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J Bacteriol. May 2011; 193(10): 2549–2556.
PMCID: PMC3133172

Monofunctional Transglycosylases Are Not Essential for Staphylococcus aureus Cell Wall Synthesis[down-pointing small open triangle]

Abstract

The polymerization of peptidoglycan is the result of two types of enzymatic activities: transglycosylation, the formation of linear glycan chains, and transpeptidation, the formation of peptide cross-bridges between the glycan strands. Staphylococcus aureus has four penicillin binding proteins (PBP1 to PBP4) with transpeptidation activity, one of which, PBP2, is a bifunctional enzyme that is also capable of catalyzing transglycosylation reactions. Additionally, two monofunctional transglycosylases have been reported in S. aureus: MGT, which has been shown to have in vitro transglycosylase activity, and a second putative transglycosylase, SgtA, identified only by sequence analysis. We have now shown that purified SgtA has in vitro transglycosylase activity and that both MGT and SgtA are not essential in S. aureus. However, in the absence of PBP2 transglycosylase activity, MGT but not SgtA becomes essential for cell viability. This indicates that S. aureus cells require one transglycosylase for survival, either PBP2 or MGT, both of which can act as the sole synthetic transglycosylase for cell wall synthesis. We have also shown that both MGT and SgtA interact with PBP2 and other enzymes involved in cell wall synthesis in a bacterial two-hybrid assay, suggesting that these enzymes may work in collaboration as part of a larger, as-yet-uncharacterized cell wall-synthetic complex.

INTRODUCTION

Staphylococcus aureus strains are currently one of the major causes of hospital-acquired bacteremia. This pathogen constitutes a major health concern due to its virulence and capacity to adaptively respond to the introduction of antimicrobial agents into the clinical environment (20). Worryingly, the epidemiology of methicillin-resistant S. aureus (MRSA) is changing, and community-associated strains of MRSA have emerged in recent years (6, 38), highlighting the importance of continued research into the mechanisms of antibiotic resistance in this organism.

The final stages of peptidoglycan (PG) polymerization involve the synthesis of long glycan chains, made of alternating β-1-4-linked N-acetylglucosamine and N-acetylmuramic acid, a reaction that is catalyzed by transglycosylase (TGase) enzymes. These carbohydrate polymers are cross-linked to each other via their peptide moieties by transpeptidase (TPase) enzymes. S. aureus has four penicillin binding proteins (PBP1 to PBP4) that are capable of catalyzing the transpeptidation reaction, which is the reaction inhibited by β-lactam antibiotics. In addition to the four native PBPs, MRSA strains have acquired a “foreign” PBP, PBP2A, which has a TPase domain displaying low affinity for β-lactams (11, 26), enabling the protein to continue cell wall synthesis in the presence of these antibiotics (11, 26). PBP2, the only bifunctional PBP, is also capable of catalyzing transglycosylation reactions, and in MRSA strains exposed to β-lactams, the synthesis of PG is performed in a cooperative manner by the TGase activity of PBP2 and the TPase activity of the acquired PBP2A (25).

Due to the resistance of S. aureus strains to β-lactams, which target the transpeptidation reaction, there is an increased interest in developing new antibiotics that inhibit the transglycosylation reaction of peptidoglycan synthesis. Moenomycins are the only well-studied example of TGase inhibitors and have been shown to be highly active against a broad range of Gram-positive organisms (14, 15, 17, 27). However, moenomycins are not yet used in humans due to their poor pharmacokinetic properties (14) and are most commonly used as growth promoters in animal feed.

