No description available.

The cellular distribution of eleven Trypanosoma brucei enzymes involved in glucose breakdown has been studied, using the following six methods of cell disruption: saponin, Triton X-100, digitonin, freezing and thawing, and grinding with the abrasives alumina and silicon carbide. By means of differential centrifugation of the homogenates of the bloodstream T. brucei obtained by these six different methods of cell lysis, it has been shown that the distribution pattern of the enzymes is greatly affected by the method of cell lysis as follows. Only three of the eleven enzymes, namely, phosphoglycerate mutase, enolase, and pyruvate kinase were completely solubilised by at least five of the methods adopted for cell disruption. As well as these three enzymes, saponin lysis which appeared to be the most severe method of treatment, led also to the complete solubilisation of phosphoglucose isomerase and partial solubilisation of glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase and glycerokinase. At the other extreme, cell lysis by grinding T. brucei with alumina or silicon carbide completely solubilised phosphoglycerate mutase, enolase and pyruvate kinase, whereas hexokinase, phosphoglucose isomerase, phosphofructose kinase, aldolase, phosphoglycerate kinase and glycerophosphate dehydrogenase were found to be concentrated in the post-nuclear fraction which sediments at 14,500 g (fraction 14.5KP). The patterns of distribution of the remaining two enzymes, glyceraldehydephosphate dehydrogenase and glycerokinase were found to be polydisperse. The post-nuclear fraction was found to be capable of metabolising glucose to give glycerophosphate without auxiliary enzyme supplementation, and this multienzyme activity proved to be very sensitive to inhibition by the trypanocidal compound, suramin. By means of Biogel column chromatography and acrylamide gel electrophoresis, it was shown that the multienzyme activity is concentrated in a particle probably bigger than a globular protein with a molecular weight of 5 million. Kinetic studies of fraction 14.5KP, in the presence or absence of Triton X-100 indicated that the particles possibly possess a limiting membrane with an inner matrix to which the component enzymes are bound. Isopycnic sucrose gradient centrifugation confirmed that the multienzyme complex is associated with large particles with a median equilibrium density of 1.22. Since the only sub¬ cellular organelles in T. brucei known to band at this density are the microbodies, it has been concluded that the probable intracellular location of the multienzyme complex is the microbodies of the bloodstream long slender form T. brucei.

No description available.

The pathway of anaerobic glycolysis of Trypanosoma brucei has been studied by the following five approaches : 1. Ensyme activity required for the various different postulated schemes for anaerobic glycolysis (hexose monophosphate aldolase, glycerol dehydrogenase, glycerophosphate : glucose or glycerophosphate : fructosesphosphate or glycerophosphate : triose or* glycerophosphate : ALP transphosphorylases) have been examined in a variety of assay conditions. Only significant activities of glycerophosphate : ALP transphosphorylase were detectable. 2. Broken cell incubation studies established that there were no significant differences in the sequence of the increase and decrease in glycolytic intermediates between aerobic and anaerobic pathways.3. Whole cell adenylate charge and glycolytic intermediates were assayed in steady-state aerobic, anaerobic and in the transitions between aerobic to anaerobic, and anaerobic to aerobic showed glucose-6-phosj)hate production to be the rate limiting step in anaerobic glucose utilization. Salicylhyaroxamic acid (0.5 mM) inhibited only glycerophosphate oxidase -and so simulated anaerobiosis. 4. The inhibitory effect of glycerol on whole cells metabolising glucose anaerobically showed it to be dependent upon the intracellular concentration of glycerophosphate. Consequently its inhibitory effect is not caused through the inhibition of glucose transport. 5. The concentration of glycerophosphate in cells metabolising glucose under glycerophosphate oxidase inhibited conditions was found to increase rapidly to a concentration that was independent of time and extracellular glycerol concentration. Furthermore it was found to be an intermediate in anaerobic glucose utilization. The results of this work were consistent with the pathway of carbohydrate metabolism under glycerophosphate oxidase inhibition which involved the production of glycerol plus ATP from ADP plus glycerophosphate catalysed by glycerokinase.

