S.aureus is highly adaptable to environmental conditions and has the ability to colonise and infect a range of tissues within the host. The ability to colonise skin requires survival mechanisms to counter an array of abiotic factors that includes epidermal and sebaceous skin lipids. This study sought to investigate the effects of cholesterol, an epidermal lipid produced in substantial quantities, on S. aureus growth and survival. Previous studies have reported that cholesterol addition reduces the growth inhibition of antimicrobial fatty acids (AFAs) and this phenomenon was investigated further to identify the underlying mechanism. The addition of ethanol-solubilised cholesterol to broth cultures of S. aureus increased bacterial survival in the presence of growth inhibitory levels of linoleic acid and the lipid sphingosine. This effect was confirmed in strains SH1000 and Newman. The pigmentation of S. aureus when grown in the presence of ethanol-solubilised cholesterol was greatly reduced. Initially this study focused on these effects being mediated by cholesterol, however ethanol concentration was not controlled effectively when designing the experiments and ethanol could also be the major mediator of pigmentation changes. It was initially hypothesised that cholesterol would affect S. aureus cell membrane properties since it is known to be incorporated when added extracellularly. From this hypothesis, studies were designed to examine factors controlling survival and pigmentation changes in response to ethanol-solubilised cholesterol. Separate screens of transposon libraries were performed to identify mutants that: i) produced pigment in the presence of ethanol-solubilised cholesterol; ii) did not show enhanced growth with ethanol-solubilised cholesterol supplementation in the presence of growth-inhibitory levels of linoleic acid. The majority of the transposon mutants identified and localised using arbitrary-primed PCR sequencing revealed insertions into genes previously associated with modulating activity of the accessory sigma factor σB. Subsequent experiments with ethanol controls indicated a clear solvent effect on pigment expression, confounding the previous hypothesis and contrary to published reports about the activation of σB. To investigate the effects of ethanol and ethanol-solubilised cholesterol on pigment expression and σB activity a series of qRT-PCR assays were established. In the presence of ethanol-solubilised cholesterol the expression of crtM was decreased, whereas, extracellular protease, aur and sspA expression were increased in the presence of 0.3 mM ethanol-solubilised cholesterol. However the effect of ethanol, as a solvent control, was substantial resulting in decreased transcription of sigB and crtM while correspondingly aur and sspA transcription were increased. These data from the study of ethanol and ethanol-solubilised cholesterol identify novel effects on the cell membrane of S. aureus that require further study to dissect the individual roles of each component and indicate that current literature reports of σB activity and regulation might be incomplete.

Cyanophages are thought to play an important role in the mortality and clonal composition of marine Synechococcus spp., and have been shown to be widespread throughout the world's oceans. However, relatively little research has been made into the molecular analysis of marine cyanophages. This study continued previous research to develop molecular probes (PCR primers) which would specifically detect cyanophages which infect marine Synechococcus spp., and be used to interrogate natural marine cyanophage populations. An attempt was made to develop a rapid technique for quantifying marine cyanophages, using competitive PCR (cPCR). For the development of cyanophage-specific PCR primers, several cyanophages which infected Synechococcus sp. strains WH7803 and WH80 18 were isolated from coastal Bermuda and the Sargasso Sea. A region of DNA had previously been found which showed homology amongst several marine cyanophages, and to T4 gene 20, which encodes a minor capsid protein. Homologues from three cyanophages were completely sequenced, and two, potentially cyanophage-specific, PCR primers were designed. The primers detected only marine cyanophages which belonged to the family Myoviridae, regardless of the geographical location of their isolation. They also detected cyanophages which infected different marine Synechococcus spp. strains, and therefore provide a more comprehensive tool than infective methods. The primers were able to detect as few as 190 cyanophages Ilr1, which would correspond to an in situ concentration of 103 PFU mH. The PCR should therefore detect most natural concentrations of marine cyanophages in surface waters, especially with prior concentration from seawater. Preliminary experiments showed that PCR products could be obtained from as little as I III of un concentrated seawater. PCR therefore provides a sensitive method for the detection of marine cyanophages, which is far more rapid than traditional infection techniques. Quantification by cPCR was attempted. An internal competitor was constructed, and a calibration curve was drawn for three cyanophages, with a loglinear relationship over ca. three orders of magnitude of cyanophage numbers. This demonstrates that rapid quantification of a known marine cyanophage is possible. However, cPCR of the three different cyanophages resulted in three different calibration curves. Hence, quantification of a marine sample containing a mixture of cyanophages was not yet possible. The cyanophage-specific primers were then applied to marine samples which were collected whilst on the AMT-2 cruise, from Port Stanley (Falkland Islands) to Plymouth (UK). Cyanophages were concentrated by tangential flow filtration, and PCR products were obtained from most of the surface samples throughout the Atlantic Ocean. Products from some of the stations were sequenced, providing novel genetic information of natural marine cyanophage populations. The results showed that cyanophage populations were highly diverse, with at least twelve genetically different cyanomyoviruses in one sample. Some sequences obtained from the same sample were clearly very similar to each other, whilst others within a sample could be as diverse as those isolated from different oceans. However, very similar sequences were obtained from some samples separated by thousands of miles, in different hemispheres, or even in different oceans.

