Two proteins putatively involved in the persistence of Mycobacterium tuberculosis infection have been investigated by heteronuclear nuclear magnetic resonance (NMR) spectroscopy and other methods. The major aspect of work describes the characterization by NMR of the Mtb thiol peroxidase TPx. TPx (165 residues) is a 36 kD symmetrical homodimer that catalyzes the reduction of a range of reactive oxygen species in vitro. Multidimensional heteronuclear NMR spectroscopy was applied to recombinant wild-type (Wt) and Cys60→Ser (C60S) TPx, both of which are clearly folded but display slightly different spectral characteristics. Triple resonance NMR experiments recorded using 2H, 13C, 15N -labelled C60S yielded assignments for the majority of the backbone resonances. 28 non-proline residues could not be assigned mostly due to the absence of cross peaks. TPxWt spectra showed little difference in peak number indicating that the C60S mutation itself does is not responsible for the missing signals. Consideration of related TPx crystal structures indicates that the missing cross peaks correspond to a region of the protein that must display conformational plasticity necessary for the mechanism of action suggesting that the spectrum is affected by conformational exchange processes. Efforts to modulate this exchange behaviour through the application of putative TPx ligands and chemical modification of the active site cysteine residues are described. In a second aspect, a preliminary characterisation of the non-canonical RNA polymerase sigma factor sigJ from Mtb was performed. Bioinformatic analysis revealed that sigJ likely contains conserved o2 and a4 DNA-binding domains fused to a unique C-terminal domain. NMR spectra of recombinant sigJ recovered from inclusion bodies and in the soluble fraction differed, though both forms yielded dispersed resonances consistent with the protein containing a globular component. However further progress was hampered by signal overlap and heterogeneous line widths. sigJ has a general capability to retard the movement of DNA in an electrophoretic mobility shift assay, though specific target sequence was identified.

The physiological role of flavocytochrome <I>b<SUB>2</SUB></I> from <I>Saccharomyes cerevisiae </I>is to couple L-lactate dehydrogenation to respiration via the ubiquitous electron carrier cytochrome <I>c</I>. For each L-lactate molecule dehydrogenated two cytochrome <I>c.</I> molecules are reduced, and as such the enzyme acts as a 'bio-electrical transformer'. The mechanism through which this process occurs can be simplified into five separate electron transfer events which form the catalytic cycle. L-lactate dehydrogenation results in the two electron reduction of flavin at the enzyme's active site. These electrons are passed individually to the <I>b<SUB>2</SUB></I>-haem (intramolecular electron transfer) and on to two cytochrome <I>c</I> molecules (intermolecular electron transfer). Using stopped-flow spectrophotometry, the intramolecular electron transfer steps have been investigated using several different experimental procedures. Electron transfer from fully reduced FMN to <I>b<SUB>2</SUB></I>-haem has been studied by monitoring both haem reduction and haem re-reduction (following oxidation by cytochrome <I>c</I>). In each situation this step proved too fast to be observed, and appeared only as a slight lag (relative to flavin reduction) in the <I>b<SUB>2</SUB></I>-haem reduction trace. Nevertheless a lower estimate for this rate constant was derived to be 1500±500 s<SUP>-1</SUP>. The second intramolecular electron transfer step takes place from flavin semiquinone to <I>b<SUB>2</SUB></I> haem and was observed as a component of the flavin oxidation process. This proved to be the slowest step in the catalytic cycle (at 120 s<SUP>-1</SUP>) and is therefore responsible for determining the overall turnover rate. The product, pyruvate, was found to be an inhibitor of the flavin oxidation reaction (K<SUB>i</SUB>= 40 ± 15 mM) consistent with reports that it acts as a non-competitive inhibitor in the steady-state. Stopped-flow studies on the cytochrome <I>c</I> reductase activity of flavocytochrome <I>b<SUB>2</SUB></I> yielded a second-order rate constant of 35 μM<SUP>-1</SUP>s<SUP>-1</SUP>, which represents the rate constant for cytochrome <I>c</I> association.

No description available.

