Doctoral Degrees (Microbial, Biochemical and Food Biotechnology)
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Item Open Access Activation of the SARS-CoV-2 spike protein by 𝘤𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 secreted proteases(University of the Free State, 2023) Mjokane, Nozethu; Sebolai, O. M.; Pohl, C. H.; Albertyn, J.; Gcilitshana, O. M. N.The thesis is not structured in a classical way. As such, it is composed of a literature review section (Chapter 1) and two research chapters (Chapters 2 and 3). A general discussion section (Chapter 4) and addendums are also included. As some chapters are in a publication format, repetition of essential information could not be avoided. Chapter 1 reviews the emergence of SARS-CoV-2 and its impact. In particular, it considers the co-infection of this virus with respiratory fungal pathogens, which are major independent risk factors that complicate COVID-19 by causing a more severe infection resulting in higher mortality than that of either infection on its own. These fungal pathogens secreted furin-like proteases to further their virulence during host invasion. In this context, the thesis argues that it is foreseeable that the virus could also access these fungal furin-like proteases and pervert them in order to activate its latent spike protein. Therefore, this set up a number of questions, which are addressed in the thesis concerning the possible activation of the viral latent spike protein by fungal furin-like proteases. In Chapter 2, it was sought to characterise 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 (𝘊.) neoformans proteases and assess if they could theoretically bind to the SARS-CoV-2 spike protein. To be specific, previous papers reporting on cryptococcal serine proteases were perused, and this made it possible to select a number of proteases, namely cryptococcal serine carboxypeptidase (CNBF4600), cryptococcal cerevisin (CNBJ2870) and cryptococcal peptidase (CNBA1340), cryptococcal peptidase (CNAG_00150) and cryptococcal cerevisin (CNAG_04625). By designing specific primers, it was possible to show that these serine proteases were expressed in 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 H99, the prototypical cryptococcal strain used in this thesis. Therefore, the expressed gene products were expected to be secreted into the culture media. This was important for the work that follows in Chapter 3. Through using the computational programme, High Ambiguity Driven protein-protein DOCKing (HADDOCK), it was possible to show that some of the selected cryptococcal serine proteases could interact with the coronavirus spike protein and yield a binding affinity greater than and comparable to furin. However, as HADDOCK is a computational programme, the predicted binding affinities might not correlate with the experimental binding affinities in solution, more so since the used 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘯𝘦𝘰𝘧𝘰𝘳𝘮𝘢𝘯𝘴 proteases structures were predicted and not solved. To account for this, Chapter 3 sought to provide enzymatic evidence using the collected culture media – in the form of supernatant. To do this, a mimetic fluorogenic peptide of the SARS-CoV-2 spike protein was designed and modified to have intra-molecular fluorescence quenching capability using 7-methoxycoumarin-4-yl acetyl (MCA) at the N-terminus and N-2,4-dinitrophenyl (DNP) at the C-terminus. The assay was performed using the cryptococcal supernatant. For reference, recombinant furin was included as this is the serine protease present in humans that catalyses the activation of the spike protein. Here, it was determined that cryptococcal serine proteases present in the supernatant could cleave the mimetic spike protein at S1/S2 site with biochemical efficiency comparable to furin. To test the veracity of these data, SARS-CoV-2 pseudovirion containing a full-length spike protein was used. It was possible to show that the pseudovirion could be transduced into HEK-293T cells in the presence of the cryptococcal supernatant. Chapter 4 takes into account the obtained results and provides a summary of the major observations. Of note, the thesis theorises that yeast kexin proteases are responsible for the observed activity. This is because there is a functional homology between yeast kexin proteases and furin (both are convertases); thus, it is reasonable that the supernatant (which contains yeast kexin proteases) could activate the latent SARS-CoV-2 spike protein. The thesis further proves that other respiratory fungal pathogens have yeast kexin proteases that activate the spike protein. This evidence is documented in Addendum no. 1. All things considered, the findings point to the regulation of protease activity as