The most important mechanism of trimethoprim (Tp) resistance is the plasmid-encoded production of an additional Tp resistant dihydrofolate reductase (DHFR). The epidemiology of plasmid-mediated resistance to Tp has been studied by biochemical typing of the enzymes responsible. In recent years several new Tp resistant DHFRs have been identified. The type III enzyme, considered the rarest of the plasmid-mediated DHRFs was isolated once in New Zealand in 1979 and never subsequently detected. However the biochemical properties of a Tp resistant DHFR isolated in Nottingham were examined as DNA gene probing had suggested that the enzyme was a type III and the biochemical properties confirmed this. The enzymes responsible for Tp resistance in two outbreaks of <i>Shigella</i> in the United States were examined. Detailed biochemical analysis suggested that the two enzymes were different from each other but similar to the type III. Therefore the three enzymes were subsequently renamed types IIIa, IIIb and IIIc. The properties of the type IIIb enzyme were very similar to the original type IIIa; however, sequence analysis of the N-terminal of this protein showed that it was quite distinct from the type IIIa. The type IV DHFR was isolated in South India in 1984 and is the only inducible plasmid-mediated DHFR. Examination of the induction process suggested that the resistance mechanism of the enzyme was similar to chromosomal hyperproduction where resistance is achieved not because of the insensitivity of the DHFR, but because it is produced in such an amount that it 'swamps' the inhibiting Tp. Purification and sequence analysis of the type IV DHFR revealed that the enzyme was similar to the chromosomal DHFR and that it was complexed with NS1 an <i>E.coli</i> DNA binding protein. The biochemical properties of the type V DHFR, which was isolated in Sri Lanka in 1985, were similar to those of the type 1 enzyme, with the exception that the type V has an unusually low molecular mass when measured in Sephadex. On native polyacrylamide gel electrophoresis however the two enzymes co-migrate, this biochemical similarity suggests the two enzymes are closely related. The efficiency of plasmid-mediated resistance to Tp has compromised the use of this drug in many parts of the world, from bichemical studies it is clear that plasmid-mediated enzymes continue to evolve.

No description available.

A previous discovery showed that a spontaneous mutant in the aroD gene of the RNAi bacteria HT115(DE3), caused a significant increase in C. elegans longevity. In this thesis, I aimed to confirm this finding and attempt to determine whether chemical inhibition of folate synthesis in other E. coli strains would also increase C. elegans lifespan, and uncover the mechanism via which inhibited folate synthesis increases animal lifespan. In order to confirm that the aroD mutant E. coli increase lifespan I investigated effects of using different media types. Data show that the aroD mutant E. coli shows variable effects on C. elegans lifespan with different media types due to the varying folate content of media used. I found that the aroD mutant E. coli effect on lifespan was maximal using a high purity agar with peptone. I then attempted to mimic the effect on other E. coli bacterial strains, such as OP50 commonly used as the E. coli food source for C. elegans, by using sulfamethoxazole as a chemical intervention to inhibit folate synthesis. Sulfamethoxazole increased C. elegans lifespan in a dose dependent manner. Interestingly, even though SMX is used as an antibiotic, the highest concentration used did not appear to decrease growth of the E. coli lawn substantially. Liquid chromatography coupled to mass spectrometry was used to measure and confirm the folate levels in both E. coli and C. elegans. In order to uncover the specific mechanism by which inhibition of folate synthesis increases C. elegans lifespan, I needed to distinguish whether folate status of E. coli itself, folate status of C. elegans, or an interaction of folate status in both organisms was responsible for the increase in lifespan. The drug methotrexate (MTX) and the C. elegans gcp-2.1 mutant were used to investigate the effect of inhibiting the worm folate cycle, and impeding animal folate uptake. Results suggest that animal folate status does not play a role in the extended longevity. SMX and kanamycin have similar but not additive effects on lifespan, suggesting a common mechanism for both drugs. This is surprising as kanamycin prevents bacterial proliferation, whilst SMX does not. Bacterial accumulation assays suggested that bacterial accumulation may be a marker of ageing not a cause in this system. Together the data presented here show that it may be possible to use chemical interventions to treat the mammalian gut flora, inhibiting excessive microbial folate synthesis and potentially slow ageing, without disrupting microbial ecology.

Staphylococcus aureus is a major pathogen causing both community and hospital-acquired infections. The diversity of diseases caused by this organism can be attributed to its ability to colonize a range of niches and to adapt to the stressful environments of the host. As part of this, successful utilization of host nutrients is crucial for pathogenesis. Sulfur is an essential element required for many cellular components. S.aureus can use glutathione as sole sulfur source and as it cannot synthesize this molecule, it must acquire it from the host. Glutathione utilization is facilitated by gammaglutamyltranspeptidase (GGT) in many organisms. To analyse the role of GGT in S.aureus, the putative ggt gene was identified and insertionally inactivated. The ggt mutant was still able to grow on glutathione, which suggests a novel alternative pathway for catabolism. The role of a putative glutathione transporter was also investigated. Mutant strains, although still able to grow on glutathione showed a stress defect, in particular to tellurite. S.aureus is well known as having high level tellurite resistance. Resistance occurs via reduction leading to cytoplasmic deposits of tellurium. Purification of tellurite reductase activities resulted in the identification of alkylhydroperoxidase subunit F (AhpF) and thioredoxin reductase (TrxB). The relative roles of these two enzymes in tellurite reduction was examined.

