directly upstream and 591 bp directly downstream of the mvaA gene) surrounding a central selectable cassette containing the 1,234-bp ermC gene from pE194 (17). The final full-length products were digested and cloned into the BamHI site of a pBluescript II KS(+) derivative containing the tetracycline resistance gene (tetK) from pT181 (21). The resulting plasmids were introduced into S. aureus RN4220 by electroporation with selection for resistance to erythromycin. All of the colonies isolated, which were also resistant to tetracycline (cointegrants), were examined for target-specific plasmid integration by diagnostic PCR with primer pairs based on plasmid- and locus-specific sequences. Bona fide plasmid cointegrants were transferred back to S. aureus RN4220 and NCTC 8325-4 by phage 11 transduction to allow for a second recombination event that could potentially resolve to generate an allelic-replacement mutation. Transductants resistant to erythromycin and sensitive to tetracycline were examined for allelic replacement by PCR.

Murine hematogenous pyelonephritis infection model. Cells from overnight cultures of S. aureus NCTC 8325-4 and the mvaA null mutant grown in TSB with 10 mM mevalonate as required were centrifuged, washed twice in sterile phosphate-buffered saline, and adjusted to an A₆₀₀ of 0.2. Female CD-1 mice weighing 18 to 20 g (Charles River, Quebec, Canada) were inoculated by tail vein injection with 0.2 ml of a suspension containing approximately 10⁷ bacteria. Mice were monitored twice daily for signs of illness. All animals were euthanatized by carbon dioxide overdose 5 days after inoculation. Both kidneys were removed using an aseptic technique and homogenized together in 1 ml of PBS, and the number of viable bacteria was determined by plating on TSA, which was supplemented with 10 mM mevalonate for the mvaA null mutant.

Construction of the HMG-CoA reductase expression plasmid. The mvaA gene was amplified from S. aureus WCUH29 chromosomal DNA using primers which introduced NheI and BamHI sites at the ends of the amplified fragment. The 1,278-bp PCR fragment was cloned using the pCR-Zero Blunt cloning kit (Invitrogen) and, using the introduced restriction sites, was subcloned into pET28 in frame with the N-terminal histidine tag and thrombin cleavage site to form pJO1. The insert was sequenced to verify that no errors had been introduced during PCR amplification.

Expression and purification of HMG-CoA reductase. E. coli BL21(DE3) cells containing pJO1 were grown to mid-log phase, induced by addition of 1 mM isopropyl--D-thiogalactopyranoside (IPTG), and harvested 2.5 h postinduction. To confirm expression, total cell lysates were prepared as outlined by Sambrook et al. (38) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purification of HMG-CoA reductase employed a 500-ml culture of E. coli BL21(DE3) transformed with pJO1. Following induction, the cells were harvested by centrifugation and HMG-CoA reductase was purified using the HisTrap kit (Amersham Pharmacia Biotech) essentially as described in the manufacturer's instructions, but with the following modifications. Cell lysis was performed by addition of 0.2 mg of lysozyme per ml and incubation for 20 min at 37°C. Lysates were frozen in a dry ice-ethanol bath, thawed, and sonicated three times for 10 s, with 10 s between bursts. The freezing-and-sonication procedure was repeated four times. Protein was eluted from the column with 10, 50, 100, and 500 mM imidazole. Purified HMG-CoA reductase in a solution of 50 mM KH₂PO₄ (pH 7.5), 200 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM EDTA, and 50% (wt/vol) glycerol was stored at 20°C. Protein concentrations were determined by using the Bradford assay (10) using bovine serum albumin as a standard.

Assays of HMG-CoA reductase activities. Spectrophotometric assays of HMG-CoA reductase activity employed a Hewlett-Packard model 8452 diode array spectrophotometer whose cell compartment was maintained at 37°C during measurements at 340 nm of the oxidation or reduction of NAD(P)H. Assays were conducted in a final volume of 200 µl. Standard assay conditions for each reaction studied were as follows.

Reaction 1, reductive deacylation of HMG-CoA to mevalonate. Assays using NADPH as the coenzyme contained 0.25 mM NADPH, 0.25 mM (R,S)-HMG-CoA, 50 mM NaCl, 1 mM EDTA, 5 mM DTT, and 25 mM KH₂PO₄ (pH 7.5). Assays using NADH as the coenzyme contained 0.25 mM NADH, 0.25 mM (R,S)-HMG-CoA, 50 mM NaCl, 1 mM EDTA, 5 mM DTT, and 25 mM KH₂PO₄ (pH 6.0).

