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The UK faces a continuing threat from the terrorist use of Chemical, Biological, Radiological and Nuclear (CBRN) materials. As such, it maintains and extensive counter-CBRN research programme. The nature of biological threat agents necessitates their handling in highly secure microbiological containment facilities. Therefore safe surrogates, which mimic their key characteristics, are used to assess their likely impact in real-world scenarios. To further develop the tools available for use with surrogates, this work provided unfinished genome sequence data for two important bacteria, E. coli MRE162 and B. atrophaeus (or BG) and used it to design novel real-time PCR assays for their detection. The E. coli PCR assay was converted and optimised for use with propidium monoazide (PMA), providing a novel viability assay for the absolute quantification of live and VBNC E. coli. The E. coli sequence data was further used in mapping transcriptome data that was generated for decontaminant-treated and untreated E. coli MRE162. The aim being to identify genes expressing during decontamination stress, which should represent ideal candidates for the design of mRNA-based viability assays for the detection of the surrogate following incomplete decontamination. The adoption of B. thuringiensis HD-1 Cry- as the surrogate of choice in UK Defence research necessitated the design of further real-time PCR assays, again with the aim of conversion to viability assays. Work towards identifying suitable conditions to generate samples of decontaminant stressed B. thuringiensis HD-1 Cry- for transcriptome sequencing was also undertaken.

There is increasing evidence that microbial interactions can happen within microbial communities resulting in an increase of the pathogen resistance to antimicrobial therapies. However, the mechanisms of these interactions remain elusive at the present day. Previously, it has been shown that in a polymicrobial community, β-lactam resistant bacteria can protect other non-resistant bacteria from the action of β-lactam drugs without any gene transfer between the resistant and non-resistant bacteria. This phenomenon was named as "sheltering effect" and occurs when the resistant bacteria releases proteins that give protection to the non-resistant bacteria living in the same environment. Klebsiella pneumoniae is one of the world's most dangerous multidrug resistant pathogens. Infections from this bacterium are seriously threatening the public health due to their great ability to quickly become resistant to every antibiotic available today. Even more importantly, K. pneumoniae is found in the blood of patients with polymicrobial infections. This doctoral project focused on the investigation of the sheltering effect in this pathogen. Studies carried out here were designed to elucidate the causes of the variability of this phenomenon observed among different clinical isolates of K. pneumoniae. Furthermore, the role of the Sec-dependent pathway and of the outer membrane vesicles (OMVs) in the sheltering effect was investigated. The present study also examined how the sheltering effect changes in response to different concentrations of β-lactam in the medium. Lastly the presence of sheltering effect in presence of drugs different from the β-lactams was also investigated, The results obtained by these tests also allowed the development of methods for the direct quantification of the sheltering effect based on the features observed on an agar plate. The causes of the variability in the sheltering effect detected on plate were investigated by carrying out Random Amplified Polymorphic DNA (RAPD), Polymerase Chain Reaction (PCR) and Whole Genome Sequencing (WGS) on the genome of the isolates to find a relationship between their genetic features and their potential of sheltering effect. Minimum Inhibitory Concentration (MIC) tests were also performed in the attempt to find a relationship between their resistance profiles and their sheltering effect potential. The results obtained suggest that the sheltering effect is a widespread and variable phenomenon in K. pneumoniae. The protective effect is likely due to the extracellular release of OMV-associated β-lactamases after Sec-mediated translocation of these enzymes from the cytosol to the periplasm. The results also suggest that the sheltering effect diminishes in response to increasing concentrations of β-lactam in the medium. Furthermore, the sheltering effect was not observed in presence of drugs different from the β-lactams. Lastly, no relationship was found between the sheltering effect potential of the isolates and their genetic and resistance features analysed in this project. Therefore more studies will be necessary to elucidate the causes of this variability.

Colonisation and chronic lung infection by the opportunistic pathogen Pseudomonas aeruginosa (PA) is the leading cause of morbidity and mortality in cystic fibrosis patients. A critical key determinant of PA pathogenicity is the switch from planktonic to the biofilm mode of growth, which facilitates chronic infections and makes PA eradication extremely difficult. Biofilms are aggregates of microorganisms in which the cells are enclosed in an extracellular polymeric substance (EPS) that contains proteins, extracellular DNA (eDNA) and exopolysaccharides. Psl and Pel are two important exopolysaccharides utilised by PA to construct biofilms. While Psl is composed of D-mannose, D-glucose, and L-rhamnose, Pel contains N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc). C-type lectins receptors (CLRs) are pattern recognition receptors (PRRs) that recognise carbohydrate motifs and are expressed by innate immune cells, including dendritic cells (DCs), macrophages and neutrophils. The overarching aim of this thesis is to investigate the interplay between PA and innate immune cells and how it is affected by biofilm formation. We hypothesise that engagement of CLRs through recognition of carbohydrate structures within PA biofilms could modulate the immune response and interfere with innate immune cells function, hence facilitating the persistence of infection. Previously, our lab demonstrated that PA biofilms and biofilm-associated carbohydrates purified from the Pel-deficient mutant ΔwspF Δpel, engage two CLRs, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209) and Mannose receptor (MR, CD206). In this study, we show that Dectin-2 (CLEC6A) is another CLRs that binds PA biofilms and purified biofilm carbohydrates; Dectin-2 binds to biofilms formed by PA strains with different EPS composition as well as biofilms formed by PA wound isolates. Similarly to MR and DC-SIGN, Dectin-2 also recognised purified biofilm-associated carbohydrates. High and low molecular fractions of purified biofilm carbohydrates were tested in this study and Dectin-2, together with DC-SIGN and MR preferentially bound to the HMW preparations. Biofilm-associated HMW carbohydrates did not induce Dectin-2 signalling on huDectin-2 reporter cells or HEK-Blue™ mDectin-2 cells but showed antagonistic activity blocking the response of muDectin-2 reporter cells to the Dectin-2 ligand zymosan. To microscopically examine the distribution of CLRs ligands within the biofilms and to study the interaction of immune cells with biofilm, we optimised biofilm generation in substrates suitable for microscopic examination. Confocal analysis shows DC-SIGN, Dectin-2 and, weakly, MR ligands within PAO1 biofilms concentrated into discrete clusters with additional DC-SIGN ligands dispersed among bacteria aggregates. Monocyte-derived DCs (moDCs) (DC-SIGN+ and MR+) cultured with biofilms formed by strains that overexpress Psl (ΔwspF and ΔwspF Δpel) tend to display round morphology and similar findings were noted when moDCs were incubated with purified biofilm carbohydrates. In a different set of experiments cytokine production by human moDCs in response to biofilms was tested and no differences were seen among biofilms with different carbohydrate compositions or after addition of purified biofilm carbohydrates. Taken together, these data indicate that DC-SIGN, MR and Dectin-2 are receptors for PA biofilms and biofilm-associated carbohydrates. The results of this research support the idea that CLRs engagements upon biofilm recognition by innate immune cells might modulate immune cells function. As the first responder cells of the innate immune system, neutrophils possess a unique set of migration features. Several studies found that neutrophils were not able to eradicate infection caused by biofilm-forming PA, which might indicate that biofilm formation was hindering neutrophil's activity. Here, we describe an in vitro assay that allows us to examine neutrophil behaviour, using live and confocal microscopy, following their interaction with PA, as a planktonic or biofilm cultures. Using live microscopy analysis, we observed that (1) biofilms reduced neutrophil velocity, net distance covered and the mean square displacement. Using confocal microscopy analysis, we observed that (2) neutrophils appeared unable to release granules when incubated with biofilms. Finally, (3) preliminary observations indicated that cells exhibit higher circularity when incubated with biofilm formed by strains expressing Psl or Psl-deficient biofilms in the presence of purified biofilm carbohydrates. The findings from this study have gone some way towards enhancing our understanding of the potential impact of biofilm formation on innate immune cells which might open new avenues for therapeutic approaches.

Static liquid microcosms have been used to study evolutionary and ecological dynamics of bacterial populations, where metabolic activity produces a heterogenous environment with a low-O2 region in the liquid column, and a high-O2 region directly below the air-liquid (A-L) interface. This system has been used to study adaptive radiation in Pseudomonas fluorescens SBW25, where non-biofilm forming wild-type populations diverge and biofilm-forming mutants known as Wrinkly Spreaders (WS) dominate the A-L interface where they are better able to exploit the ecological opportunity created by the high-O2 region. Although this system is well understood, it is not clear why biofilm-formation is such a successful strategy for colonising the high-O2 region. Similarly, this model system needs to be developed to reflect the complexity of microbial communities, where multiple species exist together and selective pressures may alter community composition, dynamics and emergent properties. Such better developed models can help understand changes in diverse natural occurring communities, enhancing our understanding of the progression of human infections, changes in ecologically important communities in agriculture, food production, and technology in applying microbial communities to mitigate the effects of anthropogenic pressures. In this thesis I show that A-L interface biofilm-formation is the best strategy for colonising the high-O2 region, where biofilms retain cells in position and overcome physical displacement including Brownian motion and bioconvection currents which otherwise would move aerotaxic wild-type cells away from the high-O2 region. WS mutant cells also possess an additional mechanism to associate and penetrate the A-L interface, where the production of additional surface-active agents further lower surface tension allowing cells to break through the interface. Biofilm-formation can then initiate above the interface and explain the dry phenotype and strength of WS biofilms as the biofilm is situated at the air-side of the interface. These key adaptive changes of the WS mutant allows highly efficient cell localisation at the high-O2 region to be achieved. However, biofilm strength is not directly related to fitness in static liquid microcosms. Other A-L interface biofilm-forming mutants within the SBW25 lineage can out-compete the WS mutant, producing weaker biofilms but maximising productivity by also significantly colonising the low-O2 liquid column compared to the WS mutant, suggesting colonising both regions provides a fitness advantage in microcosms. Ecosystem engineering was further explored to fully capture the ecological dynamics of diversifying SBW25 populations. Initial colonists not only generate O2 gradients but they were found to further alter the chemical environment through the uptake of nutrients and production of secondary metabolites and toxic waste products, which effect the diversification, biofilm-characteristics and fitness of evolved WS mutants. This additional aspect to ecosystem engineering within the microcosms system was also reflected in community-level work. I developed the microcosm model system for biofilm-forming communities using a soil-wash as the inoculum. The effects of heterogenous and O2 -limiting conditions on selection within bacterial communities were investigated and short-term serial-transfer experiments revealed changes in community productivity and biofilm characteristics. Productivity decreased in communities subject to longer incubation periods reflecting a tragedy of the commons with nutrient depletion and toxic waste-accumulation restricting growth. Final-transfer communities were stratified but retained phenotypic plasticity as isolates could form A-L interface biofilms as well as colonise the liquid column. Motility and cell localisation assays revealed isolates could migrate between both regions. This suggests a resource allocation trade-off between fast but competitive growth within the A-L interface biofilm and high-O2 region and slower but less competitive growth in the low-O2 liquid column, with community members maximising productivity by utilising the entire ecosystem. My research contributes to the growing body of knowledge aiming to understand the evolutionary and ecological processes driving change in biofilm-forming populations and communities in static liquid microcosms. It shows the value of continuing to ask deeper questions surrounding biofilm-formation within model systems, and the importance of developing model systems to reflect the complexity of naturally occurring microbial communities. A-L interface biofilm-formation has been the main subject of interest within this research, however extending my focus to the liquid column has shown the importance of colonising the region below the biofilm to improve productivity, competitive fitness and community resilience. This suggests understanding the influence of non-biofilm space in biofilm-forming communities is important in understanding the complex dynamics and persistence of multi-species microbial communities.

