No description available.

The energetics of Escherichia coli W, growing aerobically in carbon-, oxygen-, ammonium-and sulphate-limited continuous cultures were investigated. Room temperature difference spectra of respiratory membranes prepared from the cultures revealed the presence of cytochrome b and cytochrome oxidase o. In addition, cytochromes a1 and d were present in oxygen-limited cultures. Endogenous?H+/o quotients of whole cells from all of the cultures were consistent with the presence of respiratory chains with two proton translocating segments between NADH and oxygen. Glycerol-limited cultures at 30.0°C yielded Yomax2 (the maximum molar growth yield with respect to oxygen) and Ymaxatp (the maximum molar growth yield with respect to ATP equivalents) values of 50.9 g cells.mole 02-1 and 12.7 g cells.mole ATP equivalents respectively, together with a value for M (the energy requirement for maintenance purposes) of 2.32 mmole ATP equivalents.h-1.g cells-1. When the temperature of growth was decreased to 20.3°C, YmaxATP remained relatively unchanged at 11.7 g cells.mole ATP equivalents-1 but M decreased to 0.4 mmole ATP equivalents. h-1. g cells-1. Increasing the temperature of growth to 42.3°C caused YmaxATP to decrease to 7.6 g cells.mole ATP equivalents-1 and M to increase to 7.4 mmole ATP equivalents.h-1.g cells-1. Replacement of glycerol by other limiting carbon substrates caused YmaxATP and M to alter within the range 7.1 (acetate) to 13.9 (glucose) g cells.mole ATP equivalents-1 and 1.9 (glucose) to 6.7 (malate) mmole ATP equivalents.h-1.g cells-1 respectively. The carbon substrate-dependent variations in YmaxATP are probably due to differing energy requirements for macromolecular biosynthesis. Oxygen-limited (glycerol-grown) cultures exhibited similar YmaxATP and K values to those of glycerol-limited cultures. YmaxATP values for ammonium- and sulphate-limited cultures were generally similar to those for carbon-limited growth with the same carbon substrate but values for M were substantially higher (viz 14.9-30.8 mmole ATP equivalents.h-1 .g cells-1). It is concluded that neither YmaxATP nor M are constant values for E. coli W. YmaxATP was always considerably lower than the theoretical value, calculated for macromolecular biosynthesis; the magnitude and possible nature of energy-requiring processes which contribute to this difference are discussed.

No description available.

Flavocytochrome <I>c</I> (Fcc) is a novel, soluble periplasmic fumarate reductase from the Gram-negative bacterium <I>Shewanella putrefaciens. </I>A null mutant (<I>Δfcc</I>) which lacked the gene coding for the Fcc was constructed. The resultant strain was devoid of fumarate reductase activity and could not grow on fumarate as the sole terminal electron acceptor. Complementation of this mutation with recombinant Fcc restored the ability to grow anaerobically with fumarate. No other phenotype could be found for the <I>Δfcc</I> strain. This work demonstrates that flavocytochrome <I>c</I> is essential for fumarate respiration and furthermore is the sole terminal reductase for fumarate respiration. The <I>fcc </I>null mutant also allowed the expression of recombinant forms of <I>fcc</I> coding for the individual domains of the protein. The <I>in vivo</I> interaction between the haem and flavin domains of Fcc was investigated by expressing recombinant forms of the genes coding for these domains in the same cell. This indicated that the tether between the two domains, while not absolutely essential, was important for wild-type enzyme function. Studies using <I>lacZ</I> reporter genes fused to the <I>fcc</I> promoter demonstrated that the <I>fcc</I> gene is regulated primarily at the level of transcription and is regulated hierarchically in response to the redox potential of terminal electron acceptors. Regions of the <I>fcc</I> promoter sequence important to regulation of the <I>fcc</I> gene were identified by deletion analysis. The succinate dehydrogenase operon from <I>S. putrefaciens </I>was sequenced. The inferred amino acid sequences of the flavoprotein, and iron/sulphur subunits showed high sequence identity (77%) with the corresponding subunits from <I>Escherichia coli </I>succinate dehydrogenase.

