Mycobacterium tuberculosis, the causative agent of the infectious disease tuberculosis, has a distinct lipid-rich cell wall. Several anti-TB drugs target cell wall biosynthetic pathways. A good understanding of its biosynthesis will provide helpful clues for the development of novel drug targets. A strategy based on random transposon (Tn) mutagenesis with two different screening criteria, altered colony morphology and mycobacteriophage resistance, was developed. Non-pathogenic Mycobacterium smegmatis and the fish pathogen Mycobacterium marinum were used for generating Tn-mutant libraries. From the colony morphology screen, two out of eight genes identified from M. smegmatis Tn-mutants and eleven out of twenty from M. marinum Tn-mutants with altered colony morphology were directly involved in cell wall synthesis. One mutant from each species was chosen for further study, the M. smegmatis 3D9 Tn-mutant with the only isocitrate dehydrogenase (icd) gene disrupted and the M. marinum 8G10 mutant with a Tn inserted into the promoter region of MMAR0978 with a deficiency in methoxymycolates production. Four mutants resistant to generalised transducing phage I3 were identified with a Tn insertion in a gene cluster involved in the biosynthesis of the cell wall associated glycopeptidolipids (GPLs), demonstrating the potential of using phage-resistant mutants for identifying cell wall biosynthetic genes.

The V1b vasopressin receptor (V1bR) is a receptor for a neurohypophysial hormone [arginine8] vasopressin (AVP). V1bR is a G-protein coupled receptor (GPCR) belonging to the Family A GPCR superfamily. The structures of seven α-helical transmembrane domains of this family members can be predicted based on the crystal structure of bovine rhodopsin (bRho) and human β2 adrenergic receptor (β2AR) obtained by X-ray crystallography. This study aimed to identify amino acid residues which participate in ligand binding of the V1bR by site-directed mutagenesis with the aid of molecular models of vasopressin receptors based on the crystal structure of bRho. The V1bR is a potential drug target in treating stress-related conditions such as depression, anxiety and post-traumatic stress disorders. Since it is the latest subtype identified among the mammalian neurohypophysial hormone receptors, it remains as the least studied subtype. A closely related subtype V1a receptor (V1aR) has been studied in far more detail for its potential of being a drug target in treating cardiac conditions and epilepsy. Hence, effective means of studying the V1bR can be accomplished by exploring the information already available on the V1aR and thereby defining the differences and similarities existing between the two. Detailed subtype comparisons are also fundamental for designing subtype selective drugs for effective therapy with fewer side-effects. This project was designed also to elucidate amino acid residues which determine selectivity of ligands for the V1bR over the V1aR. This study demonstrated that the charged residues Glu1.35 and Arg3.26, polar residues Gln2.61 and Tyr5.38, and cyclic residue Pro2.60 are crucial in AVP binding to the V1bR. These residues are all located at the extracellular facing surface of transmembrane domains (TMs). Also at the vicinity, charged residues Arg1.27 and Asp2.65, and aromatic Trp2.64 are shown to be important components of the AVP binding cavity. The study demonstrated that two residues Trp6.48 and Phe6.51 located at the TM6 are required for high affinity binding of non-peptide antagonists to the V1bR. The reciprocal mutagenesis between V1bR and V1aR residues revealed that Phe7.35 located at the exofacial surface of TM7 is a key residue required for a high affinity binding of a non-peptide antagonist with selectivity for the V1bR over V1aR. Also on the TM7, Met7.39 was shown to be involved in a high affinity binding of an antagonist with selectivity for the V1bR over V1aR. Two residues on the top of TM5, Leu5.39 and Thr5.42 were also shown to participate in V1bR-selective binding of non-peptide antagonists. From the perspectives of drug development, the variants of V1bR were studied in two streams: firstly to characterise pharmacological properties of single nucleotide polymorphism (SNP) variants of human V1bR; and secondly to determine differences in TM architectures between rat and human V1bRs. The study showed that a SNP variant of the V1bR with a residue substitution of Gly to Arg at position 191 is more readily expressed on the cell-surface in comparison to the wild-type. The variant was also found to generate InsP-InsP3 accumulation effectively in response to AVP stimulation. The mutagenesis involving introduction of specific rat V1bR residues into human V1bR revealed that there is a slight difference in the local environment of TM4 between the V1bRs found in the two species.

