Saccharomyces cerevisiae cells respond to nutrients in their environment by altering their metabolic and transcriptional state in order to optimise the use of available nutrients and decide which of the several developmental pathways to pursue. In the yeast S. cerevisiae, meiosis and pseudohyphal growth are two major differentiation outcomes in response to nitrogen starvation. A central component of unfolded protein response pathway, the bZIP transcription factor Hac1ip, negatively regulates meiosis and pseudohyphal growth. The present study investigates this negative regulatory mechanism at early meiotic genes by Hac1ip in nitrogen-rich conditions. Regulation of transcription by Ume6p transcriptional regulator, Rpd3p-Sin3p histone deacetylase complex and Isw2p-Itc1p chromatin remodelling complex at URS1 was also investigated here. We also tested for induction of pseudohyphal growth in diploids from SK1 genetic background in response to nitrogen starvation conditions known to induce meiosis. I constructed destabilised β-galactosidase reporters expressed from URS1- CYC1-Ub-X-lacZ reporters to analyze transcriptional activity at URS1 site of early meiotic genes in nutrient rich conditions. The data presented here successfully demonstrates Hac1ip-mediated repression at URS1 sites in nitrogen-rich conditions. URS1-CYC1-Ub-X-lacZ reporters were expressed in mitotic repression machinery mutants (ume6Δ, rpd3Δ, sin3Δ, isw2Δ and itc1Δ) under nitrogen rich conditions. The data presented here from these experiments not only corroborates their known role in repression at URS1 but also suggested regulation at additional sites in the minimal CYC1 promoter. Deletion of Sin3p suggested independent repression function separable from Rpd3p. Isw2p also acts independently of Itc1p at sites other than URS1. We also show that pseudohyphal growth was stimulated by non-fermentable carbon sources in sporulation efficient SK1 genetic background. The data also indicates that stimulation of pseudohyphal growth by non-fermentable carbon sources does not require respiration function or functional mitochondrial RTG pathway.

Forkhead-associated (FHA) domains have gained considerable prominence as ubiquitous phosphothreonine-dependent binding modules; however, their precise roles in Ser/Thr kinase pathways and mechanisms of regulation remain unclear. From experiments with Rv1827, an FHA domain–containing protein from Mycobacterium tuberculosis, a complete molecular description of an FHA-mediated Ser/Thr protein kinase signalling process is derived. First, binding of the FHA domain to each of three metabolic enzyme complexes regulates their catalytic activities but does not require priming phosphorylation. However, phosphorylation of a threonine residue within a conserved N-terminal motif of Rv1827 triggers its intramolecular association with the FHA domain of Rv1827, thus blocking its interactions with each of the three enzymes. The nuclear magnetic resonance structure of this inactivated form and further mutagenic studies show how a novel intramolecular phospho-switch blocks the access of the target enzymes to a common FHA interaction surface and how this shared surface accommodates three functionally related, but structurally diverse, binding partners. Thus a remarkable and unsuspected versatility in the FHA domain that allows for the transformation of multiple kinase inputs into various downstream regulatory signals has been revealed.

Mesophilic Aeromonas are opportunistic human pathogens which produce a wide range of virulence factors and have been isolated from both untreated and chlorinated drinking waters. The presence of these microorganisms in the distribution systems suggests that Aeromonas could display an adaptive response to oxidant present during water treatment. This adaptive response of Aeromonas would lead to interference in analysis for faecal coliforms used to determine the quality of potable drinking water, and be a potential source of intestinal disorders. The Colilert defmed substrate technology system was developed as a one-step detection of both coliforms and E. coli while suppressing non-coliform heterotrophic growth. Aeromonas species were previously shown to cause production of falsepositive reaction at high cell densities (Edberg et ai., 1988). Similar results were obtained in our study when using fresh Colilert reagents. However, results obtained during this project showed Aeromonas to mediate false-positive reactions at low cell densities (101 cells/ml in presence of salt, 102 cells/ml in absence of salt) when using Colilert reagents within 4 weeks prior to shelf-life expiry. Increased incidence in falsepositive reactions mediated by Aeromonas were shown to be dependent upon the stability of the Colilert reagent affected with age. Such Aeromonas interference would lead to over-estimation of coliforms and E. coli in potable drinking water supplies. The ability of bacteria to adapt to a wide range of stress factors such as pH, heat shock, oxidants or starvation has been extensively studied. Little is known about the response of Aeromonas to such stress conditions. During this project, it has been demonstrated that mesophilic Aeromonas display an adaptive tolerance response to a lethal oxidative challenge through pre-treatment with a sub-lethal dose of oxidant. The stress adaptation process was demonstrated to occur through synthesis of stress proteins and modulation of pre-existing catalase. Of the species studied, A. sobria was most sensitive, whereas A. caviae and A. hydrophila displayed similar responses to oxidative stress. The hypersensitivity of A. sobria did not impair the adaptive response of the organism. During our investigations, stationary phase Aeromonas cells have been shown to be more resistant than their logarithmic counterpart and suggested that excreted molecules may playa role in protecting the cells. Re-suspension of fresh cells into spent medium from a stationary phase cells revealed a higher resistance of these cells compared to those re-suspended in minimal medium. This resistance was demonstrated to be mediated by non-proteinaceous, thermo-sensitive effector molecule. A potential candidate as the effector molecule, butyryl homo serine lactone, was synthesised and assayed. Preliminary data strongly suggest that this molecule has a role to play in the stress adaptation phenomenon and might be involved in stimulating synthesis of key stress proteins.

