No description available.

Tetanus toxin exerts its primary biological effect by impairing the release of inhibitory neurotransmitters in the central nervous system; the mechanism of this blockade, however, remains unknown. Studies using adrenal medullary chromaffin cells, which are closely related to the toxin's target neurones but much more accessible to biochemical investigation, have the potential to provide information on various aspects of the intoxication process of tetanus. Nicotine-evoked (but not basal) secretion of catecholamines from intact chromaffin cells was inhibited by tetanus toxin in a dose-dependent fashion up to a maximum of 75%, half-maximal inhibition being achieved at 0.7 nM toxin (in single- or double-chain form). The time course of this inhibition was long, approximately 16 hours. Catecholamine release evoked by Ba<SUP>2+</SUP> ions was not affected by the toxin, while its effect on K<SUP>+</SUP> -evokedsecretion was never greater than 30%. Pre-incubation ofcells with ganglioside GT1, a specific ligand of the toxin, in the absence of toxin itself inhibited nicotine-evoked release of catecholamines (50&37 inhibition with 24 uM GT1), making it impossible to evaluate the role of GT1 in mediating the action of the toxin. Tetanus toxin (radiolabelled) was also shown to bind in a specific fashion to chromaffin cells. Toxin binding under less physiological conditions of pH and ionic strength was a higher capacity (B<SUB>max</SUB> 0.7-1.2 pmol/mg protein) than that found under more physiological conditions (B<SUB>max</SUB> 0.2-0.3 pmol/mg protein); this is also characteristic of toxin binding to synaptic membrances. In both cases there appeared to be at least two components to toxin binding, a higher affinity component with a K<SUB>d</SUB> value of approximately 1nM (which did not account for more than 20% of total binding capacity), and a lower affinity binding with a K<SUB>d</SUB> value of 10-25 nM. As expected, pre-incubationof chromaffin cells with ganglioside GT1 enhanced their toxin-binding capacity, but did not noticeably affect K<SUB>d</SUB> value, perhaps indicating that gangliosides mediate binding of tetanus toxin to untreated chromaffin cells. This was further suggested by the finding that neuraminidase treatment of cells markedly reduced toxin-binding capacity (by 50% at pH 7.4, 90% at pH 6.0) and also by the observation that pre-incubation of <SUP>125</SUP>I-labelled tetanus toxin with GT1 led to a reduction in its binding to chromaffin cells. Mild trypsinisation of chromaffin cells completely abolished toxin-binding under both sets of conditions, suggesting the involvement of a protein component in toxin binding also.

In this study the glycolytic enzyme pyruvate kinase was chosen as a potential phylogenetic marker. It was considered to be particularly appropriate for two reasons; first, it fulfils the criteria outlined above and secondly, there is a wealth of structural information for the enzyme from various sources available in the sequence databases. The pyruvate kinase chosen for study was that of the the thermoacidophilic archaebacterium, <i>Thermoplasma acidophilum</i>, because whilst there is a great deal of information in the databases on pyruvate kinases isolated from organisms within the eucaryal and bacterial domains, no sequence information previously existed for an archaebacterial equivalent. The aim of this study, therefore, was first to characterise the enzyme to ensure that the constancy of function requirement was fulfilled, and secondly to obtain sufficient primary structure information to facilitate a realistic evaluation of the potential of pyruvate kinase as a phylogenetic marker. The pyruvate kinase was purified to homogeneity using a series of chromatographic steps, and then characterised with respect to its physical and kinetic properties. The enzyme has a native Mr of 250 K, and a subunit Mr of 60 K. It exhibits typical Km values towards its substrates PEP and ADP, and is allosterically regulated by AMP. It is one of the most thermostable pyruvate kinases yet isolated, being active at up to 90<SUP>o</SUP>C. Initial sequencing attempts were frustrated by the chemical blockage of the N-terminus of the enzyme, and hence it was cleaved both chemically and proteolytically into peptide fragments, which were then sequenced by automated Edman degradation. The sequences of these internal peptides were then used in conjunction with a codon usage table derived from the citrate synthase gene of <i>T. acidophilum</i> to design a number of oligonucleotide probes. These probes were then a) fluorescently labelled and hybridised to Southern blots of restriction digests of <i>T. acidophilum</i> genomic DNA, and b) used as primers for the polymerase chain reaction.

