INTRODUCTION

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Bacterial cell wall biosynthesis is a complex dynamic event involving participation of a variety of enzymes (12). Peptidoglycan, a key component of bacterial cell walls, is composed of glycan strands cross-linked by peptide bridges (4, 11). Two cell wall biosynthetic enzymes that have received much attention, transpeptidase and glycosyltransferase (1, 3, 21), play critical roles in the terminal stages of peptidoglycan formation. Glycosyltransferase is responsible for elongation of the glycan strands using lipid-linked disaccharide-pentapeptide as the substrate. Transpeptidase cross-links the peptide chains attached to the glycan strands (3). A group of bifunctional high-molecular-weight (HMW) penicillin-binding proteins (PBPs) possessing both glycosyltransferase and transpeptidase activity have been identified in both gram-positive and gram-negative bacteria. The N-terminal domains of these HMW PBPs contain glycosyltransferase activity, while the C-terminal domains possess transpeptidase and PBP activities (3, 20). In addition, these bifunctional enzymes contain an N-terminal hydrophobic region responsible for membrane association (5, 7, 18).

Monofunctional enzymes possessing only glycosyltransferase or transpeptidase activity have also been identified (2, 15). For example, low-molecular weight PBPs in several bacterial species have been shown to carry only DD-carboxypeptidase activity (8, 13, 14, 19). Monofunctional and/or non-PBP-related glycosyltransferase (MGT) activities have been reported for Escherichia coli (10) and gram-positive species such as Staphylococcus aureus and Micrococcus luteus (17). Several laboratories have reported molecular cloning of mgt genes from E. coli, Hemophilus influenzae, Ralstonia eutropha, and other gram-negative bacterial species (2, 15, 18). The proteins encoded by these mgt genes show a high degree of similarity to the N-terminal glycosyltransferase domain of HMW PBPs (15, 18). In addition, purified recombinant E. coli MGT is capable of catalyzing peptidoglycan synthesis in vitro (2).

MGT might also play a key role in peptidoglycan biosynthesis in pathogenic gram-positive bacteria such as S. aureus and Streptococcus pneumoniae (16, 17). In this report, we describe the identification of a complete DNA sequence with an open reading frame (ORF) encoding an ~31-kDa MGT from the S. aureus genome. A genetically engineered soluble form of S. aureus MGT was expressed in E. coli cells and purified to homogeneity. The purified MGT was characterized with regard to protein structure and enzymatic activity. Using the antibodies developed against purified MGT protein, we demonstrated that MGT was natively expressed in S. aureus cells as a membrane-associated protein.

	MATERIALS AND METHODS

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Identification and cloning of S. aureus mgt Genomic DNAs isolated from S. aureus strains ST446 (S. aureus 27S, a methicillin-sensitive strain obtained from Richard Novick) and ST430 (a methicillin-resistant strain obtained from Henry Chambers) were used for identification of the mgt gene reported previously (2). Briefly, S. aureus genomic DNA was digested with a variety of restriction enzymes and subjected to Southern analysis using standard protocols. The hybridization probes used for these analyses were produced by PCR and covered the region encoding a portion of MGT from S. aureus (2).

Expression of recombinant S. aureus MGT proteins in E. coli DNA sequence analysis of S. aureus mgt revealed two in-frame translational start sites 16 amino acids apart at the N terminus of mgt. Considering that previously characterized MGTs had an average length of approximately 240 amino acids, we selected the second translational start codon as the initiation of translation because it produced a protein of 253 amino acids, close to the average MGT amino acid length. A DNA fragment containing S. aureus mgt was generated by PCR using the primers 5'-GAACATGGATCCCATATGAAACACGAACCTCAC-3' (primer 3) and 5'-TGCGGATCCTTAACGATTTAATTGTGACATAG-3' (primer 4) with S. aureus genomic DNA as the template. For cloning purposes, these two primers were designed to incorporate a BamHI site at the 3' end and an NdeI site at the 5' end of S. aureus mgt. PCR conditions were as described above except only 25 cycles were used. The DNA fragment generated by PCR was gel purified and subcloned into the E. coli expression vector pET-16b (Novagen, Madison, Wis.). The resulting construct (pRBP1) introduced an N-terminal His tag (10 histidine residues) to aid in protein purification.

