Autolytic Release and Osmotic Properties of 'Protoplasts' from Staphylococcus aureus

Abstract

SUMMARY: The cell wall of exponential phase Staphylococcus aureus (strain Duncan) loses its tensile strength in c. 2 hr. when the organisms are incubated a t 25" in 1.2 M-sucrose a t pH 5.8 and ionic strength 0.3. The 'protoplasts' thus released from the mechanical protection of the cell wall are stable in 1.2 M-sucrose but lyse in media of lower osmotic pressure. The mean internal osmotic pressure of the 'protoplasts' is c. 20 atmospheres; they are permeable to glycerol but not to sucrose or NaCl. The rate of 'protoplast' release varies with the rate of growth of the organisms at harvesting. After osmotic explosion of 'protoplasts ' released from slowly growing organisms the plasma membranes may be recovered as spherical shells which disintegrate and condense into small particles on washing, and 75 yo of the weight of the cell walls may be recovered as hemispherical shells.The aim of the work described in this paper was to develop a method for releasing the protoplasts of Staphylococcus aureus (strain Duncan) from the protection of the cell wall so as to facilitate the study of the structure and function of the plasma membrane (Mitchell & Moyle, 1 9 5 6~) . Since staphylococci had been reported to be somewhat sensitive to lysozyme under certain conditions (Webb, 1948;Kern, Kingkade, Kern & Behrens, 1951) we decided to investigate the possibility of releasing stable ' protoplasts ' of staphylococci by modifications of the technique of Weibull (1953) similar to those which we developed for the release of the protoplasts of Il.l;icrococcus Zysodei&icus and Sarcina Zutea (Mitchell & Moyle, 19563). METHODSGrowth and preparation of organisms. Cultures were grown at 25" in a medium containing 3 yo (w/v) tryptic digest of casein, 1 yo glucose and 0.1 yo Marmite, either in Roux bottles or using the rotated flask technique (Mitchell, 1949). The organisms were harvested by centrifugation, washed once with distilled water and suspended at a concentration corresponding to c. 100mg. dry weightlml. in distilled water.Measurement of turbidity and dry weight. The extinction of 1 cm. depth of bacterial suspension was measured at a wavelength of 700mp., using the Beckman model DU spectrophotometer. The suspensions were diluted to give readings of log,, ( l o / I ) between 0.1 and 0.2 so that, the extinction being proportional to dry weight in this range, the dry weight could be estimated by multiplying the extinction by a factor. The factors for the different organisms were determined by calibrating the extinction against the dry-weight concentration estimated by drying c. 100 mg. samples to constant weight at 105O.

Reduced Oxidative Activities in Escherichia coli and Bacillus megaterium in Relation to Other Changes during Inhibition of Growth by Streptomycin

“…The systems responsible for the oxidation of succinate and malate were chosen for further investigations ; the total activity of these systems is divided about equally between the 'ghost' and supernatant fractions, but the specific activity of the 'ghost' fraction is 3 to 6 times that of the supernatant (Storck & Wachsman, 1957). It was also found here that activities on these two substrates were more consistent and reproducible than those obtained with other substrates.…”

“…It was also found here that activities on these two substrates were more consistent and reproducible than those obtained with other substrates. Although Storck & Wachsman (1957) found that certain co-factors stimulated oxidative activities in their preparations, the addition of adenosine diphosphate, di-or triphosphopyridine nucleotide, or cytochrome c, either singly or together, resulted in no stimulation of the preparations used here.…”

“…With both organisms, protoplasts from streptomycin-inhibited organisms were stable for considerable periods of time, so that there appeared to be no leakage of the stabilizing solute into the protoplasts. The internal osmotic pressure of protoplasts was estimated by the procedure used by Mitchell 85 Moyle (1956a, 1957 in which the proportion of intact protoplasts is determined over a range of external solute concentrations ; the mean internal osmotic pressure is regarded as equal to that of the solution in which 50% of the protoplasts remain intact. This procedure gives values for the internal osmotic pressure which agree well with those obtained by more direct methods (Mitchell & Moyle, 1956 b).…”

Peptidoglycan architecture can specify division planes in Staphylococcus aureus

“…Peptidoglycan is conserved over a wide range of cell shapes (for example, spheres, spirals, rods). In the absence of a cell wall, turgor pressure drives the cell towards the most energetically favourable conformation; a sphere 12 . Two possible paradigms for the nanoscale arrangement of glycan strands, the peptidoglycan structure, have been proposed: arrangement of glycan strands in the plane of the cytoplasmic membrane 13 or in a scaff old perpendicular to the cytoplasmic membrane 14,15 .…”

Observations on the Labelling of a Strain of Staphylococcus aureus with Phosphorus-32

“…The release of non-diffusible 32P compounds on dialysis into phosphate buffer from Staphylococcus aureus may have been the result of osmotically fragile cells autolysing (Mitchell & Moyle, 1957). This release of large molecular weight materials may also be the result of degradation of RNA and other phosphorus-containing compounds present in non-viable cocci.…”