The work described in this thesis is concerned with further genetic and molecular analysis of <i>wis2⁺</i> The aim of this work was to establish a cellular function for <i>wis2⁺</i> and to explain the effect of overexpression in the triple mutant strain described above. Using an <i>in vitro</i> binding assay, a number of proteins that show a physical interaction with Wis2 were isolated. These proteins were identified by peptide sequencing. Five were found to be ribosomal proteins. Another, ~85kD, was identified as <i>S. pombe </i>Hsp90; the intensity of the interacting band is increased, and the interaction is more consistent, if the extract is made from <i>S. pombe </i>cells which have previously been heat shocked. Genetic analysis revealed that overexpressing of <i>wis2⁺</i> lowers the restrictive temperature of a number of <i>cdc2</i> mutant alleles which arrest at G2-M. This effect of enhancing a cell cycle block is in contrast to the situation in the <i>cdc25-22 wee1-10 win1-1 </i>background where the G2-M block is relieved by <i>wis2⁺</i> overexpression. An extensive genetic screen was conducted to isolate mutations that are synthetically lethal with <i>wis2</i><i>D, </i>with the aim of identifying functionally redundant genes or downstream targets of <i>wis2</i>. No synthetic lethal interactions were identified. Indirect immunofluorescence studies established that Wis2 is localised exclusively in the cytoplasm. Consistent with this, a consensus nuclear export signal was identified in the C-terminus of Wis2. Structure-function analysis showed that overexpression of the C-terminal domain of Wis2 alone has the same activity as full length Wis2 <i>in vivo</i>. Overexpression of the N-terminus alone had no observable effect in the genetic backgrounds tested. The C-terminal domain of Wis2 was shown to be responsible for the interaction with Hsp90 in the <i>in vitro</i> binding experiment; this is consistent with results from other systems where the TPR domain was found to be responsible for Hsp90 binding.

The biology and evolution of many arthropod species cannot be understood without also considering their bacterial symbionts. In order to proliferate, many endosymbionts manipulate the reproduction of their hosts, and do so in a multitude of ways. The phenotypes that result from this bacterial manipulation have profound implications for the evolution of their hosts. These interactions will both influence, and be influenced by the abundance of the bacterial symbiont in arthropods. One of the best-studied bacterial manipulations is male-killing, where endosymbionts kill their male hosts in order to distribute resources towards infected females (the only individuals who will transmit the infection). Ladybirds beetles are known to harbour male-killing symbionts, and so the incidence of bacteria was investigated in the whole of this family. It was found that endosymbionts infected over half of the host species, mainly at low prevalence, which indicates that symbiont incidence and diversity may be currently under-estimated. In addition, multiple symbionts were found in the same population, lending strength to the hypothesis that they are being maintained by balancing selection with host resistance genes. The data was combined with a world-wide screen and other data from the literature, and used to estimate the distribution of across-species prevalences of the bacteria Wolbachia, Rickettsia and Cardinium in wild arthropod populations. A newly developed likelihood approach was used to find a best fit distribution, and properties of the distribution then used to predict how these symbionts manage to invade and spread through populations. The analysis revealed that the skew toward low prevalence infections may apply quite generally, suggesting that much of the diversity of endosymbionts will be missed from screens that test only a few individuals. In addition, the analysis highlighted differences in the incidence levels of different bacteria, and heterogeneity in prevalence distributions between clades of host species. Contemporary patterns of endosymbiont abundance must also be understood within the long-term evolutionary context, best investigated with a phylogenetic approach. Comparison of Rickettsia and Wolbachia phylogenies with those of their hosts indicate that these symbionts frequently switch horizontally between related hosts. Rickettsia have been less thoroughly investigated than Wolbachia, but these arthropod endosymbionts can also infect and cause serious diseases in humans and other mammals. In this study, 20 new strains of arthropod Rickettsia are identified and multiple genes sequenced to produce a robust phylogeny of the whole genus. Rickettsia are devised of two main clades, one of which primarily infects arthropods and the other infect a diverse range of protists, leeches, unidentified hosts from metagenomic samples, and some arthropods. Strategies such as male-killing and parthenogenesis induction appear to be recent innovations. Arthropod Rickettsia generally group basal to medically important strains, but some also cluster within the strains that infect vertebrates. There is increasing evidence against the traditional view that intracellular symbionts are refractory to recombination. Recombination does occur in Rickettsia but seems to be uncommon. However, there is strong evidence of large scale horizontal gene transfer events. Numerous conjugation genes were also discovered which indicates that plasmids may be common throughout the whole genus.

