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Ho Yuen Sze. / Thesis submitted in: December 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 103-116). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vi / List of Figures and Tables --- p.x / Abbreviations --- p.xiv / Chapter 1. --- Introduction / Chapter 1.1 --- General --- p.1 / Chapter 1.2 --- Mechanism of lipolysis --- p.3 / Chapter 1.3. --- Distinction between lipases and esterases --- p.5 / Chapter 1.4. --- Interface activation --- p.7 / Chapter 1.5 --- Substrate specificity of lipases --- p.10 / Chapter 1.6 --- Classification and properties of bacterial lipolytic enzymes --- p.13 / Chapter 1.7 --- Assays used to measure lipase activity --- p.15 / Chapter 1.7.1 --- Titrimetric method --- p.15 / Chapter 1.7.2 --- Colorimetric methods --- p.16 / Chapter 1.7.2.1 --- p-Nitrophenyl ester assay --- p.16 / Chapter 1.7.2.2 --- Chain reaction assay --- p.18 / Chapter 1.8. --- Factors affecting lipase synthesis and secretion --- p.20 / Chapter 1.9. --- Biotechnological applications of lipases --- p.21 / Chapter 1.9.1 --- Lipases in the dairy industry --- p.21 / Chapter 1.9.2 --- Lipases in household detergents --- p.23 / Chapter 1.9.3 --- Lipases in the oleochemical industry --- p.24 / Chapter 1.9.4 --- Synthesis of pharmaceuticals and agrochemicals --- p.25 / Chapter 1.10 --- Meiothermus --- p.26 / Chapter 1.11 --- Aims of study --- p.29 / Chapter 2. --- Methods and Materials / Chapter 2.1. --- Microbial strains --- p.30 / Chapter 2.2. --- Cultivation --- p.32 / Chapter 2.3. --- Preparation of samples for lipase assay --- p.33 / Chapter 2.4. --- Enzyme assays --- p.33 / Chapter 2.5. --- Characterization of lipases from selected strains --- p.35 / Chapter 2.5.1 --- Optimal pH for lipase activity --- p.35 / Chapter 2.5.2 --- Optimal temperature for lipase activity --- p.36 / Chapter 2.5.3 --- Effect of temperature on lipase stability --- p.36 / Chapter 2.5.4 --- Effect of pH on lipase stability --- p.36 / Chapter 2.5.5 --- Substrate specificity --- p.36 / Chapter 2.6. --- Purification of lipases I and II from Meiothermus strain 17R --- p.37 / Chapter 2.6.1. --- Pigment removal --- p.37 / Chapter 2.6.2. --- DEAE-Sepharose column chromatography --- p.38 / Chapter 2.6.3. --- Gel filtration chromatography --- p.38 / Chapter 2.6.4. --- FPLC Mono P chromatography --- p.38 / Chapter 2.6.5. --- Preparative polyaerylamide gel electrophoresis --- p.39 / Chapter 2.7. --- Purification of lipase III from Meiothermus strain 17R --- p.40 / Chapter 2.7.1 --- Phenyl-Sepharose chromatography --- p.40 / Chapter 2.8. --- Other enzyme purification methods used in this study --- p.40 / Chapter 2.8.1 --- Ammonium sulphate fractionation --- p.40 / Chapter 2.8.2 --- Cation exchange chromatography --- p.41 / Chapter 2.8.3 --- Heparin-Sepharose affinity chromatography --- p.41 / Chapter 2.9. --- Partial characterization of lipases I and II from Meiothermus strain 17R --- p.41 / Chapter 2.9.1 --- Molecular mass determination --- p.41 / Chapter 2.9.2 --- Isoelectric point determinations --- p.42 / Chapter 2.10. --- N-terminal amino acid sequencing of lipases I and II --- p.43 / Chapter 2.11. --- Molecular cloning of lipase II --- p.43 / Chapter 2.11.1 --- Isolation of genomic DNA --- p.43 / Chapter 2.11.2 --- Design of degenerate primers --- p.44 / Chapter 2.11.3 --- PCR amplification of genomic DNA of lipase II --- p.44 / Chapter 3. --- Results / Chapter 3.1 --- Screening of Meiothermus spp. and Thermus spp --- p.46 / Chapter 3.2 --- Characterization of selected strains --- p.50 / Chapter 3.2.1 --- Optimum pH --- p.50 / Chapter 3.2.2 --- Optimum temperature --- p.50 / Chapter 3.2.3 --- Relationship between the release of p-nitrophenol from p- nitrophenyl caprate and enzyme concentration under standard assay conditions --- p.50 / Chapter 3.2.4 --- Effect of pH on lipase stability --- p.58 / Chapter 3.2.5. --- "Temperature stability of lipases from Meiothermus strains 11R, 17Rand 12RB" --- p.58 / Chapter 3.2.6 --- Substrate specificity --- p.59 / Chapter 3.2.7 --- Ejfect of culture supplementation on lipase production --- p.69 / Chapter 3.3. --- Purification of cell-associated lipase activity from Meiothermus strain 17R --- p.71 / Chapter 3.3.1 --- Ammonium sulphate fractionation --- p.71 / Chapter 3.3.2 --- Purificationof lipase I and lipase II using column chromatography --- p.72 / Chapter 3.3.3 --- Purification of lipase III --- p.81 / Chapter 3.4. --- N-terminal amino acid sequencing of lipase I and lipase II from Meiothermus strain 17R --- p.85 / Chapter 3.5. --- Molecular studies of lipase II from Meiothermus strain 17R --- p.86 / Chapter 3.5.1 --- Extraction of genomic DNA --- p.86 / Chapter 3.5.2 --- Cloning of a fragment encoding part of lip II --- p.86 / Chapter 4. --- Discussion / Chapter 4.1 --- Screening of Meiothermus spp. and Thermus spp --- p.89 / Chapter 4.2 --- "Characterizations of lipase from Meiothermus 11R, 17R and 12RB" --- p.90 / Chapter 4.3. --- Biochemical and molecular studies of lipases from Meiothermus strain 17R --- p.94 / Chapter 4.3.1 --- Production of lipases --- p.94 / Chapter 4.3.2 --- Purification of lipases --- p.96 / Chapter 4.3.3 --- Characterization of purified lipases --- p.97 / Chapter 4.3.4 --- Amino acid sequencing of lipase --- p.100 / Chapter 5. --- Summary and suggestions --- p.101 / Chapter 6. --- Appendix --- p.102 / Chapter 7. --- References --- p.103

This thesis presents a systematic investigation of bacterial species associated with wine grapes cultivated in Australian vineyards during 2001-2004. Grapes, sampled throughout cultivation, were analysed for bacterial species using a combination of cultural and molecular methods. Red (Shiraz, Cabernet Sauvignon, Merlot) and white (Chardonnay, Semillon, Sauvignon Blanc) grape varieties were examined. Factors affecting the bacterial ecology of grapes were considered. The bacterial populations of mature undamaged grapes at harvest were consistently low at 102-103 CFU/g. Higher populations (103-106 CFU/g) were found on grapes at earlier stages of maturity and correlated with application of Bacillus thuringiensis as a biological pesticide. B. thuringiensis was the most prevalent bacterial species on wine grapes throughout cultivation, as determined by plate culture, enrichment culture and PCR-DGGE. B. thuringiensis carried over into wine processing but did not grow in juice or wine and did not adversely affect the growth of Saccharomyces cerevisiae or Oenococcus oeni in liquid culture. B. thuringiensis inhibited the growth of several spoilage and mycotoxigenic fungi found on grapes. Curtobacterium flaccumfaciens was the second most prevalent species detected on wine grapes. Its populations rarely exceeded 103-104 CFU/g. Other bacteria (Arthrobacter, Bacillus, Microbacterium, Pantoea, Pseudomonas, Sphingomonas) were sporadically found on grapes. Lactic acid bacteria and acetic acid bacteria were rarely detected on undamaged grapes by culture and PCR-DGGE methods. A greater incidence of lactic acid bacteria was detected by specific enrichment procedures, especially on damaged grape berries. Species found were Lactobacillus plantarum, Lactobacillus mali, Lactobacillus lindneri and Lactobacillus kunkeei. The malolactic organism, O. oeni, was never isolated from any grape sample, raising questions about its enological origin. Enrichment cultures also revealed the presence of other bacteria (e.g. Sporolactobacillus inulinus, Asaia siamensis) not previously found on wine grapes. Atypical, hot and dry conditions during cultivation may account for the low populations of bacteria found on wine grapes. This factor combined with the overwhelming presence of B. thuringiensis prevented meaningful comparisons of data to determine influences of vineyard location, grape variety, grape maturity, climate and viticultural practices on the bacterial ecology of grapes. More systematic and controlled studies of these variables are required.

