The inhibition of mucopeptide synthesis by benzylpenicillin in relation to irreversible fixation of the antibiotic by staphylococci

Abstract

1. Benzylpenicillin is irreversibly fixed to staphyloccoci by a reaction that obeys second-order kinetics, whereas the progress of inhibition of mucopeptide synthesis obeys first-order kinetics after a short lag during which the antibiotic has no effect. 2. When the micro-organisms are saturated with benzylpenicillin they can still make mucopeptide in solutions containing chloramphenicol at a normal rate after a lag period. 3. About 90% of the benzylpenicillin stays fixed to the cells after mucopeptide synthesis has reached its maximum and constant rate. 4. During the phase when mucopeptide synthesis by cells saturated with benzylpenicillin is accelerating, a small number of additional sites that fix benzylpenicillin is revealed. The number of these sites reaches a maximum and constant value at about the same time as mucopeptide biosynthesis reaches a maximum and constant rate. 5. Staphylococci saturated with benzylpenicillin are exceedingly sensitive to fresh additions of the antibiotic. 6. The degree of inhibition of mucopeptide synthesis caused by these small amounts of antibiotic agrees with the degree of substitution by benzylpenicillin of the newly revealed or ;sensitive' sites. 7. Since these sensitive sites are revealed during incubation of the bacteria with chloramphenicol it is unlikely that they are due to newly formed protein. 8. On the basis of these results, a hypothesis for the inhibition by penicillin of the cross-linking reaction in the terminal stages of mucopeptide synthesis is suggested.

Penicillin-Resistant Mechanisms in Pseudomonas aeruginosa : Binding of Penicillin to Pseudomonas aeruginosa KM 338

“…The binding followed saturation type kinetics (specific binding), and its saturation was achieved at low concentration (2 nmol/ mil) of the antibiotic, as has been shown (16,26) for penicillin-susceptible organisms. However, the binding to whole cells of this organism was entirely of the nonsaturation type (nonspecific), although binding to penicillin-susceptible organisms was specific as previously desciKbed (4,5,8,15,20,24,26), and saturation was achieved at low concentration of penicillin G, as with the membrane fractions (16,17). About 3 nmol of penicillin G was specifically bound per g of the membrane protein at saturation.…”

“…However, the binding was entirely nonspecific. The amount bound to whole cells was directly proportional to the concentration up to a level of at least 750 nmol/ml, although binding to the membrane fraction from this organism and to the whole cells of penicillin-susceptible organisms was specific as described above and previously (4,5,8,15,20,24,26). The binding of [14C]benzylpenicilloic acid to the cells was also nonspecific and of lower affinity compared with penicillin G.…”

“…All the organisms tested were grown in Trypticase soy broth (BBL) at 37 C for 18 h. These broth cultures were disrupted with an ultrasonic disintegrator (60 W, 20 kcycle/s. Ohtake Seisakusho Co., Ltd., Tokyo, Japan) for 10 min, and the resulting suspensions of each disrupted culture were used as the enzyme source.…”

Correlation Between the Binding of β-Lactam Antibiotics to Staphylococcus aureus and Their Physical-Chemical Properties

“…The washing procedures employed were judged to be adequate to remove noncovalently bound "4C-penicillin G for the following reasons: (i) cells first saturated with "2C-penicillin G and then incubated with 14C-penicillin bound 5 counts per min per ml (corrected for reduction of specific activity), whereas a control sample incubated with "-C-penicillin G bound 498 (10,17). Rogers (17) reported that the binding reaction is temperature-sensitive and obeys second-order reaction kinetics for the first 20 min. These results were confirmed in this laboratory ( Fig.…”

Mucopeptide Synthesis by rod Mutants of Bacillus subtilis

“…More than 2000 counts were collected per sample. For counting liquid samples a dioxane-based scintillation fluid was used (Rogers, 1967). Estimation of the specijk activity of amino sugars.…”