In Mycobacterium tuberculosis there are 2 distinct groEL homologues which encode the chaperonins Cpn60.1 and Cpn60.2, with the latter predicted to be the main house-keeping chaperonin. Phylogenetic analysis has revealed that the genes for the duplicated chaperonins diverged a long time ago. This implies that the duplicated chaperonins have evolved for different cellular functions. Interestingly, while most chaperonins occur as stable large complexes with a characteristic double ring structure of 14 subunits, the Mycobacterial chaperonins are very unstable and appear to form much smaller complexes. Given that the large structures formed by most chaperonins are vital to their mechanism of action, it is unclear why the oligomers are so much less stable in Mycobacteria. In this study I present detailed functional and oligomeric analysis of the M. tuberculosis chaperonins. Using various biological techniques, including complementation assays, site directed mutagenesis, and domain swap experiments; I have been able to demonstrate that both chaperonins have evolved to fulfil alternate functions within the cell. I have shown that while Cpn60.2 is fully functional in E. coli, Cpn60.1 is not. However, M. tuberculosis Cpn60.1 was able to function in M. smegmatis and complement for the loss of its endogenous chaperonin. Domain swap experiments between Cpn60.2 and E. coli GroEL have provided further evidence to support the hypothesis that they function in a similar manner. Using analytical ultracentrifugation on purified proteins, I have been able to show that Cpn60.2 and its chimeric protein do form larger oligomeric complexes in vitro under certain conditions and in the presence of ATP.

The main aim of the project was to study the effect of flow rate (shear stress), under different nutrient conditions on pre-attached bacterial biofilms. The combined effect of flow rate and nutrient supply had not previously been systematically studied, and the relationship between physiological activity, attachment and detachment, growth at a surface, and flow rate appeared to be very complex. Special experimental flow rigs were built for this project. Thin biofilms (monolayers of cells) were attached to test sections and these were placed in the flow rig. The biofilms were then subjected to different shear stresses at a known glucose concentration. The numbers of cells were small compared to the total volume of medium in the flow rig, and so the change in the substrate concentration during an experiment was insignificant. Radiotracers could therefore be used to measure substrate assimilation at various flow rates and substrate concentrations. It was hoped to produce a model which would explain the experimental data which was obtained, and assess how flow rate influences biofilm development and substrate uptake. However, the experimental results suggest that flow rate haslittle influence on glucose uptake by Pseudomonas fluorescens (the species used for this work), and that glucose uptake at various flow rates depends only upon the glucose concentration present in the medium. It is possible that the explanation for this lies in the complex mechanism of glucose catabolism which is reported for this species. A considerable part of this project was spent in establishing appropriate experimental techniques in order to make the required measurements for analysis of the biofilms in the flow rig.

Assimilation and respiration by a marine Pseudomonas sp. was evaluated to determine whether the activity of bacteria attached to solid surfaces (substrata) differed from that of free-living bacteria. Bacteria were allowed to attach to glass and plastic substrata with a range of wettabilities, evaluated by measuring water contact angles. Amino acid assimilation was determined by microautoradiography and liquid scintillation counting, and respiratory activity was determined from 14CO2. evolution and electron transport system (ETS) activity evaluated by tetrazolium staining. The uptake kinetics of leucine demonstrated thatfree-living cells had a smaller half-saturation constant than any of the surface-associated populations but a somewhat greater maximum velocity of uptake than the cells associated with all but the most hydrophobic substratum. Pre-attachment incubation of free-living bacteria with 3H-leucine resulted in the subsequent attachment to all the substrata of approximately a four fold higher percentage of labelled bacteria as compared with populations that remained unattached. When incubated with amino acids at a concentration of l0μg C l-1 after attachment, the proportion of attached bacteria that assimilated amino acids and the rate of assimilation per cell, was greater than, or similar to, that of free-living bacteria. However, the proportion of attached bacteria that demonstrated ETS activity and the rate of 14CO2 respired per surface-associated cell, was less than, or similar to, that of free-living bacteria. There was little relationship between substratum wettability and the activity of attached bacteria at low substrate concentrations (≤ 10μg C l-1) but in the presence of higher leucine concentrations the proportion of attached bacteria that assimilated leucine and demonstrated ETS activity increased with substratum hydrophilicity. The relative activities of attached and free-living bacteria depended upon the substrate, its concentration and the substratum properties. However, the efficiency of substrate utilisation by attached bacteria was generally greater than that for free-living bacteria.

