This phenylalanine located near to the catalytic site of E2 has been found to be responsible for substrate specificity and its substitution could be directly responsible for decreased enzymatic activity in this case. No major structural changes were observed in this mutant core. In summary, our recombinant PDC model has proved to be of considerable benefit in enabling us to gain a more informed insight into the molecular mechanisms of pathogenesis underlying these rare E2-linked mutations, particularly in the case of the E35-E2 mutant. In the absence of the recombinant model, such detailed investigations would have proved impossible owing to the lack of access to human tissue from individual patients. As a corollary to the main aim of the thesis, a preliminary attempt was made to create an equivalent recombinant OGDC model. OGDC is also a mitochondrial assembly that is involved in the TCA cycle and is increasingly implicated in the aetiology of various neurodegenerative diseases linked to oxidative stress including Alzheimer’s and Parkinson’s disease. The basic organisation of the OGDC is directed by the self-assembly of 24 copies of dihydrolipoyl succinyltransferase (E2o) to form a cubic core, to which multiple copies of 2-oxoglutarate dehydrogenase (E1o) and dihydrolipoamide dehydrogenase (E3) bind non-covalently. The mammalian E2o is unusual in lacking any obvious E3 or E1o binding domain. In this study, E2o and E3 were successfully overexpressed and purified. Initially it was confirmed that E2o and E3 do not interact with each other on gel filtration although stable association of all 3 constituent enzymes occurs in the native complex. Full-length E1o was also cloned successfully although it proved impossible to achieve detectable expression in our E. coli BL21 host system. Previous studies employing subunit-specific proteolysis have identified the extreme N-terminal segment of E1o as a key region involved in the maintenance of complex stability and integrity and is required for E2 and E3 binding. To investigate this region in more detail, three N-terminal E1o fragments of decreasing size were overexpressed, one in His-tag form (193 amino acids) and two as E1o-GST fusion proteins (166 and 83 amino acids). In co-expression, purification and gel filtration studies, it was found that all these N-terminal truncates of E1o appeared capable of interacting with E2o although problems were encountered with rapid degradation and unambiguous identification in some cases. However, Western blotting revealed conclusively that even the shortest N-terminal E1o fragment (83 amino acids) was able to enter into a stable association with E2o. Owing to time constraints and difficulties with rapid degradation and/or solubility of the E1o truncates, it remains to be determined whether this N-terminal region of E1o can also interact with E2o in a post-translational fashion and whether it is directly involved in mediating E3 binding. However, this type of approach should continue to provide additional insights in the unique subunit organisation of OGDC and is an important step towards creating a recombinant model of OGDC. This will be invaluable for future studies on an important metabolic assembly that has been increasingly implicated in disorders linked to oxidative stress and neurodegeneration.

Streptococcus pneumoniae is a genetically diverse organism that varies substantially in its genomic content from one strain to another. Current therapeutic strategies in the management of pneumococcal disease include treatment with antibiotics and prevention by vaccination. However, due to the highly competent nature of the bacterium the prevalence of antibiotic resistance and vaccine escape is increasing. The pneumococcus causes a wide range of diseases, and this can be attributed to both the succeptibility of the human host and the genetic background of the infecting strain. The study of the contribution of variations in the genome of S.pneumoniae is clearly important in understanding the behaviour of this organism, and managing the burden of disease relating to this organism. S.pneumoniae strains are able to acquire DNA from other strains, and also from other closely related species, who occupy the same niche in the human host. One region of genomic diversity in the pneumococcus encodes a large serine rich repeat protein, glycoysltransferases and secretion proteins, some of which are homologous to the Sec secretion pathway. Similar loci have been characterised and found to be important in the virulence of other gram positive bacteria, including S.gordonii and S.parasanguinis. The presence of this locus was investigated in a diverse population of pneumococcal isolates, and shown to be present in a wide variety of isolates. The RNA of genes in the locus was found to be expressed. Expression of the SRR protein, encoded by SP1772, was investigated; a role in biofilm formation was identified utilising an isogenic mutant in SP1772 of TIGR4. In addition, the gene encoding the SRR was found to be able to recombine within a single strain of S.pneumoniae, suggesting this region of the genome is not only variable in its presence in the pneumococcal population but also able to adapt to the environment it is in.