a viable approach to control the activation of the spike protein by either mammalian protease or fungal proteases. To this end, protease inhibitors could be used to control unwanted proteolysis. Addendum no. 2 attempted to show this. Here, it was possible to show that the South African-based medicinal plant Artemisia tea infusion extract and its active compound artemisinin could control the activation of the mimetic SARS-CoV-2 spike protein by furin but not the supernatant. The latter highlights the need to purify the supernatant and isolate yeast kexin proteases. The idea of exploring the control of unwanted proteolysis is also an interventional measure considered by Pfizer, the pharmaceutical company. This American multinational pharmaceutical and biotechnology corporation successfully piloted Paxlovid to control SARS-CoV-2. This drug contains an anti-protease (PF-07321332) that inhibits the protease (SARS-CoV-2 3CLp) responsible for viral replication.Item Open Access Role of polyunsaturated fatty acids during infection of Caenorhabditis elegans(University of the Free State, 2023) Mokoena, Nthabiseng Zelda; Pohl-Albertyn, C. H.; Albertyn, J.During polymicrobial infection, interactions between different microbial species can alter host responses and/or microbial virulence and pathogenesis, often complicating patient treatment and resolution of infection. Polyunsaturated fatty acids (PUFAs) are not only crucial for normal function in mammalian systems, but are also proposed to act as endogenous antimicrobial molecules. Thus, we adopted Caenorhabditis elegans to mimic the Pseudomonas aeruginosa and Candida albicans polymicrobial infections found in humans. We determined the influence of arachidonic acid (AA) and eicosopentaenoic acid (EPA) supplementation on mono- and polymicrobial infection, fatty acid (FA) composition and egg retention of the nematodes, as well as on expression of FA metabolic genes. Supplementation with either AA or EPA in mono- and polymicrobial infections resulted in changes in FA profiles and PUFA biosynthesis pathway. We discovered that the degree of egg retention elicited by C. albicans and P. aeruginosa varied, with C. elegans exposed to both C. albicans and P. aeruginosa showing the highest level of egg retention compared to nematodes infected by either C. albicans or P. aeruginosa alone. Interestingly, the AA supplemented infected nematodes showed an increased level of egg retention, while EPA supplemented infected nematodes showed a significant decrease. Using the C. elegans model, we determined the effects of AA and EPA supplementation on the survival of nematodes with mono- and polymicrobial infection. We showed that the survival of the infected nematodes was influenced by PUFA-supplementation. EPA supplementation effectively reduced C. albicans virulence and inhibited hyphal formation, thus leading to a partial rescue of pathogen susceptibility. However, this was not the case for P. aeruginosa infections. In fact, EPA supplemented nematodes infected with P. aeruginosa were more susceptible. Notably hyphal formation is an important component of Candida pathogenesis in mammals. Furthermore, polymicrobial infection resulted in synergistic virulence. However, C. albicans did not produce any hyphae in the co-infection of either AA, EPA supplemented or unsupplemented nematodes, this suggests that the increase in pathogenesis may be associated with increased P. aeruginosa pathogenesis. To further test the role of EPA in hyphal formation of C. albicans, we hypothesised that cytochrome P450 (CYP450) metabolises EPA to 17,18-epoxyeicosatetraenoic acid (17,18-EpETE), inhibiting C. albicans hyphal formation. We showed that 17,18-EpETE inhibits C. albicans hyphal formation in vitro and in vivo in C. elegans and that inhibitors of mammalian EPA-metabolising CYP450 enzymes, 17-octadecynoic acid (17-ODYA) and 6-(2-propargyloxyphenyl) hexanoic acid (PPOH) restored C. albicans hyphal formation in vivo. Lastly, the role of EPA on the physiology of C. elegans as well as C. albicans in vivo was investigated using gene expression analyses. Among the up-regulated genes, we observed several genes with potential roles in lipid metabolism, hyphal formation, detoxification, stress response and immune response. For instance, we observed an up-regulation of several involved in the synthesis of FAs, including fat-3, fat-4, fat-6, cyp-29A2 and cyp-37A1. Other up-regulated genes were those involved in immune response, such as cyp-37B1, daf-16, fipr-22, ilys-2, lys-5, lys-6, spp-12 and fat-3. Interestingly common genes involved in hyphal formation, such as CAS5, CRZ1, CTA4, ERG11, FCR1, SNQ2, TAC1, TEC1, YOR1 and ZCF3 were also up-regulated. Overall, the benefits of EPA supplementation