The viability of using natural manganese oxide (MnOx)-containing wastes as amendments for contaminated land remediation was examined. The success of MnOx as a viable strategy was determined via the impact that their addition had upon microbial soil functioning, in addition to their ability to immobilise and/or transform inorganic and organic contaminants within industrially polluted soils. Contaminated soils were obtained from two former industrial sites that are polluted with PAHs and metals. The intrinsic microbial functioning of these soils was assessed using a suite of microbial indicators reportedly sensitive to contamination [basal respiration (BR), potential nitrification (PNR), denitrification enzyme activity rates (DEA), microbial biomass carbon (MBC), metabolic quotient (qCO2), microbial quotient (qmic)]. The diversity and community structure of key populations related to microbial indicators were assessed using culture-independent community analysis [polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE)] to target 16S rRNA and functional genes (amoA, nirS/K). These measurements were combined with a suite of physico-chemical analyses [pH, total organic carbon (TOC), soil organic matter (SOM), moisture content (MC), water holding capacity (WHC), extractable metal and PAH concentrations] to provide a combined geochemical and molecular microbial approach. Contaminated soils were compared to a range of soils from land types defined as non-contaminated to provide a robust evaluation and define suitable microbial indicators for use in assessing soil microbial function in contaminated land and its remediation. Results showed that long term metal and PAH pollution had resulted in a microbial populations exhibiting extremely suppressed rates of BR and DEA, indicative of pollutants being bioavailable within the soil. Functional gene profiling revealed that inherent denitrifying and ammonia-oxidising community structures were significantly affected by contamination. Microbial functional processes of BR, DEA and PNR were determined to be superior indicators through their ease of use with standardised rapid and high throughput methods that could infer contaminant availability. A preliminary assessment of MnOx addition upon microbial soil functioning was investigated though 6 month microcosm trials employing BR, DEA and PNR as indictors. Microcosms employed two natural MnOx-containing wastes (mine tailings and coated sands) in a range of 0-30 % total MnOx, which were added to a low level metal contaminated soil. MnOx was found to stimulate PNR and DEA. Results implied that MnOx is not detrimental to soil microbial functioning and is capable of removing inhibitors of N-cycling, via a chemical rather than biological mechanism, ascribed to the immobilisation of bioavailable toxic metal ions by the MnOx. The potential of MnOx amendment as a viable remediation strategy was investigated through the use of 9 month outdoor lysimeter trials. Measurement of extractable PAHs, along with extractable and bioaccessible Pb and As in metal, PAH and mixed contaminated soils showed no positive effects of using a 10 % by weight MnOx-coated sand amendment for remediation. Analysis of soil microbial functional indicators (BR, DEA, PNR) showed that MnOx amendment had no detrimental effects upon the function of microbial populations in the aforementioned soils. Mn(II)-oxidising bacteria were isolated from contaminated soils and MnOx-containing wastes. This suggested that sustained and biologically enhanced redistribution of MnOx was possible in MnOx-amended soils, which may play an important role in pollutant transformation. This study provided the first demonstration of species within the genera Amycolatopsis, Sacchorothrix, Lentzea and Micromonospora as being capable of Mn(II) oxidation.

No description available.

The growth and biogenesis of the outer membrane of E.coli B/r has been studied by investigating both the pattern of synthesis of outer membrane protein in the cell cycle, and the kinetics of synthesis and assembly of the major outer membrane protein species, the 36.5K porin. To facilitate this study the detergent sarkosyl was used to separate the outer membrane from the cytoplasmic membrane of E.coli; comparison of these fractions with those obtained by centrifugation revealed that they were qualitatively identical. Analysis of the cell cycle revealed that the rate of synthesis of outer membrane protein was constant through the cycle, with an abrupt doubling in rate occurring late in the cycle. Further experiments revealed that the rate of synthesis of outer membrane protein is not differentially affected by treatments which affect DNA synthesis, and it is therefore concluded that the cell cycle doubling in rate of synthesis is unrelated to the DNA replication cycle. In other experiments the time was measured for the 36.5K porin to be translated, enter the cell envelope, and become associated with the sarkosyl-insoluble outer membrane. This was achieved by a pulse-chase method. The porin associated with the envelope as soon as it was completed; however, it took significantly longer to reach the outer membrane, indicating the existence of a sarkosyl-soluble intermediate form of the mature porin. It is concluded that this may be either a true cytoplasmic membrane intermediate, or a nascent form present in atypical regions of the outer membrane. Finally, it was shown that an outer membrane protein whose synthesis has been reported to occur periodically in the cell cycle is, in fact, a protein involved in ferric-enterochelin uptake, and it is concluded that its apparent periodic synthesis is an artefact caused by the methods used to generate synchronous cultures.

Since industrialisation global CO2 emissions have increased, and as a consequence oceanic pH is predicted to drop by 0.3-0.4 units before the end of the century - a process coined ‘ocean acidification’ (OA). There is significant interest therefore in how pH changes will affect the oceans’ biota and integral processes. This thesis investigates microbial community organisation and functioning in response to predicted end of century CO2 concentrations using an elevated CO2 (~750ppm), large volume (11,000 L) contained seawater mesocosm. This thesis utilises RNA stable isotope probing (SIP) technologies, in conjunction with quantitative reverse transcriptase PCR (RT-qPCR), to investigate the response of microbial communities to elevated CO2. This thesis finds little evidence of changes occurring in bacterial abundance or community composition with elevated CO2, under both phytoplankton pre-bloom/bloom and post-bloom conditions. It is proposed that they represent a community resistant to the changes imposed. In contrast, significant differences were observed between treatments for a number of key eukaryote community members. These findings were investigated in the context of functional change, using the uptake of two key substrates (bicarbonate and glucose) as analogues for photosynthesis and respiration respectively. Unlike community abundance, distinct changes in carbon assimilation were detected in dominant members of the picoplankton. In conclusion the data presented suggest that although current microbial communities hold the capacity to respond to elevated CO2, future responses will likely be taxa specific and controlled by wider community dynamics.

No description available.