Access to renewable energy and reduction of carbon emissions represent two major issues facing humankind in the twenty first century and beyond. The underlying driving forces behind both are multi-faceted and often intrinsically connected, ranging from environmental concerns over climate change to improving economic security through self-sustaining energy production. Possible solutions to reliance on non-renewable, carbon-emitting fossil fuels have been explored over recent decades, with significant interest placed on biofuels. Due to ease of integration into liquid-based petrochemical fuel infrastructure, these renewable alternatives have been a consistent topic of both industrial and academic interest. Despite offering renewable energy, conventional crop-based biofuel production has faced criticism due to consumption of land, water and other resources associated with agriculture. Acetogens provide a solution to conventional biofuel production due to their utilisation of carbon monoxide and carbon dioxide gas as carbon and energy sources, rather than plant matter. This allows generation of a range of chemical products from a broad range of sources, including industrial waste gases and gasified solid waste. Acetogens offer the double benefit of both renewable energy production, and carbon emission sequestation. This study outlines the development of genetic tools to provide a foundation for using synthetic biology approaches to improve performance of acetogens as industrial chassis. Specifically, development of tools and techniques for the acetogen Clostridium autoethanogenum are described, with further applications of such technology to other Clostridia.

Phosphotransferase systems (PTS) represent an important method of sugar uptake in bacteria and have been well described in the past. However, research into PTS within the genus Clostridium has been mainly restricted to the non-pathogens. Analysis of the genome of Clostridium difficile 630 revealed over 40 intact PTS; this is over three times as many as in other pathogenic Clostridia, such as Clostridium perfringens and Clostridium botulinum. Previously, carbon catabolite repression has been shown to affect toxin production in C. difficile. Being capable of utilising different carbohydrates efficiently could be important for C. difficile to adapt to, grow, and survive in the human gut. So far, little work has been done to corroborate the role of individual PTS in carbohydrate uptake, sensing of environmental stimuli and regulation of virulence, i.e. toxin expression. A deeper understanding of the PTS in C. difficile, and their importance in virulence, could lead to the development of new drug targets. The aim of this study is to characterise the main PTS of C. difficile, determine their role in carbohydrate uptake, and their effect on regulation of virulence. To date, we have chosen the main candidates thought to be involved in mannitol and sorbitol uptake, and have inactivated these PTS using the ClosTron and in-frame deletion methods. Phenotypic characterisation of these mutants was undertaken to prove their role in uptake of the relevant sugar and to determine their role in virulence regulation. This study has demonstrated, by growth assays and HPLC, that the operons at CD630_0762-8 and CD630_2331-4 respectively encode PTS specific for sorbitol and mannitol uptake. In the case of the mannitol operon, it has been proved (through the use of cytotoxicity assays, which showed reduced bacterial toxicity in the presence of the sugar) that the suppression of toxin synthesis in the presence of mannitol is dependent upon uptake of the substrate via this operon. With sorbitol, toxin levels are, seemingly, not directly reliant upon uptake of the sugar, resulting in, mainly, an increase of toxin in sorbitol. Presently, it is not possible to say whether these systems have a distinct role or not in the motility of the organism.

