S.pneumoniae is the leading cause of bacterial pneumonia and meningitis. Pneumonia alone has been estimated to kill more children under the age of five than that caused by AIDS, malaria and tuberculosis combined. The current vaccines which are used to prevent pneumococcal infection only protect against a small number of the 90+ serotypes currently identified. Current issues which may prevent the long term use of these vaccines is capsular switching, a phenomenon observed where some strains are able to escape the vaccine through switching their capsule genes. Further serotype replacement has been shown to occur since the introduction of the PCV7 vaccine, where serotypes not protected against by the vaccine have caused a higher incidence of invasive pneumococcal disease compared to the pre vaccine era. One strategy to avoid this is via the use of a multi-component protein based vaccine which is serotype independent. The pneumococcus is normally found as a harmless commensal yet can also cause invasive disease as stated above, the pneumococcus is also the leading cause of otitis media. The ability for the pathogen to occupy a number of different niches and evade host defences is attributed to its large cache of virulence factors, including numerous cell surface adhesins. The ability of the bacteria to regulate genes required for adaptation to a specified niche is vital for survival. In this study a number of signalling systems that are able to modulate gene expression (specifically virulence factors) to facilitate adaptation to varying environmental conditions are assessed to determine the genes they regulate. Further key environmental signals are evaluated to determine the effect they have on regulation of important cell surface adhesins. The main systems used to modulate global expression changes are two-component signal transduction systems (TCS). 13 TCS and one orphan response regulator are encoded in the pneumococcal genome. Little information is available with regards to the importance of each system, whether each system regulates its own separate collection of genes and the extent to which cross regulation may occur between these systems. This study used whole genome expression analysis data obtained through microarray analysis of single and double TCS mutants to assess the potential cross regulation of two chosen systems. A number of the systems have also been shown to regulate the same islet, which encodes a pilus. Measuring expression of the islet itself enabled the role of the systems shown to regulate the islet to be assessed for potential interactions between the systems and whether a hierarchy exists. The pneumococcus is highly genetically variable due to its ability to become naturally competent, taking up DNA from the environment and recombining it into its genomic DNA to aid genetic variation and survival. The new era of whole genome sequencing has begun to shed light on just how variable this pathogen is. Although a number of TCS have been shown to regulate pilus expression, with the use of whole genome sequencing of two closely related strains (one contains reduced pili expression levels) a number of other factors have also been identified which have been shown to alter pilus expression, this includes a serine/ threonine protein kinase, pyruvate oxidase and lactate oxidase. Further the pneumococcus has been shown to respond to exogenously added hydrogen peroxide which increases pilus expression levels. Levels of hydrogen peroxide may act as a key environmental cue to signal to the bacterium that they are present in the nasopharynx and require increased levels of cell surface adhesins.

This study explores the handling and analyzing of big data in the field of bioinformatics. The focus has been on improving the analysis of public domain data for Affymetrix GeneChips which are a widely used technology for measuring gene expression. Methods to determine the bias in gene expression due to G-stacks associated with runs of guanine in probes have been explored via the use of a grid and various types of cloud computing. An attempt has been made to find the best way of storing and analyzing big data used in bioinformatics. A grid and various types of cloud computing have been employed. The experience gained in using a grid and different clouds has been reported. In the case of Windows Azure, a public cloud has been employed in a new way to demonstrate the use of the R statistical language for research in bioinformatics. This work has studied the G-stack bias in a broad range of GeneChip data from public repositories. A wide scale survey has been carried out to determine the extent of the Gstack bias in four different chips across three different species. The study commenced with the human GeneChip HG U133A. A second human GeneChip HG U133 Plus2 was then examined, followed by a plant chip, Arabidopsis thaliana, and then a bacterium chip, Pseudomonas aeruginosa. Comparisons have also been made between the use of widely recognised algorithms RMA and PLIER for the normalization stage of extracting gene expression from GeneChip data.