may be two-fold, by inhibiting virulence factors of C. albicans and stimulating the immune response of the host. Thus, PUFA supplementation might be useful in the treatment of infections in patients caused by C. albicans and P. aeruginosa.Item Open Access The production of 3-hydroxy fatty acids by the yeast Dipodascopsis uninucleata and its implications(University of the Free State, 1999-11) Venter, Pierre; Kock, J. L. F.; Coetzee, D. J.English: In 1991, a novel eicosanoid namely 3-hydroxy-5, 8, 11, 14-eicosatetraenoic acid (3-HETE) was uncovered in the yeast Dipodascopsis uninucleata by van Dyk and co-workers. Strikingly, the production of this compound was found to be sensitive to low concentrations of aspirin and indomethacin. With this as background, a study was conducted that unveiled the possible biochemical pathway used by this yeast for the production of 3-HETE. Here, various fatty acids were fed to D. uninucleata, and the extracted samples analysed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography - mass spectrometry. It was found that 3-hydroxylation of fatty acids in D. uninucleata requires a 5Z, BZ - diene system either directly or following initial incomplete l3-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy - 5Z, BZ, 11Z, 14Z - eicosatetraenoic acid (3R - HETE), which rules out normal l3-oxidation as a biosynthetic route. Consequently, studies on the biological dynamics and distribution of 3-hydroxy oxylipins in D. uninucleata followed. The occurrence of oxylipins was mapped by immunofluorescence microscopy (IF) in fixed cells, with or without cell walls, using an antibody raised against 3R-HETE. This antibody turned out to cross-react with other 3-hydroxy oxylipins. These compounds were detected in situ in gametangia, asci, as well as between released aggregating ascospores. Aspirin (1mM), which is known to suppress the formation of 3-hydroxy oxylipins from exogenous polyenoic fatty acids, inhibited the occurrence of immunoreactive material as well as cell aggregation, suggesting a prominent regulatory role of 3-hydroxy oxylipins for the latter. Since these oxylipins are associated with the aggregation of sexual cells in D. uninuc/eata, the next step was to screen for the presence of these compounds in aggregating cells of other yeasts such as the biotechnological important Saccharomyces cerevisiae. It was found that oxylipins such as 3-hydroxy-8:0 and 3-hydroxy-10:0 are produced over the growth cycle of the flocculating yeast Saccharomyces cerevisiae ATCC 26602. Using oxylipin specific antibodies in IF studies, it was demonstrated that these compounds are synthesized continuously from an early stage of growth and are associated with the cell wall and are present between flocculating cells. Similar results were obtained with a NewFlo phenotype flocculent brewing yeast strain. This implicated the involvement of oxylipins in cell aggregation. Further investigations using scanning- and transmission electronmicroscopy, indicated that changes in the depositing of lipid rich osmiophilic layers in the yeast followed the same pattern as the IF results. Immunogold studies verified the presence of oxylipins in these osmiophilic layers. It was uncovered that the oxylipin containing osmiophilic layers play an important role in cell aggregation. Surprisingly, further investigations implicated the presence of aspirin sensitive 3-hydroxy oxylipins in the LPS layer of the Gram-negative bacterium Escherichia co/i. In this study it was found that aspirin, at moderate concentrations, influences the biosynthesis of the endotoxic 3-hydroxylated myristic acid in the Lipid A of Gram-negative bacteria. This discovery therefore suggests an important role for aspirin as a therapeutic agent in the treatment of LPS mediated diseasesItem Open Access Determining the canning quality of small seeded white beans (Phaseolus vulgaris L.)(University of the Free State, 1999-05) De Lange, Anna Francina; Osthoff, G; Labuschagne, M. T.English: It is important to the producers, processors and consumers in south Africa to have dry bean cultivars with acceptable canning quality. Therefore, dry bean breeders needs sui table screening methods to evaluate the various lines at an early stage (F4) when only small amounts of seed are available. A micro-canning method to evaluate canning beans .in tomato sauce has been developed and compared to the commercial processing procedures with comparable results. External factors that influenced the canning quality like the water quality were investigated. This micro-canning method could therefore be used to investigate the effect of genotype and environment interactions that significantly influenced the canning quality. The objectives of this study were to: