Production and Purification of a Staphylococcus epidermidis Bacteriocin

Abstract

Liquid cultures of Staphylococcus epidermidis 1580 contained rather small amounts of a bacteriocin, staphylococcin 1580, which was found both in the supernatant fluid and in the cell pellet. It could be extracted from the cells with 5% NaCl solution. The staphylococcin production could not be induced by ultraviolet irradiation or treatment with mitomycin C. Bacteria grown on semisolid medium produced a much larger amount of the compound with a high specific activity. The staphylococcin was purified by ammonium sulfate precipitation, ultracentrifugation, and chromatography on Sephadex columns. The purified material was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was between 150,000 and 400,000. The bacteriocin was composed of protein, carbohydrate, and lipid and consisted of subunits exhibiting a molecular weight of about 20,000.

Isolation and partial characterization of Bac201: A plasmid-associated bacteriocin-like inhibitory substance fromStaphylococcus aureus AB201

“…In order to produce enhanced quantities of Bac201, procedure of TOR- REBLANCA et al (1989) was followed. The induction was also tried with various concentrations of mitomycin C (0.1, 0.5 and 1.0 Pg/ml) (JETTEN et al 1972), 0.5% mannitol and 2% yeast extract in separate attempts (TAGG et al 1976). Cell bound bacteriocin was eluted by using NaCl (0.5, 1.0, 2.5, 5.0 M), and 0.5 M EDTA (MURIANA and KLAENHAMMER 1991); SDS (0.05, 0.1 and 1.0%) (JETTEN et al 1972) under appropriate conditions.…”

“…The induction was also tried with various concentrations of mitomycin C (0.1, 0.5 and 1.0 Pg/ml) (JETTEN et al 1972), 0.5% mannitol and 2% yeast extract in separate attempts (TAGG et al 1976). Cell bound bacteriocin was eluted by using NaCl (0.5, 1.0, 2.5, 5.0 M), and 0.5 M EDTA (MURIANA and KLAENHAMMER 1991); SDS (0.05, 0.1 and 1.0%) (JETTEN et al 1972) under appropriate conditions. Preliminary purification of Bac201: Bac201 was precipitated from culture supernatant (obtained by centrifuging overnight growth culture of Staphylococcus aureus AB201 at 8000 u g for 15 min at 4 ºC), by slowly adding ammonium sulfate (with constant stirring at 4 qC) to obtain 50% saturation.…”

“…Many of the numerous large bacteriocin complexes reported in the early literature (TAGG et al 1976) may be small peptides, because of insufficient purification procedures. This was exemplified for staphylococcin 1580 which was described as a bacteriocin of several hundred kilodaltons (JETTEN et al 1972) but is identical to the lantibiotic epidermin (SAHL 1994 b).…”

“…Each dissociates into subunits with molecular weights of about 10 kDa after SDS treatment of a higher molecular weight form. Staphylococcin 1580 isolated from Staphylococcus epidermidis was found to be a protein-lipid-carbohydrate complex with a molecular weight between 150 kDa and 400 kDa in polyacrylamide gels (JETTEN et al 1972). In SDS the bacteriocin dissociated into subunits exhibiting a molecular weight between 10 and 25 kDa.…”

“…Instability of staphylococcin production particularly after treatment with curing agents such as acridine orange or ethidium bromide suggests that several staphylococcins are plasmid determined (JETTEN et al 1972). WARREN et al (1974) have shown that exfoliative toxin responsible for scalded skin syndrome by Staphylococcus aureus strain of phage type 71, and the bacteriocin production by this strain are both determined by a plasmid with a molecular weight of 33 u 10 6 .…”