On the process of cellular division in Escherichia coli: isolation and characterization of penicillin-binding proteins 1a, 1b, and 3.
Multiple mutants of Escherichia coli defective in penicillin-binding proteins (PBPs) were constructed, and into these strains Co1E1 plasmids carrying the genes for PBP-1a, -1b, or -3 were introduced. From these plasmid-carrying strains, PBP-1a and -1b were purified by ampicillin-Sepharose affinity chromatography and PBP-3 by cephalexin-Sepharose affinity chromatography. Improved purification was achieved by differential elution with NH2OH. Purified PBP-1b synthesized murein when added to the membrane fraction of a PBP-1b-defective mutant, which by itself failed to support murein synthesis in vitro. The PBP-1b preparation was able to synthesize murein from the lipid intermediate extracted with chloroform/methanol but was unable to utilize UDP-linked precursors for murein synthesis. Murein synthesis was inhibited by vancomysin, ristocetin, moenomycin, and enduracidin, but not by beta-lactam antibiotics. The synthesized murein was shown to contain crosslinked muropeptides. Their crosslinking was abolished by action of beta-lactam antibiotics. The PBP-1a and -3 preparations showed substantially no activity for murein synthesis in the same reaction system. None of the three PBPs showed D-alanine carboxypeptidase activity with UDP-N-acetylmuramoyl-pentapeptide as substrate or endopeptidase activity with bis(disaccharide-peptide) as substrate.[1]References
- On the process of cellular division in Escherichia coli: isolation and characterization of penicillin-binding proteins 1a, 1b, and 3. Tamura, T., Suzuki, H., Nishimura, Y., Mizoguchi, J., Hirota, Y. Proc. Natl. Acad. Sci. U.S.A. (1980) [Pubmed]
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