Gor, M. C. | Search bioRxiv

bioRxiv2020

Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity

Plantacyclin B21AG is a circular bacteriocin produced by Lactobacillus plantarum B21 which displays antimicrobial activity against various Gram-positive bacteria including foodborne pathogens, Listeria monocytogenes and Clostridium perfringens. It is a 58-amino acid cyclised antimicrobial peptide, with the N and C termini covalently linked together. The circular peptide backbone contributes to remarkable stability, conferring partial proteolytic resistance and structural integrity under a wide temperature and pH range. Here, we report the first crystal structure of a circular bacteriocin from food grade Lactobacillus. The protein was crystallised using the hanging drop vapour diffusion method and the structure solved to a resolution of 1.8 Å. Sequence alignment against 17 previously characterised circular bacteriocins revealed the presence of conserved charged and aromatic residues. Alanine substitution mutagenesis validated the importance of these residues. Minimum inhibitory concentration analysis of these Ala mutants showed Phe8Ala and Trp45Ala mutants displayed a 48- and 32-fold reduction in activity, compared to wild type. Lys19Ala mutant displayed the weakest activity, with a 128-fold reduction. These experiments demonstrate the importance of aromatic and cationic residues for the antimicrobial activity of plantacyclin B21AG and by extension, other circular bacteriocins sharing these evolutionarily conserved residues.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Discovery and characterisation of novel circular bacteriocin plantacyclin B21AG from Lactobacillus plantarum B21

Lactobacillus plantarum B21 isolated from Vietnamese sausage (nem chua) has previously displayed broad antimicrobial activity against gram positive bacteria including foodborne pathogens Listeria monocytogenes and Clostridium perfringens. This study successfully identified the antimicrobial agent as plantacyclin B21AG, a 5668 Da circular bacteriocin demonstrating high thermostability, resistance to a wide range of pH, proteolytic resistance and temporal stability. We report a reverse genetics approach used to identify and characterise plantacyclin B21AG. The bacteriocin was purified from culture supernatant by a short process consisting of concentration, n-butanol extraction and cation exchange chromatography. A de novo peptide sequencing using LC-MS/MS techniques identified two putative peptide fragments which were mapped to the genome of Lactobacillus plantarum B21. This revealed an ORF corresponding to a putative circular bacteriocin with a 33-amino acid leader peptide and 58-amino acid mature peptide found on native plasmid pB21AG01. The corresponding gene cluster, consisted of seven genes associated with post-translational circularisation, immunity and secretion. The robust nature of plantacyclin B21AG, its antimicrobial activity and associated machinery for cyclisation make it an interesting biotechnological target for further development, and application as a food-safe antimicrobial.

biochemistry↗