This study set out to analyse C. pseudotuberculosis within an environment relevant to that which would be encountered within its natural host. The impact of the availability of iron within the growth environment of numerous bacteria has been widely reported, and an equivalent investigation was conducted to determine whether the same was true of C. pseudotuberculosis. To this end, a novel chemically-defined medium was designed, which supported the growth of C. pseudotuberculosis, but in which the concentration of specific growth factors could be manipulated. Subsequently, iron was shown to be essential for C. pseudotuberculosis growth, and analysis of secreted protein profiles revealed differential expression between low- and high-iron growth conditions. Furthermore, growth experiments conducted in the defined medium revealed that C. pseudotuberculosis is capable of obtaining iron from the host iron-binding proteins, transferrin and lactoferrin. The results presented in this thesis confirm the importance of iron to C. pseudotuberculosis, and reveal the existence of an iron-dependent regulator which is involved in regulating the expression of multiple target genes.

Several species of bacteria exhibit multicellular behaviour, with individuals cells cooperatively working together within a colony. Often this has communal benefit since multiple cells acting in unison can accomplish far more than an individual cell can and the rewards can be shared by many cells. Myxobacteria are one of the most complex of the multicellular bacteria, exhibiting a number of different spatial phenotypes. Colonies engage in multiple emergent behaviours in response to starvation culminating in the formation of massive, multicellular fruiting bodies. In this thesis, experimental work and theoretical modelling are used to investigate emergent behaviour in myxobacteria. Computational models were created using FABCell, an open source software modelling tool developed as part of the research to facilitate modelling large biological systems. The research described here provides novel insights into emergent behaviour and suggests potential mechanisms for allowing myxobacterial cells to go from a vegetative state into a fruiting body. A differential equation model of the Frz signalling pathway, a key component in the regulation of cell motility, is developed. This is combined with a three-dimensional model describing the physical characteristics of cells using Monte Carlo methods, which allows thousands of cells to be simulated. The unified model explains how cells can ripple, stream, aggregate and form fruiting bodies. Importantly, the model copes with the transition between stages showing it is possible for the important myxobacteria control systems to adapt and display multiple behaviours.

Landfills are a major anthropogenic source of methane and understanding the factors influencing the activity and diversity of methane oxidizing bacteria (methanotrophs) in landfill cover soil is critical to devise better landfill cover soil management strategies. A detailed study was carried out to investigate the effect of earthworms on soil methane oxidation potential and community structure of active methanotrophs in a landfill cover soil. Earthworms were found increase soil methane oxidation potential by 15% ± 7%. However, no substantial shifts in the community structure of active methanotrophs were observed. A Bacteroidetes-related bacterium was identified only in active bacterial community of earthworm-incubated landfill cover soil. However, its role in methane cycling is uncertain. In a subsequent study, a larger experimental system was used to simulate in situ landfill conditions and also to mimic the in situ environmental heterogeneity. A mRNA-based microarray analysis revealed that earthworm activity in landfill cover soil stimulates activity and diversity of Type I methanotrophs compared to Type II methanotrophs. Understanding spatio-temporal distribution pattern of microorganisms and the factors influencing their distribution pattern are integral for a better understanding of microbial functions in ecosystems. A pmoA-based microarray analysis of methanotroph community structure in a landfill cover soil revealed a temporal shift in methanotroph populations across different seasons. In the case of spatial distribution, only minor differences in methanotroph community structure were observed with no recognizable patterns. Correlation analysis between soil abiotic parameters (total C, N, NH4 +, NO3 - and water content) and distribution of methanotrophs revealed a lack of conclusive evidence for any distinct correlation pattern between measured abiotic parameters and methanotroph community structure, suggesting that complex interactions of several physic-chemical parameters shape methanotroph diversity and activity in landfill cover soils. A study was designed to investigate the shift in functional diversity of methanotrophs when microniches created by soil aggregates are physically altered. mRNA-based analysis of the bacterial transcription activity revealed an effect of physical disruption on active methanotrophs. The result emphasized that a change in a particular microbial niche need not be accompanied by an immediate change to the bacterial functional diversity and it depends on the ability of the bacterial communities to respond to the perturbation and perform the ecosystem function. DNA-SIP and mRNA based microarray techniques were compared for the assessment of active methanotroph community structure. Results from this study indicated that assessment of active methanotroph community structure by both the techniques were congruent. This suggested that the mRNA based microarray technique could be used to study active methanotroph community structure in situations where SIP experiments are not practical. However, both DNA-SIP and mRNA-microarray have their advantages and limitations and the selection of appropriate technique to assess active community structure depends on the nature of the study.

