Clostridium difficile has become the major cause of healthcare acquired diarrhoea in the world. The disease caused by this pathogen is largely mediated by the production of toxins. The C. difficile genome contains an incomplete accessory gene regulator (agr) locus, designated agr1. In Staphylococcus aureus, it has been shown that the agr locus regulates virulence factors through Quorum Sensing (QS). In addition to the incomplete agr1 locus found in all of the C. difficile strains sequenced to date, the strains belonging to the BI/NAP1/027 group possess an additional agr operon, designated agr2. These strains have been found to produce higher toxin levels and a more severe disease in patients. The studies described in this thesis have shown that the two agr systems present in the Clostridium difficile BI/NAP1/027 strain R20291 are involved in the regulation of spore formation and toxin production. The agr1 locus (agrB1, agrD1) is responsible for the production of the autoinducing peptide (AIP) AgrD1, while the second agr2 locus (agrC, agrA, agrB2, agrD2) mediates production of the AgrD2. Initial mutational analysis using a University of Nottingham isolate (R20291 NM) suggested that AgrD1 both positively regulates sporulation and negatively regulates toxin production, whereas AgrD2 positively regulates toxin production, but negatively regulates spore formation. It was subsequently discovered that strain R20291 NM exhibited significantly different phenotypes to R20291 BW. The former possessed a single polar flagellum, whereas R20291 BW was peritrichously flagellated. The NM strain exhibited impaired motility and increased biofilm formation, demonstrated different growth rates, produced greater quantities of toxins and exhibited a relative delay in the onset of sporulation compared to R20291 BW. The equivalent agr mutants made in R20291 BW indicated that the regulatory control exerted by AgrD1 in sporulation was broadly the same, while AgrD2 does not seem to play any role in the regulation of spores, contrasting with observations made in the agrB2 mutant created in R20291 NM. Surprisingly, the effects of the agr mutants made in R20291 BW on toxin production were opposite to those observed in the NM strain. The subtle differences in the behaviour of the two R20291 isolates was most likely due to the presence of mutations in R20291 NM revealed by whole genome sequencing. Of the four genes affected, a mutation in the anti-sigma factor RsbW, was considered the most likely culprit. An investigation of its possible role in toxin production and sporulation was therefore undertaken through complete ClosTron-mediated inactivation of rbsW in both R20291 isolates and the creation of a sigB ClosTron mutant in R20291 BW. The sporulation and toxin production phenotypes of the rbsW mutants of the two strains mirrored that of their respective agr2 mutants. Thus, inactivation of rbsW brought forward the onset of sporulation in R20291 NM, but had no effect on the initiation of sporulation in R20291 BW, while toxin production in R20291 NM was reduced, but increased in R20291 BW. Motility and biofilm formation in the rbsW mutants of both strains was unaffected. These data suggest that interference with the RbsW/SigB interaction preferentially affects AgrD2-mediated regulatory processes. In contrast, however, inactivation of SigB in R20291 BW caused a delay in the initiation of sporulation, but did not affect toxin production. Mutation of sigB, however, did not affect motility or biofilm formation, although, in common with other bacteria, the resistance of the R20291 BW sigB mutant to oxidative stress was reduced. The picture that emerges is of a complex regulatory interrelationship between the two agr QS systems and SigB in this important nosocomial pathogen, a relationship that has been subtly subverted in strain R20291 NM. These findings emphasise the importance of knowing the genome sequence of strains under investigation if valid conclusions are to be drawn.