Many bacteria contain multiple bifunctional PBPs that are capable of transpeptidation and transglycosylation, known as class A PBPs (10). Surprisingly, in some cases, such as the Gram-positive organisms Bacillus subtilis (21), Enterococcus faecalis (1), and Enterococcus faecium (28), it has been shown that strains lacking all class A PBPs are viable and have only slight differences in peptidoglycan composition. In contrast, in Streptococcus pneumoniae, the deletion of class A PBP1a and PBP2a was lethal (13), and in S. aureus, the only class A PBP, PBP2, is essential for viability in methicillin-susceptible strains and in methicillin-resistant strains exposed to β-lactam antibiotics (24). In addition to the transglycosylase activity supplied by the class A PBPs, a number of bacterial species have been found to possess membrane-bound enzymes capable of catalyzing glycan chain formation, known as monofunctional transglycosylases (Mgts) (7, 8, 30, 36), most of which were identified by sequence homology (30). However, in some species, for example, B. subtilis and Enterococcus spp., no identifiable Mgts have been found (1, 18, 28), while in others, such as Brucella abortus, Escherichia coli, and Haemophilus influenzae, Mgts were shown to be dispensable (5, 29, 36). It therefore seems that B. subtilis and Enterococcus spp. are able to grow in the absence of transglycosylases or that additional enzymes with transglycosylase activity are yet to be identified in these organisms.

As elongation of the nascent PG chains in S. aureus is not completely abolished following the inactivation of the TGase domain of PBP2, the only class A PBP in this organism, it was suggested that other transglycosylases play a catalytic role in the elongation reaction (25). Indeed, genes encoding two putative Mgts, sgtA and sgtB (also referred to as mgt in the literature), were identified in S. aureus strains by whole-genome analysis, and the predicted products were shown to have significant similarity to known TGases of family 51 (7, 19). The mgt gene encodes a protein known in the literature as MGT, and the sgtA gene encodes a previously uncharacterized protein, SgtA (19, 36). Chromosomal searches for TGase motifs in S. aureus suggest that PBP2, MGT, and SgtA are the only enzymes functioning as TGases in this organism. MGT has recently been the subject of structural and kinetic studies (12, 32, 36). It was shown that the enzyme catalyzes the elongation of PG chains in a metal ion-dependent manner (12, 32). The expression of the mgt gene is upregulated in the presence of cell wall-targeting antibiotics (33); however, to date there are few data regarding the contribution of this protein to transglycosylase activity in vivo and to antibiotic resistance in S. aureus. The second monofunctional transglycosylase, SgtA, has remained largely uncharacterized.

Here, we show that SgtA has in vitro TGase activity and that the mgt and sgtA genes are nonessential in the background of MRSA strain COL and methicillin-susceptible S. aureus (MSSA) strain NCTC8325-4. However, in the absence of PBP2 TGase activity, MGT becomes essential for viability, while SgtA remains nonessential. Both proteins interact with PBP2 and other cell wall-synthetic enzymes in a bacterial two-hybrid assay, suggesting that they may be part of a larger cell wall-synthetic complex.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The bacterial strains and plasmids used in this study are listed in Tables Tables11 and and2,2, respectively, and primers are listed in Table 3. S. aureus strains were grown at 37°C in Tryptic soy broth medium (TSB; Difco) or on Tryptic soy agar (TSA; Difco) supplemented with appropriate antibiotics when required (erythromycin at 10 μg/ml, chloramphenicol at 10 μg/ml, or tetracycline at 5 μg/ml; Sigma-Aldrich) or with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG; VWR). E. coli strains were grown at 30°C or 37°C in Luria-Bertani broth medium (LB broth; Difco), on LB agar (Difco), or on MacConkey agar (Difco) supplemented with 100 μg/ml ampicillin (Sigma-Aldrich) or 50 μg/ml kanamycin (Sigma-Aldrich), 40 μg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal; VWR), and 0.5 mM IPTG when required.

Table 1.
Strains used in this study
Table 2.
Plasmids used in this study
Table 3.
Primers used in this study

Construction of S. aureus strains.

