α-Glucans are typically used for carbon storage in bacteria, however, they are also a major component of the mycobacterial capsule. In this context, glucans have been implicated in the evasion of recognition by macrophages. A novel α-glucan synthesis pathway, called the GlgE pathway, is a potential source of the capsular glucan in mycobacteria. The pathway converts the disaccharide trehalose into α-glucan by the action of four enzymes: TreS, Pep2, GlgE and GlgB. Functional redundancy between other glucan synthesis pathways has made characterising the GlgE pathway difficult in mycobacteria. The genes encoding the enzymes of the pathway are found in 14% of sequenced bacterial genomes, suggesting the pathway is relatively wide-spread amongst bacteria. α-Glucans have also been isolated in a number of streptomycetes, which are distant relatives of mycobacteria. In this study Streptomyces venezuelae was used to show for the first time that the GlgE pathway is responsible for glucan synthesis in vivo, as was predicted by the annotations in the genome. A ΔglgE mutant was devoid of α-glucan and instead accumulated α-maltose 1-phosphate, which was associated with a developmental phenotype. The spores produced by the ΔglgE strain had normal levels of trehalose and had compromised resistance to abiotic stresses, with the exception of desiccation resistance, which was comparable to wild-type. In this study, another mutant from S. venezuelae ΔotsA was also investigated, which was hypothesised to be feeding the substrate trehalose into the GlgE pathway from glucose. However, under normal laboratory growth conditions, no phenotype was observed and the strain was able to accrue α-glucan. Surprisingly, a developmental phenotype arose when ΔotsA was grown on a galactose carbon source. This developmental phenotype was associated with the accumulation of GDP-glucose. Recombinant S. venezuelae OtsA was produced heterologously and the substrate preference for this enzyme was found to be GDP-glucose, as predicted by the phenotypic study of the ΔotsA strain. The crystal structure of S. venezuelae OtsA was solved to a resolution of 1.95 Å and the unusual substrate specificity was rationalised by comparison with a solved Escherichia coli OtsA structure, which has substrate specificity for UDP-glucose, and a pseudo-glycosyltransferase VldE, which uses GTP during catalysis. Four key resides were identified as being important for the substrate specificity of S. venezuelae OtsA: Ser345, Phe342, Glu341 and Asp340. Finally, TreS was found to be stereospecific for the α anomer of maltose, the appropriate anomer for Pep2, which confirms the prediction of an α-retaining mechanism.

Nod factor (NF) can induce two separate calcium responses in legume root hairs. Nuclear-associated calcium spiking is central to the symbiosis signalling (Sym) pathway, which is necessary for the activation of genes required for nodule formation and bacterial infection. In addition NF activates a tip-focused calcium influx, which is less-well studied but is thought to be involved in bacterial infection. NF also activates ROS transient production at the tip of root hair cells. In this thesis I used fluorescent probes (Ca2+-sensitive Cameleons YC2.1 and YC3.6 and the ROS-sensitive CM-H2DCFDA dye) to characterise the NF-induced calcium influx and ROS transient responses in Medicago truncatula. Along with being spatially and temporally co-incident, the responses require similar concentrations of NF to be activated, are inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and are dependent on the NF receptor NFP but independent of the Sym pathway components DMI1 and DMI2. These results suggest the NF-induced calcium influx and ROS transient are part of a common signalling pathway during bacterial infection. ROP signalling is associated with ROS production and calcium influx during developmental root hair elongation. I assessed the role of ROPs during rhizobial infection in M. truncatula and found a ROP-activating protein, MtGAP1, was upregulated in root hairs during bacterial infection and is involved in normal root hair curling and infection thread development. Two pieces of evidence directly link ROP signalling with the NF-induced calcium influx: gap1 mutants were hypersensitive for induction of the calcium influx, and there was a reduction in the number of calcium influx responses in ROP9 RNAi knockdown lines. Drawing parallels between developmental root hair elongation and bacterial infection I propose a model for the regulation of ROP signalling by NF leading to root hair curling, the activation of the calcium influx and ROS transient, and infection thread formation.

