Nitrogenase-catalyzed nitrogen fixation is a biochemically and genetically complex process requiring the participation of a number of different nif (nitrogen fixation) gene products. The nifF (electron transport), nifA (nif gene regulation) and nifE (FeMo-cofactor biosynthesis) genes from <i>Azotobacter vinelandii</i> were genetically analyzed. The nucleotide sequence of the nifF gene, which encodes a flavodoxin, was determined. Specific mutation strains indicated that in <i>A vinelandii</i> flavodoxin is not the unique physiological electron donor to nitrogenase. The nifF gene appears to be constitutively expressed but under nitrogen fixing conditions nifF gene expression is stimulated. / Master of Science

Increased mutagenesis is a hallmark of cancers. On the other hand, this can trigger the generation of polymorphisms and lead to evolution. Lately, it has become clear that one of the major sources of increased mutation rates in the genome is chromosomal break formation and repair. A variety of factors can contribute to the generation of breaks in the genome. A paradoxical source of breaks is the sequence composition of the genomic DNA itself. Eukaryotic and prokaryotic genomes contain sequence motifs capable of adopting secondary structures often found to be potent inducers of double strand breaks culminating into rearrangements. These regions are therefore termed fragile sequence motifs. Here, we demonstrate that in addition to being responsible for triggering chromosomal rearrangements, inverted repeats and GAA/TTC repeats are also potent sources of mutagenesis. Repeat-induced mutagenesis extends up to 8 kb on either side of the break point. Remarkably, error-prone repair of the break by Polζ reconstitutes the repeats making them a long term source of mutagenesis. Despite its negative connotations for genome stability, the mechanisms underlying the unstable nature of double strand break repair pathways are not known. Previous studies have demonstrated that break induced replication (BIR), a mechanism employed to repair broken chromosomes with only one repairable end, is highly mutagenic, undergoes frequent template switching and often yields half-crossovers. In the work presented here, we show that the instabilities inherent to BIR can be attributed to its unusual mode of synthesis. We determined that BIR proceeds via a migrating bubble with long stretches of single-stranded DNA and culminates with conservative inheritance of the newly synthesized DNA. We propose that the mechanisms described here might be important for generation of repair-associated mutagenesis in higher organisms. Secondary structure forming repeats like inverted repeats have been found to be enriched in cancer cells. These motifs often constitute chromosomal rearrangement hot-spots and demonstrate the phenomenon of kataegis. This study provides a mechanistic insight into how such breakage-prone motifs contribute to hypermutability of cancer genomes.

Lau Kin Chong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 149-158). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.xii / List of Figures --- p.xiii / List of Appendices --- p.xv / Abbreviations --- p.xvi / Chapter 1 --- Introduction / Chapter 1.1 --- Mucopolysaccharidosis type II as a lysosomal storage disease --- p.1 / Chapter 1.1.1 --- Prevalence of MPS II --- p.2 / Chapter 1.1.2 --- Pathophysiology of MPS II --- p.4 / Chapter 1.1.3 --- Clinical features of MPS II --- p.4 / Chapter 1.1.4 --- Clinical management of MPS II --- p.6 / Chapter 1.1.4.1 --- Diagnostic methods for MPS II --- p.6 / Chapter 1.1.4.2 --- Treatments for MPS II --- p.7 / Chapter 1.2 --- Iduronate-2-sulfatase protein (IDS) --- p.9 / Chapter 1.2.1 --- Role in GAG degradation --- p.9 / Chapter 1.2.2 --- Post-translational modifications --- p.11 / Chapter 1.2.2.1 --- Formylglycine formation --- p.11 / Chapter 1.2.2.2 --- Glycosylation --- p.12 / Chapter 1.2.2.3 --- Proteolysis --- p.12 / Chapter 1.2.3 --- Iduronate-2-sulfatase gene (IDS) --- p.14 / Chapter 1.2.3.1 --- Properties of IDS mutations --- p.15 / Chapter 1.2.3.2 --- Methylation patterns are correlated with transitional mutations --- p.17 / Chapter 1.2.3.3 --- Genotype-phenotype correlations between IDS gene and MPS II --- p.19 / Chapter 1.3 --- In this study --- p.21 / Chapter 1.3.1 --- Mutational analysis --- p.21 / Chapter 1.3.2 --- In vitro expression of mutant IDS --- p.22 / Chapter 1.3.3 --- Maturation of IDS polypeptides --- p.23 / Chapter 2 --- Materials & Methods / Chapter 2.1 --- Mutation screening for MPS II patients --- p.24 / Chapter 2.1.1 --- Patients --- p.24 / Chapter 2.1.2 --- Genomic DNA extraction --- p.24 / Chapter 2.1.2.1 --- Materials --- p.24 / Chapter 2.1.2.2 --- Methods --- p.25 / Chapter 2.1.3 --- IDS exons amplification by Polymerase Chain Reaction (PCR) --- p.26 / Chapter 2.1.3.1 --- Materials --- p.26 / Chapter 2.1.3.1.1 --- PCR --- p.26 / Chapter 2.1.3.1.2 --- Agarose gel electrophoresis --- p.27 / Chapter 2.1.3.1.3 --- PCR fragments purification --- p.29 / Chapter 2.1.3.2 --- Methods --- p.29 / Chapter 2.1.3.2.1 --- Amplifying IDS exons by PCR --- p.29 / Chapter 2.1.3.2.2 --- Purifying PCR fragments --- p.30 / Chapter 2.1.4 --- DNA sequencing for detecting IDS mutations --- p.30 / Chapter 2.1.4.1 --- Materials --- p.30 / Chapter 2.1.4.2 --- Methods --- p.30 / Chapter 2.1.4.2.1 --- Sequencing reaction --- p.30 / Chapter 2.1.4.2.2 --- Purifying sequencing products --- p.31 / Chapter 2.1.4.2.3 --- Analyzing sequencing results --- p.31 / Chapter 2.1.5 --- Fragment restriction endonuclease analysis --- p.31 / Chapter 2.1.5.1 --- Materials --- p.31 / Chapter 2.1.5.2 --- Methods --- p.32 / Chapter 2.2 --- Isolation of IDS cDNA from peripheral blood --- p.34 / Chapter 2.2.1 --- Materials --- p.34 / Chapter 2.2.1.1 --- Total RNA extraction --- p.34 / Chapter 2.2.1.2 --- Reverse-transcriptase PCR (RT-PCR) --- p.35 / Chapter 2.2.1.3 --- PCR for amplifying IDS cDNA --- p.35 / Chapter 2.2.2 --- Methods --- p.37 / Chapter 2.2.2.1 --- Extracting total RNA by QIAamp RNeasy Mini Kit --- p.37 / Chapter 2.2.2.2 --- Converting IDS mRNA into cDNA by RT-PCR --- p.38 / Chapter 2.2.2.3 --- Isolating IDS cDNA by PCR --- p.39 / Chapter 2.2.2.4 --- Isolating firefly luciferase gene by PCR --- p.39 / Chapter 2.3 --- Introducing IDS cDNA into Gateway Cloning System --- p.40 / Chapter 2.3.1 --- Materials --- p.40 / Chapter 2.3.1.1 --- Directional cloning --- p.40 / Chapter 2.3.1.2 --- LB medium/ agar with antibiotics preparation --- p.42 / Chapter 2.3.1.3 --- Plasmids purification from transformed cells --- p.42 / Chapter 2.3.1.4 --- Validation of IDS inserted plasmids --- p.43 / Chapter 2.3.2 --- Methods --- p.43 / Chapter 2.3.2.1 --- TOPO cloning reaction --- p.43 / Chapter 2.3.2.2 --- Transformation --- p.44 / Chapter 2.3.2.3 --- Small-scale plasmids preparation by QIAprep Miniprep Kit --- p.44 / Chapter 2.3.2.4 --- Sequencing the plasmids --- p.45 / Chapter 2.3.2.5 --- QuikChange II XL site-directed mutagenesis --- p.46 / Chapter 2.3.2.5.1 --- Synthesizing mutant strand with desired mutations --- p.46 / Chapter 2.3.2.5.2 --- Digesting parental strand --- p.46 / Chapter 2.3.2.5.3 --- Transformation --- p.47 / Chapter 2.3.2.6 --- Swapping IDS gene from entry clone to expression vectors --- p.47 / Chapter 2.3.2.6.1 --- LR clonase reaction --- p.47 / Chapter 2.3.2.6.2 --- Transformation --- p.48 / Chapter 2.4 --- Introducing IDS cDNA into RTS pIVEX Wheat Germ vector --- p.49 / Chapter 2.4.1 --- Materials --- p.49 / Chapter 2.4.1.1 --- Restriction digestion --- p.49 / Chapter 2.4.1.2 --- Purification of digested products --- p.50 / Chapter 2.4.1.3 --- Ligation of the IDS insert into pIVE´Xؤ1.3_WG --- p.50 / Chapter 2.4.2 --- Methods --- p.50 / Chapter 2.4.2.1 --- Restriction digestion to create sticky ends --- p.50 / Chapter 2.4.2.2 --- Purifying the digested products --- p.51 / Chapter 2.4.2.3 --- Ligating the IDS insert into pIVE´Xؤ1.3_WG --- p.51 / Chapter 2.4.2.4 --- Transformation --- p.51 / Chapter 2.5 --- Transient expression study of IDS constructs --- p.53 / Chapter 2.5.1 --- Materials --- p.53 / Chapter 2.5.2 --- Methods --- p.55 / Chapter 2.5.2.1 --- Cell culturing --- p.55 / Chapter 2.5.2.2 --- Transfecting IDS constructs by lipofection procedures --- p.55 / Chapter 2.5.2.3 --- Harvesting COS-7 cells --- p.56 / Chapter 2.5.2.4 --- Total RNA extraction from transfected COS-7 cells --- p.57 / Chapter 2.5.2.5 --- RT-PCR showing IDS mRNA stability --- p.58 / Chapter 2.5.2.6 --- Endocytosis of expressed IDS products into COS-7 cells --- p.58 / Chapter 2.6 --- Synthesizing IDS by cell-free in vitro expression systems --- p.59 / Chapter 2.6.1 --- Materials --- p.59 / Chapter 2.6.1.1 --- DNA templates for expression --- p.59 / Chapter 2.6.1.2 --- Commercial cell-free expression kits --- p.60 / Chapter 2.6.1.3 --- Supplements --- p.61 / Chapter 2.6.2 --- Methods --- p.64 / Chapter 2.6.2.1 --- Cell-free expression by ExpressWay plus expression system --- p.64 / Chapter 2.6.2.2 --- Cell-free expression by RTS 100 E.coli HY Kit --- p.64 / Chapter 2.6.2.3 --- Cell-free expression by RTS 100 Wheat Germ CECF Kit --- p.64 / Chapter 2.6.2.4 --- Cell-free expression by TnT Coupled Wheat Germ Extract Systems --- p.65 / Chapter 2.6.2.5 --- Cell-free expression by TNT Coupled Reticulocyte Lysate Systems --- p.66 / Chapter 2.7 --- Investigations of IDS protein expression --- p.67 / Chapter 2.7.1 --- Materials --- p.67 / Chapter 2.7.1.1 --- Isolation of Histidine-tagged proteins --- p.67 / Chapter 2.7.1.2 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis/ SDS-PAGE --- p.67 / Chapter 2.7.1.3 --- Fluorometric activity assay for IDS --- p.69 / Chapter 2.7.1.4 --- Luciferase activity assay --- p.72 / Chapter 2.7.2 --- Methods --- p.72 / Chapter 2.7.2.1 --- Isolating His-tagged IDS from cell-free expression products --- p.72 / Chapter 2.7.2.2 --- Protein staining of expression products --- p.73 / Chapter 2.7.2.2.1 --- Preparation of protein separating gel --- p.73 / Chapter 2.7.2.2.2 --- Preparation of proteins for SDS-PAGE --- p.73 / Chapter 2.7.2.2.3 --- SDS-PAGE analysis --- p.73 / Chapter 2.7.2.3 --- Fluorometric enzyme assay for IDS proteins --- p.74 / Chapter 2.7.2.4 --- Luciferase activity assay --- p.75 / Chapter 3 --- Results / Chapter 3.1 --- Mutational analysis of MPS II and carrier detection --- p.76 / Chapter 3.2 --- Investigating IDS mutants by transient expression --- p.86 / Chapter 3.2.1 --- Fluorometric enzyme assay for measuring IDS activity --- p.86 / Chapter 3.2.2 --- Source of IDS gene for transient expression in COS-7 cells --- p.89 / Chapter 3.2.3 --- In vitro expression of IDS and its mutants in COS-7 cells --- p.92 / Chapter 3.2.3.1 --- Analysis of transient expression in terms of IDS activity --- p.92 / Chapter 3.2.3.2 --- Analysis of IDS mRNA stability in COS-7 cells --- p.95 / Chapter 3.2.3.3 --- Analysis of IDS protein stability in COS-7 cells --- p.95 / Chapter 3.3 --- Cell-free in vitro expression for investigating the IDS mutants --- p.98 / Chapter 3.3.1 --- The five cell-free systems