Antibacterial peptides from marine bacillus species

The indiscriminate use of antibiotics in healthcare and agriculture over the past decades has led to a high incidence of antibiotic-resistant pathogens worldwide. One of the solutions that could be considered to combat this crisis is the identification of novel antibiotics or other alternatives. However, the discovery pipeline for novel antimicrobials, identified from organisms in the terrestrial environment, is sparse. Therefore, we have taken an alternative approach to this issue by screening marine bacteria to seek novel bacteriocins for further antibiotic development. Bacteriocins are a family of ribosomally synthesized antimicrobial peptides; some of which are currently applied as safe food preservatives (nisin) and they are also gaining interest as promising alternatives to conventional antibiotics. The Vietnam Sea has a variety of marine ecosystems with diverse marine species, but there has been little exploration of this source for novel active compounds. Therefore, we undertook a discovery program aimed at identifying novel bacteriocins from bacterial isolates recovered from the Vietnam Sea.<br><br> In stage one, 64 spore-forming bacterial isolates that exhibited antimicrobial activity were identified after sampling and bacterial isolation (Chapter 3). Inspection of their antimicrobial spectra resulted in the short-listing of 23 isolates for further analysis. Based on 16S ribosomal RNA sequences, these 23 isolates were identified as 22 Bacillus species and 1 Paenibacillus species (Paenibacillus polymyxa), including ubiquitous species (Bacillus subtilis, Bacillus amyloliquefaciens), and floral species (Bacillus halotolerans, Bacillus safensis, Bacillus pacificus, Paenibacillus polymyxa, Bacillus licheniformis). They exhibited strong antibacterial activity against a range of human and veterinary pathogens and food-poisoning bacteria with dominance of proteinaceous antimicrobial substances. Inspection of the breadth and strength of antimicrobial activities informed the selection of 6 isolates for whole genome sequencing in the second stage of the study.<br><br> The six isolates subjected to whole genome sequencing were; four B. amyloliquefaciens isolates (#06, #08, #11, #13), one B. halotolerans (#01) isolate, and one P. polymyxa (#23) isolate (Chapter 4). Bioinformatic analysis identified genes encoding 61 putative antimicrobial peptides within the genomes of the six isolates, including 41 mostly characterised non-ribosomally synthesized peptides (lipopeptides, polyketides and bacilysin) and 20 bacteriocins. Amongst the 20 putative bacteriocins found, there were 13 different types of bacteriocins. The putative bacteriocin encoding genes clusters included those for 6 characterised bacteriocins (mersacidin, paenicidin A, plantazolicin, LCI, amylocyclicin, subtilosin A), and 7 uncharacterised/ unique bacteriocins (2 thiopeptides, 1 two-component lantibiotic, 1 sactipeptide, 2 lantibiotic, and 1 lassopeptide). <br><br> In the third stage of the study (Chapters 5 and 6); two isolates were selected for antimicrobial purification. Purification of antimicrobial peptides from isolate #11 resulted in the identification of amylocyclicin ([m/z]= 6,381), iturin/surfactin ([m/z]= 1,058) and an undefined peptide ([m/z]= 1,473). The undefined peptide ([m/z]= 1,473), was suspected to be a novel thiopeptide due to the similarity of MW. However, the amino acid sequence of this peptide couldn¿t be elucidated by an N-terminal sequencing method. Antimicrobial purification from isolate #23 identified polymyxin ([m/z]= 1168.7), tridecaptin ([m/z]= 1550.8), and paenicidin A ([m/z]= 3290.4), but no novel bacteriocins. <br><br> In the fourth stage of the study, an attempt was made to heterologously produce the novel sactipeptide which was one of three putative novel bacteriocins predicted from the genome of isolate #23. The technique employed E. coli/B. subtilis shuttle plasmid, pDLL202, for plasmid construction and a heterologous B. subtilis host for bacteriocin expression. A 15,487 bp fusion plasmid (pDLL202/G23sac) was constructed, carrying an 8,556 bp¿sactipeptide gene cluster derived from isolate #23 and the 6,938 bp- plasmid backbone derived from plasmid pDL202. The Bacilllus transformants successfully expressed the sactipeptide. The sactipeptide had a molecular weight (MW) of 3,404 Da and depressed growth of Gram-positive bacteria. Taken together, the marine Bacillus/Paenibacillus are abundant reservoirs of genes coding novel bacteriocins for further discovery. These antimicrobial compounds produced by marine Bacillus/Paenibacillus can be developed into therapeutic antimicrobial drugs against antibiotic-resistant pathogens, particularly against antibiotic-resistant Gram-positive bacteria.