Isolated Peptidoglycan Glycosyltransferases from Different Organisms Produce Different Glycan Chain Lengths

Abstract

Peptidoglycan is an essential component of bacterial cell wall. The glycan strands of peptidoglycan are synthesized by enzymes called peptidoglycan glycosyltransferases (PGTs). Using a highresolution SDS-PAGE assay, we compared the glycan strand lengths of four different PGTs from three different organisms (Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus). We report that each enzyme makes a polymer having an intrinsic characteristic length that is independent of the enzyme:substrate ratio. The glycan strand lengths vary considerably depending on the enzyme. These results indicate that each enzyme must have some mechanism, as yet unknown, for controlling product length. The observation that different PGTs produce different length glycan chains may have implications for their cellular roles and for the three dimensional structure of bacterial peptidoglycan.Bacterial cells are surrounded by a polymer matrix comprising crosslinked strands of peptidoglycan (PG). This matrix, called the sacculus, functions as an exoskeleton, maintaining cell shape and enabling the plasma membrane to withstand high internal osmotic pressures. 1 The three dimensional architecture of PG is not yet clear, but is presumed to depend, among other things, on the lengths of the glycan strands, which are synthesized by processive enzymes called peptidoglycan glycosyltransferases (PGTs). 2 Numerous studies have evaluated lengths of glycan strands from digested bacterial sacculi, and a range of values have been reported even for digests from the same organism. 3 Because substrates and appropriate analytical methods were not available until recently, there have been no systematic studies comparing the lengths of glycan strands produced by different PGTs in vitro. Using a high-resolution gel electrophoresis assay recently developed in our laboratory, 2b we report here a comparative study of the glycan strand length distributions produced by four different PGTs, Escherichia coli PBP1A (E. coli PBP1A), Escherichia coli PBP1B (E. coli PBP1B), Enterococcus faecalis PBP2A (E. faecalis PBP2A), and Staphylococcus aureus PBP2 (S. aureus PBP2) (Fig 1). We show that different PGTs produce glycan chains having a characteristic intrinsic length distribution. The intrinsic lengths are a function of the particular PGT but are independent of enzyme:substrate ratios. There is a correlation between the intrinsic in vitro product lengths kahne@chemistry.harvard.edu; suzanne_walker@hms.harvard.edu. and the longest strands isolated from sacculi. The implications of these observations for the architecture of the bacterial cell wall are discussed. NIH Public AccessThe four PGTs we studied were overexpressed, purified and subjected under similar conditions to reaction with heptaprenyl-[ 14 C]-Lipid II (1) (Fig 1a). 2,4,5,6 Unexpectedly, we found that the four PGTs produced glycan chains of different limiting lengths (i.e., the size beyond which the length does not increase even if reaction times are extended and additional substrate is added) ...

Moenomycin Resistance Mutations in Staphylococcus aureus Reduce Peptidoglycan Chain Length and Cause Aberrant Cell Division

“…The full-length mutant protein was expressed and purified, and its ability to polymerize synthetic Lipid II was compared to that of wild type full-length PBP2 (28, 29). Reaction mixtures were analyzed by SDS-PAGE, which separates products to single disaccharide resolution (Figure 2B) (30, 31). As demonstrated previously, the wild type enzyme reacted to produce long polymers.…”

SagB Glucosaminidase Is a Determinant of Staphylococcus aureus Glycan Chain Length, Antibiotic Susceptibility, and Protein Secretion

“…Studying purified PGTs from E. coli (PBP1A), S. aureus (PBP2), and Enterococcus faecalis, Wang et al observed that each PGT generated polymers with characteristic lengths, independently of enzyme/substrate ratios, and proposed that PGTs may rely on an intrinsic mechanism for controlling product length (64). Mutations in the structural gene for mltG, encoding a lytic transglycosylase of the YceG family (Pfam02618) in E. coli, increase the length of glycan strands in the peptidoglycan (52).…”

Peptidoglycan Hydrolases of Escherichia coli

“…However, a multiply defective mutant lacking six (Stl70, MltA, MltB, MltC, MltD, and MltE) out of the seven identified LTs exhibited no significant decrease of the 1,6-anhydro-MurNAc muropeptide content (92). Moreover, the PG material synthesized in vitro by purified glycosyltransferases remained bound to undecaprenyl pyrophosphate (8,300). Thus, neither glycosyltransferases nor most LTs appear to be directly involved in the termination step.…”