Bramley apple microflora was shown to consist of Aspergillus, Cladosporium, Fusarium, Penicillium and other fungi. Some of these fungi were used for studies to determine biophysical activities of key cellular stressors present during cider fermentations: sugars, ethanol, and glycerol. Generally, these compounds acted as stressors, but glycerol was found to enhance metabolic activity and cell division at low water activity. Paradoxically, however, glycerol also exerted chaotropic activity when present at high concentrations and this ultimately limited the biotic activity of fungi, regardless of species, as assessed by studies of differentiation and cell division during germination. Other studies, based on comparisons between these fungi, the cider yeast Saccharomyces cerevisiae, and several species of bacteria indicate that the microbial lag phase, which is often regarded as an indicator of cellular stress, is in reality often inconsistent with the level of stress. The work carried out here also produced a novel assay in which to investigate the effect glycerol has on the survivability of microbes in highly stressful conditions and provided novel insights, which are pertinent to the wider microbiology field. For instance, the work provided evidence that cellular metabolism, differentiation, and cell division can occur below the previous water activity window of life.

Bacteria in our environment contribute to Earth's landscapes through mineral deposits via a process known as bacteria-induced calcite precipitation (BICP). Over recent years, such bacteria have been at the basis of innovative biotechnologies arising within civil engineering sectors, finding application for example in self-healing concrete where encapsulated bacteria (such as those of the genus Bacillus) facilitate the repair of cracks that appear during aging of built structures. BICP occurs as a product of bacterial metabolism, which creates a microenvironment that favours the precipitation of calcium cations and carbonate anions in the form of mineral calcite. This process is dependent on changes in pH, availability of cell surface nucleation sites, and ion concentrations. Current approaches using this technology in industrial applications require bacteria that are both capable of BICP, as well as possessing specific growth characteristics required for the respective application (e.g. pH/salt tolerance). This project explored the genetic optimisation of BICP using Bacillus subtilis as a model Gram-positive and industrially relevant organism. Genetic engineering was coupled with functional characterisation of the resulting strains, quantitative and qualitative assessment of BICP under laboratory conditions and electron microscopy imaging of resulting crystals. This work identified key molecular components needed for BICP to occur and a way to mobilise these into better-suited chassis organisms for application predominantly in the context of improving selfhealing concrete. Results showed that heterologous expression of the ureolytic pathway and modulation of biofilm production offer mechanisms whereby BICP can be engineered into a nonprecipitating strain. In contrast modulation of bacterial surface charge was found to be ineffective in improving BICP. A plasmid suitable for mobilisation to a broad host range of Gram-positive bacteria was also developed to facilitate the future shuttling of BICP-promoting genes to application relevant bacteria. The results presented here provide a systematic exploration of the genetic components that drive BICP and will help pave the way for the rational design or selection of better precipitators for application. The ultimate goal is for these fundamental findings to contribute to the formulation of bio-concrete that increases the lifespan of cementitious structures and consequently decreases the maintenance costs and carbon dioxide release associated with concrete production and building.

The evaluation of how Bacteria respond to oil-contamination, and the application of dispersants and biosurfactants, in North Sea seawater microcosms is the focus of Chapter Two. Analysis revealed that dispersants and biosurfactants, which significantly reduced the interfacial tension between oil and water, significantly increased growth of obligate hydrocarbonoclastic bacteria (OHCB) in 24 hours, translating into significantly enhanced alkane-biodegradation. Early sampling of microcosms revealed how the OHCB Oleispira, hitherto considered a psychrophile, can dominate bacterial communities at the relatively high temperature of 16oC. Bacterial response to oil-pollution is examined further in Chapter Three, where an in situ oil-slick is compared to a chemically dispersed oil-slick in the North Sea. Results suggest a lack of hydrocarbon-degrading bacteria (HCB) growth, even in samples with measurable hydrocarbons, could potentially be attributed to phosphorous limitation. Whilst the Ecological Index of Hydrocarbon Exposure, which quantifies the proportion of a bacterial community with hydrocarbon-biodegradation potential, revealed an extremely low score, highlighting a limited capacity for the environment, at the time of sampling, to naturally attenuate oil. Analysis of sediments contaminated by the Agia Zoni II oil-spill (Greece, 2017), in Chapter Four, demonstrated significant growth of HCB five-days post-oil-spill. Whilst the relative abundance of HCB declined as oil was removed, a legacy effect was observed, with the OHCB Alcanivorax and Cycloclasticus persisting for several months after the oil-spill. Finally, analysis of sediments around a North Sea shipwreck (HMS Royal Oak), in Chapter Five, revealed low levels of pyrogenic polycyclic aromatic hydrocarbons and little evidence of HCB, indicating sediments showed no long-term impact by previous oil-pollution from the shipwreck. This thesis not only advances our understanding of microbial response to oil-spills, remediation strategies, and shipwrecks, in a range of marine environments, but also highlights the importance of harnessing such knowledge and data to advance post-incident monitoring guidelines and models.

Gel particles including biopolymers, microgels, transparent exoploymeric particles (TEP), and other classes of exopolymeric substances (EPS) are ubiquitous in the marine environment. Despite the different names for these gel particles in the literature they represent closely related materials and will be referred to simply as marine gel particles (MGPs). MGPs have a natural ability to aggregate, forming large particles of 'marine snow' that play an important role in the carbon flux to the ocean floor and the biogeochemical cycling of carbon in the sea. MGP aggregates are also a habitat for bacteria and their metabolic activities can affect the structure and dynamics of the MGPs. Extracellular DNA (eDNA) as an abundant element in the marine environment. eDNA is a key component in the structural integrity of some biofilms, and likewise could have similar effect on the structure of MGP aggregates. Hence, it is hypothesized here that extracellular DNA (eDNA) is present in MGPs as a result of bacterial growth and lysis. Therefore, the aim of this study was to investigate the presence of eDNA in MGPs. In addition, to characterise the bacterial community associated with MGPs and to examine their functional potential focusing on the occurrence of nuclease genes. A further aim was to investigate the production of deoxyribonuclease (DNase) by marine bacteria isolated from free living and attached bacteria. Seawater samples collected from the North Sea and filtered through a 100-μm sieve and a 0.4 μm polycarbonate filters to collect MGPs. eDNA occurrence was probed by bioimaging with cell-impermeant fluorescent DNA dyes YOYO-1 and TOTO-3. For the bacterial community structure and function analysis, MGPs were subjected to total DNA extraction and sequencing of the V4 region of the 16S rRNA gene using illumina MiSeq. Marine bacterial isolates were also investigated for DNase secretion on methyl green DNase test agar, with genome sequencing being carried out for the most productive DNase isolate. Results of the bioimaging analysis and quantification of the MGP composition demonstrated for the first time the presence of eDNA in MGPs. Proteobacteria dominated the bacterial community structure within MGPs, where Pseudoalteromonas and Vibrio were the most abundant genera. The bioinformatics based functional predictions of the bacterial community using KEGG analysis also affirmed the presence of numerous nuclease genes. The results from studies of isolated strains reported for the first time DNase secreting bacteria associated with MGPs in the North Sea. Additionally, there are 43 nuclease genes present in the genome of the prolific DNase producer Serratia marcescens. In conclusion, the co- occurrence of eDNA and DNases in the MGPs indicate important implications for understanding the dynamics and properties of MGP in the world's oceans. This work can contribute to a further understanding of the role of the bacterial activities in MGPs formation, degradation and sedimentation processes.

Greenhouse gas emissions and feed efficiency in ruminant livestock are pertinent and important topics, ones which have not suffered from lack of attention as ample research has endeavoured to further our understanding of the complex rumen microbial ecosystem. Despite this, some populations remain understudied. This is the key motivation behind the studies herein, which contribute to the understanding of the niche bacterial population of hyper ammonia producers (HAP) and bacteriophages (viruses that infect bacteria). HAP species degrade amino acids and peptides for energy, in the process producing hydrogen, carbon dioxide, and excessive amounts of ammonia. Hydrogen and carbon dioxide feed into methane production by archaea present in the rumen, whilst excess ammonia is removed from the animal host. This makes the HAPs an ideal target for potential population control, but firstly it was imperative to better understand them. This study first characterised the ammonia production phenotypes of bacterial cultures, then compared their genomes and transcriptomes to identify a signature that indicates the HAP phenotype. The work presented here has demonstrated the complexity and variability underlying the seemingly simple HAP phenotype, warranting further investigation in future work and isolation of novel HAPs in order to better understand this group before controlling the population. Phage therapy is one approach to population control that has been relatively little explored to date in the rumen. Despite phages being abundant in the rumen, there were only five genomes available of phages isolated from rumen-associated samples. This study isolated and characterised a further five novel phages that infect Butyrivibrio fibrisolvens. While the work presented here did not identify phages active against HAPs, these five Butyrivibrio phages contribute valuable information about the structure and function of the rumen ecosystem. It is suggested that continuation of this line of enquiry in future work would complement ongoing research utilising metagenomics, metatranscriptomics and metaproteomics aimed at understanding and improving rumen efficiency.

Most species of bacteria preferentially grow in sessile communities, known as biofilms, rather than as free-living planktonic cells. Biofilms can be up to 1000 times more tolerant to antimicrobials compared to their planktonic counterparts, making them a growing problem within the medical and industrial settings. Biofilms formed by Pseudomonas aeruginosa are involved in chronic infections, such as those affecting the lungs of Cystic Fibrosis patients. High or low intracellular levels of a bacterial secondary messenger, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), regulates biofilm formation and dispersal respectively. The synthesis of c-di-GMP is carried out by diguanylate cyclases (DGC) due to catalytic domains known as GGDEF domains. Whereas c-di-GMP degradation is carried out by phosphodiesterases (PDE) due to catalytic domains known as EAL domains. Low (non-toxic) concentrations of nitric oxide are known to induce a biofilm dispersal through a reduction in the c-di-GMP levels and an increase in the PDE activity. However the nitric oxide sensor and the protein responsible for the reduction in c-di-GMP levels is unknown. Previously found to be involved in an NO-induced biofilm dispersal, the bi-functional enzyme RbdA (regulation of biofilm disposal) contains a GGDEF and an EAL domain in tandem. Using an enzymatic assay measuring PDE activity, we investigate the relationship between the tandem GGDEF and EAL domains. We find that the isolated EAL domain of RbdA has a higher PDE activity, suggesting that the tandem GGDEF domain negatively influences the activity of the EAL domain. We attempt to further investigate this at the molecular level using X-ray crystallography and structure determination. The structure of the EAL domain of RbdA wasdetermined and appears to be in a primed state for substrate binding, with a single Mg2+ ion bound within the active site. After comparisons to other EAL domain structures, we suggest a schematic for substrate binding to EAL domains. We investigate an RbdA homologue, PA2072, as previous biological data indicates opposing physiological roles. By comparing the primary and secondary structures of RbdA and PA2072 we suggest that their physiological differences are caused by disparities between their periplasmic regions and/or their putative sensory PAS domains. Protein crystallisation of the PA2072 periplasmic region (a putative CHASE4 domain) and the PA2072 PAS domain were attempted but require further optimisation. The first and second PAS domain of PA0285 were predicted to bind a haem-b and FAD (flavin adenine dinucleotide) cofactor respectively. We hypothesise that NO can be sensed by a haem-bound PAS domain. Using ultraviolet-visible spectroscopy we could only identify a very weak haem-b cofactor binding to the PAS1 domain of PA0285 and so requires further investigation. However, we identified a PA0285 PAS2 : FAD binding stoichiometry of approximately 2 : 1. Here we put forward models suggesting that, NO-induced changes to the redox potential are sensed by the FAD bound PAS2 domain, leading to changes in the enzymatic output of PA0285 and potentially biofilm dispersal. Restoring the sensitivity of bacterial cells to antimicrobials by inducing a biofilm dispersal, is thought to be a novel treatment strategy. This work lays some of the foundations required to understand the molecular mechanisms that lead to a biofilm dispersal in P. aeruginosa.

