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Subject: search.filters.subject.Glycocin F

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    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
    (Massey University, 2014) Drower, Kelvin Ross
    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.
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    The bacteriostatic spectrum and inhibitory mechanism of glycocin F, a bacteriocin from Lactobacillus plantarum KW30 : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2013) Kerr, Andrew Philip
    Bacteriocins have been deemed the “microbial weapon of choice”. The ability to ribosomally synthesise these toxins means that their peptide scaffolds can be rapidly adapted to optimise stability, potency and specificity, allowing producers to outgrow closely related strains and become dominant. In some cases, a bacteriocin may inhibit a broader spectrum of microbes than just its species/genus of origin. Recently, the bacteriocin glycocin F (GccF), produced byLactobacillus plantarum KW30, was biochemically and structurally characterised. GccF isunique, as it has two covalently linked N-acetylglucosamine (GlcNAc) moieties, one O-linkedand one S-linked, that are critical for the inhibition of target cell growth. How GccF causes bacteriostasis in sensitive Lactobacillus cells was unknown. Experiments were developed and conducted to probe the antimicrobial spectrum of GccF and how thisspectrum is affected by free GlcNAc. It was found that a variety of species and strains, not just those closely related to L. plantarum KW30, were inhibited by the addition of GccF to cultures in solid or liquid media. Susceptible strains were identified in the genera Streptococcus, Enterococcus, and Bacillus. Interestingly, assays indicated that free GlcNAc plays a more dynamic role in modulating GccF activity than previously thought. The protective effect of high concentrations of GlcNAc, including the reversal of GccF-induced bacteriostasis, was confirmed for susceptible L. plantarum strains, but surprisingly addition ofrelatively low concentrations of GlcNAc prior to GccF led to a concentration-dependent increase in bacteriostasis for some other species including Enterococcus faecalis. GccF’s mechanism of action was found to be different to the bactericidal membrane-permeabilising effect of the lantibiotic nisin, as L. plantarum cells treated with GccF did not die, and there was no substantial release of ATP from cells upon GccF-induced bacteriostasis. It was also found that for Gram-negative bacteria, which are generally resistant to GccF, growth inhibition was greatly enhanced if the integrity of the outer membrane was compromised by treatment with polymyxin, or by expression of a ‘leaky’ mutant of the outer membrane secretin PulD. Thus GccF-mediated inhibition of growth is limited to Gram-positive bacteria mainly because of the barrier function of the Gram-negative outer membrane. Experiments to identify changes in E. faecalis V583 gene expression or the levels of specific proteins in response to free GlcNAc were inconclusive due to time constraints. Further research is needed to determine GccF’s exact mechanism of action. The results of experiments with GccF, with and without added GlcNAc, on a range of bacterialspecies led to a hypothetical model for the mechanism of action of GccF, specifically that GccF may be ‘hijacking’ GlcNAc-specific phosphotransferase system signalling pathways.This could disrupt normal GlcNAc metabolism, perhaps resulting in UDP-GlcNAc becominglimiting for peptidoglycan synthesis, thus preventing cell wall expansion, and normal cellgrowth and division.