Bibliography: pages 140-158. / Clostridium acetobutylicum P262 is an endospore-forming Gram-positive anaerobic bacterium, and it has been used in the industrial production of acetone and butanol for many years. The aim of this study was to characterize the upstream region of the βhbd gene which is linked to the adh1 gene, and to investigate the expression of these linked genes by transcriptional analysis. The upstream region of the βhbd gene was isolated from a gene bank of C. acetobutylicum P262, constructed using the pWE15 cosmid vector. Characterization of this upstream region was done initially at the nucleotide sequence level. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1002-bp which encoded a protein of 334 amino acid residues with a calculated Mᵣ of 35,679. This protein showed significant amino acid homology to the fixB protein of Rhizobium meliloti and Azorhizobium caulinodans and the electron transport flavoproteins from humans and rats. Studies on the expression of the three linked genes fixB, βhbd and adh1 were carried out at the transcriptional level. Northern and primer extension analyses indicated that all of the three genes were independently transcribed throughout the various stages of the acetone-butanol-ethanol (ABE) fermentation in C. acetobutylicum P262. Each of the genes produced mRNA transcripts of approximately 1.4 kb. The βhbd and adh1 genes were shown to have at least two major and one minor transcriptional start sites in C. acetobutylicum P262. Transcription was initiated at the same promoter region of the fixB gene in both C. acetobutylicum P262 and Escherichia coli. the βhbd gene was shown to have a stronger promoter region than those of the fixB and adh1 genes based on the lacZ-fusion studies in E. coli. The βhbd and adh1 genes encode the 3-hydroxybutyryl-CoA dehydrogenase (BHBD) and the NADPH-dependent alcohol dehydrogenase (ADH), respectively. The BHBD enzyme is part of the central fermentation pathway and is required for acid and solvent production, whereas the ADH is part of a branched solvent pathway and is only required for solvent production. Analysis of mRNA transcription and the identification of transcription initiation sites, indicated that each of these two genes was independently and constitutively transcribed throughout the acidogenic, sol ventogenic and sporulation stages in C. acetobutylicum P262 and in exponential E. coli cells. These results suggest that the adh1 gene is not part of a branched solvent pathway which is only induced and transcribed during the solventogenic phase.

Bibliography: pages 143-167. / The production of an extracellular collagenase and serine proteases by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37°C and by lack of oxygen. V. alginolyticus had identical growth rates at 30 and 37°C. Aeration did not affect the growth rate of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. The regulation of exoprotease production by temperature and oxygen is specific and has implications regarding the ecology of V. alginolyticus. The synthesis of a 100 000 molecular weight protein was induced in V. alginolyticus by either raising the temperature from 30 to 37°C, a lack of oxygen or (NH₄)₂SO₄. Histidine stimulated synthesis of a 52 000 molecular weight protein. The possibility that these proteins have a regulatory role in exoenzyme synthesis is discussed.

Bibliography: leaves 150-183. / As lignocellulose represents an abundant renewable resource, research is in progress to obtain a better understanding of the natural mechanisms whereby this resource is utilised. Of particular interest is the degradation of forage in the rumen and one research goal is to ultimately increase animal productivity through an improvement in lignocellulose utilisation. However, although the mechanisms behind lignocellulose utilisation are reasonably well understood, relatively little is known about the mechanisms which occur in the rumen. Thus, the aim of this thesis was to gain more insight into the mechanisms of lignocellulose utilisation which occur in the rumen. Initially this research was focused on the poorly characterised exo-acting cellulases from rumen bacteria. Preliminary enzymology studies on one cellulase from Ruminococcus flavefaciens FD-1, previously isolated in this laboratory, indicated that an exo-acting cellodextrinase, CelA, had been isolated. In this report, the enzyme was purified and biochemically characterised and was shown to be an exo-acting cellodextrinase.

Bibliography: leaves 149-181. / The aim of this study involved the characterisation of the cellulolytic properties of Clostridium acetobutylicum and the development of a genetic transfer system for this organism. The production of a carboxymethyl cellulose and a cellobiase by C acetobutylicum was demonstrated. In liquid medium the carboxymethyl cellulase was induced by molasses, and it was not repressed by glucose. Optimum carboxymethyl cellulase activity occurred at pH 4.6 and 37°C. Optimum conditions for autolysis and autoplast formation in C. acetobutylicum were defined. Autolysis-deficient mutants which produced less autolysin than the parent strain were isolated. Growth of the P262 strain and the lyt-1 mutant was inhibited by the same concentrations of wall-inhibiting antibiotics.

Bibliography: pages 134-149. / Enzyme-linked immunosorbent assay (ELISA) was used successfully to detect cauliflower mosaic virus (CaMV) in crude leaf extracts. Small serological differences between CaMV isolates could be shown by ELISA and serum cross-absorption. Serological reactivity of CaMV was found to depend on the proteolytic degradation state of the virus coat protein so making it impossible to establish definite serological relationships among the virus isolates tested. Proteolysis during purification of CaMV could not be entirely eliminated. The coat protein of CaMV was shown to be glycosylated by the specific binding of labelled Concanavalin A. The role of carbohydrate residues in CaMV serological reactivity was evaluated.