Pneumolysin is a virulence factor produced by the human pathogen Streptococcus pneumoniae. It acts in disease to damage cell membranes via pore formation and to activate the complement system directly. Pore formation is accompanied by the transition of the protein from an aqueous, monomeric conformation to a lipid-inserted, ring-shaped oligomeric state. This thesis describes the behaviour of pneumolysin in solution, investigations concerning its mechanism of pore formation, and the structure of an oligomeric form of the toxin. Pneumolysin self-interacts in solution through one of its four domains. The self-interaction leads to the formation of dimeric toxin. Consequent upon dimerization, pneumolysin oligomerizes in solution into structures apparently the same as those associated with pore formation in membranes. In addition it forms helical oligomers. Small-angle neutron scattering (SANS) suggests the existence of inter-domain flexibility in pneumolysin. The kinetics of pore formation by pneumolysin are dependent on binding and oligomerization of the toxin. SANS allows the observation of a model membrane under attack by pneumolysin, indicating changes in bilayer structure. The structure of a helical pneumolysin oligomer is described determined by electron cryomicroscopy. This thesis demonstrates that oligomerization is an innate property of pneumolysin, although it was previously thought that the monomer-oligomer transition required interaction between toxin and cholesterol. It furthermore describes a novel approach to observing the interaction between a protein and a membrane in seeking to understand the biochemistry of this important and widespread process. The determination of an oligomeric structure for pneumolysin indicates the orientation of the toxin subunit in the pore for the first time, which is very different from that previously proposed. It is also possible to understand on the basis of the oligomeric structure the relationship between self-association and pore formation by pneumolysin and related toxins.

This work reports detailed structural and mechanistic characterisation of three cytochrome P450 (CYP) enzymes from diverse mycobacteria. For Mycobacterium tuberculosis (Mtb) CYP121, a high resolution atomic structure revealed mutagenesis targets to explore structure/function relationships. Several mutants were generated and structurally characterised. The R386L variant disrupted heme coordination environment, producing an enzyme with shifted Soret absorption features and predominantly high-spin ferric heme iron. EPR and resonance Raman spectra confirmed the spin-state conversion. The P346L variant resulted in altered heme conformation due to perturbed interactions with the heme pyrrole D ring. Alterations to heme ligation state were observed from EPR studies. Ligand binding and thermodynamic studies were done on CYP121 mutants. Studies of the action of various azole based drugs (which bind tightly to CYP121 heme iron) showed that azoles had potent inhibitory effects on Mtb growth. Mycobacterium ulcerans, the aetiological agent of Buruli ulcer, produces a human toxin (mycolactone) and encodes CYP140A2 on a plasmid, with the CYP140A2 gene adjacent to other mycolactone biosynthetic genes. Purified oxidised CYP140A2 has a Soret absorption band at 418 nm and α/β bands at 567/534 nm, respectively. The Fe(II)CO complex conforms to typical P450 properties, with a characteristic Soret shift to 449 nm. Resonance Raman and EPR spectra confirm the protein to be low-spin and with a typical cysteinate- and water-ligated b-type heme iron. Azole drugs were shown to bind CYP140A2 tightly, and to be potential therapeutics. CYP140A2 catalysed hydroxylation of mycolactone precursor molecules, consistent with its proposed role in synthesis of the toxic polyketide. Mycobacterium sp HE5 CYP151A2 was shown to have a role in metabolism of secondary amines, such as morpholine. CYP151A2 was expressed and purified, and spectroscopic analyses were consistent with cysteinate- and aqua-ligated heme iron. Dithionite reduction of CYP151A2 induced non-standard spectral changes, with a blue-shifted Soret band indicative of a cysteine thiolate-to-thiol switch in heme ligation. Thiolate coordination was restored on oxidation of CYP151A2, indicating that the thiol is readily deprotonated to thiolate in the ferric form. An 419 of 186 mM-1 cm-1 was determined for oxidised CYP151A2 and (at high concentration) morpholine substrate was shown to coordinate heme iron as an inhibitor at high concentrations.