Streptomyces coelicolor inhabits a harsh soil environment, which it must adapt and respond to; therefore how S. coelicolor senses and responds to its environment is of the utmost importance. This project addresses two types of regulation system: the three-component system MtrAB-LpqB and the nitrite-sensitive repressor NsrR. Previous work involving the response regulator MtrA has resulted in the hypothesis that MtrA regulates cell division within actinomycetes. However the role of MtrAB-LpqB within S. coelicolor has not been investigated. Therefore it is important to establish whether cell division is disrupted within mutants of the sco3014-lpqB operon. Removing components of the MtrAB-LpqB system resulted in actinorhodin production and spore formation being altered. The irregular spore structure seen by scanning electron microscopy (SEM) may be due to the difference in ftsZ expression, which could account for the irregularly placed septum and spore size observed. Therefore this work confirms that MtrAB-LpqB is important for cell division within S. coelicolor. Work was also carried out on the repressor NsrR to establish its role within S. coelicolor as previous findings had primarily focused on the type of Fe-S cluster contained within NsrR. The phenotype of an nsrR mutant exhibited a low level of spore production, similar to the distinctive, development whi phenotype. Some Whi proteins function via an [4Fe-4S] cluster, which is also central to NsrR regulation meaning NsrR could be linked to the Whi proteins within S. coelicolor. To understand NsrR function further, ChIP-seq was undertaken to identify direct targets; revealing, contrary to prediction studies, NsrR represses only three targets: hmpA1, hmpA2 and nsrR. These targets were confirmed by in vitro binding studies with purified S. coelicolor NsrR. Therefore this work reveals that NsrR undergoes auto-regulation and its sole role is to regulate nitric oxide (NO) stress response via detoxification within S. coelicolor.

Dimethylsulphoniopropionate (DMSP) is the most abundant organic sulphur molecule in the oceans. Its breakdown by marine organisms is important for the global cycling of sulphur, and as a nutrient source for microbial life. In recent years, the molecular basis of DMSP catabolism by marine bacteria has begun to be unravelled, through the discovery of six different DMSP lyases and a DMSP demethylase, as well as downstream pathways. From these studies, it is becoming evident that there is great diversity in the way bacteria breakdown this important molecule. The work presented here further explores and expands our knowledge of this diversity. I have identified a novel DMSP lyase (DddK), which catalyses the cleavage of DMSP into acrylate and dimethyl sulphide (DMS) in the DMS-producing Candidatus Pelgaibacter ubique HTCC1062 - one of the most prolific bacteria on this planet. I have also shown that the γ-proteobacterium Oceanimonas doudoroffii, which has long been a study species for DMSP catabolism, has no fewer than three functional DMSP lyases - DddD, DddP1 and DddP2 - this being the first example of a species outside of the α-proteobacteria having multiple lyases. Additionally, I have presented a thorough bioinformatics analysis of the occurrence and synteny of genes associated with DMSP catabolism within sequenced members of the abundant Roseobacter clade, revealing some interesting patterns which warrant further experimental investigation. Finally, I have shown that the model marine Roseobacter species Ruegeria pomeroyi DSS-3 is able to use DMSP-derived acrylate as a sole carbon source via a fatty acid biosynthesis route, linked to propionate catabolism.

Sulfur deficiency is a relatively new problem in Europe and the studies on sulfur use efficiency are still lagging behind those on the other major nutrients such as nitrogen or phosphorus. Therefore, the main aim of this work was to improve the understanding of the sulfate assimilation pathway, its regulation and interaction with other elements. In the course of this project natural variation was used to characterise further the regulation of the pathway and to identify new regulatory components. This analysis revealed that the first two enzymes involved in sulfate reduction – ATP sulfurylase and APS reductase – are nearly equally involved in its control but through different mechanisms. Moreover, a Genome-Wide Association Study was conducted on the accumulation of nitrate, phosphate, and sulfate in more than 200 arabidopsis accessions. This analysis resulted in identification of new functions of already known genes which were not previously related to plant nutrition. Additionally, previously undescribed genes were identified disruption of which results in changes in the anion accumulation phenotype. To characterise arabidopsis response to sulfate and/or nitrate deficiency a collection of genetically divergent accessions grown under different nutrition regimes was examined for a number of morphological and metabolic traits. This analysis resulted in dissection of four different patterns of plant response to sulfate availability. Individual accessions were characterised as best adapted to nutrient deficiency. Traits such as biomass allocation or root architecture were suggested as potential targets in the process of developing new crop varieties. This analysis is unique since, to my knowledge, it is the first one which provides the characterisation of arabidopsis response to nutrient availability based on the analysis of such a large number (25) of natural accessions. The results described here provided new insight into sulfate metabolism and can be used to develop new breeding strategies and improve crop yield and quality.

