Enzymatic synthesis of cell wall mucopeptide in a particulate preparation of Escherichia coli

Araki, Yoshio; Shirai, Ryoichi; Shimada, Akira; Ishimoto, Nobutoshi; Ito, Eiji

doi:10.1016/0006-291x(66)90751-0

Cited by 54 publications

(16 citation statements)

References 5 publications

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“…It is based on the assumption that only new murein strands serve as donors for the crosslinking transpeptidation reaction. This is reasonable because the newly synthesized strands contain the pentapeptide side chains terminating in D-alanyl-D-alanine, which is required for the transpeptidation reaction (16)(17)(18)(19), and over 97% of the muropeptides inserted into the sacculus during a 3-min (14). Exponentially growing cells were washed by filtration, resuspended in minimal glucose medium without A2pm, and incubated for the 25 min required to deplete E. coli W7 of its intracellular pool of A2pm (14,15 A similar labeling experiment performed with BUG 6, which is temperature sensitive for division (20), is shown in Fig.…”

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confidence: 99%

Molecular model for elongation of the murein sacculus of Escherichia coli.

Burman¹,

Park²

1984

Proc. Natl. Acad. Sci. U.S.A.

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confidence: 99%

Molecular model for elongation of the murein sacculus of Escherichia coli.

Burman¹,

Park²

1984

Proc. Natl. Acad. Sci. U.S.A.

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“…It was prepared by alumina grinding (1,7,8) min at 37 C, the reaction was stopped by the addition of 10 ,liters of isobutyric acid-1N NH4OH (5:3) and cooled to 0 C. The entire reaction mixture plus washings was spotted on a 1-cm line at the origin of a sheet of Whatman 3MM paper which was then developed by descending chromatography with isobutyric acid-0.125 N NH4OH (5:3). Chromatograms were autoradiographed for 4 days with Kodak RB 54 X-ray film.…”

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confidence: 99%

Additional Antibiotic Inhibitors of Peptidoglycan Synthesis

Linnett

Strominger

1973

Antimicrob Agents Chemother

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Diumycin, janiemycin, nisin, and subtilin inhibited peptidoglycan synthesis catalyzed by particulate enzyme systems from Bacillus stearothermophilus and Escherichia coli . All of these, except for nisin, also induced accumulation of the lipid intermediate in peptidoglycan synthesis. Concentrations required for 50% inhibition of peptidoglycan synthesis were less than 0.1 μg/ml for diumycin and in the range of 10 to 100 μg/ml for janiemycin, nisin, and subtilin in both organisms. The discrepancy between the extremely low concentration of diumycin required to inhibit the in vitro system from E. coli and the much higher concentration required to inhibit growth of the organism is noteworthy.

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“…In Escherichia coli, and probably other microorganisms, the transpeptidase is irreversibly inactivated by penicillin (4)(5)(6). It has been hypothesized that this inactivation is due to penicilloylation (2).…”

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confidence: 99%

Isolation by Covalent Affinity Chromatography of the Penicillin-Binding Components from Membranes of Bacillus subtilis

Blumberg¹,

Strominger²

1972

Proc. Natl. Acad. Sci. U.S.A.

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An affinity chromatography technique was developed to isolate the five penicillin-binding components present in Bacillus subtilis membranes. The proteins were solubilized by the detergent Nonidet P-40, bound covalently to penicillin-substituted Sepharose, and subsequently eluted from the matrix with neutral hydroxylamine, which cleaves the penicilloyl-enzyme bond. Penicillin binding-component V, the D-alanine carboxypeptidase, makes up 1% of the total membrane protein. A modification of the above procedure enabled this enzyme to be obtained from the membrane in pure form in a single step with 50% overall recovery of enzymatic activity.Penicillin kills bacteria by preventing the last stage in cellwall biosynthesis, the cross-linking of nascent cell wall by the transpeptidase (1-3). In Escherichia coli, and probably other microorganisms, the transpeptidase is irreversibly inactivated by penicillin (4-6). It has been hypothesized that this inactivation is due to penicilloylation (2). Many strains of bacteria have been shown to bind radioactive penicillin. Moreover, the sensitivity of the organisms to penicillin correlates roughly with the rate at which penicillin binds to the cells (7-12). In some cases it may be that organisms are killed by a mechanism that does not involve covalent binding of penicillin. The transpeptidase of Bacillus megaterium is competitively rather than irreversibly inhibited by penicillins (13). Moreover, both E. coli and Streptomyces strains contain or excrete soluble D-alanine carboxypeptidases that are reversibly inhibited by penicillins (14, 15). The Streptomyces carboxypeptidase catalyzes a transpeptidation reaction using synthetic substrates (16), but it has not yet been shown that this extracellular enzyme is the physiological transpeptidase. In any case, the analysis of penicillin-binding components offers one promising approach to the study of cell-wall biosynthesis and the mode of action of penicillin.It has now become clear that there exists in a bacterial membrane not one protein that reacts irreversibly with penicillins, but several. Two lines of experimentation first suggested the occurrence of multiple penicillin-binding components. The D-alanine carboxypeptidase of B. subtilis, which is irreversibly inactivated by penicillins (17), could be inhibited in growing cells by 6-aminopenicillanic acid without killing the cells. In contrast, the cells were killed by cephalothin at concentrations that caused no inhibition of the carboxypeptidase (18). This result implied the presence of at least two penicillin-binding components, the cephalothinresistant carboxypeptidase and the cephalothin-sensitive penicillin killing site. In fact, isoelectric focusing of B. subtilis Abbreviation: SDS, sodium dodecyl sulfate. 3751 membranes to which radioactive penicillin had been bound revealed multiple peaks of radioactivity. Prior treatment with cephalothin abolished some but not all of the bands (19,20

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Enzymatic synthesis of cell wall mucopeptide in a particulate preparation of Escherichia coli

Cited by 54 publications

References 5 publications

Molecular model for elongation of the murein sacculus of Escherichia coli.

Molecular model for elongation of the murein sacculus of Escherichia coli.

Additional Antibiotic Inhibitors of Peptidoglycan Synthesis

Isolation by Covalent Affinity Chromatography of the Penicillin-Binding Components from Membranes of Bacillus subtilis

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