o Evaluate different qeriet i.c material for use as parents in the breeding program. $ To obtain a better understanding of canning quality characteristics of beans and to ensure that the most important characteristics are evaluated and the component interrelationships. • Determine the genotypic, environmental and genotype x environment interactions that influenced the canning quality. • To ascertain the patterns of interrelationships of the canning quality parameters and chemical analysis. • To investigate the patterns and relationships between standard and choice grade cultivars. e To investigate stability of locali ties between seasons as well as the clustering of different environments and seasons. Small seeded white beans, carioca and yellow haricot beans were used to determine variability in canning quality but as a result of a lower canning quality of coloured beans, only small seeded white beans were used for further investigations. As result of the investigations of different a characteristics, only the seed size, water absorption during soaking and canning, the texture and subjective visual appearance evaluations were used to determine canning quality. These characteristics were interrelated but single no parameter could explain variation' in canning quality. Canonical correlation analysis was used to determine to what extent variation of chemical components was responsible for differences in canning quality and these results indicated that mainly potassium and calcium would influence the water absorption and texture, respectively. Canonical variate analysis was used to determine the difference between unacceptable, standard and choice grade cultivars. A model was described from these analysis that could be applied to independent data sets that results in coordinates that differentiates the lines or cultivars as unacceptable, standard and choice grade. Significant interactions between genotype, environmental and seasonal effects for canning quality traits indicated that cultivar responses to variation in localities and seasons differ. Environmental effects resulted in inconsistent quality measurements since trait expression is strongly influenced by genotype x environment interactions. Results from this study suggested that the difference between cultivars could therefore be due to a complex interaction of the chemical and structural composition, which is genetically determined and influenced by the anv i ronmen t . as well as the changes that occur during processing. The Additive Main Effects and Multiplicative Interaction (AMMI) model mainly grouped KwaZulu-Natal separately as a region with poor canning quality. The rest of South Africa's localities grouped different for each season. Resulting from this investigation, several recommendations can be made: c Breeding material should be tested fo~ more than one season in order to select superior and more stable lines. o Elimination of canning quality evaluations of KwaZulu-Natal could improve the genetic progress. • Exploitation of the interactions by breeding for specific adaptation in a region of homogeneous area. Demarcating one or two areas in South Africa for the exclusive production of small white canning beans could improve the overall canning quality of the small white bean production in South Africa.Item Open Access A combined systems biology and genomics approach to the study of metabolism in Kluyveromyces marxianus(University of the Free State, 2016-10) Schabort, Du Toit Willem Petrus; Du Preez, J. C.; Kilian, S. G.English: The yeast Kluyveromyces marxianus has become an important micro-organism for industrial applications, as have other non-conventional yeasts. It has the advantages over Saccharomyces cerevisiae (baker’s yeast) in that it is more thermotolerant, has a much higher growth rate and can utilise a wider range of sugars, including the pentose D-xylose, which is found abundantly in lignocellulosic biomass. Although considerable advances have been made in engineering S. cerevisiae strains to ferment pentose sugars, their performance in this respect still does not approach that of glucose fermentation. S. cerevisiae is the model Crabtree positive yeast, meaning that it naturally ferments glucose even if oxygen is present at a high level. Crabtree negative yeasts, such as K. marxianus, have to be forced into a fermentative metabolism by imposing oxygen-limited conditions, which is impractical on industrial scale. Thus, a tremendous amount of knowledge needs to be gained regarding the regulation of metabolism in this non-conventional yeast before success could be expected in the re-programming of K. marxianus strains into xylose fermenting, Crabtree positive strains. The challenge of bringing a non-model