The bacteriostatic diglycocylated bacteriocin glycocin F targets a sugar-specific transporter : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand

Abstract

The increasing prevalence of antibiotic-resistance bacteria is threatening to end the antibiotic era established following Alexander Fleming's discovery of penicillin in 1928. Over-prescription and misuse of broad-spectrum antibiotics has hastened the development and spread of antibiotic resistance. This, combined with a lack of research and development (R&D) of new antibiotics by major pharmaceutical companies, may lead to a widespread recurrence of 'incurable' bacterial diseases. However while commercial R&D of antibiotics has waned, much research has been carried out to characterise bacteriocins, ribosomally-synthesised antimicrobial polypeptides thought to be produced by virtually all prokaryotes. Although hundreds of bacteriocins have been identified and characterised, only a handful of their cognate receptors on susceptible cells have been identified. Glycocin F is a bacteriostatic diglycosylated 43-amino acid bacteriocin produced by the Gram-positive bacterium Lactobacillus plantarum KW30 that inhibits the growth of a broad range of bacteria. The mechanism of action of glycocin F is unknown, however evidence suggested that glycocin F binds to cells via a N-acetylglucosamine (GlcNAc) specific phosphoenolpyruvate:carbohydrate-phosphotransferase system (PTS) transporter, as had been shown for lactococcin A, lactococcin B and microcin E492 that target a mannose specific PTS transporter. These other bacteriocins are, however, bactericidal suggesting that glycocin F uses a different mechanism of action to stop cell growth. To test the hypothesis that one of the putative GlcNAc-specific PTS transporters identified in glycocin F-sensitive L. plantarum strains is the primary membrane receptor for glycocin F, a GlcNAc-specific PTS transporter gene knockout mutant was generated and analysed for glycocin F sensitivity. The GlcNAc-specific PTS transporter, pts18CBA, was successfully knocked out in L. plantarum NC8 which conferred the resulting L. plantarum NC8 Δpts18CBA a degree of resistance to glycocin F confirming the GlcNAc-specific PTS transporter is a receptor of glycocin F. Additionally the genomes of wild-type (glycocin F sensitive) L. plantarum ATCC 8014, L. plantarum subsp. plantarum ATCC 14917, and multiple glycocin F- resistant mutants of these two strains were sequenced, assembled and comparatively analysed to identify changes consistent with increased resistance to glycocin F. Mutations, mapped to pts18CBA in all sequenced mutants, appeared to be deleterious to both the structure and function of PTS18CBA. A correlation of glycocin F resistance to the degree of mutation in the transmembrane domain of the pts18CBA gene was established confirming that glycocin F targets the EIIC transmembrane domain of PTS18CBA.