The F[1]F[O]-ATP synthase found in bacteria, mitochondria, and chloroplasts is responsible for synthesizing ATP from the precursors ADP and P[i]. In bacteria, the enzyme is used to synthesize ATP when the electrochemical gradient of protons ([Delta][mu]H⁺) or sodium ions ([Delta][mu]Na⁺) is high, and the phosphorylation potential is low inside the cell. Conversely, the enzyme hydrolyses ATP to generate a [Delta][mu]H⁺ or [Delta][mu]Na⁺ when these gradients are low, and the phosphorylation potential is high inside the cell. This is coupled to intracellular pH homeostasis in some fermentative bacteria. The aim of this project was to determine the role(s) of the F[1]F[O]-ATP synthase in the physiology of two anaerobic extremophiles: Clostridium paradoxum, a thermoalkaliphilic bacterium that grows over the pH range 7.5 to 10, and Thermotoga maritima, a hyperthermophilic bacterium that thrives at neutral to acidic pH. These bacteria represent good models to study membrane-bound energetic processes as a function of pH and temperature. The energetics of C. paradoxum growth was investigated in batch culture. Growth of C. paradoxum was inhibited by the F-type ATP synthase inhibitor N,N�-dicyclohexylcarbodiimide (DCCD) and monensin, suggesting an important role for an F-type ATP synthase and a chemical gradient of sodium ([Delta]pNa⁺) in the growth of this bacterium. Protonophores had no effect on the growth of C. paradoxum. Inverted membrane vesicles contained DCCD-sensitive ATPase activity and this activity was extracted using the detergent Triton X-100. The solubilized enzyme was purified 30-fold by polyethylene glycol-6000 precipitation. The purified enzyme displayed the typical subunit pattern for an F[1]F[O]-ATP synthase, but also included the presence of a stable oligomeric c-ring that could be dissociated by trichloroacetic acid treatment into its monomeric c subunits. The c-ring was purified, crystallized in 2D, and the projection map indicated that C. paradoxum contains an undecameric c-ring. The purified ATPase was stimulated by Na⁺ ions, and sodium provided protection against inhibition by DCCD that was pH-dependent. ATP synthesis in inverted membrane vesicles was driven by an artificially imposed [Delta]pNa⁺ in the presence of a transmembrane electrical potential ([Delta][phi]) and was sensitive to monensin. Cloning and sequencing of the atp operon revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., Q�⁸, E⁶�, and S⁶�). On the basis of these properties, the ATPase is a sodium-translocating enzyme that generates a [Delta][mu]Na⁺ that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). In support of this proposal are the low rates of ATP synthesis catalyzed by the enzyme and the lack of the C-terminal region of the [epsilon] subunit that has been shown to be essential for coupled ATP synthesis. To facilitate future work on this enzyme at a molecular level, we developed a heterologous over-expression/production system to produce the ATPase in E. coli DK8 ([Delta]unc) in the presence of the helper plasmid pLysRARE. In future studies, this system will be used for the purification of mutant enzyme complexes. Thermotoga maritima is an anaerobic hyperthermophilic bacterium that grows at 80�C and pH 7.0. Growth is sensitive to DCCD and monensin, but resistant to protonophores. These data suggest a [Delta]pNa⁺ is indispensable for growth and a F-type ATP synthase plays an important role under these conditions. The ATPase activity was extracted from inverted membrane vesicles using the detergent n-dodecyl β-D-maltoside. To investigate the role of the ATPase, the enzyme complex was purified 13-fold by polyethylene glycol-6000 precipitation, anionic exchange, and gel filtration chromatography. The purified enzyme was stimulated in the presence of low concentrations of Na⁺ ions, and ATP-dependent uptake of ��Na⁺ into inverted membrane vesicles was observed to be sensitive to DCCD and monensin. Furthermore, analysis of the published genome sequence revealed the presence of a sodium-binding motif in the membrane-bound c subunit (viz., E��, E⁶⁵ and T⁶⁶) and analysis of the ATPase by SDS-PAGE revealed the presence of a putative SDS-stable c-ring. On the basis of these properties, the ATPase from T. maritima appears to be a sodium-translocating enzyme. While we were unable to establish a physiological role for the ATPase, we propose that the enzyme generates a [Delta][mu]Na⁺ that could be used to drive other membrane-bound bioenergetic processes (e.g., solute transport or flagellar rotation). To improve purification and yield, the atp operon was cloned to allow the heterologous over-production of the ATPase. No expression of the ATPase (F[1]F[O]) could be achieved in E. coli BL21(DE3)-CodonPlus, C41(DE3) or E. coli DK8 ([Delta]unc). However, the overproduction of the F₁-ATPase utilizing the helper plasmid pLysRARE was succesful and the enzyme was purified 18-fold by heat precipitation, followed by anionic exchange and gel filtration chromatography, yielding a specific activity of 2.5 U/mg. This complex will be used in the future to investigate the biochemical properties of an F₁-ATPase from a hyperthermophilic anaerobe.

Includes bibliographical references. / 3 v. var. pagings. : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Comprises 171 published works on the general theme of bacterial cell surfaces and pathogenesis. Seeks to address important areas facing medical research by the application of bacterial genetics and molecular biology. The common goal is to provide a better understanding of the mechanisms of pathogenesis of the diseases and how these pathogens have evolved such that this information might be applied to vaccine design and diagnosis. / Thesis (D.Sc.)--University of Adelaide, Dept. of Microbiology and Immunology, 2000?