species such as K. marxianus to the point of identifying key regulators affecting central metabolic pathways seems formidable. The aims of this work was to firstly harness the new technology of next-generation sequencing (NGS) to create a first draft genome for K. marxianus strain UFS-Y2791 and to generate high-quality RNA-seq differential transcriptome datasets, simultaneously capturing a tremendous amount of information. Efficient analytical methods and software implementations were also developed to explore these large datasets in an efficient manner, revealing new insights into the response of this species to glucose and xylose as carbon sources. RNA-seq data revealed a striking resemblance with the pattern of glucose derepression in the xylose medium, with up-regulation of genes for alternative carbon source utilisation, especially in the peroxisomes. Subsequently, two independent approaches were taken to identify differentially active transcription factors regulating the response. The first was the enumerative method of heptamer frequency comparisons, revealing the most likely regulators of differentially expressed genes. Secondly, a likelihood statistical approach was designed that employs multiple sources of evidence, which resulted in the construction of the first genome-wide gene regulatory network for K. marxianus. The method bridges the gap between the new NGS-based methods, which can rapidly generate data on any non-model species, and the wealth of experimental data that exist for a model species such as S. cerevisiae. Gene set enrichment statistics of the transcription factor target sets showed a general pattern that the activities of differentially active transcription factors were regulated primarily by post translational modifications instead of gene regulation. The use of RNA-seq was further expanded to the elucidation of the kinases that regulate transcription factors. The chromosomal context of differential gene expression was also investigated. Clusters of genes were identified, similar to the sub-telomeric regions previously identified in S. cerevisiae, but not close to telomeres. These regions contain industrially important enzymes and the potential binding sites for differentially active transcription factors. Finally, the possible roles of cofactor balances were investigated. Flux balance analysis was demonstrated here in rationalising the genetic response observed in RNA-seq transcriptomics and to understand the complex interplay between ATP, NADPH and NADH, the cofactor specificity of the oxidative pentose phosphate pathway, as well as the role of fructose-1,6-bisphosphatase. New roles are proposed for the latter enzyme, which differs from the currently accepted norm. A strategy for the metabolic engineering of a future xylose fermenting K. marxianus strain is also presented. The integrated analysis presented here expands our knowledge base of this yeast species, which is set to become increasingly important in a future bio-economy.Item Open Access Expression of avian pathogenic Escherichia coli (APEC) virulence factors, Iss and HlyF, as potential sub-unit vaccine candidates(University of the Free State, 2017-06) Van der Westhuizen, Wouter Andre; Bragg, R. R.; Boucher, C. E.; Theron, C. W.Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis in poultry and leads to economic losses in the poultry industry. Due to rising concerns of antibiotic resistance and antibiotic carry-over into food, bans have been implemented on antibiotic use in animal production. The process of discovering new antibiotics and having them registered and approved can take up to eight years, and their application will most likely be limited to human-use. Alternative therapies for the control of bacterial diseases in animals, including poultry, are therefore becoming increasingly important. Alternative treatment options could include the use of bacteriophages, bacteriophage enzymes, essential oils and vaccines. Bacterial vaccines are generally based on whole bacterial cells, and in the case of commensal bacteria such as E. coli, this can lead to poor gut health. Therefore, the need for highly specific bacterial vaccines are required, such as sub-unit vaccines containing only antigens found in pathogenic strains of E. coli. Various virulence genes have been found in APEC that contribute to the pathogenicity of the strains. Five of these genes have been found to be highly prevalent in most clinical cases of avian colibacillosis. In this study, two of these genes were selected as potential candidates for sub-unit vaccine development, namely increased serum survival (iss) and haemolysin F (hlyF). The increased serum survival gene, an outer-membrane protein of APEC, was successfully expressed