Mouse and rat cells encode an abundant 30S RNA which shares many structural properties with retroviral genomic RNA. This VL30 RNA can be efficiently packaged into retrovirus particles. Mouse cells recently infected with a MuLV (VL30) pseudotype were shown to contain full length, reverse-transcribed DNA copies of both RNA species. VL30 DNA could also be synthesized in quantity using the endogenous reverse transcriptase activity of detergent-disrupted MuLV (VL30) particles. This DNA was found to be identical to that produced in vivo. Several 4.6-4.9kbp molecular clones (NVL clones) of VL30 cDNA were obtained. The retrovirus-like LTRs of each clone displayed a moderate restriction enzyme site heterogeneity, but NVL unique sequence was identical in each case. Southern blotting experiments using NVL probes showed that (a) most of the 100-200 NIH-3T3 DNA mouse VL30 elements were organized into provirus-like structures with a high degree of sequence conservation, and (b) the majority of these elements were hypermethylated and transcriptionally inactlve, whereas an expressed NVL-like sub-class could account for no more than 5% of mouse VL30 genes. NVL-related sequences in rodent DNAs other than the mouse were markedly less abundant and showed a greater sequence divergence. This was in contrast to MuLV-related sequences whose copy number and homology to a cloned MuLV probe decreased more gradually with phylogenetic distance from the mouse. Sub-genomic NVL probes showed that two rodent species had each conserved a different block of NVL-like sequence. These data indicate that each family has exhibited a different rate of sequence divergence during rodent evolution. Finally, a MuLV (VL30)-infected rat fibroblast line was shown to have received 1-2 copies per cell of a transcriptionally active NVL-like element. This suggests the possibility that evolution of each rodent VL30 family has been influenced by retrovirus-mediated transmission across the species barrier.

Production, purification and catalytic utility of lignin peroxidase (LiP) from Sporotrichum pulverulentum. The study of Lip has been hampered by the difficulty in producing this enzyme in sufficient quantities. Several strains of Phanerochaete chrvsosporium and Sporotrichum Pulverulentum were screened for LiP expression under different culture conditions to find a method of producing adequate supplies of the enzyme for the proceeding work in this thesis. A reliable method of Lip production was achieved using 750m1 agitated cultures of S. pulverulentum containing the detergent tween 80. Lip from S. pulverulentum was purified by HPLC and was found to consist of up to 14 isozymes which varied in molecular weight, pH optimum and specific activity for veratryl alcohol. However, their catalytic spectra were similar. The isozymes from S. pulverulentum had higher molecular weights and lower pI values than those published for LiP from P. chrvsosporium, which suggested that they were not as closely related as had been assumed. Lip from S. pulverulentum was able to oxidise a range of methoxy-substituted benzyl alcohols to their respective aldehydes. The susceptibility of benzyl alcohol oxidation by LiP depended upon the amount and position of methoxyl group substitution. LiP oxidation of these substrates was dependent upon how electron-rich the molecular π-orbitals of the substrates were, but steric effects may also have been important. LiP oxidation of benzyl alcohols under aerobic conditions led to additional products such as quinones, ring-cleavage products and chloro-substituted aromatics. These latter products provided evidence for the existence of LiP-derived aryl radical cations for a range of benzyl alcohol substrates, which is consistent with the peroxidative one-electron oxidation theory of Lip degradation of lignin. In addition LiP was shown to catalyse the peroxidative oneelectron oxidation of phenolics such as p-cresol and catechol to produce dimers and polymers. Lyophilised LiP was shown to be catalytically active in organic solvents such as ether and propyl acetate. An increase in enzyme stability of up to 30 times of that in water and a broadening of its catalytic spectrum was observed. Lip was also found in C. versicolor demonstrating that LiP may be a common constituent of ligninolytic white-rot fungi. In addition, other. extracellular peroxidases were present in this fungus. These peroxidases were novel compared to the extracellular peroxidases from P. chrysosporium since at least one of these could not oxidise veratryl alcohol and neither of these peroxidases were manganese-dependent.

Stirling & Dalton (1978) isolated a formaldehyde dehydrogenase (mFDH) from Methylococcus capsulatus (Bath) which required a small heat stable component for the catalysis of formaldehyde oxidation to occur. The mFDH was purified and shown to be a dimer of 57kDa subunits. The heat stable component was purified and shown to modify the function of FDH and was therefore termed a modifier protein (protein F) (Millet et. al., unpublished). In this study purification procedures for protein F and mFDH are described. The N-terminal sequence of purified mFDH and protein Fare described and data are presented which indicates that mFDH is a tetramer of 63 kDa subunits. It was known that protein F altered the function of mFDH from a specific FDH to a general aldehyde dehydrogenase. The data presented in this study demonstrate that only formaldehyde oxidation is catalysed in the presence of protein F and in its absence fonnaldehyde oxidation can not be detected. The data also show that the oxidation of a range of aldehydes and alcohols are catalysed by mFDH in the absence of protein F. The data demonstrate that formaldehyde association to mFDH is cooperative resulting in a sigmoidal plot for rate vs. formaldehyde concentration while acetaldehyde oxidation follows Michaelis-Menten kinetics. Data are also presented which demonstrate that protein F may induce a conformational change in mFDH which allows formaldehyde oxidation catalysis to occur and inhibits the oxidation of acetaldehyde. The data also show that protein F has limited effects on other dehydrogenase enzymes. An enzyme whose activity is altered by protein F was identified in a commercial sample of calf liver glucose dehydrogenase (GDH) and this was purified and shown not to be GDH. Also described is the characterisation of a second NAD+linked FDH (nFDH) isolated from Methylococcus capsulatus (Bath) which does not require a modifier protein. From the data presented it is proposed that the function of mFDH may be to aid in reducing toxic formaldehyde concentrations in the organism and the role of the nFDH could be the generation of NADH for methane and carbon assimilation.

