Peptidoglycan Synthesis in the Absence of Class A Penicillin-Binding Proteins in Bacillus subtilis

Abstract

Penicillin-binding proteins (PBPs) catalyze the final, essential reactions of peptidoglycan synthesis. Three classes of PBPs catalyze either trans-, endo-, or carboxypeptidase activities on the peptidoglycan peptide side chains. Only the class A high-molecular-weight PBPs have clearly demonstrated glycosyltransferase activities that polymerize the glycan strands, and in some species these proteins have been shown to be essential. The Bacillus subtilis genome sequence contains four genes encoding class A PBPs and no other genes with similarity to their glycosyltransferase domain. A strain lacking all four class A PBPs has been constructed and produces a peptidoglycan wall with only small structural differences from that of the wild type. The growth rate of the quadruple mutant is much lower than those of strains lacking only three of the class A PBPs, and increases in cell length and frequencies of wall abnormalities were noticeable. The viability and wall production of the quadruple-mutant strain indicate that a novel enzyme can perform the glycosyltransferase activity required for peptidoglycan synthesis. This activity was demonstrated in vitro and shown to be sensitive to the glycosyltransferase inhibitor moenomycin. In contrast, the quadruple-mutant strain was resistant to moenomycin in vivo. Exposure of the wild-type strain to moenomycin resulted in production of a phenotype similar to that of the quadruple mutant.The structural element of the bacterial cell wall is peptidoglycan (PG), and the importance of this structure is apparent in the number of antimicrobial agents targeting its components and the enzymatic reactions leading to its synthesis. The glycosyltransferase (GT) activity carrying out one of the final enzymatic reactions in PG synthesis is an attractive target, because the enzymes performing that activity are highly conserved and believed to be essential (9, 10). PG is composed of glycan strands cross-linked by peptide side chains. The glycan strands are synthesized by a GT that adds lipid-linked disaccharide pentapeptide subunits to nascent glycan strands. The pentapeptides are then utilized by a transpeptidase to crosslink adjacent glycan strands (reviewed in reference 4). Most of the proteins involved in these final enzymatic reactions are penicillin-binding proteins (PBPs), which are divided into three classes based on the presence of conserved functional domains (9, 10). Only the class A PBPs have an N-terminal domain that contains the conserved amino acid sequences found in all GTs clearly demonstrated to polymerize PG. Class B PBPs have a different N-terminal domain; and although some researchers have associated GT activity with class B PBPs (15), others have been unable to reproduce those results (2). Both classes have C-terminal penicillin-binding domains containing the transpeptidase activity that cross-links peptide side chains. Some species also have monofunctional glycosyltransferases (MGTs) that contain the conserved amino acid sequences found in the class A PBP GT domain but whic...

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Synthesis of Modified Peptidoglycan Precursor Analogues for the Inhibition of Glycosyltransferase

“…41 PG synthesis by the quadruple mutant was demonstrated in vitro and was shown to be sensitive to moenomycin, confirming the GT reaction as an antibacterial target.…”