Methylocella spp. are facultative methanotrophs, able to grow not only on one carbon molecules e.g., methane or methanol, but also on multi-carbon compounds e.g., acetate, succinate and malate. PCR-based molecular techniques were developed and validated to target the 16S rRNA genes of all the species of the genus Methylocella present in environmental samples. DNA extracted from a variety of environmental samples screened by PCR to determine the environmental distribution of Methylocella spp. Methylocella were found to be widely distributed, not only in acidic environments, but also in neutral and alkaline environments. New primers targeting the mmoX gene of Methylocella spp. that encoded the α-subunit of methane monooxygenases were designed. A SYBR® green-based real-time quantitative PCR assay was developed and validated using these Methylocella mmoX gene-targeting primers. The abundance of Methylocella spp. present in selected environmental samples was quantified using the newly developed real-time quantitative PCR assay A series of 13CH4 DNA-SIP experiments were carried either in the presence or absence of 12C acetate in microcosms containing Moor House peat soil to investigate the effect of acetate (0.5 mM) on the ability of Methylocella silvestris to oxidize methane. Methane oxidation data indicated that acetate repressed the ability of Methylocella silvestris to oxidize methane in peat soil microcosms. 13CH4 DNA-SIP experiments revealed that in presence of acetate, Methylocella silvestris did not utilize methane as a carbon source, suggesting that acetate might be the alternative source of carbon. However, when 13C-labelled acetate DNA-SIP experiments were carried out, Methylocella spp. were not found to be dominant acetate utilizers in the peat soil microcosms. Methylocella spp. seem to have been outcompeted by more efficient acetate utilizers such as Brevundimonas and Burkholderia. To identify genes that might be involved in the utilization of methane or acetate, Methylocella silvestris whole genome transcriptomics experiments were carried out. All the genes of the sMMO gene cluster were found to be highly upregulated during growth on methane. In addition to the sMMO gene cluster, a gene encoding Fur was also found to be highly upregulated during growth on methane. During growth on acetate, a gene cluster encoding glycine dehydrogenase was found to be highly upregulated. Microarray experiments carried out here provided potential candidate genes for further characterization by gene knockout based studies. Further work is also required to validate the microarray findings. A study was carried out on forest soils derived from Swedish islands that were at different successional stages. All islands were found to be positive for the presence of Methylocella spp., and identity of composition of other methanotrophs were determined using a pmoA-diagnostic microarray. All the islands were dominated by Methylococcus, Methylosinus and methanotrophs of the uncultivated RA14 clade. The diversity of methanotrophs was higher in late successional islands compared to mid and early successional islands. In addition the diversity of methanotrophs decreased as the soil depth increased.

The vast increase in the number of protein structures identified since the first high resolution protein structure was determined has shown that there are relatively few protein folds. The study of the folding of proteins has also expanded significantly since its inception. The contact-order of residues in the native structure has been implicated as important in folding. This has raised the question of whether the common folds observed in protein structures fold via common mechanisms. The protein kinase domain is a large, pharmaceutically important and conserved protein fold, of which many examples fail to fold correctly when overexpressed as recombinant proteins in Escherichia coli. The kinase domain forms an excellent area in which to study the folding of a large conserved domain with varying sequence. To study the refolding of the kinase fold a refolding screen was created, using p38α as a model protein kinase. The results of this screen were compared to the results of refolding a further four protein kinases, AKT2, KIS, PhK and TTK to determine commonalities in the folding of protein kinases. The refolding of the five protein kinases was also examined using a fractional factorial screen which examined combinations of refolding additives. In the screening of the refolding of protein kinases no factors were idenified which were common to the refolding of all five of the tested protein kinases. The equilibrium folding of a single protein kinase, TTK, was also studied. The folding of TTK was determined to proceed via different pathways on folding and unfolding, with a co-operative unfolding pathway through a molten-globule intermediate, and a non-cooperative refolding pathway via an intermediate with different secondary structure content to the unfolding intermediate. The difference between the folding properties of protein kinases determined in the screen and through the analysis of the equilibrium folding of TTK suggest that there may not be a common protein kinase folding mechanism.