S. aureus strain LH607, a protein A-deficient mutant in which the spa gene was inactivated by the insertion of a tetracycline cassette (S. Foster), was used to transduce the mutation to COL using phage 80α (23), resulting in strain COL spa. To construct S. aureus Δmgt, ΔsgtA, and Δmgt ΔsgtA null mutants, we amplified 1-kb DNA fragments from S. aureus COL genomic DNA that corresponded to the upstream (primers MGT-P1 and MGT-P2 for MGT and primers SgtA-P1 and SgtA-P2 for SgtA) (Table 3) and downstream (primers MGT-P3 and MGT-P4 for MGT and primers SgtA-P3 and SgtA-P4 for SgtA) regions of the mgt or sgtA gene, respectively. The resulting PCR products were joined by overlap PCR using primers MGT-P1 and MGT-P4 for mgt and SgtA-P1 and SgtA-P4 for sgtA. The overlap PCR products were digested with BamHI and EcoRI (MGT) or BglII and BamHI (SgtA) and cloned into the thermosensitive plasmid pMAD (2), producing plasmids pΔmgt and pΔsgtA. The plasmids were sequenced and introduced into RN4220 by electroporation (35). Following electroporation, the plasmids were transduced using phage 80α to the NCTC and COL spa strains, using a previously described method (23). Insertion and excision of pΔmgt or pΔsgtA into the chromosome were performed as previously described (2), resulting in strains NCTCΔmgt, NCTCΔsgtA, COLΔmgt, and COLΔsgtA. The double null mutant strains, NCTCΔmgtΔsgtA and COLΔmgtΔsgtA, were created by transducing the plasmid pΔsgtA to strains NCTCΔmgt and COLΔmgt, respectively, followed by excision of sgtA. Deletion of the target genes was verified by PCR and Southern blotting. The point mutation in the TGase domain PBP2 (Glu114Gln) was transduced from COLTG42 (25) to the NCTC, NCTCΔmgt, NCTCΔsgtA, NCTCΔmgtΔsgtA, COL spa, COLΔmgt, COLΔsgtA, and COLΔmgtΔsgtA strains using phage 80α, and the mutations in the resulting strains were verified by PCR and digest as previously described (25).

S. aureus strain RNpPBP2iII, in which pbp2 is under the control of the IPTG-inducible promoter Pspac (24) was used as donor to sequentially transduce plasmids pPBP2i (integrated in the chromosome) and pMGPII into the control and mutant strains described above, using phage 80α as previously described (23) except that 0.5 mM IPTG was added to the medium. Transductants were selected with erythromycin (10 μg/ml) for pPBP2i and chloramphenicol (10 μg/ml) for pMGPII. The resulting strains were designated COLIPBP2i, COLIPBP2iΔmgt, COLIPBP2iΔsgtA, and COLIPBP2iΔmgtΔsgtA.

Growth analysis of S. aureus strains.

The growth of parental and transglycosylase mutant strains in liquid culture was analyzed by diluting overnight cultures 1:200 into fresh medium (TSB) supplemented with 10 μg/ml erythromycin for COLTG2-1 and COLTG42 strains. Cultures were incubated at 37°C with shaking, and the optical density at 600 nm (OD600) was monitored.

The growth of strains with pbp2 under the control of the IPTG-inducible promoter Pspac was tested on solid medium using TSA plates supplemented with appropriate antibiotics (10 μg/ml erythromycin and 10 μg/ml chloramphenicol) in the presence and absence of 0.5 mM IPTG. The same strains were grown in liquid culture, TSB supplemented with appropriate antibiotics and 0.5 mM IPTG, overnight at 37°C. The cells were harvested the following day and washed three times with fresh TSB lacking IPTG. These cells were then used to inoculate medium with and without IPTG, the cultures were incubated at 37°C with agitation, and the OD600 was followed. After 4 h, the cultures were diluted to an OD600 of approximately 0.1 into fresh medium either with or without IPTG, and growth was monitored for a further 8 h.

Cloning, expression, and purification of His-PBP2.