Every host is colonized by a variety of microbes, some of which can protect their hosts from pathogen infection. Harbouring protective microbes (PMs) has evolutionary implications which often result in these microbes being costly for the host. Here I have investigated the evolutionary and ecological conditions under which the interaction between a host and a protective microbe are established. For this I have used populations of Caenorhabditis elegans worm hosts, bacteria possessing protective traits (Enterococcus faecalis) and pathogenic bacteria (Staphylococcus aureus). I have experimentally coevolved the host and protective microbe and infected the coevolving system at different intervals and host generations. Furthermore, I have investigated how host sex and mating status affects the interaction of the host with the surrounding bacteria. More generally, to assess how host dynamics are affected by protective microbes, I constructed SIR models in the absence and presence of the protective microbe. My results indicate that even the rare presence of the pathogen is enough to drive the evolution of microbe mediated protection, and that this is independent of the interval or initial pathogen presence. I find that both sexes use the benefits of the PM to increase their reproductive success, even though females invest more in egg production and males more in mate searching behaviour. The SIR models indicate that the protective microbe stabilises host dynamics under a range of different parameters once the cost-benefit ratio is greater than one. Overall these results suggest, that protective microbes have high potential to influence and stabilize host dynamics, even though the two host sexes might benefit differently from the provided protection.

The use of nuclear energy has been of great importance to the United Kingdom, with Sellafield being the largest nuclear site used for both power production and more recently reprocessing activities. This project, via collaboration between the Geomicrobiology Group at the University of Manchester and Sellafield Limited, aimed to investigate the microbial ecology of a spent fuel storage hyper-alkaline indoor pond (INP) in Sellafield. The main pre-reprocessing storage pond at the Sellafield site is the Indoor pond (INP), a concrete walled indoor pond filled with demineralised water, responsible for receiving, storing and mechanically processing spent nuclear fuel (SNF) from Magnox and Advanced Gas-cooled Reactor (AGR) stations from across the UK. Samples were taken from the INP at different spatial locations and depths, encompassing main ponds (MP), subponds (SP) and a feeding tank (FT). The present study intended to identify the microbial communities present in the INP and associated structures to determine if they were stable during a prolonged operational period. A more academic focus of the PhD was to understand the metabolic processes that underpin microbial colonisation and adaptation in the pond. In order to achieve these objectives, first the microbial communities from the indoor alkaline storage pond (INP) were identified to create a microbial database consisting of population density and diversity of microorganisms present. Here traditional culturing approaches were trialled but were considered ineffective for the specialised 'extremophilic' organisms present in the INP. Therefore, the bulk of the microbial analyses focused on DNA sequencing, focusing initially on amplification and sequencing of two commonly used genetic marker genes, the 16S rRNA and 18S rRNA genes that can be used to identify prokaryotic (bacteria and archaea) and eukaryotic (algae and other higher organisms). Finally, a much wider range of genes were targeted to help identify key processes that support microbial colonisation, via high-throughput 'metagenomic' sequencing and analyses. Overall, these findings are discussed in relation to microbial survival in hyper-alkaline, oligotrophic and radioactive extreme environments, and microbial adaptation over time observed during the thirty months of analysis. Organisms identified by 16S and 18S rRNA gene Illumina sequencing were predominantly Proteobacteria, mainly Alpha and Beta in the feeding tank (FT), main pond (MP) and Subpond (SP) sample sites. The presence of the alkali tolerant hydrogen-oxidising bacterium Hydrogenophaga sp. solely in the INP main ponds and subponds suggested the metabolism of hydrogen is occurring within the INP which could be generated by radiolysis of water. Metagenomic analysis revealed that genes related to membrane transport, oxidative and osmotic stress functions were more abundant on the FT possibly due to the presence of Na+ ions. Genes related to DNA metabolism (including DNA repair and defence systems) as well as genes related to respiration functions (hydrogenases) were more abundant on the MP and SP which reinforces the proposed microbial utilization of H2 as an energy source. In order to have a broader picture of the bacterial strategies to cope with extreme environmental conditions (hyper-alkaline, oligotrophic and radioactive background), few selected samples from an open-air pond, the First Generation Magnox Pond (FGMSP) and its auxiliary pond (Aux), were analysed and compared to the indoor system (INP). Results showed that genes associated to photosynthesis were more abundant on the open-air ponds, revealing that light exposure was a key energy source that promoted microbial colonisation. Additionally the final part of this research intended to identify virus-host interactions and its influence on key metabolic processes. Metagenomic analysis revealed the presence of phages inserted on bacteria affiliated to order Burkholderiales; surprisingly phages did not seem to affect metabolic responses and promote activation defence systems (CRISPR). In conclusion, microbiological and genomic analysis showed that the despite the low nutrient (oligotrophic) nature of the indoor alkaline pond, coupled with the radioactive inventory, a stable microbial community is able to survive at relatively low energy levels, using alternative energy sources, potentially hydrogen, to cope with challenging environmental conditions.

The Conwy River in North Wales, UK, is a large freshwater system that has been the subject of numerous studies over many years. Even so, the river’s microbiome is poorly understood. It is widely known that in this system, as with all lotic systems, dissolved organic matter (DOM) is the primary source of energy for these resident microorganisms. Therefore, understanding how these species utilize DOM can allow scientists to make better predictions concerning the river’s water quality. However, there are two major scientific challenges that must be considered. Firstly, due to the anthropogenic inputs (from agriculture and wastewater treatment) in tandem with the variation in flow rate and weather events, make it inherently difficult to properly model this system. Secondly, the vast majority of bacteria and archaea cannot be cultured under typical in vitro conditions. Evidence also suggests that many microbial species in aquatic systems have evaded detection due to their ability to pass through ultra-small filters (<0.45 μm pore sizes), i.e. filterable microorganisms. The term filterable microorganism can refer to one of the following: (1) small-bodied cells (less than 0.1 μm3 volume), (2) shrunken cells (due to limited nutrients or senescence), and (3) large cells that squeeze through small filters (<0.45 μm pore sizes). Their exact role in freshwater systems remain largely unknown. The purpose of this thesis was to uncover the taxonomic identity, overall function, and role in DOM cycling of filterable species residing in the Conwy River while also comparing them to the native lotic community (i.e. unfiltered population). We utilised 16S rRNA single amplicon sequencing and shotgun sequencing to conduct a phylogenetic analysis of both ultra-filtered (passed through a 0.22 μm pore size filter) and unfiltered river water to understand the phylogenies and relative phyla distributions as well as determining which clusters of orthologous groups (COGs) were present. The distribution of COGs of both microcosms were compared to other environments and bacterial genomes to (1) assess similarity, and (2) determine if organism complexity is related to environment (i.e. are more complex organisms found in nutrient rich environments, etc). Next, we examined how either microbial community utilised dissolved organic carbon (DOC) via multi-omics and 14C radio-isotope tracking in order to determine whether DOC influenced these populations or whether the residing species showed any particular preference to a DOC type. The major findings indicated that, the dominant phyla (listed in decreasing abundance) in the whole community were Proteobacteria, Bacteriodetes, Actinobacteria, and Firmicutes. Whereas, the filtered community contained more Firmicutes than Bacteriodetes and Actinobacteria. We also we detected the presence of several candidate phyla, most notably “Candidatus Parcubacteria”. There were more COGs in the filtered community that fall under the functional categories of replication, recombination, repair, and cell wall/membrane/envelope biogenesis comparatively to the entire population. Clustering metagenomes against single genomes revealed that the filtered community’s COG distribution was closely related to COG distribution of organisms with limited/streamlined genomes. The filtered microbiome also metabolized DOC at a slower rate than the whole community and was confirmed to be a taxonomically unique subset within the greater system. Changes within each community were not influenced by the addition of DOC and neither system had a preference in DOC type. Overall, the results obtained from this body of work demonstrate that the filtered microcosm was a unique population nestled within the general microbial community. They differ in taxonomic makeup and their usage of low-molecular weight DOC, suggested that they may have different functional roles in freshwater ecosystems. By exploring the complex microbiome of the Conwy River, researchers can gain a better understanding of water quality, ecosystem management, and the nature of filterable microorganisms.

I investigated aspects of a ‘payback’ hypothesis that postulates that virus infection triggers changes in host plants that render them more attractive to pollinators. This builds on previous work, which showed that in tomato Cucumber mosaic virus (CMV) infection enhanced emission of volatile organic compounds (VOCs) that attracted bumblebees and enhanced pollination of infected plants. To test if this hypothesis is tenable with other viruses and with plants other than tomato, and to determine if pollinators might derive some advantage by visiting flowers of infected plants, I used two common bean (Phaseolus vulgaris) varieties, and three bean-infecting viruses (a CMV bean isolate, Bean common mosaic virus, and Bean common mosaic necrosis virus). Commercially produced bumblebees (Bombus terrestris) and wild bees were studied, respectively, under glasshouse conditions and in the field. My data indicates that viruses can pay back susceptible hosts by attracting pollinators through changes in host-emitted VOCs and rewarding pollinators through greater nectar quantity and sucrose concentration. The enhanced pollinator attraction correlated with a recovery in seed production in virus-infected bean plants. Virus infection delayed the onset of flowering and decreased flower numbers, but it also caused bee-perceptible changes to flower petal colour, increased nectar volumes and nectar sucrose concentration. Changes in nectar volume/concentration are likely to encourage bee visitation. Gas chromatography-mass spectrometry revealed that headspace VOCs emitted by virus-infected plants were qualitatively distinct from those of mock-inoculated plants and that virus-infected plants emitted greater quantities of VOCs. In free-choice olfactometry assays, bumblebees displayed an innate preference for VOCs emitted by non-flowering BCMNV-infected plants and both non-flowering and flowering BCMV and CMV-infected plants over those from mock-inoculated plants. Where bumblebees showed no innate preference, as was the case for flowering BCMNV-infected plants, differential conditional assays showed that bumblebees were nevertheless able to perceive differences between the VOCs emitted by BCMNV-infected plants and mock-inoculated plants. I examined pollination and seed production in virus-infected bean under glasshouse conditions using B. terrestris and in the Cambridge University Botanic Garden using CMV-infected plants exposed to naturally occurring bees. Under Garden conditions, I found that common carder bees (B. pascuorum) were the main bean flower pollinators, while B. terrestris and honey bees acted as nectar thieves. Under both conditions, virus-infected plants showed a recovery of seed numbers to levels similar to those from uninfected plants if pollinators were allowed access. These observations of virus-induced effects on plant-pollinator interactions support the idea that viruses may act mutualistically with plants by making infected plants more attractive to pollinators and suggest that pollinators may also derive benefits from visiting infected plants.

Heterochromatin is a condensed conformation of eukaryotic DNA, which plays an essential role in genome homeostasis. In depth research across various species showed that RNA interference (RNAi) is one of the pathways that is important for heterochromatin formation. To date RNAi-driven heterochromatin assembly has been extensively studied in the fission yeast Schizosaccharomyces pombe. At centromeres in S. pombe, RNAi is required for heterochromatin establishment and maintenance. Unlike heterochromatin maintenance, RNAi-driven heterochromatin establishment at the centromeres is not very well understood. The interconnectedness between the RNAi and chromatin modification pathways that are required to establish heterochromatin makes it difficult to elucidate which pathway acts first. Recent studies also showed that heterochromatin establishment requires additional factors dispensable for maintenance. Thus exploring novel factors required for the de novo heterochromatin formation can help us to understand the order and the mechanism of heterochromatin establishment. In this study I developed and tested two assays ⎯ plasmid-based and cross-based assays ⎯ designed to identify novel establishment-specific factors genome-wide. While the plasmid based assay proved unreliable, the cross-based establishment assay was shown to effectively identify establishment-specific factors. It also provided new insights into heterochromatin establishment dynamics.