Reaction 2, reduction of mevaldehyde to mevalonate. Assays contained 0.5 mM NADPH, 8.0 mM (R,S)-mevaldehyde, 50 mM NaCl, 1 mM EDTA, 5 mM DTT, and 25 mM KH₂PO₄ (pH 7.0).

Reaction 3, oxidative acylation of mevaldehyde to HMG-CoA. Assays contained 0.5 mM NADP⁺, 5.0 mM coenzyme A, 8.0 mM (R,S)-mevaldehyde, 50 mM NaCl, 1 mM EDTA, 5 mM DTT, and 100 mM Tris-HCl (pH 9.0).

Reaction 4, oxidative acylation of mevalonate to HMG-CoA. Assays contained 5 mM NADP⁺, 5.0 mM coenzyme A, 6.0 mM (R,S)-mevalonate, 50 mM NaCl, 1 mM EDTA, 5 mM DTT, and 100 mM Tris-HCl (pH 9.0).

	RESULTS

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Essentiality of the mvaA gene in S. aureus. The genes involved in the synthesis of IPP via the mevalonate pathway are essential for in vitro growth in Streptococcus pneumoniae (41). Therefore, it was of interest to determine whether S. pneumoniae is representative of the gram-positive cocci and if HMG-CoA reductase is essential in other pathogens. Attempts were made to delete mvaA from the chromosome of S. aureus using an allelic-replacement mutagenesis strategy. For nonessential genes the cointegrant can be readily resolved using phage transduction to isolate the mutant of interest at a frequency of 0.5 to 5%. In this case, however, it proved impossible to resolve the cointegrant structure and isolate a mutant on standard growth media, strongly suggesting that mvaA is essential for in vitro growth.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Chromosomal analysis of the S. aureus mvaA null mutant. (A) The chromosome of the mvaA null mutant contains an allelic replacement of mvaA (dotted block arrow) with the ermC gene (gray block arrow). PCR primers (small arrows) were designed to anneal to regions flanking mvaA. (B) Products of the expected size were amplified using chromosomal DNA from the mutant (2,288 bp [lane 3]) or wild type (2,371 bp [lane 8]). Replacement of mvaA with ermC resulted in the elimination of two HindIII sites and the introduction of three SspI sites in the chromosome. Amplified fragments were digested with either HindIII or SspI, and products of the expected size were obtained. Lane 1, the mutant PCR product incubated with HindIII (undigested); lane 2, the mutant PCR product cut with SspI (1,518 and 659 bp and two smaller bands); lane 6, wild-type PCR product cut with HindIII (894, 776, and 618 bp); lane 7, wild-type PCR product incubated with SspI (undigested); lanes 4 and 5, 100-bp and 1-kb DNA markers, respectively.

Auxotrophy of mvaA null mutants. The minimal concentration of mevalonate required for the growth of the S. aureus mvaA null mutant was investigated in TSA containing mevalonate at 0.01, 0.1, and 1 mM and in TSA without supplementation. Addition of mevalonate to the medium enabled the mutant to grow, although the rate of growth was lower at lower concentrations of mevalonate. In the absence of added mevalonate, the mutant showed no growth after 60 h on rich medium (TSA).

Virulence attenuation of the mvaA null mutant. The mvaA mutation was transferred to the pathogenic strain S. aureus NCTC 8325-4 by phage 11 transduction. The resulting mutants were auxotrophic for mevalonate. Five mice were inoculated intravenously with either S. aureus NCTC 8325-4 or the mvaA null mutant and sacrificed 5 days postinfection (none of the mice died during the infection). Viable S. aureus NCTC 8325-4 harboring the deletion could not be recovered from the kidneys (i.e., the level was below the limit of detection of 1.6 log₁₀ CFU/mouse) when plated in the presence of mevalonate, in contrast to the wild type, S. aureus NCTC 8325-4, which was present at 5.17 ± 0.17 log₁₀ CFU/mouse (mean ± standard deviation for five mice). The results indicate that the auxotrophic mvaA null mutant has greatly reduced virulence in the hematogenous pyelonephritis infection model.

