Influence of organic anions on the liberation of penicillinase from Staphylococcus aureus

1969

J Bacteriol

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In Staphylococcus aureus, penicillinase remaining cell-bound (60 to 75 % of original total) after treatment with citrate does not become exopenicillinase. Exopenicillinase in these cells appears only under conditions permitting de novo penicillinase synthesis. By use of "4C-labeled cells, it was shown that exopenicillinase consists of newly synthesized molecules which have not equilibrated with preformed membranebound enzyme.

Section: Resultsmentioning

confidence: 96%

“…Cloxacillin was a commercial product of Commonwealth Serum Laboratories, Victoria, Australia. Other materials and methods used were as previously described (2,3).…”

mentioning

confidence: 99%

Exopenicillinase Synthesis in Staphylococcus aureus

1969

J Bacteriol

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“…In previous studies of the synthesis and liberation of penicillinase (EC 3.5.2.6) by cells of Staphylococcus aureus (3,4), we showed that there is a fraction of cell-bound penicillinase, comprising up to 40% of the total, which can be liberated by incubation of the cells for short periods at 37 C in 0.15 M sodium citrate. This fraction of cell-bound exopenicillinase is replenished only under conditions permitting de novo penicillinase synthesis and is the most recently synthesized enzyme (5).…”

mentioning

confidence: 91%

Formation and Secretion of Induced Penicillinase in Protoplasts of Staphylococcus aureus

Antimicrob Agents Chemother

1973

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Conditions suitable for induced formation and secretion of penicillinase (EC 3.5.2.6) in protoplasts of Staphylococcus aureus were determined. No requirement was found for cells to be exposed to inducer prior to formation of protoplasts. Neither cell wall components nor mesosomes appeared to be necessary for induction or secretion. In medium containing 1.1 M sucrose about half of the formed enzyme was soluble, whereas in medium containing 0.37 M sodium succinate only about 10% of the penicillinase remained protoplast-bound. Low concentrations of polyanions (dextran sulfate and potassium polyvinyl-sulfonate) inhibited the formation of induced penicillinase, as did 4-acetamido-4'-isothiocyano-stilbene 2,2'-disulfonic acid. None of these compounds inhibited the activity of native penicillinase, and none would be expected to pass through the protoplast membrane. Penicillinase, denatured in 4 M urea, could be renatured by dilution in the presence of benzylpenicillin, and the above three inhibitors interfered strongly with this process. The results are taken as evidence that penicillinase may be secreted through the protoplast membrane in an incompletely folded form.In previous studies of the synthesis and liberation of penicillinase (EC 3.5.2.6) by cells of Staphylococcus aureus (3, 4), we showed that there is a fraction of cell-bound penicillinase, comprising up to 40% of the total, which can be liberated by incubation of the cells for short periods at 37 C in 0.15 M sodium citrate. This fraction of cell-bound exopenicillinase is replenished only under conditions permitting de novo penicillinase synthesis and is the most recently synthesized enzyme (5). No transformation of old remaining cell-bound penicillinase to exopenicillinase takes place. The bacterial cell wall has been implicated in the formation of active exoenzymes by several authors. Spheroplasts of Streptococcus faecalis were unable to produce extracellular proteinase until some cell wall component had first been resynthesized (9), and protoplasts of Bacillus subtilis failed to synthesize extracellular amylase and protease under conditions permitting the unimpaired incorporation of "4C-valine into a trichloroacetic acid-insoluble product (14).Induced enzyme synthesis in protoplasts of bacteria has been shown only in very few cases. It is not too clear from the literature whether true protoplasts of B. cereus have the capacity to produce inducible penicillinase. On the one hand, Sheinin (23) reported that protoplasts of B. cereus 569 could be directly induced to form penicillinase at a rate comparable to that of whole cells, whereas Duerksen (7) was not able to demonstrate induced formation of penicillinase in protoplasts of the same organism unless the cells used to produce these protoplasts had been preinduced. In an attempt to resolve this discrepancy, Wainwright and Martin (25), using spheroplasts of this organism, obtained complex results. The interpretation was further complicated by a high and variable rate of basal synthesis of penicillinase by no...

“…Incubation mixtures for incorporation of radioactive precursors were the same as induction mixtures (7) with the omission of inducer, but with the following additions: for protein synthesis, 0.1 mg of amino acid mixture (4) containing 0.1 MCi of "4C-protein hydrolysate; for nucleic acid synthesis, 0.1 ml of 3 mM "C-uracil (0.1 MCi/smol) or 0.1 ml of 3 mM 3H-thymidine (16.7 ACi/umol); for lipid synthesis, 0.1 ml of 25 mM '4C-sodium acetate (1 IACi/umol). Radioactive compounds were added after temperature equilibration at 37 C. At the end of the incubation period samples for the measurement of radioactivity incorporated into nucleic acids and proteins were treated as previously described (3,5) and counted in a Nuclear-Chicago gas-flow counter.…”

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confidence: 99%

Preparation of Metabolically Active Staphylococcus aureus Protoplasts by Use of the Aeromonas hydrophila Lytic Enzyme

1973

J Bacteriol

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Stable, metabolically active protoplasts of Staphylococcus aureus have been prepared by the use of a staphylolytic enzyme produced by Aeromonas hydrophila. Respiratory and glycolytic rates and synthesis of nucleic acids, protein, and lipid in these protoplasts, stabilized variously in 1.1 M sucrose, 0.37 M sodium succinate, or 0.37 M sodium sulfate, have been shown to be comparable with the same parameters in intact cells under the same conditions.