in E. coli BL21 (DE3) using the pET28b(+) vector system, although the protein was mostly water-insoluble due to hydrophobic N -and C-terminals. The sequence of iss was then modified to create a truncated version of the gene encoding the hydrophilic region of the gene, also the potential epitope of the Iss antigen, to improve solubility during over-expression. Water-soluble truncated Iss was produced in conjunction with the full Iss protein, solubilised using a zwitterionic detergent, purified using the hexahistidine regions flanking the inserted gene in the pET28b(+) vector system. The purified proteins were mixed with adjuvant and injected into chickens raise antibodies against the two expressed proteins. The antibodies obtained from the chickens were used to perform western blotting and ELISA and both proteins were confirmed to be immunogenic. Furthermore, the obtained serum was cross-reacted with the full and truncated forms of the protein, indicating potentially similar epitopes, showing promise of using the highly water-soluble truncated Iss protein for potential future vaccine development. Yarrowia lipolytica heterologous protein expression was also attempted with the full iss gene. However, no expressed protein was detected using SDS-PAGE, so alternative methods of expression, purification and isolation were attempted. An enterokinase proteolytic cleavage site was introduced between the iss gene and the GPI-anchor sequence, the secretion vector pINA1317 and the C-terminus hexahistidine region bearing pINA1317 secretion vector were used as alternatives methods to express the full iss gene sequence. These methods failed to produce the desired results. Western blotting using antiserum raised against E. coli expressed protein could also not detect any expressed Iss protein. It was then concluded that a yet unknown issue is preventing adequate, if not any, expression of the iss gene in Y. lipolytica and further research is required. During the study, published literature indicated that the putative avian haemolysin F, was not a haemolysin but an enzyme involved in outer-membrane vesicle (OMV) biogenesis. This was validated by our failure to detect haemolytic activity after hlyF-overexpression and the presence of OMVs observed by TEM. As this was very recent research into the involvement of this gene in OMV biogenesis, the impact of the expression of the hlyF gene was investigated regarding the regulation of outer-membrane proteins which are scavenged during OMV release. Relative quantitative PCR was used to compare hlyF-expressing and non-expressing cells, and it was shown that ompA expression is increased during hlyF expression, while ompF expression remained nearly the same, which could lead to osmotic-stress susceptible cells during hlyF induction. It was concluded that a decrease in ompA expression is not involved in the mechanism of hlyF-induced OMV biogenesis, contrary to one of the biogenesis mechanisms described in literature. Sub-unit bacterial vaccines could be the future method of preventing bacterial diseases in the poultry industry. Alternative methods such as bacteriophage therapy might not be possible due to the non-linear pharmacokinetics observed, which will hinder registration of these products. Sub-unit vaccines should elicit highly specific immune responses to virulence-related antigens and not target commensal bacteria. Various antigens accessible to the immune system of the host are outer-membrane or cell wall-associated proteins, protein expression of these antigens could pose problems such as high costs for production and problems during the development of expression systems for these proteins, even complete failure of expression. Knowledge and correct characterisation of virulence-related proteins is also essential, as seen with the HlyF protein expressed in this study which is not directly suitable for vaccine production as previously thought. Future work is required in the optimisation of protein expression parameters, and chicken challenge studies with APEC will need to be conducted to determine if protective immunity is gained by vaccination with the E. coli-expressed Iss proteins. It will also be highly advantageous if the problems encountered during protein expression in Y. lipolytica could solved and the yeast-expressed Iss protein tested during challenge studies with APEC.Item Open Access Molecular and physiological aspects of alcohol dehydrogenases in the ethanol metabolism Saccharomyces cerevisiae(University of the Free State, 2007-05) De Smidt, Olga; Albertyn, J.; Du Preez, J. C.English: When Saccharomyces cerevisiae is grown on a fermentable carbon source such as glucose, the fermentative alcohol dehydrogenase, ADH I , catalyses the regeneration