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    Identification of dominant lactic acid bacteria and yeast in rice sourdough produced in New Zealand
    (Elsevier BV, 2021-10-21) Yang Q; Rutherfurd-Markwick K; Mutukumira AN
    This study characterised a commercial New Zealand gluten free (GF) rice sourdough and its starter culture composition. Acidity of the mother sourdough, dough before proofing and dough after proofing was determined during the production of rice sourdough bread, and colour was measured for the baked bread. Yeast and lactic acid bacteria (LAB) were enumerated in the rice sourdough samples and representative colonies characterised using API kits and sequenced by the Internal Transcribed Spacer and 16 S rRNA region. Sourdough LAB isolates were identified as Lactobacillus (L.) papraplantarum DSM 10667 and L. fermentarum CIP 102980 and the yeast isolates as Saccharomyces (S.) cerevisiae CBS 1171. Dough acidity increased significantly (p < 0.05) during fermentation due to the metabolic activities of the sourdough cultures. After baking, the colour of the rice sourdough bread crust was similar to that of unleavened wheat bread (golden brown). The improved colour of the rice sourdough bread crust may be a result of combined use of sourdough technique and optimal baking conditions. The results of this study may allow bakers to improve the overall quality of GF rice sourdough baked bread by selecting suitable fermentation and baking parameters.
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    Understanding the mechanism of action of the glycosylated bacteriocin glycocin F : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatu, New Zealand
    (Massey University, 2019) Bisset, Sean William
    With the increasing threat posed by antibiotic-resistant bacteria, efforts must be made to find new antimicrobial agents. One growing area of promise is the bacteriocins, which are a diverse group of antimicrobial peptides produced by bacteria. This thesis focuses on determining the mechanism of action of one of these peptides, glycocin F (GccF). GccF is produced by the bacterium Lactobacillus plantarum and is modified with two N-acetyl glucosamine (GlcNAc) sugar moieties, one located on an interhelical loop region, and the other at the end of a flexible C-terminal ‘tail’. It has also been shown to exhibit a unique effect on susceptible bacteria, putting them into a reversible state of hybernation as opposed to outright killing them. However, little is known about the roles of the structural features of GccF, how it triggers bacteriostasis in target cells, or even what part(s) of bacterial cells it targets. This work addresses these questions using three main approaches: studying the structure-function relationship of different parts of GccF with chemically synthesised analogues; looking at the transcriptional response of a pathogenic bacteria, Enterococcus faecalis, to GccF; and trying to identify binding partners of GccF and its respective immunity protein, GccH. The results presented here highlight different roles of the GlcNAcs attached to GccF, with both the interhelical loop and presence of GlcNAc on this loop being vital for activity, while the sugar at the C-terminal position is important, but not crucial for the peptide’s activity. Additionally, a role of the GlcNAc phosphotransferase system on the mechanism of GccF is strongly indicated, with evidence from both the transcriptional studies and the protein interaction studies of GccF’s immunity protein. Taken together, the results allow for two theoretical models of GccF’s mechanism of action to be proposed. These models presented here should serve to increase the understanding of other glycocin-class bacteriocins and their mechanisms of action, and possibly contribute towards the creation of a blueprint for a new class of antimicrobial agents.
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    The role of the N-acetylglucosamine phosphoenolpyruvate phosphotransferase system from Lactobacillus plantarum 8014 in the mechanism of action of glycocin F : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2017) Bailie, Marc Alex
    The rise in antibiotic-resistant bacteria is becoming a severe public health problem because of the shortage of new antibiotics to combat existing resistant bacterial pathogens. Should this trend of increasing bacterial drug resistance continue, the previously treatable conditions may once again become fatal. Using broad-spectrum antibiotics causes collateral damage to the commensal microbiota of the host leading to complications and a greater susceptibility to opportunistic pathogenic infection. As a result, narrow spectrum antibacterials effective against specific pathogens, are becoming increasingly sought after. Among the many alternative classes of narrow-spectrum antibiotics, is a diverse group of ribosomally-synthesised antimicrobial peptides known as bacteriocins. Glycocin F (GccF), a rare and uniquely diglycosylated bacteriocin produced by Lactobacillus plantarum KW80, appears to target a specific N-acetylglucosamine (GlcNAc) phosphotransferase system (PTS) and causes almost instant bacteriostasis by an as yet unknown mechanism. This thesis demonstrates how the GlcNAc-PTS is involved in the GccF mechanism of action and that the gccH gene provides immunity to GccF. Using transgenic and gene editing techniques, regions of the GlcNAc-PTS were either removed or altered to prevent normal function before being tested in vivo. The results demonstrated that only the EIIC domain of the GlcNAc-PTS is required in the GccF mechanism of action and that it acts like a "lure" that attracts the bacteriocin to the main target that is as yet unknown. Furthermore, the immunity gene was discovered, and using PTS knockout cell lines the immunity mechanism was shown to act independently of the GlcNAc-PTS. This work will form the foundation for the work needed to unravel the bacteriostatic mechanism of action of GccF, which may lead to the development a novel antimicrobial agent.
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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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    Investigating the bacteriocin library Lactobacillus plantarum A-1 : presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Manuwatū Campus, Palmerston North, New Zealand
    (Massey University, 2014) Main, Patrick
    Bacteriocins are a highly diverse group of ribosomally synthesised, antimicrobial polypeptides produced by nearly all bacterial and archaeal species. Individual bacteriocins typically exhibit a narrow phylogenetic range of activity, but collectively inhibit a wide range of species through a variety of mechanisms. Glycocins are uncommon bacteriocins with rare, S-linked glycosidic bond;, currently there are only four characterised glycocins. Preliminary characterisation of the bacteriocin ASM1 from Lactobacillus plantarum A-1 was reported by Hata et al. in 2010. ASM1 is structurally similar to GccF, a glycocin produced by L. plantarum KW30. Like GccF, ASM1 has two covalently linked N-acetylglucosamine moieties, one of which is attached through a rare S-glycosidic bond. Due to its structural similarity, it was hypothesised that ASM1 would have similar inhibitory activity to GccF. Experiments showed that the two bacteriocins have almost identical inhibitory activity and both glycocins rely on their GlcNAc moieties to inhibit target cells. The range of species inhibited by ASM1 was shown to be wider than previously thought. The inhibitory activity, however, varied considerably even between strains in a species. The ASM1 gene cluster was established by sequencing and Southern hybridisation to be located on a 11,905 bp plasmid pA1_ASM1. An in silico analysis of the ASM1 gene cluster showed it to have the same operonic organisation similar as the GccF cluster, and overall DNA sequence identity of 75%. A second active bacteriocin gene cluster was detected in the L. plantarum A-1 genome. A synthetic peptide, named ASM2, corresponding to this bacteriocin was partially characterised. ASM2 has 82% amino acid sequence identity to the recently identified bactofencin A produced by L. salivarius DPC6502. A brief in silico analysis of proteins from the A-1 bacteriocin library and their orthologues provided some evolutionary context for the glycocins of Lactobacillus and gave hints as to the evolutionary history of the four characterised glycocins. ASM1 is one of four characterised glycocins and this work has increased the overall knowledge of glycocins. Identification of a novel secondary bacteriocin in L. plantarum A-1 shows the complexity of bacteriocin systems and provides many avenues for future research regarding bacteria that produce multiple bacteriocins.
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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.
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    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
    (Massey University, 2011) Poulson, Evelyn Marianne
    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.