This thesis presents a systematic investigation of bacterial species associated with wine grapes cultivated in Australian vineyards during 2001-2004. Grapes, sampled throughout cultivation, were analysed for bacterial species using a combination of cultural and molecular methods. Red (Shiraz, Cabernet Sauvignon, Merlot) and white (Chardonnay, Semillon, Sauvignon Blanc) grape varieties were examined. Factors affecting the bacterial ecology of grapes were considered. The bacterial populations of mature undamaged grapes at harvest were consistently low at 102-103 CFU/g. Higher populations (103-106 CFU/g) were found on grapes at earlier stages of maturity and correlated with application of Bacillus thuringiensis as a biological pesticide. B. thuringiensis was the most prevalent bacterial species on wine grapes throughout cultivation, as determined by plate culture, enrichment culture and PCR-DGGE. B. thuringiensis carried over into wine processing but did not grow in juice or wine and did not adversely affect the growth of Saccharomyces cerevisiae or Oenococcus oeni in liquid culture. B. thuringiensis inhibited the growth of several spoilage and mycotoxigenic fungi found on grapes. Curtobacterium flaccumfaciens was the second most prevalent species detected on wine grapes. Its populations rarely exceeded 103-104 CFU/g. Other bacteria (Arthrobacter, Bacillus, Microbacterium, Pantoea, Pseudomonas, Sphingomonas) were sporadically found on grapes. Lactic acid bacteria and acetic acid bacteria were rarely detected on undamaged grapes by culture and PCR-DGGE methods. A greater incidence of lactic acid bacteria was detected by specific enrichment procedures, especially on damaged grape berries. Species found were Lactobacillus plantarum, Lactobacillus mali, Lactobacillus lindneri and Lactobacillus kunkeei. The malolactic organism, O. oeni, was never isolated from any grape sample, raising questions about its enological origin. Enrichment cultures also revealed the presence of other bacteria (e.g. Sporolactobacillus inulinus, Asaia siamensis) not previously found on wine grapes. Atypical, hot and dry conditions during cultivation may account for the low populations of bacteria found on wine grapes. This factor combined with the overwhelming presence of B. thuringiensis prevented meaningful comparisons of data to determine influences of vineyard location, grape variety, grape maturity, climate and viticultural practices on the bacterial ecology of grapes. More systematic and controlled studies of these variables are required.

This thesis presents a systematic investigation of bacterial species associated with wine grapes cultivated in Australian vineyards during 2001-2004. Grapes, sampled throughout cultivation, were analysed for bacterial species using a combination of cultural and molecular methods. Red (Shiraz, Cabernet Sauvignon, Merlot) and white (Chardonnay, Semillon, Sauvignon Blanc) grape varieties were examined. Factors affecting the bacterial ecology of grapes were considered. The bacterial populations of mature undamaged grapes at harvest were consistently low at 102-103 CFU/g. Higher populations (103-106 CFU/g) were found on grapes at earlier stages of maturity and correlated with application of Bacillus thuringiensis as a biological pesticide. B. thuringiensis was the most prevalent bacterial species on wine grapes throughout cultivation, as determined by plate culture, enrichment culture and PCR-DGGE. B. thuringiensis carried over into wine processing but did not grow in juice or wine and did not adversely affect the growth of Saccharomyces cerevisiae or Oenococcus oeni in liquid culture. B. thuringiensis inhibited the growth of several spoilage and mycotoxigenic fungi found on grapes. Curtobacterium flaccumfaciens was the second most prevalent species detected on wine grapes. Its populations rarely exceeded 103-104 CFU/g. Other bacteria (Arthrobacter, Bacillus, Microbacterium, Pantoea, Pseudomonas, Sphingomonas) were sporadically found on grapes. Lactic acid bacteria and acetic acid bacteria were rarely detected on undamaged grapes by culture and PCR-DGGE methods. A greater incidence of lactic acid bacteria was detected by specific enrichment procedures, especially on damaged grape berries. Species found were Lactobacillus plantarum, Lactobacillus mali, Lactobacillus lindneri and Lactobacillus kunkeei. The malolactic organism, O. oeni, was never isolated from any grape sample, raising questions about its enological origin. Enrichment cultures also revealed the presence of other bacteria (e.g. Sporolactobacillus inulinus, Asaia siamensis) not previously found on wine grapes. Atypical, hot and dry conditions during cultivation may account for the low populations of bacteria found on wine grapes. This factor combined with the overwhelming presence of B. thuringiensis prevented meaningful comparisons of data to determine influences of vineyard location, grape variety, grape maturity, climate and viticultural practices on the bacterial ecology of grapes. More systematic and controlled studies of these variables are required.