Synthesis of peptidoglycan by high molecular weight penicillin-binding proteins of Bacillus subtilis and Bacillus stearothermophilus.
The high molecular weight penicillin-binding proteins (PBP(s) ) Bacillus subtilis PBPs 1, 2, and 4 and Bacillus stearothermophilus PBPs 1-4 were shown to catalyze peptidoglycan synthesis from the undecaprenol-containing lipid intermediate substrate in two assay systems. In a filter paper assay system, high levels of substrate polymerization occurred when reaction mixtures were incubated on Whatman 3MM filter paper. The pH optimum for peptidoglycan synthesis was 7.5 for B. subtilis PBPs 1, 2, and 4 and 8.5 for B. stearothermophilus PBPs 1-4. Polymerization was Mg2+-independent and was unaffected by sulfhydryl reagents. Reconstitution with membrane lipids or addition of detergent (optimal concentration, 0.1%) was necessary for synthesis to occur. Bacitracin, penicillin, and cephalothin did not affect polymerization while vancomycin, ristocetin, moenomycin, and macarbomycin were strong inhibitors. In a test tube assay system, optimal synthesis occurred either in the presence of 10% ethylene glycol, 10% glycerol, and 8% methanol or in the presence of 10% N-acetylglucosamine. The products of lysozyme digestion of the synthesized peptidoglycan were analyzed by gel filtration and paper chromatography. B. stearothermophilus PBPs 1-4 synthesized a peptidoglycan product that was 5-7% cross-linked. No evidence for cross-linking was apparent in the peptidoglycan product of B. subtilis PBPs 1, 2, and 4.
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D002268:Carboxypeptidases / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000378:metabolism*; D006602:Hexosyltransferases; D007700:Kinetics; D002267:Muramoylpentapeptide Carboxypeptidase / Q000378:metabolism*; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism*; D010457:Peptidoglycan / Q000096:biosynthesis*; D010458:Peptidyl Transferases; D013045:Species Specificity
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010457:Peptidoglycan; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002268:Carboxypeptidases; D002267:Muramoylpentapeptide Carboxypeptidase