Limited fossil fuel resources and the environmental impacts of climate change are motivating the development of sustainable processes for the production of fuels and chemicals from renewable resources. The development of alternative energy sources, such as biofuels, will strengthen energy security and reduce dependence on fossil fuels. n-butanol is a biofuel and platform chemical. n-butanol is an advanced fuel with high energy content, compatible with existing infrastructure. Here the ability of microorganisms to use renewable resources for biofuel synthesis is exploited. In this work use of the thermophilic bacterium Geobacillus thermoglucosidasius is explored for the production of n-butanol. Geobacillus is considered a promising industrial process organism due to its high optimum growth temperature and ability to assimilate various substrates including both hexose and pentose sugars. As a relatively novel process organism, first the development of molecular tools was required to enable subsequent engineering of the host metabolism. Here four reporter assays were developed, three of which can be used simultaneously, providing for extensive analysis of qualitative and quantitative gene expression within the cell. A range of promoters and RBS’ were screened. Extension of the Geobacillus vector series, pMTL60000, with new component parts for each module enabled co-transformation of two plasmids into G. thermoglucosidasius. The molecular tools developed were then applied in Geobacillus metabolic engineering work, with the aim of producing the target molecule n-butanol. Initially a CoA dependent n-butanol pathway, based on naturally occurring production by ABE fermentation, was considered. Following introduction of the pathway further metabolic engineering was employed to improve pathway flux, creating a driving force through the pathway and increasing the substrate pool. Production of 0.137 mM (10.166 mg/l) n-butanol demonstrated proof of concept. Next, the use of genes native to Geobacillus were investigated for improved enzyme compatibility. This approach did not generate n-butanol here. Finally a CoA independent pathway utilising the host’s native fatty acid biosynthesis pathway was considered. Using this approach, butyric acid was produced. Butyric acid can subsequently be further converted to n-butanol however this was not demonstrated here. In addition to metabolite pathway introduction, host strain engineering was carried out with the aim of adaptation towards industrially desired properties. Directed evolution resulted in selection of a strain with an increased n-butanol tolerance of 2.5% (v/v). Such modifications resulted in an improved process organism for biotechnological application. This work provides the first reported production of n-butanol in thermophilic and aerobic conditions. Multiple approaches to n-butanol production are evaluated here. Use of heterologous and native genes are considered. Both CoA and ACP dependent pathways were introduced. Each approach presented advantages and drawbacks. A system compatible for use in Geobacillus has demonstrated proof of concept n-butanol production. Further development is required to increase production to industrially feasible quantities.

Clostridium difficile is an anaerobic, Gram-positive, endospore-forming, pathogenic bacterium which is the leading cause of antibiotic-associated diarrhoea, and causes a significant burden to healthcare facilities and communities, worldwide. Bacterial endospores are one of the most resilient forms of life, able to withstand exposures to wet-heat, desiccation, UV radiation, oxygen, and some disinfectants, which would otherwise kill the vegetative cell form. Thus, endospores of C. difficile are able to persist in the environment and contaminate surfaces within healthcare settings. Once ingested, these spores pass into the anaerobic lower intestines and in susceptible individuals find favourable conditions in which to germinate, generating the toxin-producing vegetative cells responsible for C. difficile associated disease. Consequently, spores are the infectious agent of this disease and both sporulation and germination processes are essential for disease. Whilst these processes have been well studied in Bacillus subtilis, it is only recently, with the development of appropriate reverse genetics tools for clostridia, that the mechanisms of sporulation and germination have begun to be described for C. difficile. This study uses the currently available mutagenesis tools of ClosTron and allelic exchange to generate mutant spores lacking spore-specific proteins, and through a range of assays characterises the sporulation, germination and resistance properties of these mutants, to understand the roles of these proteins in C. difficile endospores. Furthermore, these genetics tools are established in a novel C. difficile strain with beneficial properties for studying the processes of sporulation and germination in vitro. Finally, this study establishes CRISPR/Cas9 genome editing in C. difficile for the first time to overcome the major pitfalls associated with the previously available reverse genetics tools. This mutagenesis method was found to offer fast, highly efficient genome editing of two different C. difficile strains and will be the method of choice for future studies in C. difficile.

Vitamins are micronutrients essential for life in an organism which must be obtained from diet or environment. Standard media often contains ten B-vitamins, which act as cofactors or precursors for co-enzymes. Clostridium autoethanogenum and Clostridium ljungdahlii are two obligately anaerobic acetogens which can utilize syngas (carbon dioxide, hydrogen and carbon monoxide) as a carbon source, generated from industrial waste gases or gasification of hydrocarbons. Clostridium autoethanogenum and Clostridium ljungdahlii use the Wood-Ljungdahl pathway to generate Acetyl-CoA from syngas. Acetyl-coA can be used by the bacterium for growth, production of energy and acetate, ethanol and 2,3-butanediol production. To increase the industrial attractiveness of producing chemicals via this route, the process must be as economical as possible and one way to increase the economic viability is to only add essential media components. This study attempted to define the exact B-Vitamin requirements of the two species, to show that the two species require only three “vitamins” – biotin, pantothenate and thiamine. Strains were created which have missing biotin and pantothenate pathway genes added in order to confer prototrophy for the vitamins and to determine the effects the addition of these genes had on viability, growth profile and product profile. A Continuous Stirred Tank reactor experiment was conducted in order to determine the effects of pantothenate, and therefore, Acetyl-CoA limitation on a continuously growing culture which mimicked an industrial reactor. Increased efficiency of the media could lead to a more economically attractive process for the sustainable production of the platform chemical than from fossil fuels leading us one step closer to decoupling our civilisation from oil.

