Shigeo Tamaki

Dual enzyme activities of cell wall peptidoglycan synthesis, peptidoglycan transglycosylase and penicillin-sensitive transpeptidase, in purified preparations of Escherichia coli penicillin-binding protein 1A

Summary Two different cell wall peptidoglycan synthetase systems are carried by penicillin-binding proteins 1A and 1B purified from Escherichia coli. Both systems consist of two enzyme activities carrying out successive reactions of peptidoglycan synthesis from the lipid-linked precursor, N-acetylglucosaminyl-N-acetylmuramyl(-pentapeptide)-diphosphate-undecaprenol, namely, those of peptidoglycan transglycosylase and β-lactam antibiotic-sensitive transpeptidase. The activities of the two enzyme systems differ in optimal conditions and sensitivities to β-lactam antibiotics. The properties of purified PBP-1A are reported in this paper.

A mecillinam-sensitive peptidoglycan crosslinking reaction in Escherichia, coli☆

Abstract The amidinopenicillin, mecillinam, induces the formation of spherical cells of Escherichia coli by inactivation of penicillin-binding protein 2 (PBP2). A mecillinam-sensitive peptidoglycan crosslinking reaction has been demonstrated in particulate membrane preparations from this organism. The activity was detected in membranes that contained elevated levels of PBP2 and in which crosslinking reactions due to all other PBPs had been inactivated with the cephamycin antibiotic, cefmetazole. The particulate membrane preparation catalyzed synthesis of peptidoglycan that was up to 20% crosslinked from nucleotide precursors. Crosslinkage of the peptidoglycan was inhibited 50% by 0.2 μg mecillinam per ml but was not inhibited by much higher concentrations of cephamycins, which have very low affinity for PBP2. The crosslinking reaction appears to be due to the transpeptidase activity of PBP2, which is implicated in the mechanism of cell shape determination, and is the killing target for mecillinam.

Formation of hyper-crosslinked peptidoglycan with multiple crosslinkages by a penicillin-binding protein, 1A, of Escherichia coli

Abstract Hyper-crosslinked peptidoglycan was synthesized in vitro by purified penicillin-binding protein 1A of Escherichia coli . The peptidoglycan formed was crosslinked up to 39%. About half the crosslinks were novel three-handed crossbridges whereas the other half were two-handed crossbridges that are the major constituents of normally crosslinked peptidoglycan of E. coli . The structure of the three-handed crossbridge constructed among three peptide side-chains of - l -alanyl- d -glutamyl-meso-diaminopimelyl- d -alanyl- d -alanine was deduced from several criteria. Probably penicillin-binding protein 1A is responsible for hyper-crosslinking of E. coli peptidoglycan in vivo .

New cephamycin antibiotic, CS-1170: binding affinity to penicillin-binding proteins and inhibition of peptidoglycan cross-linking reactions in Escherichia coli.

The binding activity of CS-1170, a new cephamycin antibiotic, to penicillin-binding proteins (PBPs) in Escherichia coli and Proteus species and the potency of this antibiotic in vitro to inhibit enzymes involved in peptidoglycan cross-linking in E. coli were tested. Similar experiments were carried out with the 7α-H analog of CS-1170, R-45656, and the results were compared with those obtained with CS-1170. CS-1170 showed high affinities (compared with that of penicillin G) for E. coli PBP-1A, -1Bs, and -3, the PBPs of higher molecular weight, but not PBP-2. It also inhibited the in vitro peptidoglycan cross linking reaction and concomitant release of d-alanine at very low concentrations (approximately its minimal inhibitory concentration). This antibiotic also showed very high affinity for PBP-4, -5, and -6, the PBPs of lower molecular weight, and at extremely low concentrations it inhibited d-alanine carboxypeptidases IA and IB, corresponding to PBP-5/6 and PBP-4, respectively. CS-1170 seemed to be resistant to the β-lactamase activity of PBP-5 and -6 in E. coli and Proteus species. R-45656 showed as high an affinity for PBP-1A, -1Bs, and -3 as CS-1170, but unlike CS-1170, it had low affinities for PBP-4, -5, and -6. The concentrations of R-45656 required for inhibition of d-alanine carboxypeptidases IA and IB were also much higher than those of CS-1170. R-45656 showed rather low activities in inhibiting the in vitro cross-linking reaction of peptidoglycan and concomitant release of d-alanine. Synergism was observed in 9 of 22 strains examined between CS-1170 and mecillinam, which bound specifically to PBP-2. Images

Further Studies onrodAMutant: a Round Morphological Mutant ofEscherichia coliK-12 with Wild-Type

Escherichia coli rodA mutant AOS151 grows as round cells at 30 and 42°C (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol., 115, 436–442 (1973)). The mutant was found to be resistant to mecillinam at both temperatures. lip+ transductants were prepared by Pl phage transduction via strain AOS151, the cotransduction frequency of round morphology (Rod−) at 42°C with the lip gene being about 90%. At 42°C all 54 Rod− transductants tested were resistant to mecillinam. At 30°C all but two of these Rod− (at 42°C)-type transductants were rod-shaped, and all were sensitive to mecillinam; the two strains grew as ovoid cells. The original rodA mutant AOS151 probably involves an additional mutation(s), that expresses the round cell shape at lower temperature, whereas the rodA51 mutation alone seems to result in temperature-sensitive expression of round cell morphology and mecillinam resistance. rodA mutant cells cultured at either 30 or 42°C had wild-type penicillin-binding protein 2, judging from penic...