Purification of recombinant His-MGT E. coli cells (40 g) transformed with plasmid pRBP2 were resuspended in 400 ml of buffer A (25 mM HEPES [pH 7.5], 0.15 M NaCl, protease inhibitor tablets [Roche], and 200 U of DNase). Cells were then lysed by sonication. The lysate was clarified by centrifugation at 100,000 × g for 40 min at 4°C. The supernatant was loaded onto a 5-ml Pharmacia HiTrap chelating Sepharose column (Ni²⁺ charged), preequilibrated with buffer B (25 mM HEPES [pH 7.5] and 0.5 M NaCl). After the column was washed with 0.1 M imidazole in buffer B to the baseline absorbance, the bound proteins were eluted using a linear gradient of 0.1 to 0.7 M imidazole in buffer B. Fractions containing the 26-kDa histidine-tagged MGT protein (His-MGT) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Generation of antibodies and Western blot analysis To develop polyclonal antibodies for S. aureus MGT, purified His-MGT was first heat denatured and then subjected to SDS-PAGE analysis. Gel slices containing the ~26-kDa His-MGT protein were used for immunization of New Zealand White rabbits. Antisera were collected after two booster injections. The quality of the harvested antisera was analyzed by enzyme-linked immunosorbent assays. For Western blot analysis, protein samples were subjected to SDS-PAGE and transferred to nitrocellulose membrane filters. The filters were first incubated with 5% milk in phosphate-buffered saline buffer, then with either the preimmune serum or the polyclonal antibodies against His-MGT (diluted 1:2,000), and finally with the peroxidase-conjugated secondary antibodies (diluted 1:2,000). The cross-linked proteins were detected using ECL immunodetection reagents (Amersham).

Analyses of purified His-MGT Circular dichroism (CD) spectra of purified S. aureus His-MGT were recorded with an Aviv 62DS spectropolarimeter. Denatured His-MGT was generated by adding guanidine hydrochloride (6 M final concentration) directly to the protein sample. Both native and denatured His-MGT proteins were analyzed at a concentration of 0.5 mg/ml. The displayed spectra were obtained from the averages of values for five scans as described previously (22), with the signal being corrected for background using the enzyme buffer solution.

Enzymatic activity of purified recombinant S. aureus MGT Cell membranes of Aerococcus viridans (ATCC 10400) were prepared as described previously (9) and used to measure glycan polymerization by monitoring incorporation of ¹⁴C-N-acetylglucosamine into trichloroacetic acid (TCA)-precipitable material. To ensure measurement of glycan synthesis, we slightly modified the published procedure (2) as specified below. A typical MGT enzymatic reaction was performed at either pH 6.1 or pH 8.0 as required in a total volume of 70 µl containing the following components: the A. viridans membrane fraction (50 µg), 0.38 mM [¹²C]- or [¹⁴C]UDP-N-acetylglucosamine (specific activity, ~4,000 cpm/nmol), 0.33 mM UDP-N-acetylmuramylpentapeptide, 50 mM MgCl₂, 0.21 mM KCl, 0.83 mM NH₄Cl, 250 µg of penicillin G per ml, 50 mM Tris-HCl, 50 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], 6.7 mM ATP, and 16 µg of purified S. aureus His-MGT. Reaction mixtures were incubated at 23°C for 1 h and quenched with 1 ml of ice-cold 10% TCA. Precipitates were collected on Whatman GF/C glass fiber filter papers and washed with 5% TCA. Samples were then counted in a liquid scintillation counter after the addition of a toluene-based cocktail as described previously (2). The data shown are averages of values from three separate experiments. The sensitivity of MGT activity to moenomycin A or lysozyme was determined under the same assay conditions.

	RESULTS

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Identification and cloning of S. aureus mgt An ORF (ORF2) encoding a portion of a putative MGT was identified in S. aureus previously (2). However, two motifs (RKXXE and KXXXLXXYXN, where X is any amino acid) typically associated with glycosyltransferase were absent in the deduced amino acid sequence of ORF2 (2). A close examination of the DNA sequence revealed the presence of a Sau3A restriction site 6 nucleotides upstream from the stop codon at the end of ORF2, raising the possibility that the missing motifs noted above may have been due to a cloning artifact, which frequently happens in genomic library constructions.

View larger version (42K): [in this window] [in a new window]   FIG. 1.   Amino acid sequence comparisons of MGTs and the glycosyltransferase domain of PBPs. Numbers in boxes are percentages of amino acid identity between the two indicated proteins. Amino acid sequence comparisons were done with the GAP program from the Genetics Computer Group sequence analysis software package from the University of Wisconsin Department of Genetics. Database accession numbers are provided at the end of each protein description. Only the N-terminal (transglycosylase) domains of PBPs were used in these comparisons. Abbreviations are as follows: Sau, S. aureus; Eco, E. coli; Hin, H. influenzae; Reu, R. eutropha; Kpn, Klebsiella pneumoniae; Ngo, Neisseria gonorrhoeae; Spn, Streptococcus pneumoniae; and Bsu, Bacillus subtilis.

