Production, purification and some properties of Bac201, a bacteriocin-like inhibitory substance produced by Staphylococcus aureus AB201

Staphylococcus aureus AB201, a clinical isolate from wound pus, produced a bacteriocin-like inhibitory substance termed as Bac201, that was inhibitory to Streptococcus agalactiae, Enterococcus faecalis, Acinetobacter calcoaceticus, Neisseria meningitidis and a number of staphylococcal species. It was purified to homogeneity by ammonium sulfate precipitation, gel filtration (BioSil-SEC-125), and reversed-phase high performance liquid chromatography (Vydac C4). The native Bac201 was sized at approximately 170-kDa as determined by GF HPLC. Fraction-I (native Bac201), having antibacterial activity was also examined by transmission electron microscopy and appeared as globular structure showing resemblance with phage-like objects. The purification of Bac201 resulted in 466-fold increase in specific activity and recovery of 0.94% of total antibacterial activity. The purified Bac201 migrated as single band on SDS-PAGE with an estimated molecular mass of about 41-kDa. Bac201 was sensitive to proteolytic enzymes, resistant to heat and organic solvents, and active over a wide range of pH (2.5-10). The amino acid composition revealed a general resemblance with other reported high molecular mass bacteriocins and predominance of glycine (39%), proline (13%) and alanine (8%) residues. Further results showed that Bac201 has a bactericidal effect on sensitive cells which is not produced by either cell lysis or apparent loss of membrane permeability.