of NAD+ from NADH and produces ethanol from acetaldehyde. When the fermentable carbon source is depleted, a variety of other enzymes are derepressed in order to utilise the previously excreted ethanol via oxidative respiration and gluconeogenesis . To provide both the carbon source and energy for this system, the yeast cell requires an efficient method for oxidising this previously excreted ethanol. ADH II is a catabolite repressible isoenzyme which primarily functions in the cell to oxidise ethanol to acetaldehyde, which can be metabolised via the tricarboxylic acid cycle or act as intermediate product in gluconeogenesis. ADH III is a mitochondrial isoenzyme participating in the respiratory metabolism by forming part of the ethanol-acetaldehyde shuttle that is important for shuttling mitochondrial reducing equivalents to the cytosol under anaerobic conditions. The physiological roles and regulation of ADH1, ADH2, ADH3, ADH4 and ADH5 were investigated by monitoring transcription levels in chemostat and batch cultivations with Northern blotting and real-time RT-PCR. ADH I was shown to be the key enzyme in the reduction of acetaldehyde to ethanol and also demonstrated ample ability to oxidise ethanol. ADH2 transcription was inhibited by glucose and ethanol in chemostat cultures pulsed with both these carbon sources, but only glucose repression was evident in batch cultures. Northern blot analysis showed that the ADH3 gene was induced during the ethanol phase of the pulses suggested that the mitochondrial ADH III enzyme could also be involved in the first step in ethanol utilisation. The growth kinetics of a strain expressing only ADH III demonstrated that the ADH3 gene product could fulfil the same function as ADH II. ADH4 transcription was detected for the first time in batch cultures and was shown not to be involved in the production or assimilation of ethanol. ADH5 transcription was also demonstrated for the first time and data suggest that ADH V is not involved in ethanol production in a adh1-adh4 deletion mutant.Item Open Access Biology of botryosphaeria dothidea and sphaeropsis sapinea as endophytes of eucalypts and pines in South Africa(University of the Free State, 2001-12) Smith, Hendrik; Wingfield, M. J.; Coutinho, T. A.; Crous, P.W.English: Botryosphaeria dothidea and Sphaeropsis sapinea both very important pathogens in the South African forestry context. These fungi are well established in the country and contribute substantially to annual losses incurred. Currently very little can be done to control the fungi and the damage they cause. The understanding of their respective disease etiologies is thus of great importance to develop relevant counter measures. The overall aim of this dissertation was to investigate various poorly understood aspects of these two fungi and to try and relate the results to practical contributions towards controlling the impact the two pathogens have. Studies have been conducted during the course of five years and each study represents an independent research investigation. The introductory chapter presents a review of the literature pertaining to all aspects of biology, history and taxonomy of B. dothidea and S. sapinea. The two fungi are clearly very similar in all these aspects and perhaps the only clear difference is that S. sapinea is restricted to pines in South Africa. Many other similarities and some differences between these two important pathogens are highlighted and many of these have provided the background for further investigations. In chapter two the presence of B. dothidea and S. sapinea lS demonstrated as symptomless endophytes in healthy, pine and eucalypt tissue. Botryosphaeria dothidea was found to be common in all the Eucalyptus spp. tested, occurring at high percentages in symptomless leaves of Eucalptus smithii, E. camaldulensis, E. grandis and E. nitens. Sphaeropsis sapinea was, in contrast, only present in young, green Pinus patuIa and P. radiata cones, but virtually absent from the cones of P. elliottii and P. taeda. Botryosphaeria dothidea is associated with die-back and canker diseases of eucalypts in South Africa. Despite this fact, little is known about the infection process. The fungus is known to occur endophytically in leaves of various Eucalyptus species in South Africa. In chapter three I consider the ability of B. dothidea to infect apparently healthy Eucalyptus leaves and the subsequent location and structure of these infections once inside leaf tissue. Scanning electron microscopy revealed that conidia of B. dothidea can infect healthy leaves through stomata. These infections ultimately reside amongst mesophyll cells and constitute a number of individual infections per leaf. Two morphologically similar fungi are