Expression of mvaA and purification of S. aureus HMG-CoA reductase. The mvaA gene of S. aureus was cloned into pET28 in frame with the N-terminal histidine tag and thrombin cleavage site. A soluble protein was expressed in E. coli BL21(DE3) to high levels following induction with 1 mM IPTG and was readily purified on a nickel-chelating column, the majority eluting in the 500 mM imidazole fraction (Fig. 2). Typical yields were 30 mg of over 90% homogenous protein per liter of induced culture. The mvaA gene encodes a putative protein of 425 residues with a calculated molecular weight of 46,180. Matrix-assisted laser desorption ionization mass spectrometry analysis indicated that the purified protein had a molecular weight of 48,503, which corresponds to the full-length HMG-CoA reductase fusion protein minus the terminal methionine residue. N-terminal sequencing of the purified protein confirmed that this residue had been removed and that the N-terminal sequence was otherwise intact.

View larger version (85K): [in this window] [in a new window]   FIG. 2.   Expression of S. aureus mvaA and purification of the gene product. A protein of the anticipated size was absent from induced E. coli BL21(DE3) cells containing the parent vector (lane 1) but was expressed in cells containing pJO1 (lane 2). Lane 3, crude cytosol; lane 4, material that failed to bind to the nickel column. Bound protein was eluted with imidazole at concentrations of 10 mM (lane 5), 50 mM (lane 6), 100 mM (lane 7), and 500 mM (lane 8). Lanes 9 and 10 contain 20 and 4 µg of purified protein, respectively.

Biochemical characterization. The enzymatic activity of the purified protein was determined by measuring the oxidation or reduction of NAD(P)H at 340 nm. Firstly, the temperature dependency of S. aureus HMG-CoA reductase for the deacylation of HMG-CoA was investigated. Optimal activity for the catalysis of reaction 1 was observed between 35 and 45°C and decreased precipitously above 45°C (data not shown). All subsequent assays were therefore conducted at 37°C.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   Effect of hydrogen ion concentration on activity. The effects of the indicated concentrations of hydrogen ion on the rates of the indicated reactions were assayed under standard conditions other than pH using either NADP(H) () or NAD(H) () as the coenzyme. Assays were conducted in a solution of 50 mM KCl, 25 mM K-PO4, and 100 mM Tris-HCl adjusted to the indicated pH values (x axis). (Top left) Reaction 1, reductive deacylation of HMG-CoA to mevalonate; (top right) reaction 2, reduction of mevaldehyde to mevalonate; (lower left) reaction 3, oxidative acylation of mevaldehyde to HMG-CoA; (lower right) reaction 4, oxidative acylation of mevalonate to HMG-CoA. eu, enzyme units.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Inhibition by a statin drug. Double-reciprocal plot for inhibition of reaction 1, the reductive deacylation of HMG-CoA to mevalonate, using NADPH as the coenzyme. Analyses were conducted at pH 7.5 and 37°C at the indicated concentrations of HMG-CoA in the presence of 0 µM () or 500 µM () fluvastatin. All reactions were initiated by adding NADPH. eu, enzyme units.

	DISCUSSION

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We demonstrated that the mvaA gene is essential for the growth of S. aureus and that mvaA null mutants could be readily isolated on media supplemented with mevalonate. These results support previous observations that mvaA is essential in S. pneumoniae (41) and strongly suggest that the gene is essential in other gram-positive cocci which use the mevalonate pathway for the synthesis of IPP, as the GAP-pyruvate pathway has not been found in these organisms. The S. aureus mvaA null mutant was severely attenuated in the mouse hematogenous pyelonephritis infection model. Although the plasma mevalonate concentration in mice has not been measured, the concentrations in humans and rats, 0.02 to 0.08 µM and 0.08 to 0.50 µM, respectively (32), are well below that required to support the growth of the mvaA null mutant.