The dynamics of protein turnover is central to the regulation of protein expression. The steady-state level of a protein is the net outcome of the change in its rate of synthesis and degradation. Different biological states or perturbations cause changes in the expression of specific proteins, which can be assessed by proteomic analysis to reveal links between genotype and phenotype. Unlike other conventional proteomic methods, which measure the amount of proteins in the system at a specific point in time, pulse-chase stable isotope labelling by amino acids in cell culture (pcSILAC) can reveal changes in the rates of protein synthesis and degradation over time. The causative agent of Leishmaniasis, Leishmania, has a digenetic lifestyle involving an extracellular flagellated promastigote living in the mid-gut of the sand fly vector and an aflagellated intracellular amastigote residing in the macrophage of the mammalian host. As they live in a different niche their protein expression could give insight into their adaptation and survival. The intricate interaction between the human host and the Leishmania parasite is key to pathology and may present new targets for chemotherapeutic development. Employing high-resolution mass spectrometry coupled with pulse-chase SILAC technique, we delved into the investigation of proteome changes in L. mexicana-infected macrophages. The first part of the thesis discusses the quantitative proteomic analysis of L. mexicana promastigote and amastigote stage. In this work, stable isotope dimethyl labelling was employed to differentially labelled promastigotes and axenic amastigotes. Our results revealed transformation from promastigote to amastigote were accompanied by: i) reduced glycolytic and gluconeogenesis pathway, ii) increased fatty acid oxidation, iii) increased mitochondrial respiration, iv) reduced expression of proteins that may have flagellar role (e.g. flagellar connector protein, flagellum targeting protein KHARON1), v) reduced stress response proteins, vi) increased protein synthesis, and vii) increased proteolytic proteins. The findings reported here substantially advance our knowledge on the differences of protein expression in different life cycle stage of L. mexicana and could be useful in finding drug targets. Another part of the thesis discusses the establishment and application of pulse-chase SILAC. In this work, a human macrophage-like cell line (THP-1) was grown in media containing L-Arg-13C6 and L-Lys-13C6 until isotope incorporation of >98% was achieved. Media was then replaced with light arginine and lysine so that light amino acids were pulsed into cells for 24 and 48 hours. In other words, protein synthesis is ‘chased’ with unlabelled amino acids. Synchronous with the switch from pulse to chase, the macrophages were infected with L. mexicana. This approach provides the ability to monitor the rates of heavy-label loss, hence determining protein degradation rates and half-lives. At 24-hour post-infection, when compared to mock-infected cells, 2016 proteins were identified, 761 were quantified, and 51 were significantly modulated at p-value < 0.05. Interestingly, proteins involved in glycolysis were markedly downregulated in synthesis after infection while oxidative phosphorylation and fatty acid β-oxidation had increased synthesis, suggesting a subversion of host cell metabolism by Leishmania which proposed to play a key role in microbial growth and persistence. Additionally, pro-apoptotic proteins such as apoptosis regulator BAX and caspase 3 had increased translation in cells infected for 24 hour. This was accompanied by the overexpression of STAT1 which could result in modulation of apoptotic pathways. These characteristics advocate that THP-1 cells most likely exhibit an M2 macrophage phenotype following 24-hour infection. Temporal proteomic data revealed some striking changes in metabolisms of the host at 24 and 48-hour post-infection. After 48 hours of infection, 2104 proteins were identified, and 84 were significantly modulated post-infection at p-value < 0.05. After 48 hours of infection, relative