Molecular characterisation of aureocin A70, a multi-peptide bacteriocin isolated from Staphylococcus aureus

Abstract

Staphylococcus aureus A70 produces a heat-stable bacteriocin designated aureocin A70. Aureocin A70 is encoded within a mobilisable 8 kb plasmid, pRJ6, and is active against Listeria monocytogenes. Experiments of transposition mutagenesis and gene cloning had shown that aureocin A70 production and immunity were associated with the HindIII-A and B fragments of pRJ6. Therefore, a 6332 bp region of the plasmid, encompassing both these fragments, was sequenced using a concatenation DNA sequencing procedure. DNA sequence and genetic analyses revealed the presence of three transcriptional units that appear to be involved in bacteriocin activity. The first transcriptional unit contains a single gene, aurT, which encodes a protein that resembles an ATP-dependent transporter, similar to those involved in lantibiotic export. AurT is required for aureocin A70 production and it appears to be essential for mobilisation of pRJ6. The second putative operon contains two open reading frames (ORFs); the first gene, orfA, is predicted to encode a protein similar to small repressor proteins found in some Archaea, whose function remains to be elucidated. The second gene, orfB, codes for an 138 amino acid residue protein which shares a number of characteristics (high pI and hydrophobicity profile) with proteins associated with immunity, needed for self-protection against bacteriocin. Four other genes are present in the third operon, aurABCD. aurABCD encode four related peptides that are small (30–31 amino acid residues), strongly cationic (pI of 9.85 to 10.04) and highly hydrophobic. Theses peptides also have a high content of small amino acid residues like glycine and alanine, and no cysteine residue. Tn917-lac insertional mutations, which affected aureocin A70 activity, reside within operon aurABCD. Analysis of purified bacteriocin preparations by mass spectrometry demonstrated that all four peptides encoded by aurABCD operon are produced, expressed and excreted without post-translational modifications. Thus, aureocin A70 is a multi-peptide non-lantibiotic bacteriocin, which is transported without processing.