Carotenoids are produced by all photosynthetic organisms and a large number of bacteria and fungi. They are responsible for a lot of pigmentation in nature, as well as often playing an essential role in the provision of light protection to cells and as precursors of vitamin A in higher organisms. Myxobacton is the primary carotenoid ester generated in the photoresistant bacterium Myxococcus xanthus. It is created through a complex light-regulated gene expression cascade and acts to protect the bacteria from blue light and the resultant generation of damaging singlet oxygen species in the presence of porphyrins. The final stage in its production is a ketonisation, and the enzyme responsible for this stage was unknown in M. xanthus. We propose a possible location for the gene encoding such a ketolase, crtW. The gene is found located within a four-gene operon separate from the other known carotenoid biosynthetic genes, and appears to have two alternative promoter regions. The additional genes in the operon were found to encode a putative MutT/Nudix family hydrolase and a periplasmic, molybdopterin and haeme-dependent oxidoreductase, YedYZ. It is also shown that crtW transcription is independent of cell exposure to blue light and that the product is an inner membrane, integral membrane protein probable ketolase. The evolutionary origins of crtW are considered in conjunction with a number of other carotenoid biosynthetic enzymes, suggesting that the gene was one of the last to be acquired by M. xanthus.

Microbial ecology is the younger sub-discipline of ecology, but its scope is clearly immense. Microorganisms’ specificities represent a major hurdle to the application of traditional ecological approaches. Recently, the opportunity to apply ecological principles to microorganisms was suggested. It is assumed that existing principles might apply, but that they are probably also driven by specific principles. Microorganisms have varying metabolic abilities and are classified as generalists or specialists. We hypothesised that contrasting metabolic properties may drive genotypic distribution. Model generalist and specialist genera were selected and distribution patterns along the salinity gradient of the River Colne estuary studied. Two models were studied in detail, Desulfobulbus a versatile sulphate-reducing bacterial (SRB) genus and Methanosaeta, a strict aceticlastic methanogenic archaea (MA). Isolation of Methanosaeta was attempted in order to link phenotypes to genotypic distribution. Two other models were also studied: Methanosarcina the most versatile MA genus and Desulfobacter a metabolically restricted SRB genus. Denaturing gel gradient electrophoresis (DGGE) and clone libraries analyses were used to determine genotypic distribution patterns. Methanosaeta have proven to be highly recalcitrant to isolation. Adjustments to commonly used anaerobic culturing methods allowed the obtention of Methanosaeta colonies. In contrast to previous studies, colonies were successfully transferred into liquid medium, and growth of pure clonal cultures confirmed. Desulfobulbus genotypic distribution pattern was previously shown to be sequential along the estuary. Contrastingly, Methanosaeta genotypic distribution pattern was found to be monotonic. Furthermore, active genotypes distribution was also found monotonic, with an apparent general increase in activity with decreasing salinity. Distribution patterns of the four different genera confirmed this trend. The generalists were both shown to have sequential distribution patterns. Contrastingly, the specialists were both shown to have monotonic distribution patterns. These results confirm the hypothesis that genotypic distribution patterns microbial communities structure are strongly driven by microorganisms’ metabolic properties and adaptative potential.

Genomic libraries of S. lavendulae and S. subrutilus were constructed in S. lividans using the technique of shotgun cloning. S. lividans is a genetically well characterised recipient for heterologous DNA, but plasmid deletions or the entry of plasmids with small inserts occurred during transformation of the DNA into S. lividans This was not due to a straightforward restriction-modif ication system as this possibility was checkea using the KC301 phage. Several gene libraries were produced, using high and low copy number plasmids, and the resultant transformants screened. The nature of deoxynojirimycin (DNJ) and its lack of microbial activity prevented use of a bioassay. However, the inhibition of cc-glucosidase by DNJ was exploited by development of a quantitative assay system f or DNJ and nojirimycin (NOJ). The assay could not only detect DNJ and NOJ-producing clones, but could also assess the titre of DNJ and NOJ in culture broths. The assay was used to demonstrate differential production of DNJ and NOJ by selected StreRtOIFYces strains of cluster 61 (the S-lavendulae species group). The assay also confirmed the effectiveness of using microtitre plates as an effective screening procedure. The microtitre screening programme generated further data and statistical treatment of the results delimited the number of isolates for further examination. No DNJ-producing colony was detected and examination of the size of the DNA inserts showed almost all were too small to contain the DNJ gene cluster. Additionally, blocked DNJ production mutants were characterised by the feeding of NOJ, one of the mutants successfully converted NOJ to DNJ.