The <I>PMR1</I> gene of <I>Saccharomyces cerevisiae</I> is predicted to encode a P-type Ca<SUP>2+</SUP> ATPase (Rudolph <I>et al</I>., 1989). This protein has been reported as localizing to a novel Golgi-like organelle (Antebi and Fink, 1992). Consistent with Pmr1p's proposed Golgi distribution is the fact that the pleiotropic phenotype of null mutants results in defects in various Golgi processes (<I>e.g</I>. outer chain glycosylation, proteolytic processing, vacuolar sorting). These defects are reversible by addition of Ca<SUP>2+</SUP> to the extracellular medium, supporting the proposed function as a Ca<SUP>2+</SUP> pump (Rudolph et al., 1989; Antebi and Fink, 1992). Work described in this thesis was carried out to investigate the nature of the <I>pmr1</I> phenotype and to further characterize the Pmr1p containing organelle(s). It is demonstrated that the <I>pmr1</I> phenotype is not due to a complete bypass of the Golgi (as proposed by Rudolph <I>et al</I>., 1989) since Kex2p processing of a secreted protein is detected in a <I>pmr1</I> mutant. There does however appear to be a change in Golgi organization in <I>pmr1</I> mutants. When the organelles containing Kex2p were isolated from <I>pmr1</I> and <I>PMR1</I> strains the profiles of other marker enzymes recovered was significantly altered. In particular the enzyme GDPase, an early Golgi marker, colocalizes with Kex2p, a <I>trans</I>-Golgi network marker, in <I>pmr1</I> strains. Furthermore DPAP A (Ste13p) no longer colocalizes with Kex2p in <I>pmr1</I> strains. This evidence suggests that protein targeting and/or retention is altered in <I>pmr1</I> strains as a result of aberrant Ca<SUP>2+</SUP> homeostasis. Another possibility is that some gross reorganization of Golgi structure has occurred. These changes in enzyme localization were not reversible by addition of Ca<SUP>2+</SUP> to the growth media. Pmr1p was tagged with protein A to allow isolation of organelles containing the fusion protein with IgG-Sepharose. However, after characterization of the recovered material it became clear that the fusion protein had localized to the vacuole and not the Golgi. This is probably due to the tag interfering with proper Golgi retention. This result is consistent with the vacuolar default model for membrane proteins in yeast (Roberts <I>et al</I>., 1992; Nothwher <I>et al</I>., 1993).

The majority of <I>Aeromonas</I> spp. are innately resistant to ampicillin, therefore the aim of this thesis was to examine the resistance mechanism of <I>A.salmonicida</I> subspp. <I>achromogenes</I> and <I>A.hydrophila</I> to β-lactam antibiotics. Previous researchers had revealed the presence of two β-lactamases in the motile <I>Aeromonas</I> spp., a penicillinase with carbapenemase activity and a cephalosporinase. However, it was demonstrated in this thesis, that these β-lactamases had not been purified completely and that three β-lactamases could be purified from <I>A.salmonicida</I> subspp. <I>achromogenes</I> and <I>A.hydrophila</I>. Anion and cation exchange chromatography were employed to separate the β-lactamases of the strains which had been induced with sub-MIC doses of ampicillin, cefoxitin or imipenem. Substrate profiles of these purified β-lactamases revealed the presence of a cephalosporinase, a penicillinase and a highly unusual carbapenemase which cannot be detected with nitrocephin. Inhibitor profiles showed that the cephalosporinase and the penicillinase were serine-based, whereas the carbapenemase was a metallo-enzyme but was unusually sensitive to zinc and may be a new class of metallo-β-lactamase. The cephalosporinase is probably a class C β-lactamase whereas the penicillinase is probably a class A β-lactamase. An ampicillin-sensitive species, <I>A.salmonicida</I> subspp. <I>salmonicida</I> was also examined and found to contain a carbapenemase and a cephalosporinase, but no penicillinase, which will explain its sensitivity to ampicillin. The expression of these two β-lactamases appears to be co-regulated as the cephalosporinase was only expressed in the uninduced strain and the carbapenemase activity was much higher post-induction. During this PhD., the first quinolone-resistant clinical isolate of <I>A.hydrophila</I> was found. The gene encoding the α subunit of the DNA gyrase was therefore amplified and sequenced and compared with the sequence of a sensitive strain. A mutation was found in the codon of amino acid 83 which affected the ability of the quinolone drugs to bind to the DNA gyrase. Five laboratory mutants of <I>A.hydrophila</I> were also selected with either nalidixic acid or ciprofloxacin and the changes in the sequences of the <I>gyrA</I> genes were also determined to occur at amino acid 83 and/or 87.