A truncated pbp2 gene, excluding the first 174 bp, was amplified from S. aureus COL genomic DNA by PCR using primers PBP2P39N and PBP2P40B. The PCR fragment was cloned into the expression vector pET28a (Novagen) with NheI and BamHI. The resulting plasmid, pETPBP2t, was introduced into E. coli BL21(DE3) for expression of His6-PBP2 lacking its N-terminal cytoplasmic region (M1 to R30) and transmembrane region (T31 to Y57). Cultures were incubated at 37°C until an OD600 of 0.6, and protein expression was induced with 1 mM IPTG for 7 h at 30°C. Cells were harvested, resuspended in buffer T (20 mM Tris-HCl [pH 8]) containing 150 mM NaCl and Complete EDTA-free protease inhibitors (Roche), and broken in a French press at 16,000 pounds/square inch. After centrifugation at 48,000 × g and SDS-PAGE analysis of cell extracts, the protein was found to be present in the pellet. The protein was recovered by solubilization of the pellet in buffer T containing 150 mM NaCl, 5 M urea, 1% Sarkosyl, and 10 mM imidazole with stirring at 4°C for 1 h. The solubilized protein was applied to a His-Trap column (GE Healthcare) pre-equilibrated with buffer T containing 500 mM NaCl, 5 M urea, and 10 mM imidazole. After a wash step in the presence of 50 mM imidazole, the protein was eluted in the same buffer containing 250 mM imidazole. The purified protein was digested with thrombin to cleave the His6 tag and applied into an 8% SDS-PAGE gel. The band corresponding to the truncated PBP2, minus the His6 tag, was cut from the gel and sent to Eurogentec for polyclonal antibody production.

Detection of PBP2 in pbp2-inducible strains.

The expression of PBP2 was detected by using a polyclonal anti-PBP2 antibody. Samples were taken from cultures of the strains with inducible pbp2 after 4 h of growth at 37°C with and without IPTG; cells were harvested and broken with glass beads in a FastPrep FP120 (Thermo Electro Corporation). Unbroken cells and debris were removed by centrifugation, and the total protein content of the clarified lysate was determined by the Bradford method, using bovine serum albumin as a standard (BCA protein assay kit; Pierce). Equal amounts of total protein from each sample were separated on 8% SDS-PAGE gels at 120 V. Proteins were then transferred to Hybond-P polyvinylidene difluoride (PVDF) membrane (GE Healthcare) using a Bio-Rad semidry transfer cell according to standard Western blotting techniques and detected using a polyclonal anti-PBP2 antibody.

Determination of flavomycin and oxacillin MICs.

Population analysis profiles (PAPs) were performed by plating 25 μl of serial dilutions (100 to 10−6) of overnight cultures of control and transglycosylase mutants on TSA and TSA containing increasing concentrations of flavomycin (0 μg/ml to 2 μg/ml) or oxacillin (0 μg/ml to 1,600 μg/ml). Plates were incubated at 37°C for 24 and 48 h for flavomycin and oxacillin PAPs, respectively, and the number of CFU was counted. The log CFU/ml was plotted against the antibiotic concentration, and the MIC was defined as the antibiotic concentration required to inhibit the growth of 99.9% of cells.

Cloning, expression, and purification of His-SgtA.

The truncated sgtA gene lacking the sequence encoding the cytoplasmic N-terminal region (residues M1 to A23) and the putative transmembrane domain (residues I24 to F50) and encoding residues H51 to R301 was amplified from COL genomic DNA by PCR using primers pETSgtA-P1 and pETSgtA-P2. The resulting PCR product was digested with BamHI and EcoRI and cloned into the corresponding sites in the pET30a vector (Novagen), giving rise to pETSgtA. This construct encodes the fusion of an N-terminal His6 tag to residues H51 to R301 of SgtA.

E. coli BL21(DE3) was transformed with pETSgtA, and the transformants were grown at 37°C in LB medium containing 50 μg/ml kanamycin. When the absorbance at 600 nm reached 0.6 to 0.8, the cultures were supplemented with 1 mM IPTG and grown for an additional 3 h. Cells were harvested by centrifugation, resuspended in buffer A (50 mM sodium phosphate buffer [pH 8] containing 150 mM NaCl and Complete EDTA-free protease inhibitors [Roche]), and disrupted by sonication. After centrifugation at 48,000 × g at 4°C, the soluble fraction was applied to pre-equilibrated Talon (Clontech) resin and incubated at 4°C overnight. Bound protein was washed twice with buffer A and then once with buffer A containing 10 mM imidazole. Bound protein was eluted in a two-step manner with buffer A containing 100 mM and then 150 mM imidazole. Eluted fractions were combined and dialyzed sequentially against 50 mM sodium phosphate buffer (pH 8) containing 500 mM NaCl, 300 mM NaCl, and 150 mM NaCl. The protein (33.8 kDa) was concentrated to 0.55 mg/ml and stored at −80°C. The protein was 95% pure as visualized by SDS-PAGE and Coomassie staining.