Avian tuberculosis remains a significant economic problem for the poultry industry in many countries. An improved understanding of the interaction between host and mycobacteria is important in designing more efficient vaccine strategies. This study investigated the immune responses to M. avium (Ma.) strains isolated from different sources, Ma strains 1-2 isolated from chickens, Ma.3-5 from cattle and Ma.6-8 from humans, using human cells in vitro and avian cells in vitro and in vivo. The responses were characterised by expression levels of pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6 and chemokines CXCLi1 (K60) and CXCLi2 (IL-8) and nitric oxide production. A differential immune response was observed between the strains in terms of induction of cytokine and chemokine production in both human THP-1 and avian HD11 cells. With the exception of the avian strains 1 and 2, all strains induced increases in pro-inflammatory cytokines in THP1 cells. Two of the 3 bovine strains also had this effect in HD11 cells but the other strains had little effect. Use of specific inhibitors indicated that in THP-1 cells the extracellular regulating kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways were important in inducing pro-inflammatory cytokines whereas the p38 pathway did not appear to be involved particularly in regulating TNF-α and IL-1β production. The results also indicate an interaction between the mitogen activated protein kinases (MAPK) pathways studied in regulation of cytokine production in HD11 cells. Although layer chickens were resistant to extensive M. avium multiplication in the tissues, significant expression of pro-inflammatory cytokines was observed in the liver of infected birds towards the end of the period of observation of 21 days as granulomata had started to appear, characterised by lymphocyte accumulation. In conclusion, the findings of this study provide further understanding of the host-mycobacteria interaction and that different strains of M. avium might behave differentially regardless of their source of isolation.

Yersinia pseudotuberculosis is a mammalian enteropathogen and is the direct ancestor of Y. pestis, the causative agent of the plague. For its pathogenicity, Y. pseudotuberculosis harbours a 70 kb virulence plasmid which encodes the components of the type three secretion system (T3SS) and effector proteins. These effectors serve to evade the host immune system and induce apoptosis of mammalian cells. Consistent with many Gram-negative bacteria, Yersinia facilitate cell: cell signalling through the production and sensing of N-acylhomoserine lactones (AHLs), which functions to mediate the expression of downstream target genes. This cell-cell communication is known as quorum sensing (QS) and is facilitated by two LuxI/R-type systems in Y. pseudotuberculosis: YtbI/R and YpsI/R, and several AHL molecules. Behaviours under QS control include motility, biofilm formation, clumping and the regulation of the T3SS. Recently, QS was reported to repress the T3SS whilst the T3SS attenuated biofilm formation on Caenorhabditis elegans. Colonising both the soil/water environment and the mammalian gut, Y. pseudotuberculosis exhibits a biphasic lifestyle whereby it exerts strict temperature-dependent control over the expression of pYV-encoded genes. The switch between these two lifestyles is govered by a pair of virulence regulators: LcrF is a transcriptional activator that targets pYV-encoded genes and is key for the assembly of the T3SS. Conversely, YmoA is a histone-like protein that represses transcription of lcrF through chromatin compaction. Considering the repression of the T3SS by QS, this study set out to investigate whether this regulation is mediated by a relationship between QS and LcrF/YmoA. By using chromosomal promoter:lux fusions, QS was identified to be an activator of YmoA at both 22oC and 37oC whilst a regulatory relationship between QS and LcrF was also identified. To investigate these links further, AHL profiling of the lcrF and ymoA mutants identified YmoA as a repressor of AHL biosynthesis whilst a very subtle repression was observed in ΔlcrF, suggesting that LcrF may influence AHL synthesis indirectly. Assessing the impact of LcrF and YmoA on the QS-mediated phenotypes of Yop secretion, biofilm formation and motility extended these observations. LcrF had no effect on any of the phenotypes examined supporting the hypothesis of either an indirect mode of regulation, or no regulation at all. In contrast, YmoA influenced both motility and biofilm formation. A decreased motility of ΔymoA was observed on both semi-solid agar and in liquid whereby both the speed and the percentage of motile cells was altered. This suggests an activating role of YmoA on motility. Interestingly, QS is known to repress motility therefore it is likely that YmoA-regulation of motility occurs irrespective of QS. Comparable to that of the QS synthase mutant (ΔypsI/ytbI), biofilm was attenuated in ΔymoA yet restored when cells were cured of the virulence plasmid supporting the hypothesis that the type three-secretion injectisome disrupts biofilm formation. This attenuation of biofilm formation in ΔymoA, in conjunction with the activation of ymoA by QS, led to the hypothesis that the repression of the T3SS by QS works through YmoA. Considering these results, evidence for an interaction between QS and virulence regulators LcrF and YmoA has been confirmed. We propose a model whereby YmoA is the missing link in the QS-mediated repression of the T3SS. Activation of YmoA by QS leads to increased repression of lcrF and subsequently, of the T3SS resulting in the de-repression of this system in the absence of QS.