No description available.

It has been established that ocean acidification will adversely affect calcifying organisms but little is known about its effects on bacterioplankton and the biogeochemical processes which they catalyse. In this thesis, the impact these changes may have on microbialy driven processes is assessed through a mesocosm experiment conducted in a Norwegian fjord near Bergen in May 2006. Three mesocosms were bubbled with CO2(g) to simulate the predicted future conditions of rising atmospheric CO2 concentrations (~760ppm, pH ~7.8), while another three were treated as controls and bubbled with ambient air to represent present day conditions (~380ppm, pH ~8.15). The mesocosms were amended with nitrate and phosphate [16:1] to stimulate a phytoplankton bloom and scientific measurements and analyses were conducted over a 23 day period. At the peak of the phytoplankton bloom chlorophyll-a concentration was ~34% higher under ambient CO2 conditions compared to the high CO2. This was reflected in the flow cytometry results which showed a significant decrease of coccolithophorid and picoeukaryote cell numbers in the high CO2 treatment. Analysis of 16S rRNA gene clone libraries supported by qPCR data revealed that elevated CO2 resulted in a sharp decline in Roseobacter-like bacteria from the Alphaproteobacteria which are significant consumers of the algal osmolyte dimethylsulphoniopropionate (DMSP). Stable isotope probing using 13C labelled sodium bicarbonate revealed that the assimilation of dissolved inorganic carbon by Roseobacter-like bacteria is more prevalent than previously thought making them major contributors to global CO2 fixation. Furthermore, metagenomic analysis of the ambient and high CO2 libraries revealed a significant decrease in genes coding for DMSP demethylase in the high CO2 metagenome. This gene is responsible for the catabolism of DMSP resulting in the eventual release of methanethiol, a source of reduced sulfur for marine bacteria. However, DMSP degradation may proceed down an alternate route known as the lyase pathway resulting in the release of the climatically active gas dimethylsulfide (DMS). In conclusion, the findings of this study strongly suggest that the subsequent decline in Roseobacter species will shift the balance in the degradation of DMSP in favour of the alternate lyase pathway resulting in increased production of DMS and a decrease in the concentration of methanethiol. It is believed that the consequent loss of fixed sulfur will affect ocean productivity and that global climate patterns may change due to the scattering of solar radiation by increased atmospheric sulfur.