Bacteria are evolving strategies to survive antibiotic treatments at a pace that is not matched by the one at which new drugs are discovered. Beyond the more notorious antimicrobial resistance, other survival mechanisms such as tolerance and persistence are today thought to play a major role in infections. Whether resistant, tolerant or persistent, cells that stop growing seem to have a survival advantage over replicating ones. These cells are conventionally referred to as dormant and very little is known about their physiology and the mechanistic reasons behind their remarkable survival capabilities. Because cell growth is intimately linked to cell physiological traits such as energy availability, this work seeks to investigate cellular energetics at the single cell level using E. coli as a model system. In particular the focus is addressed to two of the most important energy parameters in all life forms: ATP concentration and proton motive force (PMF). The PMF, further than participating in ATP synthesis, fuels a number of cellular processes that play prominent roles in cellular homeostasis. Regulation of each of its two components, the pH difference across the plasma membrane and the membrane voltage, is in turn essential for cell survival. Because cytoplasmic pH can be assayed at the single cell level with the genetically encoded fluorescent sensor pHluorin and PMF as a whole can be quantified from bacterial flagellar motor speed, the focus of this work was first addressed to membrane voltage estimation techniques. Nernstian reporters have in the past been used for the purpose, but their characterization never reached the depth of detail necessary for the measurement of membrane voltage of cells in physiological conditions. Using both an experimental and mathematical approach, I explored and described the parameter landscape in which these reporters can be used as sensors and when instead they influence cell physiology. Having built and validated such a preliminary interpretative framework, I formulated an algorithm for the characterization of novel dyes with respect to their interactions with the physiology of the cell. I applied the workflow to the characterisation of a Nernstian dye that had never been used before in E. coli. Although, in my conditions, none of the Nernstian dyes available were found suitable for (Vm) estimation, the workflow I developed is in the position to offer a simple and robust method to benchmark novel dyes and test the results obtained with old ones. ATP dynamics represent another fundamental aspect of cellular energetics and measurements at the single cell level have been sought for more than a decade. The most promising approach published suffered of low signal intensities that were not compatible with the exposure times required for time series measurements. By optimizing sensor expression and performing structural modifications, I obtained improvements in the signal intensity which rendered the sensor available to time lapse measurements. To further improve signal-to-noise ratio I installed a laser in our custom-built microscope. Coupling the ATP sensor with measurements of bacterial flagellar motor speed, which correlates to PMF, and the pH sensor pHluorin, I could investigate the main energy parameters of E. coli cells in vivo, in real time and with single cell resolution. To study these physiological traits in dormant cells I first established a definition of dormancy that lies on simple axioms such as viability and growth halt. I then individuated conditions capable to sharply induce dormancy, such the presence of bacteriostatic antibiotics, quorum sensing molecules or starvation. Opposed to the classical view that sees dormancy as an energetically poor state, my results show that the observation of growth arrest alone is scarcely informative on the physiological state of the cell. Dormant cells can be both high and low in energy, depending on the conditions that induced growth halt. While highly energetical cells might be better suited at surviving antibiotics via active means, scarcely energetical ones might have less targets to offer to antibiotics to carry out their function. In any of these scenarios, this work suggests that the environmental cues that lead to dormancy are likely to dramatically alter bacteria's susceptibility to the different antibiotic classes.

Climate change is expected to alter important process operating in soil ecosystems such as microbial activity, biogeochemical cycling, and hydrological processes, and thus soil functioning and the delivery of a range of ecosystem services. Under future climate change scenarios, extreme weather events are predicted to become ever more frequent globally, with rising temperatures and concentrated rainfall events having an impact on soil functioning. It is necessary to study how changes in soil moisture status and/or temperature will affect soil respiration for predicting future changes in soil carbon (C) storage. In the scientific literature, previous studies have frequently observed a CO2 pulse from soil after a freeze-thaw or dry-wet event; however, the mechanisms underlying these effects are not well understood. The first experiment of this thesis (Chapter 3) investigated how a single freeze-thaw or dry-wet event affected microbial C dynamics using 14C tracking. Our results revealed that freeze-thaw or dry-wet events altered the allocation of C into labile and structural microbial C pools. The next experiment (Chapter 4) investigated how the C budget of an intact plant-soil system responded to freeze-thaw and dry-wet events. The presence of plants resulted in significantly greater total CO2 flux following freeze-thaw or dry-wet events in comparison to the unplanted soil. The greater CO2 efflux seen after thawing or rewetting was caused by a disruption of the microbial biomass, rather than a stimulation of soil organic matter turnover (Chapter 5). This was supported by a decrease in extracellular enzyme activity immediately after freeze-thaw or dry-wet event (Chapter 5). We also showed that soil microbes accumulated osmotic solutes (i.e. sugars and polyols) in response to extreme freeze-thaw or dry-wet events. In this thesis, the microbial community quickly responded to freezing or drying events by altering cellular metabolism (Chapter 6). The final experimental chapter (Chapter 7) investigated how future climate scenarios may affect arctic ecosystems. We monitored greenhouse gas (GHG) emissions and nutrients in soil solution throughout a year in response to a 2050 and 2100 climate warming scenario. A simulated warmer winter led to enhanced microbial decomposition of soil organic matter with increased CO2 efflux and more N becoming available to roots and associated mycorrhiza in the Arctic soils. We hypothesize that this could lead to a potential future shift in plant communities. In conclusion, this thesis present events showing that under future climate scenarios, an increase in freezethaw or dry-wet events will alter soil C and N processing in soils and disrupt biogeochemical cycling. We also conclude that the presence of plants is key in determining how ecosystems respond to these extreme events.

Antibiotic resistance is fast becoming a global health crisis, with the increase in resistant bacteria outpacing the generation and translation of new antibiotics. Bacteria at interfaces can produce a self-made architecture called a biofilm that acts as a physical barrier, significantly reducing the efficacy of antibiotics. The aim of this part of the project was to assess the material properties of developing biofilms non-invasively. Passive microrheology was used investigate biofilms produced by Staphylococcus aureus under various hydrodynamic shears and when exposed to different anti-biofilm enzymes. Biofilms grown under any shear stress were harder (i.e. had a lower creep compliance) than equivalent biofilms grown in stationary media. Furthermore, statistical analysis of the spatial arrangement of bacteria during biofilm growth revealed clustering as a function of height away from the interface surface. The cationic peptide G3 has been shown to be a potential antimicrobial peptide, however the exact mechanism of action is unknown. Two methods were used to probe the interaction of the peptide with the bacteria membrane. Firstly, a novel 3-dimensional tracking method incorporating photoswitchable fluorophores and an adaptive optics-based super-resolution imaging technique was used to investigate the spatial distribution of G3 following exposure in Staphylococcus aureus and Escherichia coli. No preferential localisation of G3 could be observed (i.e. G3 was homogeneously distributed within the cell) for both bacteria. Diffusion kinetics were approximated from short trajectories using a newly developed neural net software package; however no spatial dependences were observed suggesting a weak binding to the membrane. To further investigate the strength of the interaction between G3 and the cell membrane, solid state nuclear magnetic resonance was employed. Using a model phospholipid system, increased disorder (i.e. spatial fluctuations) was observed in bilayers exposed to G3, suggesting a transient interaction with the membrane. From this and the tracking data, we hypothesise that G3 transiently interacts broadly with the entire membrane to cause accumulative strain disruption. No significant clustering or decreases in order parameters suggesting a strong binding with the membrane were observed, however no positive control was assessed in this study.

Animal- (AAT) and human African trypanosomiasis (HAT) is endemic within sub-Saharan Africa and is caused by Trypanosoma spp. parasites vectored by biting tsetse flies. The facultative secondary symbiont, Sodalis glossinidius, has been controversially implemented in increased parasite establishment in tsetse. As the role of S. glossinidius in tsetse is not fully understood within the literature, the research presented here aimed to utilise a functional genomics approach to elucidate S. glossinidius functionality from a genetic context. Initial phenotypic-level in vitro media screening experiments confirmed S. glossinidius heterotrophy and revealed higher growth levels in the presence of glucose: S. glossinidius was unable to grow in a minimal salts medium (M9) devoid of a sufficient organic carbon source, and showed higher growth values in glucose-positive M9 variations compared to equivalent glucose-negative counterparts. This glucose utilisation was also observed with better growth between a complex medium rich in glucose (Mitsuhashi and Maramorosch Insect Medium) versus one with lower concentrations (Schneider’s Insect Medium). Subsequent genotypic-level transposon-directed insertion site sequencing (TraDIS) library selection experiments supported S. glossinidius glucose utilisation with essential gene candidacy in glycolysis, gluconeogenesis and the pentose phosphate pathway. These results, in combination within essentiality in the citric acid cycle, a wide range of carbon source metabolism pathways, and virulence-associated genes (Omp porins, flagellar components and type III secretion system constituents), experimentally confirm the sequence-inferred literature consensus that S. glossinidius has retained a functional repertoire more aligned with free-living organisms. Many of the essential gene candidates were pseudogenes, which when considered with the literature evidence that S. glossinidius is actively maintaining a core pseudogene set across lineages, experimentally supports the theory that symbionts in early stages of genome degradation associated with the free-living to symbiont lifestyle switch preference pseudogene retention. The novel TraDIS library presented here provides the currently missing tool for subsequent targeted functionality in vivo experiments, aimed at fully understanding the S. glossinidius role in the tsetse system.

Beneficial microbes have a mostly untapped potential to serve as bio-fertilizers and soil remediators in enhancing crop growth, yield and stress resistance. Understanding the molecular mechanisms of how microbes activate benefits in plants will be crucial for its application in larger-scale crop production systems. It is postulated that beneficial microbes could employ small secreted proteins (termed effectors) to reprogram and facilitate the transmission of those positive benefits in host plants. Recent studies have attempted to investigate the functions of ‘beneficial’ effectors in crop improvement. Serendipita indica, which is the beneficial fungus studied in the project, was used as a model to study the function of its effectors and reveal their beneficial activity in improving plant growth, abiotic and biotic stress resistance. The aim of this project was to investigate the roles of a selected set of S. indica effectors in single cell, whole plant and crop-based systems. In-planta analyses of S. indica effectors showed that some effectors such as SIE44, SIE76, SIE106 and SIE120 could enhance plant growth. In addition, SIE67 could be a candidate in improving plant stress resistance. In support of this, a conserved protein domain known to be important in stress resistance was identified in SIE67. In turn, SIE10 was identified as a potential effector that facilitates S. indica host colonization. To further investigate the roles of the effectors in a crop-based system, a barley transformation system was optimised to allow rapid functional analyses. This system was used to scrutinise effector functions in crucial crop improvement parameters such as abiotic and biotic stress resistance. While further confirmatory studies are needed, this research showed a first holistic functional view of effectors from beneficial microbes that could be important in sustaining crop productivity.