to levels at 24 hours of infection, host cells increased the synthesis of glycolytic enzymes and reduced oxidative phosphorylation synthesis. Further, a total of 400 newly synthesized proteins were selected based on stringent criteria to measure synthesis rates, degradation rate constant (kdeg) and half-lives. These include several ribosomal proteins, pyruvate kinase, L-lactate dehydrogenase, moesin, several glycolytic enzymes such as glucose-6-phosphate isomerase and alpha enolase, gelsolin, galectin-9, catalase and lamin-B. We found that globally kdeg values in THP-1 were low ranging from 0.01 to 0.04 h-1. Our degradation data indicated that proteins involved in mitochondrial related functions (TCA, oxidative phosphorylation) as well as other energy production pathways were more stable and have longer half-lives. For the 400 proteins, the mean half-life for uninfected 24 h, 24 hpi, uninfected 48h and 48 hpi were 21.74 h, 20.51 h, 47.39 h and 47.33 h, respectively. Intriguingly, newly synthesized proteins involved in immune responses, including HLA complexes, were rapidly degraded in infected cells, despite having decreased synthesis rates after 48 hours of infection. Collectively, most proteins in the present study had decreased kdeg and longer half-lives following longer exposure of THP-1 to L. mexicana. Our data show the potential of pulse-chase SILAC to dissect the response of macrophages to Leishmania infection. To our knowledge, no studies have reported the proteome turnover of macrophage in response to Leishmania infection.

In this work the abundance, structure, distribution and possible role of the inverted repeat sequence DNA (IR DNA) of R. vannielii was investigated. Approximately 7% of the R. vannielii genome was found to exist as inverted repeat sequences by nuclease Si digestion of heat-denatured, rapidly renatured DNA; this compared with 3% for E. coli K12 DNA. The inverted repeat sequence DNA of R. vannielii formed two size classess a heterogeneous high molecular weight class with a size range of 100-700 base pairs (bp), and a low molecular weight class comprised of fragments of 17 and 27bp. The genomic distribution of these two inverted repeat classes was investigated by DNA/DNA hybridization studies. In vitro 3=P-labelled IR DNA was used to probe Southern blots of EcoRI and Hindi 11 restriction enzyme digests of R. vannieli i DNA. Doth classes of inverted repeat DNA showed hybridization with many bands throughout the restriction digests, suggesting that the sequences were not clustered but were dispersed throughout the genome. Low molecular weight IR DNA however, hybridized to four specific bands in an Haelll digest of R. vannielii DNA suggesting that this enzyme could reveal some repetitive structure in the genome which the other enzymes used could not. Low molecular weight IR DNA was also found to hybridize throughout the high molecular weight IR DNA class, indicating that the two IR DNA classes share some sequences and may be derived from the same chromosomal loci. Competition filter-binding assays, designed to detect protein-DNA interactions, showed that about BX of high molecular weight IR DNA sequences appeared to be bound specifically by R. vannielii protein while low molecular weight IR DNA was not bound. The genomic plasticity of R. vannielii was investigated by hybridization and by a dual-label 1ing method. Although these experiments gave inconclusive results there were indications that sequence rearrangements might occur during R. vannielii swarmer cell differentiation. Attempts to clone IR DNA directly by two methods had only limited success because of difficulties in the identification of IR DNA-containing recombinant plasmids and suggested that cloning of IR DNA should be done by hybridization screening of chromosomal gene libraries. The serine hydrolases of the differentiating swarmer cell were also examined. Using an inhibitor labelling technique up to eight bands could be identified on fluorographs of SDS gradient gels and a number of changes in banding were observed to occur during swarmer cell differentiation.