Campylobacter jejuni is the leading cause of gastrointestinal disease in the developed world. Chemotactic motility is a pre-requisite for intestinal colonisation by C. jejuni. In silico analysis of the C. jejuni NCTC 11168 genome identified homologues of 10 chemotaxis receptor and two aerotaxis genes. Six of the ten putative Transducer-like proteins (Tlp 1, 2, 3, 4, 7 and 10) resemble chemoreceptors of Escherichia coli. The aim of this project is to characterise the C. jejuni Tlp1-4 chemoreceptors. The genes encoding the Tlps were inactivated using an insertional inactivation strategy. Isogenic mutants were made in tlp1, tlp2 and tlp4; a final mutant in tlp3 could not be obtained. A tlp1 complement was also constructed in this work. The tlp1 mutant showed reduced chicken colonisation ability when tested by our collaborators. Chemotactic phenotypes of the tlp mutants were determined in the swarm assay; the tlp mutants appeared defective for chemotaxis when compared with the wild-type and non-motile flaAB mutant. The Capillary assay and Hard-Agar Plug (HAP) assay were developed as methods to ascertain the ligand specificities of the Tlp chemoreceptors under study. Unfortunately, the Capillary assay proved to be insufficiently reproducible for effective use with C. jejuni. The HAP procedure was optimised using a C. jejuni wild-type motile variant. Positive chemoattractant responses were observed in NCTC 11168 for the first time towards a range of chemicals. Data derived from the modified HAP assay indicated that Tlp1 may be the receptor for serine. Chemotactic responses could not be detected in the tlp2 and tlp4 mutants in the HAP assay. The signalling domain of Tlp1 was purified using a polyhistidine tag and used to produce a polyclonal antibody. The Tlp1 primary antibody and immunofluorescence labeling has shown for the first time that the Tlps cluster at the cell poles in C. jejuni.

1. The fermentation of pasture grass at different stages of maturity has been studied during two seasons by the use of an in vitro technique. A variation in both total volatile fatty acid production and in the distribution of individual acids has been noted. Acid formation is highest at the stage of maximum leafy growth of the grass and declines with advancing maturity of the sward. Acetic acid tends to be the main end product of fermentation from grass during its early growth period, but propionic acid is formed in increasing amount as the percentage of fibre in the grass rises. The use of pure cellulose as an index of the relative activities of rumen inocula has been discussed. 2. Samples of dried and stored grasses prepared from the corresponding fresh material have, on fermentation, been shown to give rise to an altered distribution of acids. Acetic acid is formed in greatest quantity by all samples of the dried grass studied. This difference in fermentation behaviour has been shown to be related to changes, possibly in chemical composition of the soluble carbohydrate fraction, occurring during the drying process. Little further change is observed on storage. 3. The chemical composition of the dried material derived from fresh grass cut at different stages of growth has been determined, and the relationship of levels of soluble carbohydrate, fibre and protein to volatile fatty acid production discussed. Acid formation is considered to be a function, not of any major single component, but of the relative proportions and availabilities of all the constituent fractions. 4. An attempt has been made to examine the mode of formation of the individual volatile fatty acids by fractionating the dried grass. Partition on a water solubility basis indicates that the grass extract and the extracted residue each produce characteristic proportions of individual acids, and an approximate relationship between the fermentation products of whole grass and those of its simple separates has been shown to exist. It has not, however, proved possible to establish such a connection between the fermentation of isolated "pure" compounds and the fermentation of the whole grass. Cellulose has been found to react in a similar manner to the fibre fraction of grass, but fermentation of a grass extract containing water soluble carbohydrate gives rise to an acid distribution very different to that obtained from hexose sugars or from fructosan. 5. Factors determining the nature of breakdown of pasture grass and its constituents by rumen microorganisms have been discussed, and possible applications of in vitro techniques to problems of ruminant nutrition suggested.

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