The emergence of drug-resistant strains of tuberculosis has led to a demand for the development of new antibiotics. One new target is the cell wall biosynthesis enzyme UDP-Galp mutase (UGM), which aids the formation of the bacteria’s characteristic mycolic acid cell wall. LQ10 and LQ6 were discovered through a library screen. The synthesis of LQ10 was achieved along with 4 analogues. Another class of compounds, 2-aminothiazoles, were produced. Thirteen of these compounds were produced and along with the LQ10 analogues, initially gave encouraging results in silico. To test their biological activity, a fluorescent probe was synthesised for use in a high-throughput fluorescence polarization (FP) assay against UDP-Galp Mutase which was expressed from E. coli. The compounds were screened using the fluorescence polarisation assay initially at a concentration of 50 µM, 9 of which demonstrated >70 % inhibition of UGM. Two of which had inhibition greater than 90 %. These preliminary results suggest that some of these compounds are, and can be developed into potent UGM inhibitors. However, it should be noted that these are only single-point results due to limitations in the quantity of UGM available, and that these will need be repeated in triplicate to determine any errors and give more reliable values.

Mastitis remains one of the most common, costly and intractable diseases affecting the dairy cattle industry worldwide. In spite of concerted efforts meaningful progress in reducing the incidence of mastitis has been limited over the past thirty years by our partial understanding of the epidemiology of key pathogens, such as S.uberis. The aim of this thesis was to investigate the epidemiology of S.uberis mastitis by analysis of the population structure and transmission dynamics of clinical mastitis patterns within and between commercial dairy herds in England and Wales. In Chapter 3 Multi Locus Sequence Typing (MLST) was used to describe the sub-species (sequence type) genetic heterogeneity of S.uberis isolates collected from a longitudinal study of clinical mastitis on 52 farms. The clinical cases were classified according the pattern of occurrence of the sequence types in the herds. The findings suggested that a small subset of MLST sequence types were disproportionally important in the epidemiology of S.uberis mastitis, with cow-to-cow transmission of S.uberis, potentially occurring in the majority of herds in the United Kingdom; this may be the most important route of S.uberis transmission in approximately one third of herds. In Chapter 4 cow and herd level variables, including monthly recorded milk constituents, yield and parity were evaluated against the clinical case classifications defined in Chapter 3. The temporal relationships between clinical cases and classifications were also evaluated as potential predictors of transmission dynamics within a herd. The findings indicated that the time interval between clinical cases classified by genotype as potentially contagious transmission was significantly shorter than that between successive mastitis clinical cases attributed to environmental transmission. The distribution of clinical cases throughout lactation also indicated a higher proportion of potentially contagious isolates were cultured from clinical cases originating from lactation rather than the dry period compared to those attributed to environmental transmission. In Chapter 5 Matrix Assisted Laser Desorption (MALDI) mass spectroscopy was used to generate spectral profiles of S.uberis isolates cellular composition. Spectral profiles were used successfully as an alternative method of discriminating between clinical mastitis isolates associated with contagious transmission from those associated with and environmental origin of infection defined in Chapter 3. The findings of this chapter demonstrated marked variation between herds in the spectral profiles of isolates from the same clinical case classification. In Chapter 6 selected isolates of S.uberis associated with contagious transmission and persistent intramammary infections were sequenced using next generation technology and compared by core genome multi locus sequence typing (cgMLST) to evaluate the discriminatory capacity of the MLST and MALDI. The results from this chapter confirmed the importance of the herd unit in the genotypic population structure of S.uberis suggested by the results of Chapter 5 and also support the results of Chapter3 which suggest ‘low grade’ contagious transmission of S.uberis is superimposed on a ubiquitous, environmental S.uberis mastitis pattern in many herds This thesis demonstrated that the S.uberis population is complex with variation at the bacterial, cow and herd level suggesting different patterns of disease and bacterial evolution occurring in different circumstances. Categorisation of sub-species of S.uberis within herds as being transmitted via contagious or environmental routes appears to be eminently achievable using modern, high throughput technologies; this could lead to a step change in mastitis control.