The phylogeny and proteins of chemolithoautotrophic growth of thermoacidophilic Sulfolobus-like archaea were investigated. Sulfolobus-like strain HT is a high growth temperature chemolithotrophic isolate with potential application in mineral sulphide bioleaching. The gene encoding its 16S rRNA was cloned and sequenced. Phylogenetic analysis of this sequence showed strain HT segregating within the genus Sulfolobus. It showed greater similarity to the sequence of Sulfolobus shihatae, a facultative chemolithotroph, than to the sequence of Sulfolobus acidocaldarius, an obligate heterotroph. Flanking regions of the 16S rRNA gene were also sequenced, showing secondary structure similarity to those of S. acidocaldarius, implying a similar excision and processing pathway. A protein of 330 kDa, consisting of 59 kDa and 19 kDa subunits, was overexpressed during CO2-limited autotrophic growth of Sulfolohus strain LM and had previously been shown to co-purify with ATP and acetyl-CoA dependent CO2 uptake. The 59 kDa subunit was partially purified and its N-terminal amino acid sequence obtained. The gene encoding this polypeptide was cloned and sequenced. An open reading frame likely to encode the 19 kDa subunit was adjacent to this gene, forming a possible operon. Homology searches revealed that the predicted amino acid sequence of the 59 kDa subunit was similar to those of ATP-dependent biotin carboxylase enzymes, predicted active site residues being conserved. Homology searches of the predicted amino acid sequence of the ORF likely to encode the 19 kDa subunit revealed similarity to biotin carboxyl carrier proteins, with a biotin binding motif being conserved. In Sulfolobus LM, a polypeptide of 27 kDa molecular weight was overexpressed during autotrophic growth on ferrous iron in comparison with autotrophic growth on tetrathionate. This polypeptide was partially purified and its N-terminal amino acid sequence obtained. After the cloning and sequencing of the gene encoding this protein by a co-worker, homology searches were carried out. It showed homology to the alkyl hydroperoxide reductase / thiol specific antioxidant (AbpCrrSA) family of proteins, members of which are thought to play a role in protection against oxidative stress. The predicted amino acid sequence was phylogenetically analysed, segregating within a group of sequences derived from eukaryotes and archaea, which possess one conserved cysteine residue, as opposed to a group consisting of eukaryotes and eubacteria, possessing two conserved cysteine residues. A membrane bound cytochrome showing a difference spectrum alpha absorbance peak at 572 nm had previously been found to be present only during ferrous iron oxidation in thermoacidophilic archaea. This novel cytochrome was partially purified from the membrane fraction of Sulfolohus strain LM autotrophically grown on ferrous iron. It was shown to retain haem staining activity after SDS treatment, thus allowing its identification as a polypeptide of approximately 66 kDa. A procedure which may allow the N-terminal sequencing of this protein and the initiation of its molecular biological study was identified.

The morphological and associated physiological aspects of the chemoorganotrophic and photoorganotrophic appendaged (prosthecate) bacteria were considered. Initially work centred on the enrichment, isolation and enumeration of the chemoorganotrophic species Ancalomicrobium,Caulobacter and IIyphomicrobium from freshwater sources which varied in eutrophication levels. Induction and repression of the multiappendaged form of an Ancalomicrobium isolate, induced by environmental stimuli, was indicated. Additionally the morphological diversity of IIyphomicrobium found to be expressed in response to carbon source variation (methanol to methylamine) brings into doubt the validity of generic classification on the grounds of morphology. Of the appendaged photoorganotrophic (Athiorhodaceae) species isolated, the growth, and reproduction of a Rhodomicrobium species was studied in detail. This microorganism was shown to be similar physiologically to Rh.vannielii (Duchow and Douglas, 1949) but differed in that it produced exospores in profusion during the stationary phase. The formation, germination and physiological characteristics of these resting cells were examined. Information from the growth and reproduction studies, correlated with ultrastructural work, was used to formulate a model for growth and replication applicable to the vegetative cell 'and exospore. The obligatory, sequential, differential events required for growth and replication of the Rhodomicrobium. swarm cell lead to exploitation of this system as a model for the study of differentiation. A selective synchronisation procedure was formulated and the resulting synchronised swarm cell population characterised morphologically and physiologically with respect to the differential cycle. Contrary to reports in the literature no extrachromosomal (plasmid) DNA could be detected In any of the appendaged, obligate life cycled genera. In addition, sub- division of these genera by reference to their mole percent G + C content was found to be of little value.