The Gram-negative bacterial outer membrane (OM) is an asymmetric bilayer with an inner leaflet composed of phospholipid and an outer leaflet of lipopolysaccharide (LPS). The OM forms an impermeable barrier to environmental challenges including many commonly used antibiotics. It is known that OM proteins (OMPs) do not diffuse freely within the OM. Newly inserted OM proteins are inserted at mid-cell, pushing old OM proteins to the poles with cell growth. By contrast little is known about the insertion pattern of LPS. Previous studies of LPS lateral diffusion are contradictory with both mobile and immobile behaviour observed. We have investigated the spatio-temporal dynamics of LPS using fluorescence microscopy techniques. LPS is fluorescently labeled using a metabolic labeling technique to incorporate a sugar analogue into LPS, which can be fluorescently labeled with a specific bio-orthogonal click-it reaction. Using both fluorescence recovery after photobleaching and single-molecule tracking experiments we show that LPS is unable to diffuse laterally in the OM. The factors on which this immobility depends are explored and it is shown that the immobility is independent of LPS polysaccharide structural complexity and only partially dependent on metal ion mediated LPS-LPS interactions. Using pulse-labeling methods it is shown that there is a preference for insertion of new LPS at mid-cell, as was observed for OMPs. Midcell incorporation of new material combined with the lack of diffusion in the OM leads to a mechanism by which OM material may be rapidly turned over in response to environmental changes. To further explore the parameters which control the insertion of new material into the OM a model for OM growth is developed. Preliminary results suggest that targeting of new protein and LPS insertion complexes to mid-cell is sufficient to reproduce the experimentally observed insertion of new material at mid-cell.

The adhesive behaviour of extracellular polymeric substances to poly(ethylene terephthalate), a model hydrophobic surface, were measured in response to their degradation by enzymes known for their biofilm dispersion potential. By examining the physical changes in the nature of the binding, structural or adhesive roles could be established for the targets of the enzymes. Degradation of extracellular DNA (eDNA) significantly decreased the adhesive force of Micrococcus luteus biofilms with the surface, and furthermore almost completely eliminated any components of the biofilm maintaining the adhesion. This established a key structural role for eDNA. Due to the significant results observed by the targeting of eDNA, a highly potent novel DNase was investigated to understand its mechanism of action. This would allow further optimisation of the enzyme to maximise its efficiency against a major structural component of bacterial biofilms. Rapid data collection and computer software was used to construct and validate a model of the enzyme activity. This resulted in real world conditions that must be met to maximise the activity of the enzyme, as well as providing direction for additional engineering of the enzyme's behaviour. The tools and procedures developed during the study of the model bacterium, Micrococcus luteus, were used to study the adhesive properties of two pathogens, Leishmania mexicana and Staphylococcus aureus (S. aureus). Improving understanding of the adhesive mechanisms used by these pathogens allows for the development of new treatments against them. Custom MATLAB scripts enabled new data analysis of the interaction between Leishmania parasites and galactose-coated AFM tips. This helped elucidate the binding changes used by the parasite as it matures and becomes infectious. Biofilm cantilevers were modified to examine a potential skin treatment that has the potential to decrease the adhesion of S. aureus to epithelial cells. A decrease in peak adhesion of 52 % was observed by force experiments between a biofilm-coated cantilever and treated human epithelial cells.

Despite an increasing amount of evidence acknowledges the importance of eukaryotic microbes in aquatic food webs, human / wildlife health, climate and biogeochemical cycles; their diversity is still widely underinvestigated. This statement is particularly truthful in the case of heterotrophic protists, more difficult to establish as clonal cultures in lab conditions than their phototroph counterpart. Only with the recent application of molecular ecology methods, we have started to gain deeper insights in the structure of complex microbial communities where heterotrophic protists, and especially putative parasites, resulted as an abundant and diverse component in both aquatic and terrestrial ecosystems. Diatoms, on the other hand, have long being recognised as one of the most successful group of phytoplankters and their role as main primary producers at the base of the food web in aquatic ecosystems is well-established. This thesis aims at investigating the link between heterotrophic eukaryotes and diatoms, by focussing on osmotrophic oomycete and fungi, two well-known groups of pathogens sharing similar molecular weaponries and habits despite their large evolutionary distance. By developing a method to isolate and molecularly characterise single cells (SCs) we describe nine novel oomycetes infecting bloom-forming and toxic planktonic diatoms, as well as epiphytic ones, and provide a first analysis of their distribution worldwide. Oomycete parasites infecting the toxic diatom genus Pseudo-nitzschia have been investigated in the field via DNA metabarcoding, highlighting swift parasitic outbreaks and suggesting a role in the regulation of diatom blooms. The same method resulted in the first molecular identification of a transatlantic distributed chytrid infecting the spring bloom-dominating diatom Skeletonema, advocating for a high ecological relevance for this parasite. Finally we characterised a stable cultivated pathosystem involving the freshwater diatom Asterionella formosa and a chytrid parasite, and started investigating the physiology of this interaction by means of RNA sequencing and transmission electron microscopy (TEM). Overall this thesis highlights an unsuspected cryptic diversity hidden within morphologically similar parasites of diatoms underpinning a high complexity of interactions, which cannot be appreciated but by integrating microscopy observation, SC molecular analysis and investigation of cultivated pathosystems.

The ever-increasing population and resource demand are putting a stress upon the planet's resources. This increased demand places an even greater need today for the exploration of alternative, greener fuels that can aid in the alleviation of the traditionally used fossil fuels. One such method is in the production of acetone, butanol and ethanol (ABE) by the bacteria genus Clostridium spp. These gram-positive anaerobic bacteria were first characterised in the late 19th centaury and have been used throughout the 20th and 21st centauries for their solvent producing capability, most notably in the supply of weapons grade acetone during the first world war. After falling out of favour in the last half of the 20th century due to competition with cheaper and more readily available petrochemicals; interest in ABE production via Clostridium spp. has been on the rise in recent years as the ABE fermentation is investigated for its potential as a greener more renewable source of fuel production. As interest in ABE fermentation has been on the rise in recent years, so too has our understanding of the genus as a whole. Traditionally C. Acetobutylicum first described by Chaim Weizmann in the early 20th centaury has been the industrial strain of choice. However, as the overall understanding of the strains has improved other strains have been explored for their industrial relevance. These are largely split into two characterisations, autotrophs who are able to fix CO2 and CO, converting them acetyl-CoA for solvent production and heterotrophs who are able to metabolise hexose sugars in solvent production. The strain used in this study is Clostridium saccharoperbutylacetonicum N1-4(HMT). Clostridium saccharoperbutylacetonicum N1-4(HMT) is a heterotrophic Clostridium species first described by (Hongo and Ogata, 1969) . Herein we have utilised CLEAVEÔ, this is a CRISPR/Cas system developed by Green biologics ltd. CLEAVEÔ was used for the deletion of the gene gapN from the genome of Clostridium saccharoperbutylacetonicum N1-4(HMT). GapN is a cytosolic nonphosphorylating NADP-dependant GAPDH that catalyses the irreversible oxidation of glyceraldehye-3-phospate (G3P) to 3-phospholycerate. Deletion of gapN causes a reduction in acid production, an increased rate of solvent production to pre-toxic concentrations, as well as an increase in ATP and ratio of NADH:NAD+. Additionally, the deletion of gapN results in an increase in formic and lactic acid production that is believed to be as a result of pyruvate accumulation in response to the earlier shift into solventogenesis in gapN deletion strain.

Bacteria adhere to almost any surface. Medical-device biofilm-centred infections pose an enormous threat, particularly from multi-antibiotic resistant pathogens. Biofilm formation is largely understood and encompasses an initial,reversible bacterial cell surface-attachment phase followed by irreversible surface-attachment, leading to the formation of biofilms that can be up to 1,000 times more resistant to antibiotics and refractory to host immune defences. A better understanding of bacterial-surface interactions should aid human intervention into this process to reduce biofilm formation on implanted medical devices. Previously, Hook et al. screened an (meth)acrylatepolymer microarray in high-throughput for polymers that prevent biofilm formation by a selection of bacterial pathogens including Pseudomonas aeruginosa. This resulted in the discovery of poly(ethylene glycol dicyclopentenyl ether acrylate)(pEGdPEA) which has recently been approved for human use as a coating (BACTIGON®) for urinary tract catheters. However, the mechanism(s) by which pEGdPEA prevents biofilm formation is not known beyond the observation that it does not inhibit bacterial growth. This thesis describes the response of bacteria to pEGdPEA in comparison with that of a biofilm-promoting polymer, poly(neopentyl glycol propoxylate diacrylate polymer) (pNGPDA). Fluorescence and electron microscopy after 24 h of incubation with P. aeruginosa showed that little extracellular matrix formed on pEGdPEA in contrast with pNGPDA where a robust biofilm was observed. A method for spatio-temporal characterisation of bacterial motility at and above surfaces was developed using a custom designed microscope that allowed the interaction of Pseudomonas on different materials to be followed over time. This multimode 2D-3D microscope utilised several optical techniques simultaneously with the ability to record stable video data of individual bacterial cells: namely digital holography, differential interference contrast (DIC), TIRM (total internal reflection microscopy),TIRF (total internal reflection fluorescent microscopy) and widefield epifluorescence microscopy. The behaviour of large numbers of bacterial cells was characterised simultaneously from videos using high-throughput motion analysis algorithms. To study bacteria surface adhesion strength on different chemistries a method was developed using microfluidics and xurography or razor writing to form channels. P. aeruginosa cells showed weaker surface adhesion strength, moved faster and with shorter residence times on pEGdPEA compared with pNGPDA. P. aeruginosa cells were also observed, post cell-division, to leave the pEGdPEA surface with a higher frequency than from the pNGPDA surface. By using a cdrA::gfp fusion as a cyclic diguanylate (c-di-GMP) biosensor, the inability of P. aeruginosa cells interacting with pEGdPEA to increase c-di-GMP levels was observed and contrasted markedly with the rapid induction of high levels of fluorescence in cells on pNGPDA. Biofilm-forming bacterial cells are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on the extracellular polymeric substance (EPS) which is known to affect surface adhesion. Here, by combining EPS staining and bacterial tracking, P. aeruginosa was shown not to deposit exopolysaccharides on pEGdPEA, in contrast to that observed on pro-biofilm pNGPDA. These data, together with weaker adhesion strength, higher frequency of detachment events and lower c-di-GMP intracellular levels, suggest that on pEGdPEA, P. aeruginosa is unable to switch from the reversible to irreversible attachment stage. The key signalling and sensing pathways used by P. aeruginosa to respond to surfaces were investigated and the data obtained suggest that bacteria actively “decide” whether to attach to a particular surface and that the decision not to form a biofilm on pEGdPEA is likely to involve the sadB pathway. SadB is a cytoplasmic protein with an, as yet, unknown function but its upstream regulatory pathway involves c-di-GMP signalling. How this protein is involved in the ability of bacteria to maintain a surface-associated state which leads to an irreversible attachment is described. Bacteria have been shown to clump as multicellular aggregates during the process of biofilm formation either in bulk (as planktonic aggregates) or at the surface. Here differential planktonic aggregation with respect to pEGdPEA and pNGPDA is described where greater aggregation was observed on the latter. The formation of aggregates in the bulk liquid phase above these polymers was characterised using bright-field microscopy and laser diffraction analysis. The differential aggregation observed was also examined for ‘aggregation inducing’ signals by analysis of the cell-free spent media using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Comparative metabolomics enabled the identification of the aggregation-inducing signal N-(3-oxododecanoyl)-L-homoserine lactone, which was at a significantly lower concentration in spent medium from cells previously exposed to pEGdPEA compared with the pro-biofilm pNGPDA. These data provide new insights into the understanding of how bacteria, after being in contact with different surface chemistries, are able to alter their bulk liquid phase behaviour. Additionally, this highlights the potential of novel use of these polymer surfaces to investigate bacterial biofilm development and to develop new solutions for the prevention of bacterial biofilm-centred infections.