The genome of Mycobacterium tuberculosis encodes for 11 pairs of Esx family proteins such as EsxA/EsxB and EsxO/EsxP that are located in pairs within the genome. Despite the clear importance of the Esx family proteins in mycobacterial virulence and pathogenesis, the precise molecular functions and mechanisms of action for these proteins remain unknown. Initially expression vectors carrying EsxO and EsxP were constructed and used to express these proteins as inclusion products. The inclusion bodies of both proteins were successfully resolubilized and co-refolded. The final purification step by gel filtration chromatography shows that these proteins form a tight 1:1 heterodimeric complex. Analysis using circular dichroism (CD) spectroscopy of the purified refolded complex showed that it contained a high helical content (53%). The complex showed a significant resistance to heat-induced denaturation with co-operative denaturation observed that indicates a stable folded structure. In addition, significant chemical shift dispersion was seen in 1D1H NMR of the EsxO/EsxP complex, which clearly indicates a folded structure. Previous studies have shown that the EsxA/EsxB complex binds specifically to the surface of monocyte and macrophage cells. Fluorescence microscopy studies described here show specific binding of the EsxO/EsxP complex to the surface of monocyte and macrophage cells, suggesting that EsxA/EsxB and EsxO/EsxP complexes bind to specific target but distinct targets on the surface of host cells, which suggests possible roles in pathogen-host cell signalling. Further work, I investigated whether exposure to the EsxO/EsxP complex results in changes in host cell motility or gene expression. Analysis of macrophage motility over period of eight hours revealed that neither EsxA/EsxB nor EsxO/EsxP has any effect on the motility of macrophages. In addition, microarray analysis was used to identify any changes in the gene expression profile of monocyte cells when exposed to the EsxO/EsxP complex. Interestingly, after 30 minutes exposure to EsxO/EsxP complex only 6 genes showed significant change in expression, but three of these are involved in regulation of chromatin structure. After 2 hours exposure to EsxO/EsxP complex still only small numbers of genes showed significant changes, but no clustering to specific biological processes was apparent. The observation of specific cell surface binding of the EsxO/EsxP complex strongly suggests role in signaling, however the precise function of the complex remains to be elucidated.

The stability of the penicillinase plasmids of Staphylococcus aureus has been investigated both by the effects of various agents on the elimination of the plasmids and by the characterisation of mutations influencing the stability. Growth or plasmid-carrying strains in the presence of ethidium bromide enhanced the spontaneous rate of loss of plasmids in all seven strains of S.aureus tested. In one of these strains, growth in media containing acridine orange had a similar effect on plasmid loss. One of the strains grew in the presence of high concentrations of ethidium bromide. This resistance to ethidium bromide is determined by a gene located on the penicillinase plasmid and appears to be due, at least in part, to a permeability effect. Two types of mutant affected in the stability of the penicillinase plasmids have been isolated from S.aureus PS80 after treatment with ethyl methane sulphonate. In one, the mutation is present on the penicillinase plasmid and results in an inability to replicate at 42 C; replication at 30 C being unaffected. The second type of mutation is not located on the plasmid and causes instability of penicillinase plasmids of both compatibility groups but does not affect a plasmid conferring resistance to tetracycline. Reversion of the temperature-sensitive plasmid to temperature stability has been studied. Evidence is presented to show that this reversion is the result of integration of the plasmid into another replicon, probably the bacterial chromosome. Deletions encompassing a particular region or the plasmid reduce the frequency of this reversion. It is suggested that this region includes a gene specifying a protein involved in the integration process. A penicillinase plasmid that has a gene conferring resistance to erythromycin (ero) has been used to integrate a fragment of this plasmid into the chromosome of S.aureus PS80. The fragment includes only the ero gene of the known plasmid genes. After transduction into this constructed strain, the temperature-sensitive plasmid integrated at a considerably increased rate. Such an integration can be effected at sites distant from the ero. since linkage between the integrated plasmid and the chromosomal ero is not obligatory. It is suggested that the effect is due to the fragment including,in addition to the ero gene, a gene int that produces a protein involved in recombination between a specific site on the plasmid and sites on the chromosome. An integrated plasmid can be excised by a superinfecting plasmid. This excision is apparently independent of any recombination between the integrated and superinfecting plasmid. A gene xis that specifies a protein responsible for this excision is proposed, xis activity functions independently or int product. Deletion mapping involving irradiation of transducing phage normally gives the same linear sequence of plasmid genes. This implies that the circular DNA in a host is ruptured at a specific site. Deletion mapping of a plasmid without this putative site results in an altered linear sequence. It is proposed that this site be called end. The location on the plasmid of the genes specifying these functions is discussed and a tentative plasmid map presented. In view of the similarities between the integration and excision functions specified by the penicillinase plasmid and those specified by temperate phage, such as lambda, it is proposed that temperate phages are the precursors of staphylococcal penicillinase plasmids. It has proved impossible to remove all plasmid-like DNA from a penicillinase plasmid negative S.aureus PS80. Nevertheless there is some indication that the temperature-sensitive plasmid DNA can be separated from other plasmid-like DNA by centrifugation in neutral sucrose gradients.