The outer membrane, hetero-trimeric multi-heme cytochrome complex MtrCAB, enables the process of dissimilatory metal reduction (DMR) in Shewanella oneidensis. The properties of the decaheme protein MtrA as well as a truncated version of this protein were investigated using analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS) and spectropotentiometric techniques. MtrA and a truncated N-terminal MtrA construct containing the five N-terminal hemes (MtrA N) were both observed to be prolate and highly extended along one axis. Through aligning MtrA N with MtrA, the N terminus of the MtrA structure was identified. Redox titration experiments were performed on MtrA as well as N and C terminal truncations. From these titrations three distinct groups of hemes were identified; a high, a middle potential and a low potential group of hemes. The five N terminal hemes contained the high and middle potential groups of hemes; whereas the five C terminal hemes contained the middle and low potential groups of hemes. The MtrCAB complex was inserted into liposomes containing either methyl viologen, small tetra heme cytochrome (STC) or cytochrome c. Using these proteoliposomes the rate of electron transfer across MtrCAB was investigated. MtrCAB was seen to enable the reduction and oxidation of methyl viologen and STC, but only the reduction of cytochrome c. STC was reduced during the experiments, implying an electron storage role in the periplasm during respiration. Finally, the structure of MtrCAB was investigated through small angle neutron scattering (SANS). Structures of MtrCAB were highly elongated with a globular head region and a tail region. Through density matching buffers, scattering produced from detergents was removed and a prediction of the relative location of MtrB within the model made. The model produced was found to be long enough to span the periplasm enabling direct contact of MtrA with proteins on the inner membrane.

Cell migration is a highly coordinated process and any aberration in the regulatory mechanisms could result in pathological conditions such as cancer. The ability of cancer cells to disseminate to distant sites within the body has made it difficult to treat. Cancer cells also exhibit plasticity that makes them able to interconvert from an elongated, mesenchymal morphology to an amoeboid blebbing form under different physiological conditions. Blebs are spherical membrane protrusions formed by actomyosin-mediated contractility of cortical actin resulting in increased hydrostatic pressure and subsequent detachment of the membrane from the cortex. Tumour cells use blebbing as an alternative mode of migration by squeezing through preexisting gaps in the ECM, and bleb formation is believed to be mediated by the Rho-ROCK signaling pathway. However, the involvement of transmembrane water and ion channels in cell blebbing has not been examined. In the present study, the role of the transmembrane water channels, aquaporins, transmembrane ion transporters and lipid signaling enzymes in the regulation of blebbing was investigated. Using 3D matrigel matrix as an in vitro model to mimic normal extracellular matrix, and a combination of confocal and time-lapse microscopy, it was found that AQP1 knockdown by siRNA ablated blebbing of HT1080 and ACHN cells, and overexpression of AQP1-GFP not only significantly increased bleb size with a corresponding decrease in bleb numbers, but also induced bleb formation in non-blebbing cell lines. Importantly, AQP1 overexpression reduces bleb lifespan due to faster bleb retraction. This novel finding of AQP1-facilitated bleb retraction requires the activity of the Na+/H+ pump as inhibition of the ion transporter, which was found localized to intracellular vesicles, blocked bleb retraction in both cell lines. This study also demonstrated that a differential regulation of cell blebbing by AQP isoforms exists as knockdown of AQP5 had no effect on bleb formation. Data from this study also demonstrates that the lipid signaling PLD2 signals through PA in the LPA-LPAR-Rho-ROCK axis to positively regulate bleb formation in both cell lines. Taken together, this work provides a novel role of AQP1 and Na+/H+ pump in regulation of cell blebbing, and this could be exploited in the development of new therapy to treat cancer.

Landscape scale habitat restoration has the potential to reconnect habitats in fragmented landscapes. This study investigates landscape connectivity as a key to effective habitat restoration in lowland agricultural landscapes and applies these findings to transferable management recommendations. The study area is the Stonehenge World Heritage Site, UK, where landscape scale chalk grassland restoration has been implemented. Here, the ecological benefits of landscape restoration and the species, habitat and landscape characteristics that facilitate or impede the enhancement of biodiversity and landscape connectivity were investigated. Lepidoptera were used as indictors of restoration success and results showed restoration grasslands approaching the ecological conditions of the target chalk grassland habitat and increasing in biodiversity values within a decade. Restoration success is apparent for four species with a broad range of grass larval host plants (e.g. Melanargia galathea, Maniola jurtina) or with intermediate mobility (Polyommatus icarus). However, two species with specialist larval host plants and low mobility (Lysandra bellargus), are restricted to chalk grassland fragments. Studies of restoration grassland of different ages show that recent grassland restoration (1 or 2 years old) may reduce the functional isolation of chalk grassland fragments. A management experiment showed that mowing increases boundary following behaviour in two species of grassland Lepidoptera; Maniola jurtina and Zygaena filipendulae. Analysis of the landscape scale implications of the grassland restoration illustrates an increase in grassland habitat network size and in landscape connectivity, which is likely to benefit the majority of grassland associated Lepidoptera. Landscape and habitat variables can be managed to increase the success of restoration projects including the spatial targeting of receptor sites, vegetation structure and selection of seed source and management recommendations are provided that are transferrable to other species-rich grassland landscape scale restoration projects. Overall results show restoration success for some habitats and species within a decade. However, additional management is required to assist the re-colonisation of specialist species. Despite this, habitat restoration at the landscape scale can be an effective, long term approach to enhance butterfly biodiversity and landscape connectivity.