The pathogenic bacteria Streptococcus pneumoniae is one of the major causes of morbidity and mortality in humans in the world today. A Gram-positive facultative anaerobe, under natural conditions it exists as a commensal bacteria residing in the nasopharynx. Upon invasion of the body, S. pneumoniae can cause a number of diseases, which range in severity from acute otitis-media to pneumonia, septicaemia and meningitis. The main virulence factor of S. pneumoniae has been identified as the polysaccharide capsule, which coats the outside of the cell. S. pneumoniae can be categorised into 92 distinct serotypes based on capsular composition. Current available vaccines utilise a mixture of polysaccharides from the most prevalent capsular serotypes, or capsular polysaccharide conjugated to a carrier protein. Vaccine coverage is therefore serotype specific. Furthermore, vaccine efficiency is lower in those groups most at risk of infection; namely infants, the elderly and the immuno-compromised. As a result investigation is ongoing into a next generation of protein-based vaccine that can provide increased coverage and efficiency. A novel family of proteins termed Pneumococcal Histidine Triad (Pht) proteins were identified from a whole genome antigen-screen as potential candidates for inclusion in a novel protein-based pneumococcal vaccine. Animal models of infection have shown that immunisation with the Pht protein Pneumococcal Histidine Triad D (PhtD) confers protection against invasion of S. pneumoniae. PhtD was cloned, expressed, purified and subjected to crystallisation trials in an attempt to uncover the function of PhtD through determining the protein structure by macromolecular X-ray crystallography, which yielded some rudimentary crystallographic data. Biophysical analysis of PhtD using a variety of techniques including limited proteolysis and circular dichroism revealed that PhtD exclusively bound the divalent-cation Zn2+ and that Zn2+-binding induced a major conformational change in the protein structure, which proved to be a reversible process. A rationalised, targeted analysis of PhtD protein structure by limited proteolysis, Nuclear Magnetic Resonance (NMR), N-terminal sequencing and mass-spectrometry revealed localised, ordered regions of structure within the protein sequence that were highly stable. These identified protein fragments were subsequently cloned, expressed, purified and subjected to crystallisation trials. Due to their smaller and more ordered nature, it was postulated that these PhtD fragments may prove more readily crystallisable than the full-length molecule. Initial crystals have been obtained for these protein fragments and are being optimised to improve crystal size and quality. Evaluation of the PhtD proteolysis products by Western blotting with anti-PhtD antibody has revealed the dominant epitope for the PhtD protein, localised to a 15 kDa region in the C-terminal half of the PhtD protein sequence. This could be a major advancement in development of a protein based vaccine as only the 15 kDa epitope-containing region need be included in order to elicit an antibody reaction. Furthermore, the small size of the protein fragment is highly conducive to structural determination by a variety of methods. This 15 kDa epitope fragment has been cloned into an expression plasmid allowing recombinant protein expression in order to investigate further. Additionally, as training for handling of PhtD X-ray diffraction data, macromolecular X-ray crystallography was attempted with a variety of different proteins from Gram-positive bacteria. Two proteins -a novel cis-trans isomerase PpmA from S. pneumoniae, and the transketolase TktA from Lactobacillus salivarius- were successfully crystallised. Diffraction quality crystals of TktA were grown that produced X-ray diffraction to 2.3Å resolution. The structure of TktA was successfully determined using the molecular replacement method. Diffraction quality crystals of PpmA were grown that show X-ray diffraction to 2.5Å resolution, and optimisation of crystallisation conditions should yield better X-ray diffraction, allowing the structure of PpmA to be determined. The data pertaining to these proteins is also included as part of this thesis.