The genus Clostridium comprises a range of physiologically diverse species including notorious pathogens as well as industrially relevant organisms. Solventogenic Clostridia produce solvents via their unique acetone-butanol-ethanol (ABE) fermentation metabolism and encompass species of significance for future biofuel production. Typically these organisms undergo an initial acidogenic phase which is followed by a solventogenic phase as they enter stationary growth. The later phase also sees a morphological change in these organisms as they accumulate storage compounds and endospores. With an increased need for sustainable fuel production, solventogenic Clostridia offer an attractive alternative to produce biobutanol. An intrinsic problem with these organisms however is the phenomenon of spontaneous strain degeneration. This phenomenon is characterised by a reduced or complete loss in the production of solvents and the formation of spores which typically occurs when the bacteria are repeatedly subcultured in batch culture or grown in continuous fermentations. The aim of this study was to gain a further understanding of the degeneration process by investigating the genetic, ecological and evolutionary forces behind this phenomenon. To do this, Clostridium beijerinckii NCIMB 8052 was selected as a model organism due to its tendency to degenerate more rapidly than other strains. Furthermore, this strain was progenitor to a hyper-butanol producing strain making it a relevant candidate for industrial use. Through repeated subculturing of C. beijerinckii NCIMB 8052, four distinct colony morphologies emerged. A total of 71 degenerate isolates were obtained based on their colony appearance. These were phenotypically characterised in regards to their solvent, spore and granulose producing capabilities. The most severe of the colony types was unable to produce solvents or endospores. Genetic comparisons between derived isolates and their parental ancestor revealed four regions that contained more mutations than anywhere else in the genome. These regions included the master regulator gene of solvent and spore formation spo0A and two notable histidine kinases. The role of these genes was proven via insertional disruption which showed a huge reduction in sporulation capacity. Social interactions were studied by mixing degenerate isolates with the wild type in various starting frequencies. At low frequencies, isolates were much fitter than the wild type however this fitness rapidly declined as the starting frequencies increased. This frequency-dependent fitness is suggestive of social cheating. These findings led to the hypothesis that degenerate isolates have gained an increased fitness compared to the wild type and this fitness is due to the loss of Spo0A activity.

Within the human body, microorganisms reside as part of a complex and varied ecosystem, where they rarely exist in isolation. Bacteria and fungi have co-evolved to develop elaborate and intricate relationships, utilising both physical and chemical communication mechanisms. Mucorales are filamentous fungi that are the causative agents of mucormycosis in immunocompromised individuals. Key to the pathogenesis is the ability to germinate and penetrate the surrounding tissues, leading to angioinvasion, vessel thrombosis, and tissue necrosis. It is currently unknown whether Mucorales participate in polymicrobial relationships, and if so, how this affects the pathogenesis. This project analyses the relationship between Mucorales and the microorganisms they may encounter. Here we show that Pseudomonas aeruginosa culture supernatants and live bacteria inhibit Rhizopus microsporus germination through the sequestration of iron. Therefore, treatment of P. aeruginosa in a patient could result in the release of this inhibition, leaving the patient more susceptible to an underlying fungal infection. However, P. aeruginosa responds to the presence of R. microsporus by enhancing siderophore production, which increases host mortality in a zebrafish co-infection model. This project highlights the complex competition between these organisms and the possible enhanced disease pathology when R. microsporus and P. aeruginosa meet in a host.

Chlamydomonas reinhardtii possesses a single-cell-based CO2-concentrating mechanism (CCM). The CCM is an important element of algal photosynthesis, metabolism, growth and biomass production, which works by increasing the concentration of inorganic carbon (Ci) in the pyrenoid, a dense RuBisCO-packed structure within the chloroplast. This suppresses RuBisCO oxygenase activity and associated photorespiration. The enhanced efficiency of CO2 assimilation in the pyrenoid via CCM had been modelled theoretically as a requirement for successful CCM in higher plant systems. The ultimate aim of my research is to understand the biogenesis of the pyrenoid using a set of CCM mutants with pyrenoidal defects. Immunofluorescence methods and spot growth tests under different CO2 concentrations were performed on mutants with CCM defects generated by an insertional mutagenesis screen. Morphological and physiological characterisation of these mutants revealed differences in the pyrenoid morphology, the ability for RuBisCO to aggregate into the pyrenoid and the formation of thylakoidal tubule network associated with the pyrenoid. The thylakoid tubule network may be linked to the transport of inorganic carbon into the pyrenoid as part of the CCM. Further characterisation of one of the mutants gave rise to the hypothesis that the gene of interest, Cre11.g467712 (SAGA), is a multi-functional anchor protein related to the structural formation of the pyrenoid and may be another essential component of the pyrenoid.

Microbial communities are highly complex systems, yet are assembled from basic building blocks of some of the simplest organisms on Earth. We currently have ample information on many individual microbial taxa, but we lack fundamental understanding of how complexity emerges as microbial communities are assembled. As microorganisms almost always exist in complex communities, a series of experiments were implemented in order to study the factors involved in community assembly. Here, focus was placed on investigating two assembly processes described by the metacommunity concept: neutral assembly, dominated by stochastic processes, and species sorting, where the environment selects for the emerging complex community. The process of assembling a complex microbial community on different rock substrates was studied in a series of interlinked experiments. In an experiment examining colonisation of two end-member igneous rock types over the course of 1.5 years, it was hypothesised that neutral processes would dominate at the outset, with environmental selection and thus species sorting becoming more important with time. The results indicate that the opposite is true: the communities are selected for at the outset and converge through neutral processes to a more complex community as the environments become more similar over time. Other experiments were set up in order to probe different factors controlling the assembly of complex microbial communities. Microbial environmental engineering was studied by investigating microbially-mediated rock weathering and its effect on the emerging community. The role of priority effects in building a complex community from simple building blocks was investigated using strains isolated from the colonisation experiment, by mixing together single isolates with some time lag into a co-culture. Lastly, the impact of environmental perturbation on viability of communities at different stages in the assembly process was studied using stresses such as freeze-thaw and desiccation. Together, these experiments have given greater insight into the various factors that influence the assembly of a complex microbial community.

Bacterial colonies and biofilms play an important role in many aspects of human life. Communities formed by harmful bacteria are responsible for human and animal infections and food spoilage, but beneficial bacteria find applications in water treatment and other waste processing. Rather unsurprisingly, a great deal of research has been undertaken into all aspects of their nature, especially into biofilm control and prevention. Traditional approaches to control bacterial growth have focused upon using chemicals to kill bacteria or inhibit their replication, such as antibiotics. However, bacteria can quickly develop resistance to antibiotics. This is because resistant mutants that emerge spontaneously in bacterial colonies outgrow sensitive cells. This process of Darwinian selection is making many antibiotics ineffective against infections. Recent work on evolution in bacterial populations has shown the importance of the spatial effects (colony shape) upon the fixation probabilities of mutants and the overall fitness of a population. For example, the roughness of a bacterial colony's front significantly affects the fixation probability of a new mutant. These and other studies have come to recognise the significance of mechanical interactions upon the colony morphology. This thesis considers how mechanical interaction can effect the colony shapes in two comparatively simple systems of confined, immotile E. Coli colonies. The first system (reffed to as "quasi-2D") is micro-colonies (< 500 cells) initiated from a single cell and grown at the interface of agarose gel and a glass slide. The second system studied (reffed to as "submerged") is colonies initiated from cells placed within the bulk of a stiff (> 0:6% w/w) agarose gel. In the "quasi-2D" system it has been found that different micro-colonies can vary greatly and apparently randomly in shape under identical growth conditions. In the "submerged" system, colonies are found to grow into oblate spheroid-like ("smartie") shapes. Here I use computer simulations and continuum mechanics approach to understand the origin of these shapes. Both systems are initially investigated by individual based models (IBMs). All IBMs used are based upon a "null" model simulating growth, division and contact between cells, to which other interactions are added as required to reproduce the experimental results. In particular I consider: cell-cell adhesion; cell-substrate adhesion; asymmetric friction; ageing of cells friction; a shifting center of mass during growth; compression from agarose; and phenotype switching between "sticky" and "non-sticky" cell types. Most interactions (cell-substrate adhesion, asymmetric friction, and variants thereof) are found to have some significant effect upon the colony shapes, but distributions of colony shapes do not correlate well with the experimental distribution. One interaction, cell-cell adhesion, is found to have no significant effect on the colony shape within a reasonable parameter range. The remaining interactions, compression from agarose and phenotype switching are capable of replicating the experimental colony shape distributions. In the case of the second system of "smartie" colonies, the colony shape appears to be principally determined by the interaction with the agarose gel. This is demonstrated in 2D simulations via a bead and spring agarose model added to the "null" model of bacterial colony. To get qualitatively similar shaped colonies to those seen experimentally, springs that represent agarose fibres must be robust and not break easily. Additionally, the model is able to qualitatively reproduce the experimental variation in the smartie aspect ratio with agarose gel concentration. To expand on simulations of "smartie" colonies a dynamical continuum model of agarose fracture is developed. The model predicts that cavity shape is significantly affected by the colony growth law (i.e. linear, exponential or cubic in time) and by critical strain. In particular, "smartie" shapes are only expected for cubic and sub-cubic colony growth laws. Other parameters of the model such as agarose or bacterial Young's moduli are also found to affect the cavity shape but to a lesser extent. The model also qualitatively reproduces the time dependence of the experimental colony shape for a growth law fitted to an experimental growth curve.

Limiting the availability of metals in an environment is known to restrict bacterial growth and proliferation. For example, humans sequester metals to help prevent infection by pathogens, a system termed nutritional immunity. Chelators are small molecules that bind tightly to metals and thus have antibacterial properties that mimic these innate immune processes. This activity of chelators has not been studied extensively, although experiments with EDTA suggest that it disrupts bacterial membrane permeability by stripping lipopolysaccharide from the bacterial outer surface, possibly due to the stabilising Mg2+ and Ca2+. The work described here examines in detail the antibacterial effect of 11 chelators on Escherichia coli and how this relates to cellular starvation. Four distinct effects on cellular metal content were found with these chelators i) no change, ii) reduction to manganese, iii) reduction in zinc, and iv) reduction in iron combined with an increase in manganese. There was limited correlation between chelant metal affinities in solution with effects seen in cells. The chelants also exhibited variation in antibacterial efficacy, which was enhanced when used in combination, most yielding synergistic or additive effects. These chelants therefore offer significant potential as tools to probe metal homeostasis systems and as antibacterials. EDTA, DTPMP and Octopirox were studied further by screening their effects on growth using a selection of E. coli mutants. DTPMP and Octopirox have similar effects on cellular metal content, depriving cells of iron and inducing uptake of manganese; mutant data suggests that DTPMP primarily affects Fe3+ uptake, while Octopirox Fe2+. All three chelators also seem t have effects on oxidative damage and tolerance, especially EDTA which deprives cells of manganese. The E. coli transcriptional response to EDTA was also investigated by RNA-SEQ. EDTA has wide-ranging effects on cellular metabolism, upregulating genes involved in carbon utilisation, energy production, translation and transcriptional regulators, including some iron-sulphur cluster proteins. Overall, the results offer the first detailed insight into the antibacterial effect of a structurally diverse group of chelants and the first step in understanding the relationship between metal affinity and their antibacterial mechanisms of action.