MGT was purified as previously described (32).

In vitro glycosyltransferase activity assays.

[14C]lipid II was prepared as previously described (31, 34). In vitro peptidoglycan polymerization was performed by incubation of 2.5 μM d-[14C]Ala-d-[14C]Ala-labeled lipid II (0.078 μCi nmol−1) with purified His-SgtA or His-MGT (5 μM) at 30°C for 3 h in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1% Triton X-100, 10 mM CaCl2, 12% dimethyl sulfoxide (DMSO). Reactions were stopped with moenomycin (10 μM), and the products were separated by thin-layer chromatography (TLC) on silica gel plates (Fluka), using 2-propanol-ammonium hydroxide (25%)-water (6:3:1, vol/vol/vol) as the mobile phase. The TLC plates were exposed to a storage phosphor screen (GE Healthcare) for 16 h, and images were revealed using a Typhoon Trio+ imager. The images were analyzed using Image Quant TL software (GE Healthcare).

Bacterial two-hybrid assays.

To construct the plasmids for bacterial two-hybrid assays, mgt, sgtA, pbp1, pbp2, pbp3, pbpD, and mecA genes were amplified from S. aureus COL genomic DNA using Phusion DNA polymerase (Finnzymes) and primers with the prefix BTH listed in Table 3. The resulting PCR products were fused in-frame to the 3′ end of the cyaA gene fragments in the plasmids pUT18C and pKT25. The pbpD gene, however, was fused to the 5′ end of the cyaA gene in plasmids pUT18 and pKNT25 because the mature protein it encodes, PBP4, has an amphipathic C-terminal helix, unlike the other membrane-associated proteins used here, which have an N-terminal membrane anchor following a short cytoplasmic tail. Combinations of the constructs were used to cotransform the reporter strain E. coli BTH101 (cya-deficient), and transformants were grown on Luria-Bertani agar plates containing 40 μg/ml X-Gal, 100 μg/ml ampicillin, and 50 μg/ml kanamycin or on MacConkey agar containing the same antibiotics and supplemented with 1% maltose. The β-galactosidase activity in E. coli cells containing the bacterial two-hybrid plasmids was measured in cell extracts from liquid culture essentially as previously described (16). Activity is expressed in Miller units as described previously (22), where 1 Miller unit is equal to 1,000[(A420 − 1.75A550)/tvA600], where t is the reaction time in minutes and v is the volume of culture assayed in milliliters. A level of β-galactosidase activity at least 4-fold higher than that measured for BTH101 (T18/T25) cells (~500 Miller units) was considered to indicate an interaction between the fusion protein pair tested.

RESULTS AND DISCUSSION

The monofunctional transglycosylases from S. aureus are not essential for viability.

To assess whether the monofunctional transglycosylases from S. aureus, MGT and SgtA, are essential for cell viability, we deleted the mgt and sgtA genes from the chromosome of the methicillin-resistant S. aureus (MRSA) strain COL spa, leaving no antibiotic resistance markers, to produce the null mutant strains COLΔmgt and COLΔsgtA and the double null mutant strain COLΔmgtΔsgtA. The mgt and sgtA genes were also deleted from the methicillin-sensitive S. aureus (MSSA) strain NCTC8325-4. All gene deletions were confirmed by PCR and Southern blot hybridization (data not shown). Growth was analyzed in liquid culture, and all mutant strains grew like the parental strains COL spa and NCTC8325-4, as seen by the results shown in Fig. 1A for the double null mutant strains. Similarly, when we analyzed the mutant strain COLTG42, in which a point mutation (Glu114Gln) was introduced into the transglycosylase domain of PBP2, rendering it inactive, growth was not affected. These results indicate that S. aureus can grow normally in the absence of any of the three transglycosylases alone, as well as in the absence of the two Mgts when PBP2 is present.

