Patterns of transcriptional activity along circular prokaryotic chromosomes such as Escherichia coli and Bacillus subtilis have previously. been characterized through microarray-based analysis of gene expression. However, patterns across linear prokaryotic chromosomes are yet to be investigated. Given the different topological constraints (to circular DNA) imposed upon the linear chromosome the derived transcriptional patterns would be expected to be different. Here we explore transcriptional activity along the linear chromosome of Streptomyces coe/ic%r using expression data from 139 microarrays and genomic features. Representing total chromosomal expression as a spatial series of transcript abundances we observe both short and long range periodicities of transcription along the S. coelicolor chromosome with data derived from different microarray technologies (spotted and ink jet in situ-synthesized). Application of the autocorrelation function confirmed these periodicities as significant both in two and single channel analyses and allowed further analysis of chromosomal properties: Codon adaptation index, GC content, secondary structure and geme length. Another signal processing technique, namely wavelet analysis, has aided the visualization of the periodic signals along the chromosome, identifying 'hot spots' of activity related to specific responses.

A chemo-heterotrophic bacterium Burkholderia cepacia strain 2a was isolated from garden soil in Wales by Dr. A. R. W. Smith (personal communication), and it has been the subject for the biochemical and genetic studies on 2,4- dichlorophenoxyacetate (2,4-D) metabolism. The genes involved in 2,4-D metabolism were located on the plasmid pIJB1 in a composite transposon Tn5530. This plasmid was sequenced in this project for the evolutionary study of this group of plasmid. The in-silico analysis of the 99,001 bp DNA sequence of pIJB1 revealed 93 open reading frames. Over 52 % of the sequence was comprised of well conserved IncPI backbone genes; the rest of the plasmid was composed of accessory genes for 2,4-D and malonate degradation on the Tn5530 transposon, and for mercury resistance on a Tn5058-like transposon. Phylogenetic analysis against twenty-two IncP-I plasmids revealed that pIJBI has the highest evolutionary relationship with the IncPI-1) plasmids pAKD4 (from Pseudomonas putida UWCI), and pEST40II (from Achromobacter xylosoxidans ssp. denitrificans EST4002). The loss of the 2,4-D metabolising phenotype during growth in non-selective (succinate-minimal salts) medium was confirmed to be due to homologous recombination of two identical ISI071 ::IS1471 elements flanking the Tn5530 transposon. Loss of the ability of strain 2a to utilise 2,4-D was followed through seven sequential subcultures in succinateMS liquid medium at different phases of growth. A cumulative 9 % of the viable cells in the seventh sub-culture was observed to have lost the 2,4-D phenotype. The functionality of the mer resistance gene operon on pIJB 1 was investigated by observing the level of inhibition different mercury compounds have on the growth of strain 2a on 4 mM succinate-MSM agar. Preliminary observation revealed that strain 2a possesses broad-spectrum resistance to both organic and inorganic mercury compounds. Strain 2a was tested for its ability to utilise a panel of the chlorinated phenoxyalkanoates to elucidate the specificity of the 2,4-D degradation pathway. Strain 2a was unable to grow on a range of substituted phenoxyalkanoates. Preliminary evidence showed that the organism cleaved the ether linkage in several of these compounds to produce the corresponding phenols, and strain 2a was observed to utilise glyoxylate, the other product of the ether-cleavage reaction; but was unable to utilise most of the phenols, due to the narrower substrate specificity of the second enzyme of the 2,4-D pathway, 2,4-dichlorophenol monooxygenase. Succinate exerted carbon catabolite repression on 2,4-D metabolism when both carbon substrates were presented in minimal salts medium (MSM) to strain 2a together. In this medium, the duration of the lag period between succinate exhaustion and the onset of 2,4-D metabolism was influenced by the previous history of the inoculum, grown in 2,4-D MSM, provided for the culture. Cells taken at mid-log phase for inoculation reproducibly produced longer lag periods (5 h) than those taken at early or late stationary phase (3 h and 3.7 h respectively). This was attributed to the different levels of poly-β-hydroxybutyrate (Hill) accumulated during growth in succinate; the greater the accumulation inside cells, the longer the lag was sustained. However, the causal link between PHB accumulation during succinate metabolism in diauxic growth and the state of the cells taken from different growth phases in 2,4-D-MSM for inoculation was unclear. The constancy of ATP levels around the period of the inter-growth lag phase suggested that PHB utilisation may have sustained cellular energy demands and thereby delayed the onset of2,4-D utilisation. The presence of the tfdK gene within the 2,4-D dissimilatory operon on pIJBl prompted the investigation into involvement of the encoded product in 2,4-D metabolism in strain 2a In-silico comparison of the tfdK amino acid sequence with its analogue in Achromobacter xylosoxidans ssp denitrificans strain EST4002 (pEST40II) and in Cupriavidus necator strain JMP134 (pJP4) revealed 100 % and 76 % identity respectively. The chemotactic role of tfdK was implicated by testing for swarming on soft-agar containing 2,4-D in JMP134 and EST4002, but little sign of swarming was observed with strain 2a The tfdK gene expression level in the three organisms, determined using qPCR, revealed that this phenotypic difference was possibly due to a diminished level of expression of the gene in stain 2a compared with the levels in the other two strains.