Rhizosphere microorganisms were grown in batch culture with mecoprop, a selective herbicide, as the sole carbon and energy source. Growth was detected only if the mecoprop concentration was 2.08 mM or less and lag phases of up to 37 days were required before growth commenced. After consecutive subculturing the length of the lag phase decreased to less than 24 hours. Degradation of the herbicide was followed by monitoring inorganic chloride ions released from the mecoprop structure, culture absorbance increases and the disappearance of the maximum ultra-violet absorption peak of mecoprop. The culture enriched to degrade 2.08 mM was able to utilize mecoprop concentrations up to 66.7 mM, but prolonged lag phases were recorded. Analysis of the mecoprop-degrading culture revealed that it contained two Pseudomonads, an Alcaligenes species, Acinetobacter calcoaceticus and a Flavobacterium species. None of the pure cultures was able to utilize mecoprop but some combinations of two or more organisms degraded the herbicide stressing the importance of interacting communities for the degradation of xenobiotic compounds. The two Pseudomonads made up 83.5% of the community, the remaining organisms were minor components. A specific relationship existed between the Pseudomonads based on the provision of a growth factor. The community was a stable association as subculturing for over 300 days failed to reduce the complexity. Growth studies using mixed carbon sources demonstrated that the two Pseudomonads and Alcaligenes species were able to utilize mecoprop when succinate was present as a co-substrate. Pronounced two-phase growth curves were produced with mecoprop degradation occurring after initial growth on succinate. A biodegradation pathway for mecoprop was proposed based upon degradation studies of the structurally related herbicides 2,4-D, MCPA and dichlorprop by the community and the oxidation of these herbicides by mecoprop-grown cells.

An R-prime, pUU2, derived from the broad host-range plasmid R68.44+ was generated carrying the dehalogenase I gene from Pseudomonas putida PP3. This R-prime enabled its host to use 2-monoch1oropropionic acid (2MCPA) as sole carbon and energy source. The R-prime was studied by restriction endonuclease analysis. The process of R-prime formation was examined in detail and was shown to have suffered from interference from an insertion sequence originating from the PP3 chromosome. The dehalogenase I PP3 chromosomal insert was shown to interfere with the plasmid transfer functions and to be capable of translocation within the plasmid. A Pseudomonad containing the R-prime pUU2 was grown under phosphate limitation on 2MCPA in a chemostat. During 2600h. a range of mutants were isolated in which the plasmid was shown to have undergone several modifications including the acquisition of lOkb of novel DNA, the transfer of the dehalogenase I gene to the chromosome, and in some cases the concomitant loss of the plasmid. A new range of 2Mcpa+ R-prime plasmids were generated using a chemostat-derived plasmid-minus strain and R68.A5. These demonstrated that though the insertion sequence IS21 of R68.A5 may be involved in R-prime formation it need not be intimately associated with the chromosomal insert into the plasmid, at least in the case of the dehalogenase I gene. This, together with the frequency of R-prime formation, lends support to the view that the dehalogenase I gene may be on a transposable element. Restriction fragments from the R-prime pUU2 were cloned into pAT153 and pKT231. Clones were isolated capable of growth of 2MCPA and their plasmids were analysed by restriction digestion.

This work concerns the identification of the information controlling the accumulation of the influenza virus nucleoprotein (NP) in the nuclei of Xenopus oocytes. The NP accumulates in the nuclei of Xenopus oocytes whether introduced into the oocytes as the protein itself or encoded in RNA or DNA. Since no other influenza virus components are present in the oocytes injected with DNA, this accumulation appears to be a property of the NP itself. In vitro mutagenesis of the cloned NP cDNA has then been used to identify which regions of the protein are important in its nuclear accumulation. Mutant proteins lacking amino acids 327-345 of wild-type NP enter the nucleus but do not accumulate there to the same extent as the wild-type protein, suggesting that this region has a role in nuclear accumulation. This proposed location is strengthened by studies involving the production of fusion proteins in which various amino- terminal sequences of the NP gene are fused to the complete chimpanzee ꙋ1-globin sequence: when globin cDNA is injected into and expressed in oocytes the protein remains in the cytosol, however when the globin cDNA is fused to a portion of NP cDNA which includes the region encoding amino acids 327-345 the resulting fusion protein enters and accumulates in the nucleus. Fusion proteins lacking this region of the NP enter but do not accumulate in the nucleus.