Fig. 1.
Analysis of growth of transglycosylase mutants of S. aureus. (A) The growth of parental strains MSSA NCTC8325-4 and MRSA COL and double mutant strains COLΔmgtΔsgtA and NCTCΔmgtΔsgtA in liquid medium was followed by monitoring ...

MGT but not SgtA is essential for cell viability in the absence of PBP2 transglycosylase activity.

In order to investigate whether S. aureus could survive in the absence of the three known TGase enzymes, we attempted to transduce the Glu114Gln mutation in the PBP2 TGase domain to the single and double MGT/SgtA mutant strains. The PBP2 TGase mutation was successfully transduced from COLTG42 to COL spa and COLΔsgtA. In contrast, transduction of the mutation to strain COLΔmgt or COLΔmgtΔsgtA was not possible, even after several attempts. Similarly, the point mutation could also be introduced into NCTC and NCTCΔsgtA but not into NCTCΔmgt or NCTCΔmgtΔsgtA.

The fact that we were unable to construct an MGT/PBP2 double transglycosylase mutant suggests that either MGT or PBP2 must be present in order for S. aureus to survive. In order to confirm this observation, we placed pbp2 under the control of the IPTG-inducible, LacI-repressible Pspac promoter (24) in the background of the COL Mgt mutant strains by transduction. The plasmid pMGPII, which encodes LacI, was introduced into the resulting strains to enable tight regulation of the pbp2 gene (37). The resulting transformants, COLIPBP2i, COLIPBP2iΔmgt, COLIPBP2iΔsgtA, and COLIPBP2iΔmgtΔsgtA, were grown on selective plates in the presence or absence of IPTG. In the presence of 0.5 mM IPTG, all transformants grew normally. However, in the absence of IPTG, strains COLIPBP2iΔmgt and COLIPBP2iΔmgtΔsgtA failed to grow (Fig. 1B).

Analysis of the growth of the PBP2-inducible strains was also performed in liquid medium, with similar results (Fig. 1C), showing that TGase activity of PBP2 or MGT alone but not that of SgtA is sufficient for cell growth. Western blot analysis of cell extracts prepared from growing cultures showed that PBP2 protein was undetectable in cultures growing in the absence of inducer (Fig. 1D).

The fact that a strain in which SgtA is the only functioning transglycosylase could not be made indicates that this enzyme cannot replace the function of the other TGases, PBP2 and MGT. It is possible that the enzyme does not have sufficient TGase activity in vivo to enable cell survival, that it cannot use the same substrate as MGT and PBP2, or that it is expressed at different times, suggesting that MGT and SgtA may have different functions within the cell.

SgtA has glycosyltransferase activity in vitro.

Classification of SgtA as a transglycosylase was previously based only upon sequence homology to other known transglycosylases. In order to test whether this enzyme has in vitro glycosyltransferase activity, we expressed and purified a soluble form of SgtA that is devoid of its cytoplasmic and transmembrane N-terminal region. Incubation of the protein with 14C-labeled lipid II showed that the protein catalyzed glycan chain polymerization in vitro (Fig. 2, lane 2). Purified MGT has previously been shown to display optimal in vitro transglycosylase activity in the presence of MnCl2 (32), and this activity was comparable to that of S. aureus PBP2 (4). Comparison of the two Mgts, in the best conditions found for His-SgtA, shows that His-SgtA converted 5 to 10% of lipid II into polymerized peptidoglycan and His-MGT converted 66 to 78% of the substrate into polymerized PG (Fig. 2). These results provide evidence that the third transglycosylase identified in S. aureus, SgtA, has in vitro activity and show that MGT has higher glycosyltransferase activity than SgtA under these conditions. Therefore, the inability of SgtA to support growth in the absence of the TGase activities of PBP2 and MGT may be related to its low activity in vivo. Interestingly, the two Mgts have different optimal conditions for in vitro activity, as MGT displays maximum activity in the presence of Mn2+ (32), while SgtA shows a preference for Ca2+ (data not shown), suggesting these two enzymes may have different specific activities within the cell.

