Induction of L‐Phase Variant from Protoplast of Staphylococcus aureus

Abstract

Protoplasts of Staphylococcus aureus 209P and Cowan I were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature.Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCI) formed typical L-form colonies. Horse serum (0.25%) and M g2 + (lOa msr) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan I, M g2 + was not required.The active factor(s) in horse serum was heat-resistant and protein in nature.L-Phase variant induction in Staphylococcus aureus has been reported by many workers since the initial success of Diens and Sharp (2). The essential and common procedure for inducing the variants in various bacterial species is removal of the cell wall by a digesting enzyme (9) or inhibition of cell wall synthesis by penicillins (2) and the addition of an osmotic stabilizer and 5-10% serum to the culture medium. The generally used stabilizer is sucrose or sodium chloride. These are necessary to protect the fragile cells from cell bursting due to increased osmotic pressure. The addition of serum is also indispensable, but its function in the growth of L-phase variants still remains obscure.The induction of protoplast fusion by polyethylene glycol, which was first demonstrated in plant protoplasts by Kao and Michayluk (5), was extended to various microbial protoplasts (3, 7) including Staphylococcus aureus (4, 6). The cell fusion method will provide an excellent opportunity for studying genetic recombination and chromosomal mapping in Staphylococcus aureus, concerning which there has been little work done. Before investigation of protoplast fusing, however, it is necessary to determine the conditions that will allow highly efficient induction of protoplasts and L-phase variants.In this communication, a preliminary experiment on cell conversion to L-phase variants with high efficiency in Staphylococcus aureus is described. Some characteristics of the serum factor are also mentioned.

Anaerobiosis influences virulence properties of Pseudomonas aeruginosa cystic fibrosis isolates and the interaction with Staphylococcus aureus

“…The role of LasA in governing interspecies interactions may also be complicated by previous findings which demonstrate how the growth of S. aureus in the presence of P. aeruginosa LasA and staphylolysin produced by Staphylococcus staphylolyticus , selects for S. aureus colonies which lack a cell wall 66,67 . Whilst the emergence of such colonies in CF airways requires further study, selection of such a phenotype may serve to facilitate S. aureus persistence under both normoxia and anoxia in the presence of P. aeruginosa .…”

L-form-like colonies of Staphylococcus aureus induced by an extracellular lytic enzyme from Pseudomonas aeruginosa

“…), 0.002 g; nicotinic acid (Sigma), 0.004 g. This medium was normally used at pH 7.5, but when necessary, its pH was raised to 8.5 by adding 10 mM sodium phosphate buffer instead of distilled water. Secondly, the activity of the lytic enzyme was assayed in 10 mM sodium phosphate buffer (pH 8.5) containing 4% (wt/vol) NaCI, 1 M sucrose, or 0.25% (vol/vol) horse serum (heat denatured according to the method recommended by Makino [10]).…”

Salt‐Induced L‐Forms of Staphylococcus aureus

“…Antibiotics which inhibit cell wall synthesis and cell wall-lytic enzymes are effective inducing agents (3). Induction with any of these agents requires theinitial formation of cell wall-defective cells which need increased osmotic protection for growth (2,4,5). NaC1 is the most commonly used osmotic stabilizer for staphylococcal L-forms.…”