Approximately 400 tonnes of antibiotics (including synthetic antibiotics) are used every year in treating infections in farm animals, and as prophylactics against infection. Antimicrobial resistance is a crucial problem that is now of great concern in public health, with food and food producing animals as a potential route for spread of these resistances, especially resistance to cephalosporins, which is increasing. The main aim of this study was to determine the prevalence and range of multidrug resistance (MDR) and extended spectrum β-lactamase (ESBL) or ampicillin C (AmpC) β-lactamase producing Escherichia coli within a commercial dairy farm, to understand the diversity of resistance to β-lactam antibiotics, and to determine if co-carriage of other antimicrobial resistance (AMR) was associated with ESBL/AmpC producers. This would allow a better understanding of the contributions that farms and farm slurry may make to the presence of AMR in the environment, and the reservoir of resistance in agriculture. In this study, E. coli strains were isolated from a single dairy farm (East Midlands, England, United Kingdom) on two visits, a preliminary isolation using TBX agar in 2012 and more targeted isolation using antibiotic supplemented TBX media in 2014. Confirmed E. coli (126 out of 155 selected strains) were genotyped using ERIC-PCR and analysis of the ERIC profiles showed that, in comparison to the 2014 isolates, the 2012 isolates were a quite distinct genetic population. Antimicrobial sensitivity tests were performed using a disk diffusion test for all the strains against 17 antimicrobials representing seven different antimicrobial groups: β-lactams, aminoglycosides, tetracyclines, sulphonamides, chloramphenicols, nitrofuran derivatives and quinolones. Antimicrobial resistance profiling showed 92% of isolates showed resistance to at least 1 antimicrobial, of which 27.8% of the isolates were isolated without antibiotic selection, and 57.9% of the isolates were multidrug resistant to between 3 and 15 antimicrobials, of which 43.6% of the isolates were isolated using antibiotic supplemented media. Two strains showed resistance to imipenem which appeared to be an unstable phenotype and was subsequently lost. The finding was unexpected and of concern as imipenem is not used in veterinary medicine. blaCTX-M, blaTEM and blaOXA genes were detected by PCR among the cephalosporin resistant strains. No plasmid ampC genes were detected. Four strains were fully sequenced and the genetic/genomic environment surrounding β-lactamase genes and analysis of some other AMR genes showed these genes are associated with transposable elements, such as ISEcp1, ISCR2, IS26-IS26, Tn2, Tn10 or within a class I integron carried by a Tn-21 like transposon. The association of AMR genes with these transposable elements might make the dissemination rate of these genes greater. Some of the insertion sequence-AMR gene combinations are thought to be novel, such as the unique insertion of ISEcp1- blaCTX_M14 unit into the fdeC chromosomal gene. This is the first study of this type performed on this dairy farm; the data showed a diverse range of resistance genes present in the E. coli population in the farm, including resistance to historically used antimicrobials as well as cephalosporins in contemporary use, and a high level of multidrug resistance. The spread of such highly resistant strains to the environment and possibly to humans could present a real threat to human health especially if they are pathogenic.

Wheat (Triticum spp.) is a particularly important crop for food security, providing 20% of worldwide calorie intake. Food production is not meeting the projected global demand of an increase of 2.4% p.a. Improvement of resource capture in wheat could help meet this demand. Nitrogen (N) is an essential macronutrient for plant growth and development; however, nitrogen use efficiency (NUE) for cereal production is only 33%. Domestication of modern varieties of wheat may have lost potentially beneficial agronomic traits, particularly in the root system. Optimisation of root system architecture could profoundly improve nitrogen uptake efficiency (NUpE) and in turn increase the yield potential of the crop. Using ancestral wheat germplasm and mapping populations, desirable traits may be identified and bred back into commercial wheat varieties to increase yield potential. Using a high-throughput hydroponic root phenotyping system, N-dependent root traits have been identified in wheat mapping populations. Using transcriptomic analyses, the gene expression profile of a candidate N-dependent root QTL has been identified. Using a new root phenotyping system, X-ray micro-computed tomography (μCT), a three-dimensional representation of wheat roots can now be imaged in soil. A selection of the same mapping lines have been used for 3D μCT analysis based on field NUpE parameters to identify promising root traits in both seedlings and mature plants.

