The influence of the leader sequence on antimicrobial activity of Leucocin A, an antilisterial bacteriocin produced by Leuconostoc gelidum UAL187-22.

Abstract

Bacteriocin leader pepides are currently receiving much attention due to their possible functions. It is predicted that these leaders prevent cytoplasmic toxicity within the producer organism by rendering the bacteriocin inactive. Leucocin A, a class IIa bacteriocin produced by Leuconostoc gelidum UAL187-22 is synthesized with a 24 amino acid leader pepide which is cleaved during extracellular translocation. The antimicrobial activity of the leucocin A precursor, pre-leucocin A, was determined to gain insight into whether, the presence of a leader peptide has an impact on anti-listerial activity. The leucocin A and pre-leucocin A genes were generated by PCR of L. gelidum UAL187-22 plasmid DNA. Recombinant plasmids, pLcaA and pPreLcaA were isolated by cloning the amplified genes into the Escherichia coli pMAL.c2 vector, and by screening transformant colonies using blue white selection methods. The malE-LcaA and malE-preLcaA fusion genes were expressed, and resulting maltose binding fusion proteins, were purified using amylose affinity chromatography. Fractions collected, contained partially pure forms of MBP-LcaA (46.433 kDa) and MBP-preLcaA (49.088 kDa) fusion proteins. Following Factor Xa digestion, the MBP affinity tag was removed; and recombinant peptides, leucocin A and pre-leucocin A were further purified by reverese phase high performance liquid chromatography. It was determined that leucocin A was eluted with a retention time of 24.893, while pre-leucocin A was eluted with a retention time of 31.447. Fractions of pure leucocin A and pre-leucocin A were thereafter assayed for activity using a deferred antagonism assay, with Listeria monocytogenes being the indicator strain. Pre-leucocin A tested positive for antimicrobial activity. However, when compared to leucocin A it was found that the leucocin A precursor inhibits Listeria to a lesser degree than leucocin A. The relative bactericidal activities of leucocin A and pre-leucocin A was calculated at 6.0 x 10⁵ AU and 4.0 x 10⁵ AU. Taking this into consideration, it was estimated that the leucocin A precursor is ~66.667 % active as mature leucocin A. Hence the presence of a leader peptide does not have an influence on leucocin A antimicrobial activity.