involved --- p.98 / Chapter 3.3.2 --- Source of IDS gene for cell-free in vitro expression --- p.98 / Chapter 3.3.3 --- SDS-PAGE analysis of IDS protein stability in cell-free systems --- p.100 / Chapter 3.3.3.1 --- Wheat germ-based cell-free expression system (Roche) --- p.100 / Chapter 3.3.3.2 --- E.coli-based cell-free expression system (Invitrogen) --- p.102 / Chapter 3.3.3.3 --- E.coli-based cell-free expression system (Roche) --- p.102 / Chapter 3.3.4 --- In Vision His-tag In-gel stain for wild-type IDS and its mutant --- p.103 / Chapter 3.3.5 --- Analysis of IDS activity in cell-free expression systems --- p.107 / Chapter 3.3.6 --- Analysis of the cellular uptake of IDS --- p.110 / Chapter 4 --- Discussions / Chapter 4.1 --- Mutational analysis --- p.113 / Chapter 4.1.1 --- Heterogeneity of IDS mutations --- p.113 / Chapter 4.1.2 --- Role of molecular diagnosis for MPS II --- p.113 / Chapter 4.1.3 --- Two novel mutations and one reported mutation were identified --- p.115 / Chapter 4.1.3.1 --- A novel nonsense mutation: Ser369term --- p.115 / Chapter 4.1.3.2 --- A reported nonsense mutation: Gln389term --- p.115 / Chapter 4.1.3.3 --- A novel missense mutation: Leu339Pro --- p.116 / Chapter 4.2 --- Expression studies of the IDS mutants --- p.117 / Chapter 4.2.1 --- Analysis of transient expression in COS-7 cells --- p.117 / Chapter 4.2.1.1 --- Stability of mutant mRNA --- p.119 / Chapter 4.2.1.2 --- IDS catalytic activity --- p.119 / Chapter 4.2.2 --- Analysis of mutant stability by cell-free expression systems --- p.120 / Chapter 4.2.3 --- Structural analysis of amino acids alterations --- p.121 / Chapter 4.2.3.1 --- p.L339P causes conformational change --- p.122 / Chapter 4.2.3.2 --- p.L339R changes overall charge balance --- p.122 / Chapter 4.2.3.3 --- Mutations at Leu339 residue affect substrate binding --- p.123 / Chapter 4.3 --- Analysis of IDS maturation processing --- p.124 / Chapter 4.3.1 --- Active IDS modifications are not completed in lysosomes --- p.124 / Chapter 4.3.2 --- C-terminal proteolysis is essential for active IDS --- p.125 / Chapter 4.3.3 --- Functional role of glycosylation during IDS processing --- p.126 / Chapter 4.4 --- Analysis of cell-free expression systems --- p.128 / Chapter 4.4.1 --- Microbial systems using E.coli cell extracts: insoluble IDS precursors --- p.128 / Chapter 4.4.2 --- Plant system using wheat germ extracts: soluble IDS precursors --- p.129 / Chapter 4.4.3 --- Mammalian system using rabbit reticulocytes extracts: undetectable --- p.129 / Chapter 4.5 --- Role of transfecting IDS constructs --- p.131 / Chapter 4.6 --- Conclusion --- p.132 / Appendices --- p.133 / Electronic-database and computing system --- p.149 / Bibliography --- p.149

After alignment of 300 HBV sequences randomly downloaded from GenBank, we found that the frequency of A1762T and G1764A mutations in genotype C was found as high as 64%, while 34% was found for other genotypes (A, B, D to H). Besides, recent clinical studies have also shown that A1762T/G1764A mutations occur frequently in HCC patients with genotype B infection (81%, 30 of 37 patients), but were relatively lower in asymptomatic carriers (43%, 22 of 51 patients). These indicate that the contribution of A1762T/G1764A mutations to liver cancer might not be limited to genotype C. As the double mutations are present within the region of HBV Enhancer II/Basal core promoter (BCP) and cause residue substitution of HBx (Lys130Met and Val131Ile); therefore, their effects on the promoter and HBx activities were examined. / Chronic infection of hepatitis B virus (HBV) increases the risk of hepatocellular carcinoma (HCC) by more than 100-fold. However, the