M. methulotrophus is a Gram negative obligate methylotroph depending on the presence of reduced carbon compounds containing one or more carbon atoms, but containing no carbon-carbon bonds. This organism can synthesize all its cellular constituents from methanol, trimethylamine, dimethylamine, or methylamine. Conversion of methanol and the methylated amines to cell carbon involves the ultimate oxidation to formaldehyde and ammonia. While the methanol dehydrogenase is produced constitutively, the enzymes involved in the assimilation of the methylated amines are inducible. All three enzymes of the trimethylamine pathway are always induced regardless of the methylated amine substrate. Transposon mutagenesis was used to generate mutations in M.methylotrophus and an antibiotic selection procedure used to isolate mutants defective specifically in the trimethylamine pathway. Two mutants were characterised and subjected to further study. The mutant tmd 3 was unable to utilise trimethylamine, dimethylamine or methylamine as substrates and was shown to lack trimethylamine dehydrogenase by polyacrylamide gel electrophoresis. Enzyme studies confirmed the lack of the dehydrogenase within the mutant cells. The mutant mad 1, unable to use methylamine as a substrate, was shown via enzyme studies to contain trimethylamine dehydrogenase, and dimethylamine dehydrogenase, but to lack methylamine dehydrogenase activity. Molecular cloning of wild-type M. methylotrophus DNA in a broad host-range plasmid vector was used to isolate DNA fragments that could replace the mad 1 defect. An 11kb fragment was isolated that fully restored methylamine dehydrogenase activity to mad 1 cells. A 2.5kb fragment was subcloned and shown, by Southern blotting with 32P-labelled In5 DNA as a probe, to contain the site of integration of the In5 insertion, located to within a few hundred base pairs of the end. DNA sequencing, now in progress, has generated 600 base pairs of sequence from either end of the subcloned fragment, within which several regions of interest were noted.

No description available.

No description available.

No description available.

Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, Discipline of Genetics, 2004. / "May 2004" Addendum inside back page. Bibliography: p. 99-114.

The gram negative bacterium Shigella flexneri is an etiological agent of bacillary dysentery, and causes destruction of the human intestinal epithelium. S. flexneri is primarily transmitted via the fecal-oral route to its primary infective site in the colon. The bacterium invades and replicates within colonic epithelial cells, ultimately ulcerating the mucosal epithelium. To successfully establish infection, S. flexneri must quickly adapt to different environments in the host, including adjusting metabolism in response to changes in available carbon sources. In this study, the importance of the glycolytic and gluconeogenic pathways in S. flexneri pathogenesis was examined. The metabolic regulators CsrA and Cra reciprocally regulate the glycolytic and gluconeogenic pathways. The post-transcriptional regulator Cra activates expression of genes involved in gluconeogenesis and represses glycolysis. Conversely, CsrA activates glycolysis and represses gluconeogenesis. The absence of Cra increased S. flexneri attachment and invasion of cultured epithelial cells. In contrast, the csrA mutant was significantly impaired in both adherence and invasion. Both the csrA and cra mutants formed small, turbid plaques, suggesting that both regulators are required for plaque formation. The opposing phenotypes of the csrA and cra mutants suggested a correlation between invasion and glycolysis. The role of glycolysis in S. flexneri pathogenesis was confirmed by directly examining the first committed step in the pathway. The glycolytic enzyme phosphofructokinase I (PfkI, encoded by pfkA) is repressed by Cra and activated by CsrA. Glycolysis was critical for S. flexneri pathogenesis, as a mutation in pfkA rendered the bacterium noninvasive. The invasion defect of the csrA and pfkA mutants was due to reduced expression and secretion of the Shigella invasion plasmid antigen (Ipa) effectors. Expression of the master virulence regulators virF and virB was significantly reduced in the pfkA mutant, and is the principle reason for decreased invasion. The data presented show that glycolysis is required for invasion, but that plaque formation requires both glycolysis and gluconeogenesis. Because expression of the master virulence regulators is repressed in the pfkA mutant, S. flexneri may use carbon as an environmental regulator of virulence gene expression. / text