Expression and purification of recombinant MGT in E. coli A full-length MGT encoded by S. aureus mgt was expressed in E. coli cells. Although full-length MGT, with an anticipated molecular mass of ~31 kDa, could be overproduced in transformed E. coli cells, it was present mainly in the particulate fraction of the cell lysate (data not shown). These results indicated that S. aureus MGT might be a putative transmembrane or membrane-associated protein, consistent with the results of hydropathy analysis mentioned above.

View larger version (110K): [in this window] [in a new window]   FIG. 2.   Purification of recombinant His-MGT. An aliquot containing His-MGT was loaded onto a 4-to-20% gradient gel for separation, followed by Coomassie blue staining. Lane 1, whole-cell lysate; lane 2, solubilized fraction; lane 3, Ni2+ column pools; lanes 4 and 5, monomer and oligomer pools after Superdex-75 column chromatography, respectively. Lane 6 reflects the results of the Western blot analysis of purified His-MGT (50 ng) using the polyclonal anti-His-MGT antibodies as described in Materials and Methods.

Structural analysis of S. aureus MGT Although most of the overproduced His-MGT was present in the soluble fraction (Fig. 2), gel filtration analysis showed that nearly all His-MGT appeared in the void volume of the size exclusion column (Fig. 3A), suggesting that His-MGT might aggregate in solution. Addition of 0.5% CHAPS into the protein preparation decreased the aggregation level, which generated a portion of His-MGT (~35%) that eluted at the monomer position (Fig. 3A).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Self-interaction of S. aureus His-MGT. (A) Gel filtration analysis of His-MGT. Shown are the elution profiles of purified His-MGT proteins from a Superdex-75 column in the absence (dashed and dotted line) and presence (dotted line) of 0.5% CHAPS detergent. Elution positions for protein standards are marked. (B) Chemical cross-linking of purified His-MGT. Purified His-MGT (5 µg) was incubated with either the solvent dimethyl sulfonate (lane 1) or the linker disuccinimidyl suberate (lane 2) for 2 h at 4°C as described in the text. The samples were then subjected to separation by SDS-PAGE followed by Coomassie blue staining. Cross-linked complexes are indicated by arrows.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   CD spectrometric analysis of purified S. aureus His-MGT samples. The CD spectra were collected as described in the text using highly purified His-MGT samples. Filled circles, monomer; open circles, oligomer; triangles, monomer denatured by 6 M guanidine hydrochloride.

View this table: [in this window] [in a new window]   TABLE 1.   Structural analysis of His-MGT protein derived from S. aureus

Glycosyltransferase activity of purified S. aureus His-MGT To test if the purified S. aureus His-MGT was enzymatically active, a glycosyltransferase assay was performed. A previous report suggested that MGT from E. coli had a pH optimum between 6.0 and 6.5 for its catalytic activity and that the glycosyltransferase activity of HMW PBPs from E. coli preferred a pH between pH 7.5 and 8.0 (10). Accordingly, we tested the ability of a cell membrane preparation from A. viridans to incorporate a radiolabeled nucleotide-linked sugar precursor, UDP-N-acetylglucosamine, into a glycan polymer at pHs 6.1 and 8.0. These results are presented in Fig. 5. When purified S. aureus His-MGT was tested at pH 6.1, the incorporation of UDP-N-acetylglucosamine increased ~3-fold, but when it was tested at pH 8.0, only a 25% increase was seen in the reactions (Fig. 5). Addition of moenomycin A, a known glycosyltransferase inhibitor, or lysozyme, an enzyme that cleaves specific disaccharide bonds in peptidoglycan, reduced the formation of the radioactive product (Fig. 5). These results confirmed that purified His-MGT catalyzed the incorporation of the radioactive substrate into peptidoglycan.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   Peptidoglycan synthesis catalyzed by purified S. aureus His-MGT. Peptidoglycan synthesis reactions were performed as described in the text. Bar 1, control reaction; bar 2, control reaction plus S. aureus His-MGT; bar 3, control reaction plus His-MGT and 10 µg of moenomycin A per ml; bar 4, control reaction plus S. aureus His-MGT and 300 µg of lysozyme per ml.

