Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein.

Penicillin-binding protein (PBP)-2 and the RodA protein are known to function in determining the rod shape of Escherichia coli cells. Peptidoglycan biosynthetic reactions that required these two proteins were demonstrated in the membrane fraction prepared from an E. coli strain that overproduced both of these two proteins and which lacked PBP-1B activity (the major peptidoglycan synthetase activity in the normal E. coli membranes). The cross-linked peptidoglycan was synthesized from UDP-N-acetylmuramylpentapeptide and UDP-N-acetylglucosamine in the presence of a high concentration of cefmetazole that inhibited all of PBPs except PBP-2. The peptidoglycan was synthesized via a lipid intermediate and showed up to 30% cross-linking. The cross-linking reaction was strongly inhibited by the amidinopenicillin, mecillinam, and by other beta-lactam antibiotics that have a high affinity for PBP-2, but not by beta-lactams that had very low affinity for PBP-2. The formation of peptidoglycan required the presence of high levels of both PBP-2 and the RodA protein in the membranes, but it is unclear which of the two proteins was primarily responsible for the extension of the glycan chains (transglycosylation). However, the sensitivity of the cross-linking reaction to specific beta-lactam antibiotics strongly suggested that it was catalyzed by PBP-2. The transglycosylase activity of the membranes was sensitive to enramycin and vancomycin and was unusual in being stimulated greatly by a high concentration of a chelating agent.