The molecular and cellular characterisation of the first glycocin, plantaricin KW30 : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand

Abstract

Bacteriocins, typically secreted by Gram-positive and -negative bacteria, are ribosomally synthesised antimicrobial peptides which inhibit the growth of competing bacteria. We have purified a 43 amino acid bacteriocin, plantaricin KW30 (PlnKW30) produced by Lactobacillus plantarum KW30, that has little amino acid sequence similarity to any other characterised bacteriocin. The gene encoding plnKW30 is in a cluster with the genes required for maturation and export of, and immunity to, the bacteriocin. This arrangement of genes is similar to the genomic context of bacteriocin genes in other lactic acid bacteria. The plnKW30 gene cluster comprises six genes encoding a glycosyltransferase, a proteolytic ABC-transporter, two putative thioredoxins, a response regulator and PlnKW30 itself. PlnKW30 was found to possess two unusual post-translational modifications: an O-glycosylated serine and an unprecedented S-glycosylation of the C-terminal cysteine. The modified serine is located on an eight residue loop that is tethered by a disulfide bridge. Bothmodifications have been identified as N-acetylglucosamines (GlcNAc), making PlnKW30 the first described class IV bacteriocin. A post-translational modification with S-linked GlcNAc is unprecedented in bacteriocins as well as in all genera. The antimicrobial activity of PlnKW30 on L. plantarum ATCC 8014 was analysed using enzymatic dissection coupled with bioassays. It was found to be concentration dependent and both the N-and C-terminalfragments are necessary for activity. Furthermore, reduction of the disulfide bonds results in abolishment of antimicrobial activity and it appears that deglycosylation of the serine 18 decreases the antimicrobial activity by about two thirds. These results show that all posttranslational modifications contribute to the antimicrobial activity of PlnKW30. The addition of N-acetylglucosamine to cultures of the indicator strain L. plantarum ATCC 8014 protects it from the antimicrobial effect of the added PlnKW30. PlnKW30 probably targets an N-acetylglucosamine transporter in the target cell membrane, similar to the mannose phosphotransferase system targeted by lactococcin A.