Fig. 2.
Determination of in vitro transglycosylase activities of MGT and SgtA. The formation of polymerized peptidoglycan from [14C]lipid II, catalyzed by purified MGT and SgtA, was analyzed by thin-layer chromatography. Lanes: 1, lipid II incubated without enzyme; ...

Effect of transglycosylase mutations on resistance to cell wall-targeting antibiotics.

TGase mutant strains were analyzed by population analysis profiling to determine their sensitivity to the cell wall-targeting antibiotics oxacillin (a β-lactam which inhibits transpeptidation) and flavomycin (an inhibitor of transglycosylation) (14). The TGase activity of PBP2 was shown to be essential for the expression of β-lactam resistance in MRSA strains, as the COLTG42 strain has a drastically reduced MIC to the β-lactam methicillin (25). It also has increased sensitivity to moenomycin, a TGase inhibitor (25). Accordingly, COLTG42spa and the double mutant COLTG42ΔsgtA strains both showed drastically reduced MICs to both oxacillin (10- to 16-fold) and flavomycin (10-fold) (Fig. 3). Surprisingly, the Mgt mutant strains COLΔmgt and COLΔsgtA showed no reduction in resistance to oxacillin (Fig. 3A). The fact that lack of PBP2 TGase activity results in reduction of the oxacillin MIC while lack of MGT TGase activity has no effect on the oxacillin MIC indicates that although MGT can substitute for PBP2 in terms of cell viability, it cannot do so in terms of β-lactam resistance.

Fig. 3.
Oxacillin and flavomycin population analysis profiles (PAPs) of S. aureus transglycosylase mutants. (A) Oxacillin PAPs. The COLTG42spa and COLTG42ΔsgtA mutant strains showed 16-fold reductions in oxacillin MIC compared to the MIC of the wild-type ...

Strains COLΔsgtA and COLΔmgtΔsgtA showed a very mild (1.6-fold) reduction in resistance to flavomycin (Fig. 3B), while the COLΔmgt strain had a MIC like that of the wild-type strain. As the Mgt double mutant strain COLΔmgtΔsgtA grows using PBP2 as the sole transglycosylase, these results suggest that PBP2 can function in the presence of (low concentrations of) flavomycin. However, in the absence of PBP2, the effect of flavomycin upon the Mgt mutants becomes apparent, resulting in a 10-fold decrease in MIC, showing that Mgts are exquisitely sensitive to this TGase inhibitor.

MGT and SgtA interact with cell wall-synthetic enzymes in a bacterial two-hybrid assay.

In order to determine whether MGT and SgtA interact with each other directly and with other cell wall-synthetic enzymes, a bacterial two-hybrid system (BTH) was used (16). The mgt, sgtA, pbp1, pbp2, pbp3, pbpD, and mecA genes were cloned into the BTH vectors as described in Materials and Methods. Plasmids encoding the MGT and SgtA fusions were cotransformed into the reporter strain with plasmids encoding the PBP fusions, and the production of adenylate cyclase from the cya-deficient strain was assayed. The plasmids p18Zip and p25Zip, which contain two leucine zipper domains, were used as positive controls, and the empty vectors pUT18C and pKT25 were used as negative controls.

In the bacterial two-hybrid screen, MGT was found to interact with itself, SgtA, PBP1, PBP2, and PBP2A. Transformants producing interacting fusion proteins formed blue colonies on LB-X-Gal-IPTG plates (Fig. 4A), and the levels of β-galactosidase activity in permeabilized cells were 7- to 10-fold higher than those measured in the negative controls or in cells expressing noninteracting fusion pairs (Fig. 4A). These results suggest that MGT interacts specifically with the other monofunctional transglycosylase in S. aureus, SgtA, with PBP2, which also has transglycosylase activity, and with PBP1 and PBP2A, both of which are involved in peptidoglycan synthesis. MGT was also seen to interact strongly with itself in the BTH assay, suggesting that its biologically active form may be a dimer or multimer within the cell.