underlying molecular mechanism of this process is not fully understood. Several recent studies have shown that A1762T and G1764A mutations of HBV were associated with the aggressiveness of liver disease, in which inactive carriers would develop active hepatitis, and eventually liver cirrhosis and HCC. In Asia, genotypes B and C are the predominant genotypes of HBV infections. Our longitudinal five-year follow-up study of 426 chronic hepatitis B patients in Hong Kong found that the genotype C HBV (normally with A1762T/G1764A mutations) was closely associated with higher risk of HCC than genotype B HBV (non-frequent mutations with A1762T/G1764A). / In this study, systemic site-directed mutagenesis studies, promoter assays, replication capacity assays and overexpression of HBx assays were carried out to demonstrate the molecular mechanisms of these mutations for the increases risk of HCC. Three conclusions were drawn from this study. (1) A1762T and/or G1764A mutations of HBV could reduce BCP activities in a synergistic manner with 1764A contributing more. Reversed T1762A and/or A1764G mutations increase the BCP activities also in a synergistic manner with 1764G contributing more; (2) HBx could increase HBV BCP activity, HBV replication and HBsAg expression. The Lys130Met and Val131Ile mutations of HBx could further increase the above abilities while the A1762T/G1764A double mutations in the BCP region could not affect the interaction of HBx and HBV BCP; (3) The G1677T/A1679C and T1706C mutations could increase the BCP activity; The ectopic expression of HBx could further increase the BCP activity while the mutated HBx (130Met and 131Ile) has less effect on these mutated promoters. / Dong, Qingming. / Adviser: Ming-Liang He. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: December 2008. / Thesis submitted in: December 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 132-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Wong, Kin Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 86-98). / Abstract also in Chinese. / THESIS COMMITTEE --- p.i / ABSTRACT (ENGLISH) --- p.ii / ABSTRACT (CHINESE) --- p.iv / ACKNOWLEDGEMENTS --- p.v / DECLARATION --- p.vi / ABBREVIATIONS --- p.vii / TABLE OF CONTENTS --- p.x / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1. --- Antibiotics market and β-lactam antibiotics --- p.1 / Chapter 1.2. --- Semi-synthetic cephems --- p.1 / Chapter 1.3. --- Conversion of CPC to 7-ACA --- p.3 / Chapter 1.4. --- Chemical production versus enzymatic bioconversion --- p.5 / Chapter 1.5. --- Industrial two-step bioconversion of CPC --- p.11 / Chapter 1.6. --- Phylogenetics and physiological roles of DAAO --- p.15 / Chapter 1.7. --- Yeast DAAOs are suitable candidates for enzymatic bioconversion --- p.17 / Chapter 1.8. --- Structural and mechanistic studies of DAAOs --- p.18 / Chapter 1.9. --- "Modifications of pkDAAO, RgDAAO and TvDAAO" --- p.25 / Chapter 1.10. --- Objectives of the study --- p.26 / Chapter CHAPTER 2 --- HOMOLOGY MODELLING / Chapter 2.1. --- Introduction --- p.27 / Chapter 2.2. --- Methods / Chapter 2.2.1. --- Sequence alignment and selection of homologs --- p.28 / Chapter 2.2.2. --- Generation of three-dimensional TvDAAO model --- p.28 / Chapter 2.3. --- Results --- p.29 / Chapter 2.4. --- Discussion --- p.33 / Chapter CHAPTER 3 --- "MUTAGENESIS, EXPRESSION, PURIFICATION AND SCREENING OF MUTANTS" / Chapter 3.1. --- Introduction --- p.38 / Chapter 3.2. --- Materials and methods / Chapter 3.2.1. --- Cloning of TvDAAO mutants / Chapter 3.2.1.1. --- Preparation of competent E. coli --- p.39 / Chapter 3.2.1.2. --- Transformation of E. coli --- p.40 / Chapter 3.2.1.3. --- Agarose gel