Derived sequence similarities and conservation of known active-site residues and motifs (Fig. 5) suggested that the mvaA gene of S. aureus encodes a class II HMG-CoA reductase (41). Expression in E. coli of an mvaA-polyhistidine tag construct and the subsequent purification and enzymatic characterization of the gene product revealed that this was indeed the case. Inspection of the pH profiles for catalysis of all four reactions revealed that two functional groups with pK_a values around 7 and 9 must be in their protonated states for activity. We propose that these are His378 of S. aureus (His378_S) and Lys263_S (Fig. 5), the apparent cognates of active-site residues His381 of P. mevalonii (His381_P) and Lys267_P of P. mevalonii HMG-CoA reductase that have established functions in catalysis (9). We further infer that the mechanism proposed for catalysis by P. mevalonii HMG-CoA reductase (39) is valid for S. aureus HMG-CoA reductase.

View larger version (16K): [in this window] [in a new window]   FIG. 5.   Alignment of selected amino acid sequences of the class II HMG-CoA reductases from S. aureus (Sa) and P. mevalonii (Pm). Conserved paired residues E81S-E83P, K263S-K267P, D279S-D283P, and H378S-H381P, all in bold, represent active-site residues known to function in catalysis by the P. mevalonii enzyme. The conserved ENVIG, DAMGXN, and GTVGG sequences are shaded. Asp146P and Leu148P, marked with asterisks, contribute to the ability of P. mevalonii HMG-CoA reductase to discriminate against NADP(H). Additional residues that are conserved in both sequences are boxed. The sequences of S. aureus and P. mevalonii HMG-CoA reductases are 39% identical overall. Alignment was performed using the MegAlign sequence analysis software from DNASTAR Inc., Madison, Wis.

All three characterized class II HMG-CoA reductases use NAD(H) (Table 2), but only the S. aureus and A. fulgidus enzymes use either NAD(P)H or NAD(H). In this respect, the S. aureus enzyme more closely resembles the archaeal enzyme than its mesophilic, biodegradative counterpart. The ability of S. aureus HMG-CoA reductase to use either coenzyme was predicted, since Asp146_P and Leu148_P are major determinants of nucleotide coenzyme specificity for P. mevalonii HMG-CoA reductase (14). Crystal structures of the P. mevalonii enzyme revealed that Asp146_P interacts with the 2'-hydroxyl of the adenosyl ribose of NAD(H), discriminating against NADP(H) (24, 39). Consistent with the ability to use NADP(H), sequence alignments revealed no clear cognates of Asp146_P or Leu148_P in S. aureus HMG-CoA reductase. Despite significant similarity (39% identity and 57% similarity), the ability of S. aureus HMG-CoA reductase to accommodate both coenzymes suggests that significant differences in the coenzyme binding site may become apparent when the crystal structure of S. aureus HMG-CoA reductase is determined and compared to that of the P. mevalonii enzyme. Finally, we suggest that the ability to use both coenzymes may turn out to be a general property of biosynthetic class II forms of the enzyme.

Despite its ability to use NAD(H), S. aureus HMG-CoA reductase is a true biosynthetic enzyme. This is inferred from its preference for NADPH and the presence in S. aureus of genes that encode a putative HMG-CoA synthase, mevalonate kinase, phosphomevalonate kinase, and mevalonate decarboxylase (41). While most oxidoreductases exhibit a high order of coenzyme specificity, oxidoreductases that use either NADP(H) or NAD(H) include L-glutamate dehydrogenase (11, 12, 28), isocitrate dehydrogenase (13, 25), glucose-6-phosphate dehydrogenase (3, 5), 6-phosphogluconate dehydrogenase (5), aldose reductase (30), and biliverdin IX reductase (26). With the exception of liver alcohol dehydrogenase (2), coenzyme K_m values are 1 or more orders of magnitude lower for NADP(H) than for NAD(H), suggesting a preference for NADPH. The HMG-CoA reductases of A. fulgidus and S. aureus thus are among a mere handful of oxidoreductases, and the only HMG-CoA reductases, for which the K_m is essentially the same for either coenzyme.

The K_i for inhibition of S. aureus HMG-CoA reductase by a statin drug is over 4 orders of magnitude higher than the K_i for a class I eukaryotic enzyme, suggesting that the two classes can be discriminated chemically. The recent publication of the crystal structure of the catalytic portion of the human HMG-CoA reductase (18) confirms large differences in the architecture of the active sites of the human and P. mevalonii enzymes. Thus, agents selective for the inhibition of the class II bacterial enzymes might be achievable. The essential nature of this enzyme suggests that the class II HMG-CoA reductases represent a promising target for antibacterial agents directed against multidrug-resistant gram-positive cocci.