The lactic acid oxidising, sulphate-reducing bacteria were numerically predominant over the acetic acid oxidising sulphate- reducing bacteria in the sediments at station C in the River Don estuary. The numbers of acetic acid oxidising, sulphate-reducing bacteria were higher than the acetic acid oxidising, sulphur-reducing group. The anaerobic cellulolytic bacteria and Escherichia coli. were, also present in high numbers at station C. Demographic fluctuations in the numbers of these bacterial groups demonstrated periodicity influenced seasonally. High numbers of bacteria were associated with low winter temperatures, high sulphide concentrations, more negative redox potentials and high suspended solids in the river water. The numbers of anaerobic cellulolytic bacteria were highly correlated with the numbers of lactic acid oxidising, sulphate-reducing bacteria. Fluctuations in bacterial, populations demonstrated similar trends to those observed by Parkes for 1975-1976 substantiating the hypothesis of seasonal periodicity. The proportionality of the sulphur-reducing and sulphate-reducing groups of bacteria did not exhibit a fixed ratio throughout the year. Particle size analysis revealed that the sediments were homogeneous, particle size distributions were uniform with depth and for a 1.5 m square at the sampling site. Analysis of the sediment pore water showed low levels of sulphate, sodium and chloride ions and a low pool size of short-chain fatty acids. One of the proposed hypotheses influencing seasonal periodicity in the numbers of sulphur-reducing and sulphate-reducing bacteria is the direct provision of substrates for sulphater-reduction by the anaerobic cellulolytic bacteria. No evidence was obtained in this study which would refute the "temperature moderated sulphide toxicity" hypothesis forwarded by Parkes (1978) as one of the possible influences delineating bacterial periodicity. The vertical distribution of the following groups of bacteria were examined with sediment depth: the lactic, acetic, butyric and propionic acid oxidising sulphate-reducing bacteria and the anaerobic cellulolytic bacteria. The highest numbers of these groups of bacteria were in the 1 centimetre to 5 centimetre region of the core. There was a sharp decline in the numbers of bacteria from 6 centimetres to 10 centimetres (the lowest depth sampled). Isolation of the groups of sulphate-reducing and sulphur-reducing bacteria from the sediment was precluded by persistent contamination of enrichments by the facultative bacterium Escherichia coli. Defined mixed culture studies, involving the cellulolytic bacterium Clostridium papyrosolvens grown in the presence of DesuIfovibrio salexigens or Desulfotomaculum acetoxidans showed that cellulose degradation was enhanced in the presence of these sulphate-reducing bacteria. Clostridium papyrosolvens produced lactic acid, acetic acid, ethanol, carbon dioxide and hydrogen from the degradation of cellulose. Cellulose degradation by Clostridium papyrosolvens in co-culture with Desulfovibrio salexigens was enhanced by the removal of hydrogen by the sulphate-reducer. This interrelationship demonstrated interspecies hydrogen transfer, a form of protoco-operation of benefit to both partners. Cellulose degradation by Clostridium papyrosolvens in co-culture with Desulfotomaoulum acetoxidans was enhanced by the removal of acetic acid by the sulphate-reducer. This interrelationship demonstrated a nutritional link between these bacteria of benefit to both partners. The formation of composite colonies by sulphate-reducing bacteria and Escherichia coli in minimal media; the banding of sulphate-reducers in the region of the interface between agar layers containing each of the bacteria and the removal of hydrogen produced by Escherichia coli under anoxic conditions by Desulfovibrio salexigens suggests an interrelationship between these organisms. Protoco-operative interrelationships were demonstrated between Clostridium papyrosolvens and Desulfovibrio satexigens and the cellulolytic bacterium and Desulfotomaculum acetoxidans. These interrelationships are advantageous to each of the organisms and could be of significance in the environment.

Pneumolysin is a major virulence factor of Streptococcus pneumoniae. It is a 53-kDa protein with four domains which form pores in host cells and depletes serum-complement resulting in a diminished host immune response. Despite its importance, the high-resolution structure of pneumolysin is yet to be solved. This thesis seeks to address the lack of high-resolution data, enabling the identification of residues important for pore formation and binding to its cellular target ligand, cholesterol. The interactions between complement and pneumolysin were investigated, to further understand the role of pneumolysin in serum complement depletion. The structure of pneumolysin was solved using X-ray crystallography at 2Å resolution. The crystal packing mimics the pre-pore arrangement of pneumolysin, enabling the identification of intermolecular interactions that stabilise the pre-pore. Mutation experiments revealed interacting residues were not structurally important but resulted in a substantial reduction in the pore forming capability of pneumolysin, showing that they are functionally important and most likely inhibit pre-pore oligomerisation. Residues involved in the interactions are 69% conserved across five proteins of the same family implying a common mechanism. The crystal structure revealed other residues that may be functionally important with respect to domain orientation and cholesterol binding in the pre-pore, and these could be investigated in the future. Complement assays showed that pneumolysin diminished complement in the fluidphase and when immobilized on a surface. However pneumolysin did not interact with IgG and a mutation previously identified as being important (D385N) had no effect on the activity observed. Activation occurred via the alternative pathway, with contributions from the classical and/or the lectin pathway.

Armillaria, or the 'honey fungus', is an edible basidiomycete that causes Armillaria root disease on numerous species in forests gardens and agricultural environments worldwide. Armillaria research has been hindered by several factors: the lack of a reliable in vitro fruiting system for heterothallic Armillaria mellea requiring reliance upon intermittently available wild-collected isolates, existing pathogenicity assays that are laborious, time-consuming and often conducted under variable conditions and limited availability of genetic tools. Furthermore, Armillaria is diploid and contains homothallic and heterothallic subspecies but the genetic mechanisms controlling the different heterothallic and homothallic lifestyles and ploidy in this genus are unknown.