Bibliography : pages 234-264. / Some genetic systems operative in Bacteroides fragilis have been successfully investigated. Firstly the bacteriocin produced by the B.fragilis BF-1 strain was purified and partially characterized. This bacteriocin was found to be cell bound and constitutively produced by the bacteria. The purified bacteriocin was a protein with an apparent Mr of 6400-7200, and was relatively heat stable. The action of the bacteriocin resulted in the lysis of sensitive bacteria. As previous reports indicated that a bacteriocin isolated from the BF-1 strain inhibited the action of RNA-polymerase in-vivo and in-vitro, RNA-polymerase was extracted and partially purified from the bacteriocin sensitive B.fragilis BF-2 strain (described in Appendix 5). This enzyme was essentially similar to that described for other Eubacteriales, but was not inhibited in the in vitro assay by the purified bacteriocin described in this thesis.

Bibliography: leaves 190-209. / The red agar-containing macroalga G. gracilis (Stackhouse) Steentoft, Irvine et, Farnham has occurred in Saldanha Bay, South Africa for many years. However, in recent years a number of collapses in this G. gracilis population were recorded, in some instances almost erradicating the entire population. One of the causes of these collapses is thought to be bacterial disease about which there is very little known. The bacterial pathogens of this macroalga were thus investigated to determine the nature of disease occurrence and how G. gracilis responds to such infections. A large number of culturable bacterial epiphytes isolated from G. gracilis from Saldanha Bay, South Africa, and Luderitz, Namibia were characterised and compared. The number of culturable bacteria isolated from the seawater surrounding the macroalgae was significantly lower than that which occurred epiphytically on the macro algal thalli. Most of the bacteria isolated were Gram-negative, motile rods, and many were classified to genus level. Scanning electron microscopy confirmed that a large epiphytic population of coccoid and rod-shaped bacteria occurs primarily on the main thalli of the rnacroalga and that significantly fewer (and often none) reside on the thallus growth tips.

OBJECTIVE: Insulin and calcitonin are involved in bone remodeling. Our aim was to determine the effect of insulin and calcitonin when added separately and in combination to mouse calvarial cultures on osteoblast proliferation and osteoblast mediated biomineralization in medium containing ascorbic acid and beta-glycerol phosphate. Our hypothesis is that both hormones in combination will cause a synergistic increase in osteoblast proliferation and biomineralization. METHODS: Osteoblasts were isolated from neonatal mouse calvaria and cultured under conditions that allow quantification of cellular proliferation and biomineralization. In our proliferation model, osteoblasts were grown in medium containing fetal calf serum stimulated by insulin, calcitonin, both, or culture medium alone that served as control. Proliferation was measured using a hemocytometer (Neubauer cell chamber). Osteoblast biomineralization was estimated by assessing cellular calcium uptake and secreted alkaline phosphatase activity. In the biomineralization model, osteoblasts were grown in the presence of ascorbic acid and betaglycerol phosphate and stimulated by factors used in the proliferation study. Calcium uptake was measured by an Arsenazo III microplate calcium assay and alkaline phosphatase activity was measured by enzyme release from the calvarial cellular layer. Histological and quantitative histomorphometric evaluations measured mineral deposition. RESULTS: Osteoblast proliferation was significantly increased by insulin or calcitonin alone but not by both together. All treatments, especially calcitonin, significantly increased calcium uptake. Only the combination of insulin and calcitonin significantly increased alkaline phosphatase activity. Discussion: Our findings suggest that insulin and calcitonin increase proliferation and biomineralization controlled by osteoblasts.

Bibliography : pages 157-173. / The main aim of this study was to develop an economically viable bacterial leaching process for a gold-containing pyrite/arsenopyrite ore. The effect of various parameters on, and the mechanism of, bacterial leaching were investigated. Initially milled run-of-mine ore was examined. Batch tests and a continuous bacterial leach were carried out. Bacterial leaching was successful and 91-93% gold dissolution was attained in four days. The process was not economically feasible when compared to the standard flotation-roasting process.

Bibliography: leaves 89-100. / Herophyta viscosa (Baker) is a monocytyledonous resurrection plant that can tolerate extremes of dessication. Upon rewatering, it rehydrates completely and assumes its full physiological activities. Studies on changes in gene expression associated with dehydration stress tolerance were conducted. A cDNA library constructed from m RNA isolated from dehydrated (85%, 37% and 5% relative water content) X. viscosa leaves, was differently screened. Of the 192 randomly selected cDNAs screened, 30 showed higher expression levels when X. viscosa was dehydrated while 20 showed lower expession. XVLEA, XVDH and XVLEC represent three cDNAs that were upregulated during dehydration stress. XVLEA showed the highest identity at the amino acid level with a late embryogenesis abundant protein, LEA29G, from Gossipium hirsutum (30%) and LEA D-29 from cotton (50%). XVDH exhibited significant identity to dehydrin proteins from Arabidopsis thaliana (45%) and Pisum sativum (43%) at the amino acid level. It encodes a glycine-rich protein (27kDa) which is largely hydrophilic and contains a hydrophobic segment at the C-terminus. XVLEC showed 28% identity and 50% similarity to a lectin-like protein from Arabidopsis thaliana. Southern blot analysis confirmed the presence of the three cDNAs in the X.viscosa genome. Both XVLEA and XVDH transcripts were highly expressed during dehydration- (37% RWC) and rehydration (4%, 32%, 72% RWC) treatment of the plant ͌ 1.0kb was observed. However, with XVDH a transcript of ͌ 1.0 kb and 1.09 kb were observed. XVDH transcripts accumulated in X. viscosa plants in response to low temperature, heat and dehydration stresses, as well as to exogenous supply of abscisic acid, ethylene and methyl jasmonate. Localization studies of the XVDH encoded protein showed that XVDH is located in the plasma membrane-cell wall region.