Nitrogen is an essential component of the bacterial cell, thus rapid adaptation to nitrogen starvation is essential for survival. In Escherichia coli and related bacteria the response to nitrogen starvation is initially coordinated by transcriptional reprogramming, and occurs through signalling from the NtrBC two component system, where NtrC is the master transcriptional regulator required for σ54-dependent transcription. This NtrC-coordinated response has been extensively studied and is initially required for "scavenging" of alternative nitrogen sources. The yeaGH operon is one of the most highly upregulated and only uncharacterised operon expressed in an NtrC-dependent manner upon nitrogen starvation. Previous work in the laboratory revealed that the eukaryote-like serine/threonine kinase, YeaG, is required by E. coli to cope with sustained nitrogen starvation. However, the molecular basis by which YeaG achieves this remains elusive. The main objective of the work presented here was to expand on how YeaG contributes to adaptation to sustained nitrogen starvation by using a transcriptomics based approach. Results revealed that YeaG is required throughout nitrogen starvation for repression of two energetically costly pathways, flagellar and methionine biosynthesis, in a temporal manner. Understanding how YeaG affects temporal transcriptome dynamics inspired further research into how the transcriptome is affected during adaptation to sustained nitrogen starvation. Utilising global transcriptome studies, two genes, abgR and yedL, were identified to contribute towards maintenance of viability during sustained nitrogen starvation, and revealed new avenues for investigation of temporal transcriptional adaptation to stress. Overall, this study suggests that adaptation to sustained nitrogen starvation is likely dynamic, and is required for optimal adaptation and maintenance of survival, whilst being prepared to resume growth once nitrogen becomes available. YeaG is required in this adaptive process to act as a temporal metabolic brake in repressing energetically costly pathways, required for survival.

A multitude of microorganisms possess the ability to metabolise molecular hydrogen (H2). The major enzyme family involved in hydrogen metabolism are Hydrogenases. These enzymes catalyse the reversible conversion of molecular hydrogen to protons and electrons (H2 ↔ 2H+ + 2e-). These enzymes have the potential to be utilised for biotechnological applications such as hydrogen fuel cells, but they also represent promising drug targets for inhibition of bacterial energy metabolism both within the gastrointestinal tract and after infection. Therefore, further understanding and discoveries made in the hydrogenase field warrants progression into applied medical and biotechnological research areas. Hydrogenases are also interesting due to their phylogeny and physiology in a large number of microbial species. These enzymes are categorised by their active site architecture. One well studied, ancient group is termed the [NiFe]-hydrogenases, which all harbour a complex NiFe(CN-)2CO active site in the 'large' catalytic subunit and usually have three iron-sulfur clusters within a 'small' electron transferring partner subunit. [NiFe]-hydrogenases have undergone massive diversification, with four major phylogenetic subgroups arising. The major part of this Thesis concerns work on a Group 4 [NiFe]-hydrogenase that functions in partnership with a formate dehydrogenase as a formate hydrogenlyase (FHL). This FHL complex generates H2 and CO2 from the disproportionation of formate (CHOO- + H+ ↔ H2 + CO2). In this Thesis, genetic and biochemical characterisation of Pectobacterium atrosepticum SCRI1043, a potato pathogen, led to the identification of a novel FHL complex. The [NiFe]-hydrogenase in this organism is similar to that of Escherichia coli Hydrogenase-4, with an extended membrane domain similar to that of respiratory Complex I. Importantly, the P. atrosepticum formate dehydrogenase is selenium-free, while previously characterised FHL complexes have selenocysteine-containing formate dehydrogenases. Using genetic and biochemical approaches it was shown that the [NiFe]-hydrogenase and a formate dehydrogenase were vital for H2 production by P. atrosepticum. Using plant infection assays it was also shown that the gene encoding the formate dehydrogenase was important for full infective ability of P. atrosepticum in potato plants and tubers. The latter part of this Thesis focuses on developing genetic tools to study this novel FHL from P. atrosepticum as well as Hydrogenase-1 and -2 from E. coli.

A biofilm is a state where bacteria are attached to a surface to form sessile communities. Cells in the sessile communities have a higher tolerance to antibiotics and sanitizers. Correspondingly, the persistence of bacteria can lead to infections and outbreaks of foodborne diseases. Listeria monocytogenes is a Gram-positive Firmicute that causes foodborne infections. Frequently, infection is caused by ingesting food that has been contaminated in processing plants. L. monocytogenes can form biofilms on machinery in food processing facilities, which further leads to outbreaks of infections. This study aimed to understand the molecular mechanism of biofilm formation by Listeria monocytogenes. It is known that mutations can be unintentionally introduced or accumulate in the chromosome of laboratory-adapted strains and affect the phenotypes displayed by the strains. Therefore, four isolates of a commonly used laboratory-adapted L. monocytogenes strain, EGDe, were collected. It was hypothesised that these variants may have different biofilm-forming abilities. Through the detection of mutations in the genome, the molecular mechanism of biofilm formation by L. monocytogenes could therefore be uncovered. In this project, I first examined five different phenotypes displayed by the EGDe collection and resequenced the genome. Variations in the phenotypes and genotypes between the isolates were discovered. I next examined the biofilm-forming ability of these four isolates using crystal violet staining and scanning electron microscopy. Among the four EGDe isolates, reduced biofilm formation was detected in one EGDe isolate compared. The analysis of the genomic profiles showed that this isolate contains nonsense SNPs in lmo0184, rsbU, and rmlA. To examine the effect of each SNP on biofilm formation, I constructed single gene deletions in the defined reference strain. The biofilm formed by the mutant strains revealed two genes involved in biofilm development: rsbU and rmlA. RsbU is the upstream regulator of SigB, a sigma factor. Either ΔrsbU or ΔsigB mutant strains had an 'enhanced' profile of biofilm formation. On the other hand, removing RmlA, which is the first enzyme for TDP-L-rhamnose biosynthesis, displayed a reduction in the amount of biofilm formed. Further analysis of biofilm formation of the rmlT mutant strain, which still contains TDP-L-rhamnose production but not the rhamnose decorated wall teichoic acid (WTA), showed that the decorated WTA is required for adhesion of cells to surface. Finally, deletion of rmlA can override the effect of rsbU deletion on biofilm formation. In brief, I identified that (1) removing rsbU or sigB induces biofilm formation and (2) sugar decorated WTA enhances cell-to-surface interactions.

Biofilms are communities of microorganisms attached to a surface and encompassed within a self-produced extracellular matrix. The matrix functions to shield the interior cells from biotic and abiotic stresses and to retain nutrients and signalling molecules. The multiple functions of the biofilm matrix are mediated by the constituent parts which may include a combination of complex polysaccharides, lipids, nucleic acids and proteins. Some of the proteins found within the matrix are polymeric, with examples including the flagellum, pilus and amyloid-fibres. There is a growing list of proteins in the matrix that are categorised as "amyloid-like", including the Enterobacteriacae Curli, Staphylococcus aureus Bap, Pseudomonas Fap and Bacillus subtilis TasA protein fibres that give structure to the respective biofilms. However, the categorisation of 'amyloid-like' to fibre-forming proteins of the biofilm matrix requires careful consideration. This thesis presents evidence that the B. subtilis TasA fibres are not amyloid in form. Through the comparison of the biochemistry and structure of a functional TasA fibre to the canonical amyloid fibre, we propose a linear assembly of TasA monomers within the fibre. The ongoing collaboration of molecular microbiology at the University of Dundee and protein biophysics at the University of Edinburgh throughout this project has also led to the discovery and characterisation of TasA surface activity and preliminary investigations of the molecular mechanism imparting surface activity are also presented.

Biofilm formation is a significant mechanism by which pathogens are able to evade the human immune system and to be unresponsive to antimicrobial treatments. Indeed, biofilm formation is a major contributor to antimicrobial resistance (AMR), a current and growing clinical problem around the world. Herein, the synthesis of linear and cyclic polyamines containing different spatial distances between the amino groups were designed on the basis of the chemical structures of certain naturally occurring polyamines. The complete reduction of aliphatic nitrile functional groups to primary amines can be performed via catalytic hydrogenation using Raney nickel catalyst under 1 atm pressure of hydrogen and in basic media. The NMR spectroscopic data of the synthesised polyamines that have not been fully assigned, or indeed are mis-assigned in the literature, were fully and typically unambiguously assigned. Then their biological activities in preventing biofilm formation and for the dispersal of existing biofilms, with and without combination with the antibiotic vancomycin, were investigated. Two novel polyamines 33 and 35 show activity in preventing biofilm formation in NCTC 6571 and MSSA 15981 strains. In the NCTC 6571 strain, each of these two polyamines shows high activity when used in combination with vancomycin achieving higher activity in the killing of bacterial cells inside preformed biofilms than the use of vancomycin alone. Polyamine 49 shows antibacterial activity and prevents biofilm formation in the above two S. aureus strains and in the MRSA 252 strain, and shows high activity in the killing of bacterial cells within preformed NCTC 6571 biofilms in combination with vancomycin. These results are a contribution to the fight against AMR. Whilst simple linear polyamines do not exhibit the biological activity some have claimed in this research area, these three polyamines 33, 35, and 49 certainly do show potential, at least in a bacterial strain dependent manner.

Resistance to the bactericidal effects of bile is crucial for the survival of Staphylococcus aureus in the human gut. This study was conducted to identify and characterize components of the bacteria, which allow it to resist bile acids. A comparative study was used to investigate the natural protein diversity within the Staphylococcus in relation to bile resistance. Imaging of one-dimension gel electrophoresis showed a unique protein band in samples prepared from bile-treated S. aureus. Mass spectrometry and database analysis showed the protein to be FadB. which has a role in lipid metabolism in Escherichia coli. It is hypothesized that fadB was responsible for the observed bile salt resistance phenotype; to test this, a ΔfadB strain was created in S. aureus SH1000. The mutant phenotype showed a significant decrease in viability upon exposure to bile acids in comparison with the parental wild type. Furthermore, survival of S. aureus ΔfadB was attenuated in an in vitro human colonic model, implicating fadB in S. aureus colonization of the human intestine. Moreover, upregulated expression of fadB was detected upon exposure to bile salts. To further confirm the role of FadB in bile salt resistance, the gene was cloned under the control of an inducible promoter, which enabled arabinose-dose dependent expression of fadB in E. coli JW113 as a heterologous host, confirming a bile resistant phenotype. Recombinant FadB was purified and shown to have affinity for cholic acid and might possess an ability to modify bile salts.

Individuals across the tree of life make costly contributions to resources that benefit the group as a whole. However, such ‘public goods’ come with a problem; a selfish individual could refrain from contributing to public goods, instead leeching off the contributions of others. How does cooperation stay stable in the face of such exploitation? This problem of the maintenance of cooperation is commonly understood through the ‘tragedy of the commons’, with resolutions to the problem largely focused on avoiding the individuals who can undermine cooperation – cheaters. In this thesis, I counter the perspective of cooperation being most vulnerable to ‘cheater’ individuals who contribute nothing, aiming instead to highlight the problem caused by the strategic (i.e. conditional and quantitative) behaviour of all individuals. To this end, I use the model organism of the social amoeba D. discoideum as an empirical system to test new models of strategic behaviour, and back an argument for the importance of conditional and quantitative contributions in the evolution of cooperation in public goods. I develop a theoretical framework of the public goods game, and empirically test its utility to predict social behaviour in simple and complex social groups, finding a close match between model predictions and empirical data (Chapters 1-2). Further, I demonstrate the important consequences of strategic contributions for how we think about conflict in public goods (Chapter 3) and how genetic self-recognition (in D. discoideum and beyond) can occur through the ‘greenbeard’ effect, which has previously been considered highly unlikely to occur in nature (Chapter 4). My work in this thesis combines theory and data to demonstrate that cooperation and conflict can be misunderstood by a binary ‘cooperate’ vs ‘cheat’ perspective, and are instead better understood through the more complex idea of conditional and quantitative strategies of all individuals shaping the patterns of cooperation and conflict we see in nature.