Presence of MGT in actively growing S. aureus cells Western blot analysis was used to detect the expression of MGT in actively growing S. aureus cells. Crude cell lysates of S. aureus grown to log phase were probed with polyclonal antibodies specifically developed against purified His-MGT. As seen in Fig. 6, three major proteins, with molecular masses of approximately 42, 32, and 30 kDa, reacted with the antibodies against S. aureus MGT. The 42-kDa protein was assumed to be well-known membrane-bound protein A because this protein was reactive to the preimmune serum (Fig. 6, lanes 3 and 4) as well as the antibodies developed against other unrelated proteins (data not shown). The second protein had a molecular mass of ~32 kDa, matching the predicted size of a full-length S. aureus MGT protein (Fig. 6, lane 1). Our data also revealed that these proteins were found mainly in the particulate fraction of cell lysates (Fig. 6), consistent with the expected localization for both protein A and MGT. The identity of the ~30-kDa protein reacting with the antibodies was not clear; it might represent a proteolytic fragment of MGT.

View larger version (104K): [in this window] [in a new window]   FIG. 6.   Detection of MGT in S. aureus cells. S. aureus cells (0.7 g) grown to log phase were collected and lysed by sonication. The whole-cell lysate was centrifuged at 75,000 × g for 30 min to generate a supernatant fraction (lanes 2 and 4) and particulate fraction (lanes 1 and 3). After separation by SDS-PAGE, Western blotting was performed using either polyclonal antibodies against purified His-MGT (lanes 1 and 2) or preimmune serum (lanes 3 and 4) as described in the text.

	DISCUSSION

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Glycosyltransferase activity derived from either bifunctional PBPs or the more recently identified MGTs may represent a useful target for the development of potent antibiotics. One of our efforts in searching for novel glycosyltransferase enzymes from pathogenic bacteria included the identification of a complete DNA sequence (mgt) encoding a putative MGT in S. aureus. To the best of our knowledge, this is the first fully characterized mgt reported for gram-positive bacteria.

Our results suggested that S. aureus MGT possessed glycan synthesis activity that was sensitive to assay conditions such as pH, in agreement with results published previously for E. coli MGT (2). Consistent with our expectations, the glycan-synthesizing activity of purified S. aureus MGT was inhibited by moenomycin A and the product was sensitive to lysozyme treatment. The observed MGT activity was low under the conditions tested; this may have resulted from the use of a truncated enzyme, a lack of cofactors, and/or simply the measurement methods used (2, 17).

It has been reported by Park and Matsuhashi that peptidoglycan synthesis in S. aureus is catalyzed mainly by non-PBP-associated transglycosylases (17). To date, it is not clear how much this MGT contributes to the non-PBP-related transglycosylase activity reported for S. aureus cells (17). Final conclusions on this point, as well as its essentiality in S. aureus cells, remain to be determined in future experiments. It is worth noting that gene disruption of an H. influenzae MGT was not a lethal event (data not shown). The knockout results obtained from H. influenzae were not surprising because the glycosyltransferase activity involved in cell wall biosynthesis might be contributed in a redundant fashion by several HMW PBPs plus MGTs. Therefore, one might anticipate that S. aureus MGT is not essential to S. aureus. However, since the purified MGT had catalytic activity and this activity is sensitive to moenomycin A, which also inhibits other cell wall biosynthetic glycosyltransferases, it is reasonable to think that this enzyme can be used in in vitro assays to find inhibitors of glycosyltransferases in general.

Purified S. aureus His-MGT demonstrated self-interaction in solution even though the hydrophobic N-terminal domain had been deleted. This self-interaction was not the result of covalent disulfide (S---S) bridges because this protein did not contain cysteine residues. Oligomerized His-MGT possessed percentages of secondary structural elements, such as the -helix and -sheet, different from those of the monomeric form. However, other major elements such as -turns, important for establishing the overall three-dimensional structure of a protein, and a random coil, which provides dynamic flexibility to the protein, were present to similar degrees in the His-MGT monomer and oligomers. This observation may explain the similar enzymatic activities of these different enzyme preparations. Apparently, His-MGT's self-interaction did not significantly affect the active-site conformation. We have not examined the state of the full-length MGT protein in S. aureus with regard to this phenomenon. Thus, the significance of MGT self-interaction is not clear at this time.

The mere presence of an ORF within an organism does not constitute proof that the encoded protein is expressed. With S. aureus mgt, we were able to detect a membrane-associated protein of the appropriate molecular weight with polyclonal antibodies exhibiting specificity for S. aureus MGT (Fig. 6). These results suggested that S. aureus mgt is not a silent gene and that MGT is actively expressed in S. aureus, most likely as a membrane-associated protein. These data, together with its measurable enzymatic activity, support the notion that this protein may play a role in bacterial cell wall biosynthesis. In this regard, the S. aureus MGT may represent a model for further characterization of non-PBP-related transglycosylases as well as for development of novel antibiotics.