Abstract
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.
Highlights
The high molecular weight penicillin-binding pro- biosynthesis has been successfullydemonstrated in mancyellteins (PBP(s))Bacillus subtilis PBPs 1, 2, and 4 and free systems and with some purified
The high molecular weight penicillin-binding proteins isolated from B. subtilis and B. stearothermophilus, together with a small amount of cross-linked peptidoglycan in the case of B. stearothermophilus PBPs, have, been shown to catalyze peptidoglycan synthesis either in afilter paper assay or ina conventional test tube assay when certain organic solvents were added to thereaction mixture
The pH optima for peptidoglycan synthesis andother properties were identical withthose previously reported for the cell-free particulate enzyme systems from these organisms [33,40]
Summary
Peptidoglycan synthesiswas observed with increased amounts of the high molecular PBPs ata constant detergent concen-. High molecular weight penicillin-binding proteins from B. tration of 0.375% Triton X-100. Stearothermophilus 15952 were purified concentration to0.125% resulted in increased peptidoglycan by cephalosporin affinity chromatographayfter detergent-salt synthesis. Similar results were obtained with B. subtilis PBPs 1, 2, and 4 (data not shown). Optimal peptidoglycan synthesis weight PBP mixtures consist of four proteins: PBP 1 PBPs 1-4 would proceed only if ( a ) incubation was carried out on Whatman3MM chromatography paper or (b)certain sugars and solvents were included in the reaction mixture of a test tubeassay.
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