electrophoresis and gel-purification --- p.41 / Chapter 3.2.1.4. --- Plasmid extraction --- p.42 / Chapter 3.2.1.5. --- Site-directed mutagenesis of TvDAAO --- p.42 / Chapter 3.2.2. --- Heterologous expression and purification of mutants / Chapter 3.2.2.1 --- Shake flask fermentation --- p.45 / Chapter 3.2.2.2. --- Cell harvest and disruption --- p.45 / Chapter 3.2.2.3. --- Purification of WT and mutants --- p.47 / Chapter 3.2.2.4. --- Determination of protein concentration --- p.47 / Chapter 3.2.2.5. --- SDS-PAGE --- p.48 / Chapter 3.2.3. --- Screening of mutants --- p.48 / Chapter 3.3. --- Results / Chapter 3.3.1. --- Preparation of purified TvDAAO mutants --- p.50 / Chapter 3.3.2. --- Evaluation of activity and thermostability --- p.50 / Chapter 3.4. --- Discussion --- p.53 / Chapter CHAPTER 4 --- ENZYME KINETICS / Chapter 4.1. --- Introduction --- p.57 / Chapter 4.2. --- Materials and methods / Chapter 4.2.1. --- Standard assay --- p.58 / Chapter 4.2.2. --- Determination of kinetic parameters --- p.59 / Chapter 4.2.3. --- Inhibitory studies --- p.59 / Chapter 4.2.4. --- Effects of pH --- p.60 / Chapter 4.2.5. --- Heat treatments --- p.60 / Chapter 4.2.6. --- CD measurements --- p.61 / Chapter 4.3. --- Results / Chapter 4.3.1. --- Time progress curve analysis --- p.61 / Chapter 4.3.2. --- Kinetics of WT and mutants --- p.62 / Chapter 4.3.3. --- Temperature-dependent and time-dependent thermostability --- p.67 / Chapter 4.3.4. --- Secondary structure measurements --- p.71 / Chapter 4.4. --- Discussion --- p.71 / Chapter CHAPTER 5 --- CONCLUSIONS AND PERSEPECTIVES --- p.83 / BIBLIOGRAPHY --- p.86

Thesis (Ph. D.)--University of Missouri--Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 156-180). Also issued on the Internet.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy Ganta / Ehrlichia chaffeensis a tick transmitted Anaplasmataceae family pathogen responsible for human monocytic ehrlichiosis. Differential gene expression appears to be an important pathogen adaptation mechanism for its survival in dual hosts. One of the ways to test this hypothesis is by performing mutational analysis that aids in altering the expression of genes. Mutagenesis is also a useful tool to study the effects of a gene function in an organism. Focus of my research has been to prepare several modified Himar transposon mutagenesis constructs for their value in introducing mutations in E. chaffeensis genome. While the work is in progress, research team from our group used existing Himar transposon mutagenesis plasmids and was able to create mutations in E. chaffeensis. Multiple mutations were identified by Southern blot analysis. I redirected my research efforts towards mapping the genomic insertion sites by performing the semi-random two step PCR (ST-PCR) method, followed by DNA sequence analysis. In this method, the first PCR is performed with genomic DNA as the template with a primer specific to the insertion segment and the second primer containing an anchored degenerate sequence segment. The product from the first PCR is used in the second PCR with nested transposon insertion primer and a primer designed to bind to the known sequence portion of degenerate primer segment. This method aided in identifying the genomic locations of four E. chaffeensis mutants and also was valuable in confirming four other sites mapped previously by the rescue cloning method. This is the first mutational analysis study in the genome of an Ehrlichia species. Mapping the genomic transposon insertion sites is the first critical step needed for the continued research to define the importance of the mutations in understanding the pathogenesis caused by the organism.