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen which causes pneumonia, septicaemia and otitis media infections. The pneumococcus asymptomatically colonises the upper respiratory tract, which may act as a reservoir for subsequent infection of the lower respiratory tract. Current vaccines available include a polysaccharide vaccine (Pneumovax), and a conjugate vaccine (Prevenar). However, problems with both vaccines exist; Pneumovax does not elicit protection in children under the age of 2 years, the elderly or people who are immunocompromised, and Prevenar is an expensive vaccine that has limited serotype coverage. Serotype replacement has also become a growing problem. Therefore, new vaccine targets which can elicit broad cross-serotype protection are required. Pneumolysin and neuraminidase A are proteins that are highly conserved, in all serotypes of pneumococci. Throughout the research presented in this thesis, MF1 outbred mice and Balb/c inbred mice were immunised with PdB or neuraminidase A protein. Following I.P. or subcutaneous immunisation the mice were challenged using a variety of doses of virulent passaged pneumococci by the intraperitoneal, intravenous or intranasal routes. Immunisation with neuraminidase A elicited specific anti-neuraminidase antibodies. However, these antibodies were unable to protect the mice from acute pneumococcal challenge. Immunisation with PdB elicited a high titre of anti-PdB antibodies which were able to inhibit PLY activity in vitro. However, the antibodies were unable to protect the mice from acute pneumococcal challenge, or invasive pneumococcal challenge. Following colonisation challenge a significant drop in pneumococci recovered from nasopharynx of PdB immunised mice was seen. This significant drop in bacterial numbers correlated with a significant increase in the titre of specific IgG antibodies, as well as a significant increase in the number of specific IgG and IgA producing B cells, present in the cervical lymph nodes of PdB immunised mice, in comparison to that of the controls. Immunisation with neuraminidase A was unable to protect the mice from intraperitoneal and intranasal pneumococcal challenge, therefore, may not be necessary to include in future protein vaccines developed. Immunisation with PdB was able to significantly reduce nasopharyngeal colonisation, but was unable to clear colonisation. Therefore, PdB should be considered, along with other protective pneumococcal proteins, for inclusion in any future protein vaccine developed against pneumococcal colonisation.

This thesis describes the mathematical modeling of the Bacterial Flagellar Motor (BFM) system, for both the torque-generation and switching mechanisms.

Oxic soils are generally classed as a sink for atmospheric methane (CH4), as methanotrophy far outweighs internal production. However, CH4 production can be stimulated in such soils by exposure to wet conditions, or even cause them to act as a net CH4 source. The work detailed in this thesis was carried out with the aims of investigating the nature and distributions of the microbial communities controlling CH4 cycling in waterlogged aerobic soils, and assessing their potential to respond to marginal increases in soil moisture and aeration conditions caused by climate-change induced precipitation. The concentration of archaeol in its free and conjugated forms was investigated within three oxic soils using gas chromatography/mass spectrometry (GC/MS) in combination with selected ion monitoring (SIM). Archaeol was proposed to be a biomarker for methanogenic biomass in such soils on the basis that trends in abundance differed from Thaumarchaeota-derived crenarchaeol. Phospholipid and glycosidic archaeol seemed to derive from extant and fossilised methanogen biomass, respectively. Observed trends in concentrations were attributed to carbon contents and soil moisture, due to their association with the development of anoxic microniches and substrate availability. The presence and distribution of glycerol dialkyl glycerol tetraethers (GDGTs), detennined by high performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (HPLC/ APCI-MS), were investigated within two highmoisture, oxic soils. As with archaeol, trends in distribution were driven by organic matter content and soil moisture. Bacteria-derived GDGTs dominated at both sites. Isoprenoid GDGTs I and II-IV derived from a mutual archaeal source, potentially methanogens; however, distributions did not reflect archaeol concentrations.