The archaeon Haloferax volcanii is used to study DNA replication and repair, and it is unique amongst cellular organisms as it is able to grow in the absence of DNA replication origins. There are four DNA replication origins on the main circular chromosome (including the integrated mega-plasmid pHV4) and one on each of the other mega-plasmids pHV1 and pHV3. Replication origins are normally required for the initiation of DNA replication, however H. volcanii is able to grow faster when all chromosomal origins have been deleted. Therefore, H. volcanii must utilise other methods of DNA replication such as recombination-dependent replication. The origin found on pHV3 cannot be deleted from the episomal mega-plasmid, whereas the origin can be deleted from episomal pHV4. The pHV3 mega- plasmid can be integrated onto the main chromosome, which allows the pHV3 origin to be deleted from the chromosome. The pHV1 mega-plasmid origin can be deleted from the episomal mega-plasmid, and the entire mega-plasmid can be lost from the H. volcanii cell. This generates a viable, healthy strain, which shows that the pHV1 mega-plasmid is non- essential. It was also found that the pHV1 mega-plasmid exists in H. volcanii as a 6x concatemer which is ~510 kb in size, which may explain the reason for being able to delete the origin. To further investigate the mechanisms that recombination-dependent replication may use, replication machinery (MCM and GINS) were tagged and expressed. Due to time constraints, interactions were not seen. The mcm gene was put under the control of a tryptophan inducible promoter. A strain lacking chromosomal origins and therefore primarily using recombination-dependent replication was shown to require more MCM than a wild-type strain.

Johne’s disease (JD) is a severe wasting disease of ruminants, characterised by chronic enteritis, reduction in milk yield, and severe weight loss despite a maintained appetite. The causative agent is Mycobacterium avium subspecies paratuberculosis (MAP), a slow growing pathogen that can take up to 18 weeks for detection on solid culture. Control programmes rely on sensitive diagnostics to identify infected animals quickly so they can be either removed from the herd or managed differently to control the spread of disease. Unfortunately, the Gold Standard of detection is culture, which due to decontamination procedures, has low sensitivity. Enzyme-linked immunosorbent assays (ELISA) are used more often than faecal culture within control programmes as they are cheaper and quicker than culture methods. However, they only detect the animal’s immune response, rather than the causative agent. This can cause some issues with diagnosis as the immune response can be affected by other variables. Therefore, to effectively control disease, a new detection method needs to be developed. In this series of studies, phage-PCR was used within large scale on-farm sampling to establish its performance against the Gold Standard (liquid culture with ESP-trek) and MAP specific antibody milk ELISA (ab-ELISA). Phage-PCR is thought to be more sensitive than other methods due to its low limit of detection. It is also rapid and relatively inexpensive. Results suggest that phage-PCR can detect more animals shedding MAP into their milk than other methods, or in the least a different group of animals than the other methods. There was some evidence that animals who have had an ab-ELISA positive result in the last year are shedding less MAP into their milk, suggesting that the immune response is helping to control the disease in the short-term. However, this was not observed beyond one year. Phage-PCR had a better agreement with faecal culture than milk culture or ab-ELISA, but this was limited. There was also evidence that early detection could be achieved, as some animals were identified as faecal shedding with phage-PCR before they had seroconversion and detected with a-ELISA. However, it must be noted that these animals may not be infected and just passaging MAP through the GI tract from the contaminated environment. An investigation into the prevalence of MAP in pasteurised milk using phage-PCR was also carried out. There is thought to be an association between MAP and Crohn’s Disease, with milk highlighted by some as a key transmission vector. There was an increase in the proportion of samples containing viable MAP when compared to other surveys within the literature. However, this was thought to be due to the lower limit of detection that phage-PCR provides, rather than an increase in prevalence. Phage-PCR can be used effectively for large-scale on-farm sampling to identify animals shedding MAP into the milk. However, some changes to the assay and sample processing will have to be undertaken before this can be used within industry as its current format is laborious and not suited to automation. Until then, it could be used as a tool to further research and understanding into JD in dairy cattle.