Haemophilus influenzae is an important human pathogen, responsible for respiratory infections, such as otitis media, bronchitis and epiglottitis, as well as invasive disease. Despite being the first free-­‐living organism to have its whole genome sequenced, there have been only a few published studies investigating its transcriptional profile using next-­‐generation sequencing (NGS). The work presented in this thesis aimed to use NGS to improve the understanding of how H. influenzae behaves during natural infection and to identify novel RNA structures with potentially important roles in pathogenesis. The whole transcriptome of H. influenzae during infection-­‐relevant conditions was analysed using high-­‐throughput RNA sequencing. For the first time, the transcriptional profile of H. influenzae during stationary phase and nutritional stress was determined on a whole-­‐genome scale. Differential gene expression analysis of an invasive strain, R2866, and a laboratory strain, Rd KW20, revealed differences in their transcriptional response, particularly during oxidative stress and iron starvation. Importantly, a new systematic and robust bioinformatic tool, "toRNAdo", was developed to identify non-­‐coding RNA elements from the bacterial transcriptomic data. It enabled discovery of a repertoire of novel putative intergenic and antisense non-­‐coding RNAs in H. influenzae. In addition, the first fully sequenced genome of a free-­‐living organism, the Rd KW20 strain of H. influenzae, was re-­‐sequenced and re-­‐ annotated for the first time. This enabled identification of multiple nucleotide-­‐ level differences between original and re-­‐sequenced genomes of Rd KW20. The work presented here facilitates future characterisation of novel RNA elements, with potentially important regulatory roles in pathogenesis in H. influenzae, and has implications for defining a model bacterial strain. Importantly, the findings present significant insight into the pathogenic lifestyle of H. influenzae. They provide the basis for further work, where novel vaccine and antibiotic targets may get developed.

Turnip mosaic virus (TuMV, family Potyviridae, genus Potyvirus) has the widest host range amongst potyviruses. Globally it was said to be the second most important virus infecting field vegetables. Brassica juncea (Oriental mustard, family Brassicaceae), is an amphidiploid plant species with the genome AABB, comprising the genomes of the two diploid species, Brassica rapa (AA) and Brassica nigra (BB). It is widely grown and has various uses including as a leaf, stem, or root vegetable, oilseed crop, forage crop, condiment and biofumigant. Most B. juncea cultivars are very susceptible to TuMV, resulting in severe losses. Research on TuMV resistance and the mapping and identification of natural resistance genes would be very useful in order to speed up breeding resistant crops through marker-assisted selection. Sources of resistance to TuMV have been identified in B. juncea. The specificity of the resistances has been determined. A B. juncea DH line for which there is genomic information has been challenged with TuMV and found to be susceptible. This line has been used as a susceptible parent in crosses with resistant plants derived from different sources to develop segregating populations for mapping the resistance gene(s). Two BC1 populations (222 plants and 205 plants) and one F2 population (159 plants) have been phenotyped and segregation ratios were not significantly different from a Mendelian model based on the action of two recessive genes. Parental lines and selected plants in the two BC1 populations have been analysed by SNPs genotyping using the Illumina Infinium Chip. Genetic linkage maps have been constructed and QTLs have been mapped. Additionally, attempts are being made to identify a dominant TuMV resistance gene present in both Brassica napus and B. rapa. Inter-specific crosses have been made in order to introgress this gene into B. juncea. Resynthesised B. juncea plants possessing this dominant resistance have been produced through embryo rescue and polyploidy induction of F1 plants from crosses between resistant B. rapa and susceptible B. nigra plants. BC2 plants have also been developed by crossing B. rapa and B. napus plants possessing the dominant TuMV resistance with a susceptible B. juncea plant line.