Trypanosoma brucei is a protistan parasite of mammals that evades its host’s immune responses by antigenic variation, which in T. brucei involves the periodic switching of the Variant Surface Glycoprotein (VSG) coat to antigenically distinct variants. The T. brucei genome contains a huge archive of silent VSG genes that are expressed from specialised expression sites, only one of which is actively transcribed at any one time. Copying of silent VSG genes into the active expression site has been shown to occur by homologous recombination, as mutation of the RAD51 recombinase and a distantly related gene, RAD51-3, impairs this process. BRCA2 is a protein that binds and regulates the function of Rad51 during homologous recombination. Mutation of BRCA2 in bloodstream form T. brucei leads to increased sensitivity to DNA damaging agents, and impairments in homologous recombination, RAD51 subnuclear foci formation and VSG switching, suggesting that it too acts in recombination-repair and antigenic variation. Beyond these phenotypes, an accumulation of putative gross chromosomal rearrangements in the megabase chromosomes of the T. brucei genome and a novel replication phenotype were also observed, and the basis of both these processes was unclear. T. brucei BRCA2 is highly unusual relative to orthologues in other eukaryotes, as the protein contains an expansion in the number of RAD51-binding BRC repeat motifs, which are arranged in a tandem repeat array that has not been observed elsewhere. In order to examine the function of BRCA2 in the maintenance of genome stability in T. brucei, brca2 homozygous mutants were generated in procyclic form TREU 927 and Lister 427 cells. Analysis of genomic stability by Southern blotting and pulsed field agarose gel electrophoresis revealed that BRCA2’s function in the maintenance of genome stability appears to be either bloodstream form-specific, or plays a more substantial role in this life cycle stage. To examine the function of the BRC repeat expansion, cell lines containing variants of BRCA2 with reduced numbers of BRC repeats were generated, expressed in brca2 homozygous mutants and phenotype analysis carried out. Growth and DNA repair were restored by the expression of virtually all variants, suggesting the BRC repeat expansion is not an adaptation for general genome maintenance, though the repair activity of a variant with a single BRC repeat appeared to differ between bloodstream and procyclic form parasites. In contrast to this, a striking correlation between BRC repeat number and the regulation of RAD51 subnuclear dynamics was observed, showing that the BRC array expansion has important functional significance. GST pull-down analysis was used to examine the domains of T. brucei BRCA2 that interact with RAD51, revealing an extent of interaction not apparent in BRCA2 orthologues in other organisms. This complexity of interaction was further analysed by immunolocalisation of BRCA2 and RAD51, before and after DNA damage, which showed potentially dynamic co-localisation of the two repair factors. Finally, a putative interaction between T. brucei BRCA2 and CDC45 was tested both in vitro and in vivo, but could not be validated, suggesting it does not provide an explanation for the replication defects observed in bloodstream form brca2-/- mutant cells. All of the analyses above shed light on the function of the BRCA2 protein in the regulation of homologous recombination in T. brucei.

The stereotyped motor behaviour of the Drosophila larva provides a useful model for the development and function of neural circuitry. However, the cellular and molecular basis of this behaviour is poorly understood. Reported in this thesis is an analysis of slowmo (slmo), a gene previously shown to be expressed in the developing embryonic central nervous system of Drosophila. Null mutants of slmo are able to hatch, but the resulting larvae exhibit a progressive phenotype of defective locomotor activity. An enhancer trap, P(GAL4)c682, which is inserted in the slmo locus, reports expression in a subset of neurons within the embryonic, larval and adult central nervous systems. Inactivation of marked neurons with tetanus toxin light chain leads to severely impaired motor function, but not total paralysis. Affected embryos are capable of some sporadic movements, but are unable to hatch and normal peristaltic contraction waves are largely absent. The slmo gene is shown to encode a member of a novel family of conserved proteins of unknown function. GFP fusions of Slmo are shown to localise to the mitochondria in a cultured cell line. Two novel slmo related genes, termed pre/i/ (pre!) and real-time (retm), are identified in Drosophila. The prel gene is expressed ubiquitously during embryonic development, and disruption of the gene by a P-insertion results in lethality during larval development. retm encodes a member of a novel subclass of larger Slmo related proteins which contain the conserved CRAL- TRIO domain thought to be involved in the transport of small hydrophobic ligands. GFP fusions of both Prel and Retm are also associated with the mitochondria in cell culture, suggesting this might be true all proteins of this family. Using a yeast-2-hybrid approach, the identification of candidate Slmo interacting proteins is described, providing a basis for future work on the function of Slmo.