Fig. 4.
Interactions of MGT and SgtA with other cell wall-synthesizing enzymes in a bacterial two-hybrid assay. (A) MGT interactions. Interactions were observed with MGT, SgtA, PBP1, PBP2, and PBP2A. (B) SgtA interactions. The fusion protein showed interactions ...

When SgtA was tested in the same manner, similar interactions were observed (Fig. 4B). The protein displayed strong interactions with T18-PBP2A and with itself, PBP1, PBP2, and MGT when fused to either the T18 or T25 adenylate cyclase subunits. The levels of β-galactosidase activity in permeabilized cells from all of these combinations were 7- to 17-fold higher than the levels measured in cells expressing the T18 and T25 fragments alone, indicating strong interactions in each of the coexpression strains.

These data suggest that the three TGases, MGT, SgtA, and PBP2, may work together in vivo to polymerize peptidoglycan. Interestingly the Mgts also seem to interact with the transpeptidase enzymes PBP1 and PBP2A, indicating that the key cell wall-synthetic enzymes from S. aureus may function in concert or within a complex to coordinate and catalyze the synthesis of peptidoglycan.

Concluding remarks.

Most studies of bacterial cell wall synthesis have focused upon the transpeptidation step of peptidoglycan synthesis, most probably because it is the step inhibited by β-lactam antibiotics. Less is known about the transglycosylation step or the enzymes that catalyze it. To date, there are no drugs licensed for use in humans which target this step that have therapeutic value comparable to that of β-lactams. Therefore, additional information regarding the role of TGases within the bacterial cell could be of particular importance for the treatment of MRSA strains, one of the major causes of hospital-acquired bacteremia (6), which can be resistant to most classes of available antibiotics.

This study has shown that besides MGT and PBP2, a third transglycosylase enzyme from S. aureus, SgtA, is capable of synthesizing glycan chain formation. We have shown that MGT is essential in S. aureus cells lacking a PBP2 TGase function and that cells cannot survive when SgtA is the sole transglycosylase. Importantly, although MGT can replace the transglycosylase activity of PBP2 in both MSSA and MRSA strains, it cannot replace the role of PBP2 in β-lactam resistance of MRSA strain COL, as the presence of MGT was not sufficient to allow the growth of cells exposed to high concentrations of oxacillin.

The requirement in S. aureus for the transglycosylase activity of either PBP2 or MGT for survival differs from what was observed with B. subtilis and Enterococcus spp. (both of which encode multiple class A PBPs and no homologs of known Mgts), where the deletion of all enzymes with known transglycosylase activity has little effect on viability (1, 21, 28). We have shown here that, in MRSA strain COL, the class A PBP2 is dispensable but only when MGT is present. The fact that cells cannot survive when SgtA is the lone functioning transglycosylase indicates that the activities or the specificities of the two monofunctional transglycosylases are probably different, as SgtA cannot take over the role of MGT. The three transglycosylase enzymes interact with one another and with other cell wall-synthetic enzymes in a bacterial two-hybrid assay, suggesting that they act in concert within the cell.

The transglycosylation step of peptidoglycan synthesis is an attractive target for the development of new therapeutic agents, and thus, continued investigation of the enzymes involved in this reaction is a step further in the battle against drug-resistant bacteria.

ACKNOWLEDGMENTS

We thank G. Karimova and M. Débarouillé for the generous gifts of the bacterial two-hybrid plasmids and the pMAD plasmid, respectively. We also thank Sérgio Filipe and James Yates for helpful discussions.

This work was funded by Fundação para a Ciência e Tecnologia through research grant PTDC/BIA-MIC/099151/2008 awarded to M.G.P. P.R. was supported by fellowship SFRH/BPD/23812/2005, A.M.J. by fellowship SFRH/BD/28480/2006, and H.V. by fellowship SFRH/BD/38732/2007. M.T. is a Research Associate of the National Fund for Scientific Research (F.R.S_FNRS, Belgium) and supported by the Fonds de la Recherche Fondamentale Collective (FRFC no. 2.4506.08).

Footnotes

[down-pointing small open triangle]Published ahead of print on 25 March 2011.

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