The spindle checkpoint delays the metaphase to anaphase transition in response to spindle and kinetochore defects. Genetic screens in budding yeast originally identified the Mad and Bub proteins (and later the Mps1 kinase) as being core components of this conserved regulatory pathway. This work describes the identification and phenotypic characterisation of the fission yeast open reading frame (ORF), <i>SPCC895.02. </i>Based initially upon sequence homology and supported later by biochemical data we believe <i>SPCC895.02</i> encodes the fission yeast homologue of the Mad3 protein. This thesis comprises three key parts. In the first instance evidence is presented proving that the putative <i>mad3⁺. </i>ORF does indeed encode a component of the spindle checkpoint. Fission yeast cells devoid of <i>mad3⁺ </i>are unable to arrest their cell cycle in the presence of microtubule depolymerising agents, re-replicate their DNA and furthermore precociously separate their sister chromatids when spindle integrity is compromised. Secondly, a detailed localisation study was undertaken in which the endogenous <i>mad3⁺</i> ORF was tagged with the green fluorescence protein (GRF). Mad3-GFP is recruited to unattached kinetochores early in mitosis and accumulations there upon prolonged checkpoint activation. Furthermore, Mad3-GFP kinetochore localisation was found to be dependent upon the Bub1p kinase, Bub3p and also the Mph1p (Mps1 homologue) kinase, but not upon Mad1p or Mad2p. Finally, biochemical analysis of Mad3p interactions revealed genetic and biochemical interactions with Mad2p and the checkpoint effector S1p1/Cdc20p, suggesting an important role for Mad3p in transducing the inhibitory ‘wait anaphase’ signal to the anaphase promoting complex/cyclosome (APC/C).

This study aimed to characterise the role of bacterial extracellular polymers in surface reactivity, metal adsorption and biomineralisation. This was undertaken using an EPS-producing, thermophilic, bacterial strain, Bacillus licheniformis S-86. Experimental work was undertaken comparing cells with the EPS layer intact (native cells) with cells from which the EPS layer had been extracted (EPS-free cells). The study incorporated surface characterisation by potentiometric titration, infrared analysis and electrophoretic mobility analysis. Investigation of the mechanisms of zinc and nickel adsorption to cell surfaces was undertaken by both macroscopic batch adsorption experiments, and spectroscopic (EXAFS) analysis. Surface complexation modelling of the potentiometric titration data indicated that the native and EPS-free cells contained four proton-active functional groups, with pK_a values of 3.3-3.4, 5.3-5.4, 7.4-7.5 and 9.9-10.1. These were tentatively identified as phosphodiester, carboxyl, phosphoryl and hydroxyl/amine groups respectively, and ATR-FTIR analysis supported identification of the pK_a 5.3-5.4 site as carboxylic. The site concentrations of the pK_a­3.3-3.4 and 9.9-10.1 groups were significantly lower in the EPS-free cells than in the native cells. Both the macroscopic and EXAFS metal adsorption studies indicated that the carboxyl group is of principle importance to Zn complexation, and a lack of temperature-dependent adsorption provides evidence that Zn binds by an outer-sphere mechanism. Results for Ni did not provide a conclusive explanation of the binding mechanism. Biomineralisation experiments indicated that the presence of EPS affects both CaCO₃ morphology and polymorphism. The metastable polymorph vaterite appears less stable in the presence of EPS. The results of this study have shown that EPS, and potentially the associated dissolved organic carbon, can significantly affect the surface reactivity of bacterial cells.

The original aim of this thesis was to utilise <I>Vibrio harveyi </I>luciferase as a reporter of the expression of cell division genes during the cell cycle. Several plasmids expressing <I>luxAB </I>genes from <I>ftsZ</I> promoters were constructed. To achieve maximal luciferase expression, the ribosome binding site in front of the <I>luxA</I> gene was improved, which led to increased expression of luciferase. The level of expression of the improved luciferase reporter from plasmids was sufficiently high to be detected in single cells, although not high enough to be used in lower copy number constructs. However, luciferase activity showed significant fluctuations that did not appear to be linked to cell cycle events. These fluctuations made the detection of any cell cycle related changes in luciferase expression impossible. Another direction of this thesis is represented by the studies on the topology of the cell shape determining RodA protein. The ampicillin resistance levels were measured in 52 fusions with the topology probe BlaM made to different parts of the RodA protein. The combination of the BlaM fusion data and computer modelling argue for a topological structure of this protein consisting of ten transmembrane segments with both ends of the protein located in the cytoplasm. An important feature of the protein appears to be a large periplasmic loop - a highly conserved part of the protein possibly interacting with other members of the shape controlling system. The topology of the protein as well as the location of conserved regions suggests a possible function as a transporter of peptidoglycan precursors. During the course of RodA characterisation a mutation was isolated that permits stable growth of a <I>rodA</I> null strain in rich media. The cause of this stabilisation appears to be a simultaneous increase in the amounts of FtsZ and FtsA proteins.