by Deng Yu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 111-120). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / List of Abbreviations --- p.viii / List of Tables --- p.ix / List of Figures --- p.x / Chapter 1 --- Introduction / Chapter 1.1 --- Primary nitrogen metabolism in fungi --- p.1 / Chapter 1.1.1 --- Ammonium assimilation --- p.4 / Chapter 1.1.2 --- Regulation of ammonium assimilating enzymes --- p.8 / Chapter 1.2 --- Relevant central carbon metabolism in fungi --- p.11 / Chapter 1.2.1 --- Glyoxylate cycle and isocitrate metabolism --- p.11 / Chapter 1.2.2 --- GABA shunt --- p.15 / Chapter 1.3 --- Relationship between nitrogen metabolism and fungi morphogenesis --- p.15 / Chapter 1.4 --- General background of Volvariella volvacea --- p.17 / Chapter 1.5 --- Objectives of the study --- p.20 / Chapter 2 --- Materials and methods / Chapter 2.1 --- Organism --- p.22 / Chapter 2.2 --- Growth media --- p.22 / Chapter 2.2.1 --- Basal medium --- p.22 / Chapter 2.2.2 --- Solid-state cultivation --- p.23 / Chapter 2.3 --- Effect of different nitrogen sources on the mycelial growth of V volvacea in submerged culture --- p.26 / Chapter 2.4 --- Effect of different carbon and nitrogen sources and concentrations on the production of selected enzymes involved in central carbon and nitrogen metabolism --- p.27 / Chapter 2.5 --- Enzyme extraction --- p.28 / Chapter 2.6 --- Enzyme assays --- p.28 / Chapter 2.6.1 --- NAD-dependent glutamate dehydrogenase --- p.28 / Chapter 2.6.2 --- NADP-dependent glutamate dehydrogenase --- p.29 / Chapter 2.6.3 --- NAD- dependent isocitrate dehydrogenase --- p.29 / Chapter 2.6.4 --- Isocitrate lyase --- p.30 / Chapter 2.7 --- Protein determination --- p.30 / Chapter 2.8 --- Determination of optimum pH for enzyme assays --- p.31 / Chapter 2.9 --- Determination of optimum temperatures for enzyme assays --- p.31 / Chapter 2.10 --- Transfer experiments --- p.31 / Chapter 2.11 --- Enzyme stability --- p.32 / Chapter 2.12 --- Purification of NAD-dependent glutamate dehydrogenase --- p.33 / Chapter 2.12.1 --- Ammonium sulphate precipitation --- p.33 / Chapter 2.12.2 --- Ion exchange chromatography --- p.33 / Chapter 2.12.3 --- Ultrafiltrartion --- p.34 / Chapter 2.12.4 --- Gel filtration chromatography --- p.34 / Chapter 2.12.5 --- Affinity chromatography --- p.34 / Chapter 2.13 --- Electrophoresis --- p.35 / Chapter 2.13.1 --- SDS polyacrylamide gel electrophoresis --- p.35 / Chapter 2.13.2 --- Native polyacrylamide gel electrophoresis --- p.35 / Chapter 2.13.3 --- Activity staining for NAD-dependent glutamate dehydrogenase --- p.36 / Chapter 2.13.4 --- Protein staining --- p.36 / Chapter 2.14 --- NAD-dependent glutamate dehydrogenase characterization studies --- p.37 / Chapter 2.14.1 --- Effect of substrate concentration --- p.37 / Chapter 2.14.2 --- Molecular weight determination --- p.37 / Chapter 2.14.2.1 --- Molecular weight determination by gel filtration chromatography --- p.37 / Chapter 2.14.2.2 --- Molecular weight determination by native PAGE --- p.38 / Chapter 2.14.2.3 --- Protein subunit molecular weight determination by SDS- PAGE --- p.38 / Chapter 3 --- Results / Chapter 3.1 --- Effect of different nitrogen sources on the mycelial growth of V. volvacea in submerged culture --- p.39 / Chapter 3.2 --- Optimum assay conditions for NAD-dependent glutamate dehydrogenase --- p.42 / Chapter 3.3 --- Optimum assay conditions for NADP-dependent glutamate dehydrogenase --- p.46 / Chapter 3.4 --- Optimum assay conditions for NAD-dependent isocitrate dehydrogenase --- p.50 / Chapter 3.5 --- Optimum assay conditions for isocitrate lyase --- p.54 / Chapter 3.6 --- Biomass production and enzyme activities in extracts of in vegetative mycelia grown with different nitrogen and carbon sources provided at different concentrations --- p.58 / Chapter 3.6.1 --- Mycelia growth under different conditions --- p.58 / Chapter 3.6.2 --- NAD- and NADP-dependent glutamate dehydrogenases in extracts of vegetative mycelia grown with different nitrogen and carbon sources provided at different conditions --- p.58 / Chapter 3.6.3 --- NAD-dependent isocitrate dehydrogenase and isocitrate lyase in vegetative mycelia grown with different nitrogen and carbon sources provided at different conditions --- p.64 / Chapter 3.7 --- Transfer experiments --- p.67 / Chapter 3.7.1 --- Activities of glutamate dehydrogenases in extracts of myceila transferred to media containing different carbon sources --- p.67 / Chapter 3.7.2 --- Effect of different carbon sources on the glutamate dehydrogenases in submerged cultures --- p.67 / Chapter 3.8 --- Glutamate dehydrogenase activity in various parts of the fruit body during different stages of fruit body development --- p.70 / Chapter 3.9 --- Stabilization of NAD-dependent glutamate dehydrogenase activity --- p.75 / Chapter 3.10 --- Purification of NAD-dependent glutamate dehydrogenase --- p.77 / Chapter 3.10.1 --- Ammonium sulphate precipitation --- p.77 / Chapter 3.10.2 --- Partial purification by column chromatography --- p.78 / Chapter 3.10.3 --- Electrophoretic determination of the protein profiles of crude extract and partially purified samples --- p.83 / Chapter 3.11 --- Characterization of partially purified NAD-dependent glutamate dehydrogenase from V. volvacea --- p.86 / Chapter 3.11.1 --- Optimum pH and temperature --- p.86 / Chapter 3.11.2 --- Kinetic parameters --- p.86 / Chapter 3.11.3 --- Molecular weight --- p.92 / Chapter 3.11.3.1 --- Molecular weight determination by gel filtration chromatography --- p.92 / Chapter 3.11.3.2 --- Molecular weight determination by native PAGE --- p.92 / Chapter 3.11.3.3 --- Subunit molecular weight determination by SDS-PAGE --- p.92 / Chapter 4 --- Discussion / Chapter 4.1 --- Nutrient nitrogen for the growth of Volvariella volvacea --- p.97 / Chapter 4.1.1 --- Mycelial growth on simple nitrogen compounds --- p.97 / Chapter 4.1.2 --- Nutrient nitrogen in mushroom compost --- p.98 / Chapter 4.2 --- Production and regulation of selected enzymes in vegetative mycelia --- p.98 / Chapter 4.2.1 --- Production and regulation of glutamate dehydrogenases --- p.98 / Chapter 4.2.2 --- Production and regulation of isocitrate dehydrogenase and isocitrate lyase --- p.103 / Chapter 4.3 --- Glutamate dehydrogenases and fruit body development --- p.104 / Chapter 4.4 --- Purification and characterization of NAD-dependent glutamate dehydrogenase --- p.105 / Chapter 4.4.1 --- Enzyme purification --- p.105 / Chapter 4.4.2 --- Enzyme stability --- p.106 / Chapter 4.4.3 --- Enzyme properties --- p.107 / Chapter 4.5 --- Future works: nitrogen metabolism and the growth of Vohariella volvacea --- p.109 / References --- p.111