Gas fermentation has emerged as a promising technology that converts waste gases containing CO, CO2 and H2 (also known as syngas) into fuels and chemical commodities. Employed by LanzaTech Inc., Clostridium autoethanogenum is an industrial acetogen that converts gases into ethanol, 2,3-butanediol, acetate, and lactate. Metabolic engineering offers unique opportunities to eliminate side-products, synthesize novel, high-value molecules as diversification strategies, and increase productivities of natural products. However, there had been no scientific reports of genetic manipulation of this acetogen so the overall goal of this PhD project was to develop genetic tools for this gas-utilizing microorganism and construct a hyper-ethanol producing strain via metabolic engineering. The formulation of electroporation and conjugation procedures allowed exogenous DNA to be routinely introduced into the bacterial host. ClosTron mutagenesis and Allele-Coupled Exchange (ACE) techniques were fully exemplified in this bacterium during the construction of knockout, in-frame deletion, and overexpression mutants. Carbon monoxide dehydrogenases (cooS1, cooS2 and acsA) were specifically targeted to elucidate their roles in supporting CO oxidation and carbon fixation. In the ethanol formation pathway, inactivation of bi-functional aldehyde/alcohol dehydrogenases (adhE1 and adhE2) impaired growth on pure CO but elevated ethanol titres. Conversely, inactivation of the more highly expressed aldehyde:ferredoxin oxidoreductase (aor1), but not the weakly expressed aor2, significantly reduced ethanol production, highlighting the importance of aor1 in autotrophic ethanol formation. A double KO mutant of aor1 and aor2 was also generated via ClosTron mutagenesis and pyrE-mediated allelic exchange. In an effort to engineer a robust biocatalyst, the native chaperone systems groESL and/or grpE-dnaK-dnaJ were overexpressed in C. autoethanogenum, resulting in enhanced tolerance towards ethanol, heat and salts. In summary, this study demonstrated the genetic tractability of C. autoethanogenum and revealed gene targets for future metabolic engineering of a hyper-ethanol producing acetogen.