Purple bacteria are metabolically-fiexible organisms that have been used widely in the study of the fundamental processes of photosynthesis. These bacteria possess multi- component photosystems composed of a variety of pigment-proteins. In all characterized purple photosynthetic bacteria a reaction centre (RC), whose function is photochemical charge separation, is surrounded by an LH1 light harvesting pigment-protein to form a core RC-LH1 complex. In some species additional light harvesting is provided by one or more peripheral light harvesting pigment-proteins. In the best characterized purple photosynthetic bacterium, Rhodobacter sphaeroides, the RC-LH1 core complex is present in either a monomeric or dimeric form, in which either one or two RCs are surrounded by a C- or S-shaped LH1 antenna, respectively. One aim of the work described in this thesis was to carry out the first systematic analysis of how environmental (growth) conditions, changes in carotenoid composition and genetic manipulation (gene deletion and complementation in trans) affects the composition of the Rhodobacter sphaeroides photosystem, with particular focus on the compositions and relative amounts of the monomeric and dimeric forms of the RC-LH1 complex. The composition of the photosystem assembled in range of Rhodobacter species was also explored. Subsequent work focused on the influence of the LH1 and PufX components on the assembly of the dimeric form of the RC-LH1 complex. In addition, a series of site-directed mutagenesis of the PufX polypeptide was carried out to explore the roles of this protein in defining the aggregation state of the LH1 pigment-protein around the RC, enabling correct function of the RC-LH1 complex, and facilitating assembly of the dimeric form.

Type III secretion systems (T3SS) are protein injection devices used by Gram-negative bacteria to manipulate eukaryotic cells. In Shigella, the T3SS is assembled when the environmental conditions are appropriate for invasion. However, secretion is only activated when physical contact of the injection needle with the host cell generates an activation signal. The signal is transmitted to the cytoplasm where it triggers secretion. First, translocators are secreted which form a pore in the host cell membrane. Second, effector proteins are translocated into the host cell. The activation process is controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gate-keeper protein MxiC. The major tip protein IpaD provides a scaffold for pore-forming translocators. In its absence no needle tip is formed, the T3SS secretes constitutively and is unable to sense host cell contact. Using random mutagenesis combined with a genetic screen we have mapped the region of IpaD required for activation signal generation/transmission and identified an additional intracellular role for IpaD in secretion control. Thus, IpaD has a dual role in secretion regulation. The gate-keeper protein MxiC is a cytoplasmic protein that plays a key role in mediating secretion hierarchy. In its absence, the secretion of translocator proteins is decreased and effector proteins are leaked. We have used site-directed mutagenesis, genetics and analysis of native protein complexes to further characterise its function. While MxiC seems to be a predominantly cytoplasmic and monomeric protein, we show that it acts in the same intracellular pathway as IpaD to control translocator secretion. We have identified the areas of MxiC required for activation signal reception, promoting translocator secretion, blocking premature effector secretion and for regulating its own secretion. We also provide evidence that a conformational change in MxiC might be involved in its function. Taken together, our work suggests how cytoplasmic mechanisms block premature secretion of translocators and effectors and in which steps secretion activation might occur.