One-carbon metabolism is an essential process that is conserved from yeast to humans. Glycine stimulates the expression of genes in one-carbon metabolism, whereas its withdrawal causes repression of these genes. The transcription factor Bas1p and the metabolic enzyme Shm2p have been implicated in this regulation. I have shown that Bas1p physically interacts with Shm2p through co-immunoprecipitation. Using chromatin immunoprecipitation (ChIP), I have also shown that the interaction between Bas1p and Shm2p occurs at the promoter of two genes in the one-carbon metabolism regulon and that the binding of Shm2p requires Bas1p. Using a yeast-two hybrid system, I have systematically truncated Bas1p from the C-terminal end to find a region responsible for the interaction with Shm2p. My data suggest that Shm2p is directly bound to Bas1p at the promoters of glycine regulated genes where it regulates the transcriptional activity of Bas1p in response to changes in glycine levels.

One-carbon metabolism is an essential process that is conserved from yeast to humans. Glycine stimulates the expression of genes in one-carbon metabolism, whereas its withdrawal causes repression of these genes. The transcription factor Bas1p and the metabolic enzyme Shm2p have been implicated in this regulation. I have shown that Bas1p physically interacts with Shm2p through co-immunoprecipitation. Using chromatin immunoprecipitation (ChIP), I have also shown that the interaction between Bas1p and Shm2p occurs at the promoter of two genes in the one-carbon metabolism regulon and that the binding of Shm2p requires Bas1p. Using a yeast-two hybrid system, I have systematically truncated Bas1p from the C-terminal end to find a region responsible for the interaction with Shm2p. My data suggest that Shm2p is directly bound to Bas1p at the promoters of glycine regulated genes where it regulates the transcriptional activity of Bas1p in response to changes in glycine levels.

No description available.