Spironucleus is an opportunistic protozoan parasite capable of causing devastating losses in the production of both ornamental and food fish. Control of infection outbreaks is problematic due to restrictions on the use of chemotherapeutics and rapid parasite transmission amongst fish. This PhD investigated the life cycle, biochemistry and chemotherapy of Spironucleus vortens. Direct transmission of S. vortens was found to be facilitated by the trophozoite form, information which may be applied in aquaculture to prevent infection outbreaks. No S. vortens cysts were observed in vitro or in vivo and trophozoites were able to survive for prolonged periods in the faeces of angelfish. This novel finding facilitated development of a non-invasive method to quantify the degree of intestinal colonization in the host, which was then applied to determine the efficacy of new and existing chemotherapeutics against S. vortens in vivo. Garlic-derived compounds were shown to be realistic alternatives to the current drug of choice, metronidazole, in the treatment of Spironucleosis in fish. Synergy between metronidazole and the garlic-derived compound, ajoene, was also observed in vitro and in vivo. The mode of action of metronidazole and garlic-derivatives involved disruption of S. vortens intracellular redox balance, a pivotal cellular process which ensures normal cellular function and survival. Further biochemical investigations into the antioxidant defence system (consisting of glutathione, thioredoxin and superoxide dismutase) as well as the carbohydrate and amino acid metabolism of S. vortens provided greater understanding of the success of this organism as a parasite. This is reflected in its ability to withstand fluctuations in O2 and nutrition during key pathogenic stages of its life cycle, including extra-intestinal systemic infection and transmission to a new host.

The microbiological characterisation as well as the application of microbiocides for the storage and protection of mineral dispersions is of ever-increasing interest for scientists and industrialists and includes many challenges for the mineral slurry producer and user. Increasing conversion from dry pigment handling to water-based dispersions is taking place over a wide range of production applications, for example, papermaking filler products and coating formulations in both the paper and paint industries. The requirements for the delivery of preserved slurried products begins from the moment the mineral is extracted or synthetically produced. The process conditions are as important regarding bacterial colonisation and control as the delivery and storage strategy of the end-product itself. This thesis attempts to give a detailed insight into the background issues and procedures needed to provide an environment of "good housekeeping", essential in optimising the microbiological control needed for preservation and acceptable application of the pigment in its end-use. On this base, the latest research on the bacterial strains, their identification, measurement and growth dynamics in real-time are presented, and new biocide strategies, applicability and constraints are discussed. Illustrations are given throughout of the sources of microbiological contamination likely to occur during production, storage and transportation. Based on the current knowledge being gained from combining active Research and Development and on the ground applications expertise, new possibilities for optimising microbiological quality control are described.

The protein FtsZ is a tubulin-like GTPase, which plays an essential role in prokaryotic cell division. In vivo it assembles into a dynamic ring (the Z-ring) at the future site of cell division on the inner surface of the cytoplasmatic membrane. The Z-ring then serves as a scaffold which recruits all other division proteins to form the cytokinetic machinery. The constriction of the ring facilitates the separation of two daughter cells. In vitro, FtsZ polymerizes in the presence of GTP to form single-stranded protofilaments. It is assumed that FtsZ association and assembly reactions studied in vitro will play an important role in understanding the mechanism of Z-ring assembly and disassembly in vivo. In this work, we therefore have studied the dynamics of FtsZ polymerization in vitro, especially the bundling induced with YgfE. YgfE, the putative Escherichia coli ZapA orthologue, is one of these division proteins recruited by FtsZ. It acts to enhance lateral associations between FtsZ fibres to form larger bundles. In this study, we have demonstrated that YgfE exists as a tetramer in solution at the concentrations reported in this study, and the bundling activity is exerted more efficiently on preformed FtsZ protofilaments. We also investigate the importance of the tetramerisation of YgfE on function. A number of mutant forms of ZapA were generated with the aim of disrupting the dimer:dimer interface. Using those mutants we show that tetramerisation is a requirement for both FtsZ bundling, and GTPase modulation activities. In addition, a novel technique, continuous channel flow linear dichroism (LD) spectroscopy, has been first developed in this work. LD is a simple spectroscopy technique for structural characterization of long biomacromolecules in solutions. Continuous channel flow Flow LD has overcome the limitation of Couette flow LD due to the time required to assemble and fill the cell.