Antibiotic resistant bacteria can be detected in numerous environments. In recent years, agricultural practices have been demonstrated to contribute to the emergence and persistence of antibiotic resistance. Heavy rainfall events are capable of transferring microorganisms from land stores to receiving water sources. This thesis describes the increase in numbers of enterococci in a freshwater stream following heavy rainfall events in a catchment located in County Monaghan, republic of Ireland. During the summer event, an increase in the level of antibiotic resistance was observed preceding the peak of the storm. A survey of the farms surrounding the freshwater streams revealed that antibiotic resistant enterococci are prevalent in this area. Analysis of isolates from all farm and water samples using a combined approach of biochemical profiling and multilocus sequence typing (MLST) indicated that poultry farming is a major contributor to the presence of enterococci in the streams. Enterococci are a diverse genus, with some strains capable of causing nosocomial infections while some are harmless commensals or environmental strains. In addition to antibiotic resistance, detection of putative virulence factors was common among isolates of enterococci from both the farm and water environments in this catchment. The number of isolates carrying multiple virulence factors was higher than expected, indicating that some of these strains may be capable of causing infection. Mobile DNA accounts for a significant proportion of the enterococcal genome. Antibiotic resistance and virulence genes are commonly located on mobile DNA. This thesis describes the presence of pheromone-responsive plasmids among animal and environmental isolates of enterococci. The isolation of such a large number of isolates carrying these plasmids from environmental samples was a surprise, in particular from E. faecium isolates, as these plasmids are strongly associated with clinical isolates. Overall, this thesis indicates that this environment can provide a substantial reservoir and means of transfer for antibiotic resistant and potentially virulent isolates of enterococci.

Lipopolysaccharide is an essential component of outer membranes in Gram-negative bacteria and there is increasing evidence that it might be involved in mediating host invasion via a direct interaction with host cell membrane. Also, many pathogenic intracellular bacteria, such as Brucella spp., use lipid rafts-dependent entry to mimic endocytic pathway and avoid lysosoma l degradation. In th is project, solid state NMR (ssNMR) spectroscopy and fluorescence spectroscopy are used to investigate possible interactions between bacterial LPS and membranes with raft-like composition. A ssNMR technique for studying changes in lipid dynamics during LPS/ membrane interactions was first developed and J- resolved 13C MAS NMR was used to study changes to DPPC membranes in response to temperature variation and in the absence of isotopic enrichment. J- resolved 20 HETCOR NMR spectroscopy was also developed to investigate more complex lipid membranes and was applied to DOPC/cholesterol and triple mixture DOPC/ SM/Chol, a model membrane used to mimic lipid rafts. Interactions between membrane-incorporated LPS and raft- like membranes were then investigated using 13C MAS NMR as well as 31p NMR spectroscopy. Results revealed that endotoxins undergo significant mobility restrictions in the presence of sphingomyelin and cholesterol suggesting that they partition into the liquid-ordered phase. Fluorescence spectroscopy was then used to study interactions between free LPS and membranes with and without latera l domains showing a preferred affin ity of endotoxins towards bilayers with raft-like composition over plain PC membranes, in agreement with the NMR data. ; ..

Cyanobacteria are present in most water bodies and sometimes grow to large populations known as blooms. Some of these cyanobacteria are also capable of producing toxins which can be fatal to livestock and humans. The production of cyanobacterial toxins is not specles-, but gene-specific, so the prediction of toxicity based on identification of the cyanobacteria using morphological characteristics is unlikely to be reliable. A better way to predict the likely toxicity of a bloom is the use of molecular techniques to detect the genes for toxin production and this was the approach taken in the first part of this project. This consisted of an analysis of . both cyanobacterial diversity and the occurrence of toxin genes in selected water bodies in Yorkshire. The mcyE gene, specific to some Microcystis sp. Anabaena sp. and Planktothrix sp. was found in plankton samples taken from the selected sites. The second part of the project was concerned with the use of rotting barley straw to inhibit cyanobacterial growth. Control of bloom formation using chemicals such as copper sulphate can have damaging environmental consequences but an alternative is the use of barley straw, the efficacy of which has been previously confirmed. Of the five newly-isolated cyanobacteria tested in this project, Microcystis strains 50053 and 50054, and Pseudanabaena strains 50055 and 50056 were markedly susceptible to straw inhibition, while Anabaena strain 50051 was susceptible, but to a much lesser degree. It was shown that prolonged incubation of sterile straw did not render it active at inhibiting the growth of the test cyanobacteria, conclusively demonstrating for the first time that straw activation requires microbial activity. This project was also the first to examine bacterial and fungal diversity on rotting barley straw and revealed a complex population of bacteria and fungi, many of which have the capacity for lignin degradation. This observation strengthens the evidence in support of the hypothesis that lignin derivatives released during the breakdown of straw may be responsible for inhibiting cyanobacterial growth.