Cyanobacteria are major players in marine biogeochemical processes, primarily CO2 fixation via oxygenic photosynthesis, and nitrogen cycling. These phototrophs occupy a variety of oceanic niches, with their distribution and abundance being shaped by a range of abiotic (e.g. temperature, light, nutrients) and biotic factors (e.g. grazing and virus infection). Viruses infecting cyanobacteria are termed cyanophage. During evolution these cyanophages have acquired an arsenal of ‘auxilliary metabolic genes’ (AMGs), often horizontally acquired, which can influence the metabolism of the infected host thereby optimising viral production. Cyanophage infection of their host under sub-optimal growth conditions can lead to deleterious effects on infection success. For example, cyanophage S-PM2 infection of Synechococcus under P-deplete conditions causes a delayed latent phase and decreased burst size – a process termed pseudolysogeny. In this thesis I set out to provide a molecular understanding of pseudolysogeny, at the same time hypothesising that specific cyanophage AMGs help to avoid the negative effects of sub-optimal host growth conditions on the infection process. In support of this, infection by cyanophages that possess putative P-stress related AMGs do not show signs of delay (Chapter 3), and the presence of these genes in cyanophage genomes correlates well with the prevailing P conditions in the temporal and spatial niches from which these cyanophages were isolated (Chapter 4). Meanwhile, in a cyanophage lacking such nutrient stress genes, and thus entering pseudolysogeny during infection under P-deplete conditions, transcriptional profiling showed retardation in the timing of known cyanophage temporal gene expression clusters (Chapter 6). Moreover, a significant increase in expression of several cyanophage early genes involved in DNA replication was also observed under these P stress conditions compared to infection of a P-replete host. Quantitation of intracellular cyanophage DNA showed that while levels were generally lower under P-deplete conditions, the rate of DNA replication between P-replete/deplete conditions was similar. The observed increased expression of cyanophage genes involved in DNA replication during the early stages of infection may thus be an evolved response to compensate for decreased levels of intracellular phosphate experienced under these conditions (Chapter 6). Overlaid on top of specific bacterial nutrient stress responses is the ‘stringent’ response, mediated by the alarmone molecule (p)ppGpp, a process which occurs under prolonged nutrient stress and in late stationary phase. A bacterial gene mazG, encodes a pyrophosphatase which participates in (p)ppGpp homeostasis. Interestingly, a mazG orthologue is found as part of the cyanomyovirus core genome, suggesting that cyanophages attempt to alter intracellular signalling during the course of infection. The stringent response has been shown to have a particularly negative effect on phage replication, with (p)ppGpp levels in a cyanobacterial host being previously shown to be dramatically reduced under phage infection. In this thesis I show that the Synechococcus host mazG is dispensable for growth under normal laboratory conditions. However, this Synechococcus mazG mutant shows a modified cyanophage infection profile, slower and less productive, compared to the WT, under P-deplete conditions (Chapter 5). Furthermore, comparison of enzymatic activity of host and cyanophage MazG showed that the viral orthologue exhibits an increased affinity towards GTP, compared to the host protein and a general preference towards G and C nucleotides (Chapter 5), possibly reflecting the low GC content of cyanophage genomes. Thus, the cyanophage and host MazG may have additional functions in phosphate metabolism and controlling DNA integrity, a hypothesis strengthened by experimental evidence for the cyanophage mazG being over-expressed under P-deplete conditions (Chapter 6). Taken together, data presented in this thesis demonstrates a general strategy by cyanophages to acquire host genes involved in modification of central metabolism or that regulate host signalling. Furthermore, once acquired, cyanophage genes appear to have evolved divergent functions to suit specific differences in genome content, compared with their host, as well as mechanisms to regulate transcription of these genes in response to external nutrient stimuli. Thus, this study expands our view of lytic phages, and suggests sophisticated mechanisms occur for overpowering their hosts under a range of infection conditions. This new information provides a mechanistic understanding of viral infection in a ubiquitious primary producer under environmetally relevant conditions, and will undoubtedly improve our ability to understand and model biogeochemical cycling performed by these key marine phototrophs in a more accurate manner.