Characterization and heterologous expression of an antimicrobial peptide from Bacillus amyloliquefaciens CMW1

Abstract

An antibacterial peptide gene (designated as accA) was found in Bacillus amyloliquefaciens CMW1. The accA gene consists of 336 nucleotides and encodes 111 amino acids. The accA gene product, amylocyclicin CMW1, consists of a leader peptide and a mature peptide. The genome analysis indicated that the accA gene constitutes a gene cluster together with five open reading frames (accBCDEF genes) that are predicted to play roles in amylocyclicin CMW1 biosynthesis. Amylocyclicin CMW1 is similar to precursor proteins of amylocyclicin FZB42 and uberolysin A, which are known as circular hydrophobic bacteriocins. To investigate the antimicrobial activity of the accA gene product, we constructed recombinant Escherichia coli cells expressing amylocyclicin CMW1, His-tagged amylocyclicin CMW1 or His-tagged mature amylocyclicin CMW1. The culture supernatant, the cell-free extract and debris fraction were prepared with recombinant E. coli cells expressing amylocyclicin CMW1. Antibacterial activity against Bacillus subtilis JCM10629 was detected in the debris fraction, but not in the culture supernatant or cell-free extract. His-tagged mature amylocyclicin CMW1 inhibited Gram-positive bacteria but not Gram-negative bacteria. Unlike amylocyclicin FZB42, His-tagged mature amylocyclicin CMW1 exhibited antibacterial activity against Staphylococcus aureus. Electrospray ionization mass spectrometry of His-tagged mature amylocyclicin CMW1 indicated that the peptide is a linear rather than a circular peptide.