Enterohaemorrhagic Escherichia coli (EHEC) are a group of food and contact-borne pathogens responsible for haemorrhagic colitis. The bacteria can be transmitted by contaminated meat, but importantly, also by plants. The bacteria can use plants as an alternative host, where they associate with both the leaves and the roots. Colonisation in the rhizosphere of plants is thought to be the main habitat for colonisation. Four different plant species, commonly associated with EHEC outbreaks, were infected with EHEC O157:H7 isolates Sakai and TUV 93-0 over ten days to assess the colonisation potential of the bacteria in both the phyllosphere and rhizosphere of plants. The rhizosphere was found to sustain a higher population level of bacteria over time in comparison to the phyllosphere, yet both strains were unable to utilize root exudates for growth. Global gene expression changes of EHEC O157:H7 strain Sakai were measured in response to plant extracts such as leaf lysates, root exudates and leaf cell wall polysaccharides from spinach cultivar Amazon and lettuce cultivar Salinas. Microarrays analysis showed a significant change in expression of 17 % of genes on exposure to leaf lysates of spinach. A more specific response was seen to spinach leaf cell wall polysaccharides with only a 1.5 % change. In contrast, when exposed to lettuce leaf cell wall polysaccharides a higher change of 4.8 % was seen. Genes that were differentially expressed belonged to multiple functional groups, including metabolism, indicating the utilization of plant-specific polysaccharides. Several areas of further investigation have been determined from this project, including the importance of culturing bacterial strains at a relevant temperature, the proposed lack of the type III secretion system in plant colonization by EHEC O157:H7 and the utilization of plant components for growth and persistence in the plant environment.

The outcomes of host-parasite interactions are potentially affected by both the genotype and phenotype of the hosts and parasites involved, and modulated by the environmental conditions under which they interact. Anthropogenic environmental changes therefore have the potential to shift the balance in host-parasite interactions, with consequences for disease processes. This thesis examines how host factors and environmental conditions influence the outcome of host-parasite interactions in the experimentally amenable three-spined stickleback-Schistocephalus solidus model. When sticklebacks invade freshwaters, their lateral plate count typically reduces and becomes more variable. In a freshwater population with unusually high diversity in plate morphology, fish with fewer numbers of lateral plates were found to show increased susceptibility to experimental S. solidus challenge. Hypoxic conditions often arise in degraded environments and have the potential to interact with infection status. Schistocephalus solidus infected sticklebacks showed significantly reduced expression of genes (including GADD45 and LDHA) usually associated with the normal cellular hypoxic response of fish. These results suggest that S. solidus infections impair the normal cellular response to hypoxia, and may contribute to observed behavioural changes observed in infected individuals under hypoxic conditions. The results of a study investigating the effects of salinity on the development of S. solidus showed embryonic development was prevented at salinity greater than 20 ppt, indicating that the threat of infection is confined to brackish and freshwaters. Schistocephalus solidus therefore represents a novel selection pressure for sticklebacks colonising freshwaters from ancestral marine populations. Both the level of host food intake and host body size significantly affected the outcome of experimental parasite challenge. Experimentally manipulated host ration generated faster plerocercoid growth under greater levels of alimentation. The establishment of S. solidus plerocercoids was related to host size, with infections developing more frequently in smaller than larger sticklebacks. However, plerocercoids grew more quickly in larger hosts. Hence, environmental changes that affect host size, the timing of infections or the availability of food have the potential to influence parasite growth and life cycle completion rates. The overarching conclusion of the thesis is that the biology of host-parasite interactions is highly susceptible to environmental changes, which can exert their effects through direct impacts on hosts, on parasites or on the interaction between them.