Characterization and heterologous expression of an antimicrobial peptide fromBacillus amyloliquefaciensCMW1

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. Histagged 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 Histagged mature amylocyclicin CMW1 indicated that the peptide is a linear rather than a circular peptide.

Bioinformatic prospecting and phylogenetic analysis reveals 94 undescribed circular bacteriocins and key motifs

“…There are many bioactive peptides which report low stability [57], which could be stabilised with C-N terminal ligation [58] found in circular bacteriocins. During the process of this manuscript being written, circular bactercion amylocyclicin CMW1 was discovered [20]. This sequence was successfully predicted as a circular bacteriocin from this dataset, appearing in Bacillus amyloliquefaciens LL3.…”

Discovery and characterisation of novel circular bacteriocin plantacyclin B21AG fromLactobacillus plantarumB21

“…Circularisation gives them unique characteristics including high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. To date, There are 18 circular bacteriocins being reported to date, including aureocyclicin 4185 [15], enterocin NKR-5-3B [16], amylocyclicin [17], amylocyclicin CMW1 [18], enterocin AS-48 [19], bacA [20], carnocyclin A [21], circularin A [22], thermocin 458 [23], garvicin ML [24], lactocyclicin Q [25], leucocyclicin Q [26], uberolysin [27], acidocin B [28], butyrivibriocin AR10 [29], paracyclicin [30], gassericin A/reutericin 6 [31] and plantaricyclin A [32]. This group of bacteriocins are unique due to their pH and thermal stability, and the ability to resist many proteolytic enzyme activities, making them an interesting target for industry applications [33].…”

Bioinformatic prospecting identified 99 novel, misannotated and unnoticed putative circular bacteriocins

“…There are many bioactive peptides which report low stability (Espinosa-Hernández, Morales-Camacho et al 2019), which could be stabilised with C-N terminal ligation (Clark, Fischer et al 2005) found in circular bacteriocins. During the process of this manuscript being written, circular bactercion amylocyclicin CMW1 was discovered (Kurata, Yamaguchi et al 2019). This sequence was successfully predicted as a circular bacteriocin from this dataset, appearing in Bacillus amyloliquefaciens LL3.…”