Monofunctional transglycosylases are not essential for Staphylococcus aureus cell wall synthesis

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.

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 the absorbance at OD₆₀₀. The double mutant strains show growth similar to that of the parental strains. (B) Bacterial cultures were grown on solid medium (TSA) supplemented or not with 0.5 mM IPTG. Control strain COLIPBP2i (sector A) grows well with or without IPTG, COLIPBP2iΔmgt (sector B) shows no growth in the absence of IPTG, COLIPBP2iΔsgtA (sector C) grows well in the absence of IPTG, and COLIPBP2iΔmgtΔsgtA (sector D) does not grow in the absence of IPTG. (C) The growth of the control and mutant strains was monitored in liquid medium with or without IPTG by determining the OD₆₀₀. After 4 h, cultures were diluted to an OD₆₀₀ of approximately 0.1 in fresh medium with or without inducer, and the OD₆₀₀ was monitored for a further 8 h. (D) Detection of PBP2 by Western blot analysis of cell extracts prepared from liquid cultures shown in panel C after 4 h of growth. Lanes: a, COLIPBP2i; b, COLIPBP2iΔmgt; c, COLIPBP2iΔsgtA; and d, COLIPBP2iΔmgtΔsgtA.

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; 2, lipid II incubated with 5 μM SgtA; and 3, lipid II incubated with 5 μM MGT. Both MGT and SgtA catalyze the polymerization of lipid II under these experimental conditions.

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 strain COL spa⁻. (B) Flavomycin PAPs. The PBP2 TGase mutant (COLTG42spa) and the double mutant strain COLTG42ΔsgtA showed 10-fold reductions in MIC to flavomycin. The double mutant COLΔmgtΔsgtA and the COLΔsgtA mutant showed greater sensitivities to the antibiotic than the COLΔmgt mutant, which had the same MIC as the wild-type strain COL spa⁻.

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 with SgtA, MGT, PBP1, PBP2, and PBP2A. Cotransformants were qualitatively analyzed by growing strains on LB-X-Gal plates containing 0.5 mM IPTG for 30 h at 30°C. The panels below the charts show the formation of blue colonies by interacting fusion proteins and that the colonies from noninteracting pairs remained white on indicator plates. For quantitative analysis, transformants were grown in LB medium in the presence of 0.5 mM IPTG at 30°C for 16 h, followed by determination of β-galactosidase activities. Data are averages of the results of three independent experiments, and error bars indicate standard deviations.