Diminishing fossil fuel reserves and the drawbacks of conventional crop-based biofuels has catalysed recent research into the microbial conversion of lignocellulosic biomass into liquid biofuel. Fatty acids represent the most abundant form of reduced carbon chain in nature, and represent the basic building blocks for the creation of a wide-range of advanced biofuels; such as alkanes, fatty alcohols, and fatty acid methyl- and ethyl-esters. It is hoped that the use of a thermophilic platform strain, that is capable of producing fatty acid-derived biofuels at elevated temperatures, will circumvent some of the challenges faced by established mesophilic organisms such as Escherichia coli or Saccharomyces cerevisiae. Here we describe the heterologous expression of an alkane biosynthesis pathway from the thermophilic cyanobacteria Thermosynechococcus elongatus BP-1 in both E. coli and the thermophilic production organism Geobacillus thermoglucosidasius. Alkane biosynthesis in T. elongatus BP-1 is facilitated by two enzymes: fatty acyl-ACP reductase (AAR) and aldehyde deformylating oxygenase (ADO): both of which were found to demonstrate a level of activity in vivo at mesophilic and thermophilic temperatures (30 - 52°C). Expression of an alkane biosynthesis operon in G. thermoglucosidasius NCIMB 11955 resulted in the production of ~100 mg OD-1 L-1 fatty alcohols, and an inconsistent formation of minute amounts of heptadecane. Improved titres of alkane may be achievable through the identification and elimination of competing pathways, and a better understanding of n-alkane biodegradation in G. thermoglucosidasius. However, we recommend the continued pursuit of fatty alcohol production using G. thermoglucosidasius as a host. Elimination of several fatty acid degradation (fad) genes in G. thermoglucosidasius was undertaken with the hope of showing an ability to manipulate the cellular pool of fatty acyl-ACP substrates available to the alkane biosynthesis pathway. The combined elimination of two long-chain-fatty-acid—CoA ligase genes (fadD1 and fadD2) resulted in increased levels of pentadecanoic- and heptadecanoic acid. The heterologous expression of a fatty acyl-ACP thioesterase (FAT) from Clostridium thermocellum and from the Aminicenantes candidate phylum (OP-8) was also undertaken in an attempt to manipulate levels of cellular FFAs, although we postulate that observation of a differential phenotype requires the development of a strain completely defunct of long-chain-fatty-acid—CoA ligase activity. Fatty acid metabolism in G. thermoglucosidasius represents a complex myriad of multiple genes that are subject to strong homeostasis. Nevertheless, we present evidence that genetic manipulations of G. thermoglucosidasius are sufficient to bring about changes in the fatty acid profile of cells, and encourage the further genetic characterization of fatty acid metabolism in the organism through targeted gene deletions, with the hope of producing an improved platform strain for fatty alcohol and alkane biosynthesis at thermophilic temperatures.

The Gram-positive, anaerobic, acetogenic bacterium Clostridium autoethanogenum is regarded as an important biocatalyst in the current advancement of industrial gas fermentation. The ever-widening diversity of industrially-relevant acetogenic bacteria has inspired a rational approach into utilising industrial waste gases as a viable feedstock, with goals of mitigating greenhouse gas emissions, and supporting an alternative means of fuel and high-value chemical production. Combined with this effort, is the expanding repertoire of gene editing tools that have allowed for the improvement of gas fermentation processes and increased spectrum of fermentative products. Despite these advances, there remain many pertinent questions, which addressed, can further the understanding of metabolism and physiology in acetogenic bacteria. This includes cell-to-cell communication and signalling, Quorum Sensing. In this project, these questions are addressed through the study of the Agr QS system in C. autoethanogenum. Signalling peptide genes, agrD1 and agrD2 were disabled separately and more importantly, in tandem, which effectively abolished Agr signalling. Phenotypic characterisation of the double agrD mutants revealed a significant increase in ethanol at the expense of acetate output. Further observations exhibited a complete utilisation of the fructose carbon source, and the inability to fully re-assimilate CO2. These findings markedly contrasted with the wild type, and both single knock-out agrD mutants. Proteomics and enzyme activity analysis of the double AgrD mutant revealed a marked down-regulation of Wood-Ljungdahl pathway genes that included the CO2-assimilating, carbon monoxide dehydrogenase / acetyl-CoA synthase complex subunits and hydrogenases. An up-regulation of alcohol dehydrogenases was observed explaining ethanol increases, alongside an unexpected upregulation of bacterial micro-compartment clusters. These findings led to the hypothesis that the C. autoethanogenum Agr system influences the ancient Wood-Ljungdahl pathway, primarily as a means of survival by managing carbon-source utilisation and regulation.