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

NtdB is an enzyme encoded within the ntd operon in Bacillus subtilis. This operon is reported to contain a complete set of genes necessary for the biosynthesis of 3,3'-neotrehalosadiamine (NTD), a compound composed of two kanosamine subunits linked together by a 1,1'-(α,β)-linkage. Both NTD and kanosamine have reported antibiotic properties. The function of NtdB has been a matter of speculation, but has never been investigated in vitro. Using a phosphate assay and an array of substrates, NtdB was determined to be a phosphatase, specific to kanosamine-6-phosphate (K6P) (kcat = 32 ± 1 s-1, Km = 93 ± 7 µM). Site-directed mutagenesis of amino acid residues in the core and the cap domains of the enzyme identified residues important for the catalytic reaction and substrate specificity. These mutations confirmed the presence of four motifs, characteristic of members of the haloacid dehalogenase (HAD) superfamily, and allowed identification of the substrate binding site of the enzyme. KabB, a homologue of NtdB from Bacillus cereus, showed notably lower activity with K6P than NtdB. This research defines the role of NtdB as a specific K6P phosphatase and challenges the previously reported NTD biosynthesis pathway by demonstrating a novel pathway for the production of the antibiotic kanosamine.

Young children are particularly sensitive to environmental pollutants. They can directly ingest soil by putting dirty hands and objects in their mouths. The reliance on animal derived models for human health risk and exposure assessment has several limitations. In this investigation, a tool-kit was developed and optimised to facilitate more accurate, reliable and representative predictions of soil contaminants that might pose a significant hazard to young children. The tool-kit was developed and optimised using an in vitro human digestion bioassay. This procedure was followed by the optimisation of several mutagenicity bioassays to link to the bioaccessible fraction which quantified by the in vitro bioassay. The application of novel and sensitive environmental-based biosensors requires them to work in parallel with effective and proven extraction techniques. In this study, chemical analysis was used to quantify the bioaccessible (human assimilated portion) of pollutants in soils. Acute toxicity was measured using constitutively marked bioluminescent bacterial biosensors and these were indicative of the total contaminant burden. A range of mutagenic assays were applied and optimised. In the Ames assay, any compound exhibiting a greater than two-fold increase in the number of revertants colonies over the number of spontaneous revertants was considered as a mutagen. Mutagenic-responsive SOS-lux based microbial biosensors were compared to the Ames assay. Mutagenicity assessment of a broad range of environmental pollutants (i.e. B[a]P, DiB(a,h)A, B[a]A, Ni and Cu), was performed using four SOS-lux microbial biosensors; E. coli DPD1718, E. coli K12C600, S. aureus pAmiUmuC and S. aureus pAmiRecA. The results substantiated that the four biosensors were unable to be induced by these pollutants. Nevertheless, E. coli DPD1718 and E. coli K12C600 were successfully induced by Mitomycin C (MMC) in a dose response manner. The Ames assay was performed for the above pollutants in the absence and the presence of the metabolic activation S9 mix. The standard plate incorporation assay and a modification protocol for the Ames assay were applied. Results reported from the Ames assay confirmed mutagenicity responses of the tested pollutants except Cu and Ni. MMC was selected and introduced into soil samples as a case study to assess the performance of the developed tool-kit. Soils amended with MMC were extracted by the in vitro human digestion bioassay, and the mutagenicity of the bioaccessible fraction was measured using the Ames assay and the biosensors. A comparison was made between the permissible concentrations of MMC obtained from the developed tool-kit and RISC4 derived concentrations. The four microbial biosensors applied in this study were incapable to detect the mutagenicity of the tested pollutants. On the other hand, the Ames assay was more robust and sensitive to a broad range of environmental pollutants. The in vitro human digestion bioassay enabled the quantification of the human bioaccessible fraction of the tested pollutants. This fraction posed a concern due to its estimation of the doses that would reach the blood circulation and cause harm to human. While the permissible concentration of MMC measured by the developed tool-kit was less than 10 μg MMC/g, the RISC4 model calculated that it should be 40 μg MMC/g. This revealed that, in this situation, risk assessment model was less conservative than empirical study for human health risk assessment. This study enabled the assessment of the permissible concentrations of environmental pollutants that could remain in a soil and pose permissible harm to humans. This approach also enabled a comparison of modelled and empirical data to allow a measure of sensitivity to be judged. There is a need to develop bioassay techniques more able to assess the potency of hydrophobic compounds both in isolation and combination.