The species within the genus Mycobacterium are commonly detected in a variety of environments including soil, water and dust. Many species within the group are capable of causing opportunistic diseases and are hypothesised to be responsible for the reduction in BCG efficacy in tropical countries. Consequently it is important to understand the diversity and biogeography of mycobacteria in the environment. Soil and water samples were collected from a total of 42 residential sites in 9 different climatic regions. To determine community composition, community DNA was extracted and amplicon pyrosequencing was employed to target the 16S rRNA gene of the Mycobacterium genus and slow-growing mycobacteria. Quantitative PCR was employed to quantify the total abundance of Mycobacterium species and specifically members of the M. tuberculosis complex. The study revealed a greater diversity of both fast-growing and slow-growing mycobacteria than previously reported. Prevalent species in soil were closely related to the fast growers M. neglectum, M. moriokaense and the slow growers M. malmoense and M. colombiense, in contrast to water had a high abundance of sequences related to the fast growers M. aurum sp. ATCC 23070, M. neoaurum and the slow-growers M. gordonae and M. colombiense. The abundance of the Mycobacterium genus ranged from 3.35 x 101 to 8.01 x108 gene copies per gram/ml. M. bovis was detected in six environmental samples using qPCR. Biogeographical analysis demonstrated the importance of elevation and temperature for the community composition of mycobacteria in soil. A nonlinear relationship was observed between elevation and the outcome variables Mycobacterium species richness, diversity and abundance with a peak midelevation. In contrast latitude was the primary factor to explain the composition and diversity of mycobacteria in water samples. To our knowledge this is the first time that the diversity and abundance of mycobacteria has been elucidated on a large geographical scale using pyrosequencing and multivariate analyses. Results indicate ample opportunity for human exposure to mycobacteria with potentially pathogenic species in soil and water substrates. These results have implications for the risk of infection and similar biogeographical surveys on a worldwide scale may provide improved correlations with BCG vaccine efficacy.

In order to investigate the occurrence of methanotrophs in the marine environment, representative organisms have been isolated from seawater. As traditional methods for isolating and cultivating methanotrophs were found to be unsuccessful when applied to the marine environment, new techniques for methanotroph isolation were developed. These were used to enrich for methanotrophs in several marine areas. Two bacteria isolated from marine areas have been extensively characterized. Both isolates appear to be typical of Type I methanotrophs and have an absolute requirement for sodium chloride. The nitrogen assimilation of the isolates was studied in detail. Both isolates appear to have unusual nitrogen assimilation pathways. A method for detecting methylotrophs and methanotrophs without cultivation has also been developed. The polymerase chain reaction has been adapted to amplify DNA sequences specific to methylotro~ls and methanotrophs. This has been used to detect amplify DNA sequences in DNA extracted from water samples from a range of fresh water and marine environments. The methods has been demonstrated to be specific for methylotrophic and methanotrophic DNA. DNA from non-methylotrophic bacteria was not amplified. Initial studies on the application of this method for enumeration has also been studied. An investigation into the occurrence of methanotrophs in the Southern Ocean was also carried out. Methanotrophic activity was examined in two areas of the Southem Ocean. This was compared with the methane concentration in the water and total bacterial activity.