Two strains of alkaliphilic, strictly aerobic, Gram-positive, non-motile bacteria were isolated. First, an alkaliphilic bacterium (optimum growth at pH 10) designated as MAK7 was isolated from a water and sediment sample obtained from a non-extreme environment (River Lathkill in the Derbyshire Peak District). Second, an alkaliphilic Bacillus sp. was isolated as a laboratory contaminant from a culture of MAK7 grown at pH 10. Phylogenetic analysis, based on 16S rRNA gene sequences, showed that isolate MAK7 was an unclassified strain of Bacillus, most closely related to a Bacillus isolate associated with marine sponges (99.4% sequence identity). The laboratory contaminant Bacillus sp. was most closely related to the species Bacillus cereus (100% sequence similarity). Generation times of 55 and 40 minutes were observed at external pH lOin Horikoshi medium for alkaliphilic Bacillus MAK7 and B. cereus respectively. The internal pH of Bacillus MAK7 and B. cereus at pH 10 was 9.00 ± 0.08 and 8.76 ± 0.28 respectively. To cope with the reversed ~pH at pH 10, the membrane potential of both strains increased significantly. However, there was a significant overall drop in the proton motive force at pH 10 to -112 m V for Bacillus MAK7 and -97 m V for B. cereus. The optimum salinity for growth was determined to be 100 mM NaCI for Bacillus MAK7 and 400 mM NaCI for B. cereus. However, neither strain showed a Na+ or K+ requirement for growth or optimal respiration rates, which is unusual for alkaliphilic bacteria. Increasing concentrations of NaCI were inhibitory to the respiration rate of both strains, but KCl concentrations up to 400 mM did not inhibit respiration. Enzyme activities (malate dehydrogenase, fumarase and hexokinase) in crude cell-free extracts were measured to investigate the effects of alkaline pH on metabolic pathways.

No description available.

Oxygen uptake and CO2 evolution were measured in synchronous cultures of the fission yeast Schizosaccharomyces pombe, growing in a minimal medium. The rate of oxygen uptake increased abruptly at the middle of the cycle and again at the end of the cycle. During the intervening periods the rate remained constant. Oxygen uptake was measured nanometrically and polarographically. The rate of CO2 evolution doubled sharply at the tin of nuclear division (0.75 of the way through the cycle). For the remainder of the cell cycle, the rate remained constant. Similar step wise increases in the rate of CO2 evolution could be induced by adding glucose to late exponential cultures. So various attempts were made to show that the steps in the rate of CO2 evolution in synchronous cultures were not artefacts induced by the synchronisation technique. The addition of DNA synthesis inhibitors and a nuclear division inhibitor to synchronous cultures had no effect on the patterns of oxygen uptake and CO2 evolution. Similarly in an induced synchronous culture, in which DNA synthesis, nuclear division and cell division - but not "growth" - were synchronised, oxygen uptake and CO2 evolution showed a continuous increase and not a "synchronous" pattern. Evolution of 14'CO2 was measured in synchronous cultures. Various technical problems made the interpretation of these results difficult but there is a similarity between the measurement of 14CO2 evolution and CO2 evolution measured nanometrically in synchronous cultures. Experiments with protein and RNA synthesis inhibitors suggested either that an increase in oxygen uptake and CO2 evolution was dependent on continued protein and RNA synthesis; or that in the absence of either protein or RNA synthesis, the demand for energy decreased and hence the flux through the energy yielding pathways which result in oxygen uptake and CO2 evolution also decreased. Other experiments showed that optical density increase doubled in asynchronous culture at about the same time that CO2 evolution doubled in rate. Both hexokinase and ATP were shown to increase exponentially in synchronous cultures and are therefore not obvious regulators of, or obviously regulated by, oxygen uptake or CO2 evolution. The control of respiratory and fermentative activity is discussed and it is suggested that the levels of intermediates in the E pathway and the TCA cycle control the activities of these pathways.