Burkholderia thailandensis is widely used as a non-pathogenic model of Burkholderia pseudomallei, the causative agent of melioidosis, a disease with a 90% mortality rate if untreated. One of the histopathological features of melioidosis is the presence of multinucleate giant cells (MGC). MGC formation occurs when the plasma membrane of infected cells become closely positioned with other cells, which then become attached and fuse together. Many studies have described how effector systems of B. thailandensis can induce the formation of MGC. It seems that these bacterial effectors can regulate this process by affecting the expression of mammalian membrane proteins, including the tetraspanins (Tspans) superfamily. Tspans are a large family of membrane proteins that bind partner proteins to form Tspan-enriched microdomains (TEM) that have many biological roles, including cell fusion, adhesion, and bacterial infection. Mammalian cells express 33 Tspans, but their specific functions during MGC formation and pathogen infection are not fully understood. Here, we have attempted to define the roles of all Tspans and some Tspan-partner proteins in B. thailandensis infection and/or MGC formation. We found that 5 Tspans are specifically involved in MGC formation induced by B. thailandensis: Tspan-2, Tspan-5, Tspan-13, CD81, and CD9. 3 Tspan-partner proteins are also specifically involved: ADAM10, CD98, and CD172α. Using antibodies, an inhibitory peptide derived from CD9 and knockouts and knockdowns of Tspans and their partners, we attempted to elucidate the roles of these molecules in MGC formation. It was observed that CD9, CD81, CD172α, and ADAM10 have negative roles in MGC formation induced by B. thailandensis whereas Tspan-2 and Tspan-13 play positive roles and could also be required for B. thailandensis infection. CD98 also has a positive role in MGC formation but has no role in B. thailandensis infection. It was also found that the peptide derived from CD9 could reduce the total number of bacteria of B. thailandensis, and internalisation after 2 and 18hr.

The market for solvent production is predicted to reach $43.4 billion in 2018, with n-butanol having over 20% market share value where n-Butanol is the chemical precursor of several industrially important products, such as butyl-acetate, butyl-acrylate, glycol-ethers, and plasticisers. Butanol is currently produced from crude oil, and therefore in light of dwindling fossil fuel reserves, and more importantly, the need for green and clean production processes, synthesis of bio-butanol from biomass using Clostridia represents a viable and desirable alternative method. This project focuses on the metabolic and physiologic characterisation of the acetone-butanol-ethanol (ABE) producing species Clostridium saccharoperbutylacetonicum (Csb). A minimal medium for Csb was defined based on literature data, modified by the addition of glutamate to support growth. Interestingly, batch cultures using this medium showed that Csb was able to grow and produce butanol under aerobic conditions, with titres of approximately 74% of those observed under anaerobic conditions. Steady state cultures in chemostats are essential to elucidate and characterise physiological features of microorganisms. Steady state cultures of Csb were used to determine the effect of acid production on solventogenesis, bacterial growth, and energy metabolism. Studies at different pH in the range 5.5 to 6.5 showed no correlation with the onset of solventogenesis. However, the pH and the growth rate seem to influence the productivity of butanol. In those experiments, significant increases in the production rate of butanol were observed when the dilution (growth) rate increased from 0.01 h-1 to 0.03 h-1 and the pH decreased from 6.5 to 5.5. Growth is potentially linked to production rate due to an increased demand for ATP and NADH recycling. The use of genome scale metabolic models allows for the interpretation of metabolic and physiological changes upon changes in the culture conditions. A metabolic model of Csb was constructed based on the genome sequence of the microorganism and incorporating biomass synthesis equations specific for Csb which were constructed based on the analysis of the composition of the cells grown in the chemostat experiments, as opposed to current models that use biomass composition from related species (e.g. B. subtilis). The metabolic model was used to perform flux balance analysis to identify and interpret the changes in the distribution of metabolic fluxes that would explain the metabolic changes observed in Csb cultured under different conditions. This work has demonstrated the basis for the presence of monophasic solventogenesis in C. saccharoperbutylacetonicum and provided important tools (defined media, GSMN equations) to improve industrial scale production of renewable sources of carbon-based feedstocks and thus reducing reliance on crude oil.

Wood recycling is key to biogeochemical cycling and largely driven by microorganisms, with bacteria and fungi naturally coexisting together in the environment. Terrestrial isopods Oniscusasellus and Porcellio scaber have adaptations to enable them to colonise diverse terrestrial environments and scavenge on dead and decaying organic matter that is rich in cellulose. The Amazonian catfish, Panaque nigrolineatus have physiological adaptions enabling the scraping and consumption of wood, facilitating a detritivorous dietary strategy. Substrates high in lignocelluloseare difficult to degrade and as yet, it is unclear whether these organisms obtain any direct nutritional benefits from ingestion and degradation of lignocellulose. However, there are numeroussystems that rely on microbial symbioses to provide energy and other nutritional benefits for host organisms via lignocellulose decomposition. Whilst previous studies on the microbial communities of O. asellus, P. scaber and P. nigrolineatus, have focused upon the bacterial populations, the presence and role of fungi in lignocellulose degradation has not yet been examined. These studies describe the bacterial and fungal communities within the gastrointestinal tracts using next generation sequencing. The hepatopancreas of O. asellus and P. scaber was predominantly colonised by one bacterial species and had more fungal diversity. The hindgut was colonised bymore diverse bacterial and fungal communities. Due to the woodlouse inhabiting diverse environments, including those with heavy metal pollution, culture methods were used to detect antimicrobial resistance in the gastrointestinal tract of woodlice. The effects of diet on enteric fungal populations were examined in each gastrointestinal tract region of P. nigrolineatus and fungal species were found to vary in different regions of the gastrointestinal tract as a function of diet. This is the first study to investigate the bacterial and fungal communities within the hepatopancreas and hindgut from two species of woodlice, using the same individual woodlouse,using next generation sequencing. This is the first study to detect fungi in the digestive tract of anywoodlice. This study is the first to examine the fungal community in a xylivorous fish and results support the hypothesis that diet influences fungal distribution and diversity within the gastrointestinal tract of P. nigrolineatus. This study provides new insights into the microbial communities that may have a symbiotic role involved in wood degradation in the GI tracts of woodeating organisms. This study also highlights the need for further research into fungi inhabiting many diverse environments to give more complete and balanced information about the absence and presence of microorganisms.

Environmental DNA analysis using metagenomics can provide an insight into the taxonomy and functional potential of microbial communities ex situ, without the need for culturing or DNA amplification. However, metagenomics has had limited application to environmental microbial ecology, in particular, to microbial communities in proglacial regions. This thesis aims to contribute to the body of literature on environmental metagenomics through evaluating assemblers for soil microbial ecologists, and subsequently applying metagenomics to investigate microbial communities in proglacial environments. Assembly of metagenome sequencing reads can improve sequence alignment to taxonomic and functional databases, thereby improving ecological conclusions. However, limited guidance is available for assembler choice by microbial ecologists. The first study in this thesis compares assemblers for soil metagenome data, demonstrating the importance of assembler evaluation and parameterization. The guidance produced was applied to investigate microbial communities in proglacial regions, including fjords and forefields. Proglacial forefields present a unique opportunity to understand microbial colonization in land exposed by glacier retreat. Here, metagenomics was used to investigate microbial diversity and functional potential during forefield succession, alongside comparing the diversity of nitrogen-fixing bacteria between Arctic forefields. This work contributes to our understanding of Arctic microbial ecology, which has significance given the continued exposure of forefield soils during global warming. In addition, metagenomics was used to investigate microbial communities in oligotrophic, dark, saline fjord waters, fed by glacial meltwater. This work highlights the potential of metagenomics to understand uncultured microbial samples and demonstrate areas for further analysis, such as targeting novel genomes. This thesis has contributed to the literature on metagenomics by providing methodological guidance for microbial ecologists, alongside enhancing understanding of microbial diversity in proglacial regions. It is hoped that this work will inspire others to use metagenomics to explore uncultured microbial samples and to target further analysis or exploration for unique genomes.

Natural microbial communities act as metabolic conversion systems in soil, oceans, animal guts and other environments, provide essential nutrition for animals and plants, and drive global biogeochemical cycles. Such functions rely on complex interactions among microbes with different genotypes and metabolic capabilities. In order to achieve a deeper understanding of microbial communities and to further engineer synthetic communities, it is necessary to identify the metabolic interactions among key species, and characterise how these interactions are affected by different environmental factors. Deciphering the physiological basis of species-species and species-environment interactions in spatially organized microbial communities requires bottom-up approaches through assembling ecologically and functionally relevant species. To this end, the work herein focuses on a defined system to study the metabolic interactions in a spatial context between the plant-beneficial endophytic fungus Serendipita indica and the soil-dwelling model bacterium Bacillus subtilis. Focusing on the growth dynamics of S. indica under defined conditions, it was discovered that this organism was auxotrophic to thiamine, a co-factor for essential reactions in the central carbon metabolism. Furthermore, it was found that the growth of S. indica was restored in thiamine-free media when co-cultured with B. subtilis. However, the success of this auxotrophic interaction was determined by the spatial and temporal organization of this two-species synthetic community; the beneficial impact from B. subtilis to S. indica was only possible when inoculation of B. subtilis was separated from that of S. indica either in time or space. The microscopy analyses were performed and a microfluidic system was developed to investigate the real-time community interaction and fungal growth at single cell level. The time-lapse imaging IV data of interactions between S. indica and B. subtilis as well as S. indica spore germination were analysed, and obtained a fine characterisation of the growth dynamics of S. indica. The following work described the thiamine auxotrophy of S. indica, the key auxotrophic interaction between S. indica and B. subtilis and the importance of spatial and temporal organization for the success of auxotrophic interactions. These discoveries contribute to the understanding of S. indica growth, allow the controlled investigations of fungal-bacterial interactions and have implications for the engineering of functional synthetic communities with plant beneficial microbes.

Morphological features have been investigated in various automation studies to identify different types of medical images. These studies rely on this type of feature, whereas digital images can provide other different features as descriptive characteristics. Pixels can be utilized as feature sets to recognize regions of interests. This research develops pixel values to extract informative features to identify protozoan parasites of the Eimeria genus. Eimeria is a single-celled intestinal parasite which infects humans and animals. Each type of host can be infected with different Eimeria species. Coccidiosis is caused when Eimeria infects animals, which is a rapidly spreading and fatal disease. Its treatment requires the identification of which species has infected the host, but similarities between Eimeria species make identification a very challenging process. Previously, automatic identification was carried out by imitating biological measurements, but these require complex and costly computational processes to extract the desired features. Therefore, this research aims to simplify the feature extraction process considering the use of another type of feature to distinguish between Eimeria species. The features considered do not need complex extraction processes and provide high accurate results. Pixel-based features are analysed by calculating the means of image matrix columns and rows of regions of interests. Features are represented as sets of column features (CF), row features (RF), and combinations of both in (CRF). Moreover, CF, RF, and CRF are extracted from greyscale level and colour images. Therefore, six feature sets are considered, and these are optimized by utilizing five selection and reduction algorithms to minimize the feature space. Furthermore, the extraction of super-pixel feature sets is developed to simplify segmentation. For classification, three classifiers are applied. The 5-fold cross-validation is used to evaluate the results and every experiment is repeated 50 times. Consequently, the results shown are the averages of 50 runs along with values of standard deviation. The proposed method is examined by analysing two microscopic image databases of 4402 images of the 7 Eimeria species in chickens and 2902 images of the 11 Eimeria species in rabbits. The best accuracy results achieved are 96.7% (±0.89%) and 95.85% (±2.4%) for the respective datasets. Finally, the proposed method succeeds in finding simple features to identify Eimeria species, reducing the feature number by 40% of the original size, and super-pixel feature sets are established which give excellent results.