The growth of micro-organisms in foods is different from that in axenic liquid culture in laboratory media. In natural environments, including food, micro-organisms generally grow in mixed culture and in close proximity to each other, because of which antagonistic or synergistic interactions can occur. To elucidate the behaviour of bacteria within food matrices an understanding of the food structure is required, as foods are complex ecosystems on the micrometer scale. Most processed foods are emulsions and as such are highly structured heterogeneous environments. Antibody-linked probes can be used for the immuno-location of micro-organisms or their products within food matrices to demonstrate the sites at which growth occurs and elucidate the possible bacterial interactions with food components. The aim of the project was to raise antibodies to spoilage Pseudomonas species and to use the developed antibody-linked probes to follow psychrotrophic spoilage Pseudomonas within heterogeneous foods. By using antibody-linked probes the natural spoilage of milk and milk products can be followed along traditional lines examining extrinsic parameters but with the additional benefit that the major spoilage organisms can be located within the mixed natural flora. The use of antibodies in this way facilitated the study of a defined natural population and surmounted any adaptive problems associated with introduced organisms. An oil-in-water near-foodgrade model was developed to investigate the growth of Pseudomonas as it overcame some of the technical problems of using natural cream. Pseudomonas species, which grew as colonies within the near-food-grade model, were visualised using fluorescently-labeled antibody-linked probes. Pseudomonas used to raise the antisera were isolated from psychrotrophically spoiled food and characterised together with isolates retrieved from the environment. The phenotypic characterisation of Pseudomonas using classical biochemical tests and API 20NE test strips (BioMerieux) did not produce definitive identifications of the unknown isolates. Nutritional screening of the Pseudomonas isolates using commercially produced standardised test microtitration plates (Biolog MicroPlate TM), that contained 95 carbon sources, was carried out. The data produced from the test microtitration plates were analysed using numerical taxonomic methods. The relatedness of the Pseudomonas isolates was strongly influenced by the source from which the test isolates originated and did not definitively identify all of the unknown isolates tested. Molecular techniques, ribotyping and amplified ribosomal DNA restriction analysis (ARDRA), based on the genomic fingerprinting of the 16S rRNA gene were evaluated to aid the definitive identification of the Pseudomonas isolates but needed a more extensive data base to be useful. The difficulties encountered in phenotypically identifying food and environmentally isolated Pseudomonas species stems from the fact that the Pseudomonas genus is now classified according to its ribosomal DNA homology. The classification of the species within the Pseudomonas genus is still under review. Robust phenotypic criteria for the identification of all the species within the genus have not to date been defined. In this study, the association of phenotype with environmental source of isolation (whether characterised by nutritional studies or by antibody cross-reaction) demonstrates clearly that more appropriate phenotypic characterisation is required to allow identification schemes to reflect the underlying phylogeny of this group.

Streptococcus uberis is a Gram-positive, catalase-negative member of the family Streptococcaceae and is an important environmental pathogen primarily responsible for a significant amount of bovine intramammary infections. This thesis describes the sequencing and comparison of multiple strains from clinical and sub-clinical infections. Following de novo assembly, these are compared to the single reference strain (0140J). The assemblies of strains sequenced with two technologies (Illumina and SOLiD) were compared. From these assemblies, annotation allowed the comparison of gene content, the pan and core genomes and gene gain/loss of coding sequences associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Identification of sequence variants allowed identification of highly conserved and highly variant genes. Inferred intraspecies and interspecies (host-S. uberis) protein-protein interaction networks revealed pathways of bovine proteins enriched with potentially interacting pathogen proteins. These identified known and predicted pathways and also novel interaction partners. This was the first “whole-genome” comparison of multiple S. uberis strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains. These data allowed the first insight into potential evolutionary forces behind virulence differences.

Drugs that are preferentially absorbed through the stomach or the small intestine have a narrow time window for absorption since passage through this region of the gastrointestinal tract is rapid. A drug delivery system that can adhere to the gastric epithelium will substantially slow down drug transit and help overcome this problem. To achieve this, this study proposes the novel use of a glycan-binding adhesion protein from Helicobacter pylori, BabA, to create targeted drug delivery vectors that can mimic the attachment of this bacterium to the gastric epithelium. In this work, a recombinant form of BabA was expressed in the periplasmic space of Escherichia coli; it was found that after the incorporation of a C-terminal hexa-lysine tag, the expression and purification of this protein was significantly improved to amounts that enabled its subsequent characterisation and application. Recombinant BabA retained the highly selective glycan-binding properties of H. pylori and next, its crystal structure was solved in the absence and presence of Lewisb – a glycan well studied for its role in serving as a receptor for BabA. The structural models revealed that Lewisb binding occurred through a network of hydrogen bonds within a single, shallow binding pocket at the tip of a β-unit in BabA. Binding studies then confirmed that this site was also responsible for the recognition of other glycan receptors. Using this insight, recombinant BabA was conjugated to model drug delivery vectors via a linkage that favoured exposure of its glycan-binding β-unit; the binding properties of BabA successfully translated to these model BabA-vectors. The research presented in this thesis lays a strong foundation for future work to assess the in vitro and in vivo efficacy of biomimetic BabA drug carriers.