Investigation into the structure and function of the glycosylated bacteriocin GccF and the glycosyltransferase GccA from Lactobacillus plantarum KW30 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Palmerston North, New Zealand
Bacteriocins are typically antimicrobial proteins or peptides produced by Gram-positive and G–negative bacteria, that are capable of inhibiting the growth of other bacteria. Glycocin F (GccF) is a 43 amino acid bacteriocin produced by Lactobacillus plantarum KW30 which is post-translationally modified by two N-acetylglucosamine residues (GlcNAc). One of these residues is linked to a serine side-chain (O-linked), while the other is linked through the thiol sulphur at the C-terminal cysteine (S-linked). Within the gene cluster encoding GccF are a set of genes thought to be required for the maturation and secretion of GccF. The GccF gene cluster consists of six genes encoding a family 2 glycosyltransferase (GTase) thought to be responsible for the addition of either one or both of these GlcNAc groups, an ABC transporter involved in the secretion of the bacteriocin across the cellular membrane, two thioredoxin-like genes which may be responsible for the disulfide bonding pattern of GccF, a gene of unknown function, and GccF itself.
Within L. plantarum KW30 no other proteins modified by a GlcNAc residue were identified in the present study, making GccF the only known GlcNAcylated protein produced by this organism. Methods were developed to pull-down the proteins involved in the maturation and secretion of GccF, and to find its binding target(s) in strains suceptible to its activity. Although proteins were found to bind tightly to GccF during pull-down experiments, those that bound were mostly involved in glycolysis/gluconeogenesis which does not fit into the hypothesised mechanism of action for GccF. Fluorescent microscopy experiments on wild-type GccF and GccF that contained only the O-linked or S-linked GlcNAc residue found that localisation of the modified GlcNAcylated GccF on susceptible strains was different to what is seen for wild-type in that they appeared randomly along the cells, whereas wild-type GccF appeared to localise at the point of cell division and at the tips of the cells. These microscopy results show that the post-translational modifications appear to play a role in targeting of GccF to susceptible cells. Assays to detect and test the activity of the GTase found that it may be located within the cytosol of
L. plantarum KW30 instead of the membrane which is where it was proposed to be due to the presence of a predicted transmembrane spanning region identified during bioinformatic analysis.