Preparation of Metabolically Active
            <i>Staphylococcus aureus</i>
            Protoplasts by Use of the
            <i>Aeromonas hydrophila</i>
            Lytic Enzyme

Coles, N. W.; Gross, R

doi:10.1128/jb.115.3.746-751.1973

Cited by 4 publications

(1 citation statement)

References 13 publications

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“…The most straightforward demonstration of this is illustrated by the comparative 13 C CPMAS spectra of whole cells and protoplasts (figure 3a). Protoplasts are osmotically delicate cells in which the majority of the cell wall has been removed by lysostaphin treatment [56,57]. Protoplasts must be stabilized by sucrose or trehalose to prevent cell lysis from the turgor pressure of the cell.…”

Section: Detecting Cell Wall Content and Major Alterations In Whole-cell Nmr Spectramentioning

confidence: 99%

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

Romaniuk¹,

Cegelski²

2015

Phil. Trans. R. Soc. B

136

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One contribution of 12 to a theme issue 'The bacterial cell envelope'. The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of b-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms.

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Section: Detecting Cell Wall Content and Major Alterations In Whole-cell Nmr Spectramentioning

confidence: 99%

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

Romaniuk¹,

Cegelski²

2015

Phil. Trans. R. Soc. B

136

View full text Add to dashboard Cite

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Application of microbial chip for amperometric detection of metabolic alteration in bacteria

Nagamine

Kaya

Yasukawa

et al. 2005

Sensors and Actuators B: Chemical

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Formation and Secretion of Induced Penicillinase in Protoplasts of Staphylococcus aureus

Coles

Gross

1973

Antimicrob Agents Chemother

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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...

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

Cited by 4 publications

References 13 publications

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

Application of microbial chip for amperometric detection of metabolic alteration in bacteria

Formation and Secretion of Induced Penicillinase in Protoplasts of Staphylococcus aureus

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