associated with die-back and canker of eucalypts in South Africa. The one was identified as part of the Botryosphaeria dothidea-complex. In chapter four, the identity of the second fungus was determined by comparing morphology, pathogenicity and DNA sequence analysis of isolates of both taxa. Based on results obtained, Botryosphaeria eucalyptorum, and its anamorph Fusicoccum eucalyptorum, are described as a new species. I found that the teleomorph is morphologically similar to other taxa in the B. dothidea-complex, but conidial characteristics of the anamorph are distinct, as well as the sequences of the nrDNA internal transcribed spaeers ITS 1 and ITS2. As is the case with B. doth idea , the fungus is pathogenic to Eucalyptus, there do not, however, appear to be differences in pathogenicity between the two. Sphaeropsis sapinea is the most important pathogen of pines in South Africa. The fungus, which reproduces only asexually, occurs only on exotic pines. In chapter five, I investigated the diversity of the S. sapinea population in South Africa and compared it with a population from Northern Sumatra. Both populations were obtained from exotic P. patuIa plantations. The phenotypic diversity of these populations was assessed using vegetative compatibility tests. The percentage maximum genotypic diversity, based on Stoddard and Taylor's index, for the South African population was much higher than the Northern Sumatran population, thus indicating that the South African S. sapinea population was more diverse than the Northern Sumatran population. These results support the hypothesis that the population of S. sapinea in South Africa has been introduced from various parts of the world, during the last century. In chapter six, I investigated the role that latent S. sapinea infections in seed cones of P. patuIa, play in post-hail associated die-back. Pinus patuIa seed cones were found to be infected during the second year of development, with extensive colonization only occurring m the third year when cones mature, prior to seed discharge. Vegetative compatibility tests revealed that the presence of S. sapinea in individual third year seed cones is confined to a single genetic entity. Sphaeropsis sapinea colonisation of third year seed cones thus, apparently results from a single successful infection per cone. The probable role of latent infections by S. sapinea indicated that tree age and by implication, increased numbers of attached seed cones, contributes to more severe die-back after hail damage. The control of damage caused by S. sapinea is highly dependant on a dynamic hybridisation programme. Alternative species of pines is thus constantly evaluated for potential. In chapter seven, 65 families representing both the northern and southern populations of P. greggii were evaluated for their tolerance to infection and subsequent die-back caused by S. sapinea. Families were evaluated following natural infection after hail damage, as well as by artificial inoculation. Variation in tolerance occurred and was highly significant between the two provenances, with the northern provenance proving to be very tolerant. Pinus greggii trees of the southern provenances were comparable with P. patula. The potential of the families from northern origins has to be investigated further. Cultures of Cytospora isolated from Eucalyptus trees in South Africa, Congo, Thailand, Venezuela, Mexico, Uganda and Australia, as well as Cytospora-like isolates from Indonesia were compared in chapter eight. Comparisons were based on the homology of the internal transcribed spaeer regions and the 5.8S ribosomal DNA of the nuclear ribosomal DNA repeat unit. Isolates clustered into at least three unrelated groupings, with a fourth grouping that included isolates that morphologically resembled Cytospora. Results from this chapter indicated that the current description of Valsa ceratosperma encompasses several distinctly different species and needs to be further refined. Botryosphaeria dothidea and S. sapinea are two of the most important pathogens of eucalypts and pines in South Africa. The fact that they exist as symptom less endophytes in trees has added a fascinating aspect to our understanding of their role in tree diseases. In the past, they have generally been considered to be wound infecting opportunistic fungi. Results of these studies have shown that this is not so and that they are clearly able to infect healthy trees. They are unlikely to be able to infect dead or moribund tissue. The investigations presented in this dissertation have added considerable knowledge to our understanding of B. dothidea and S. sapinea and will also promote efforts to reduce disease caused by them. However, there are many questions that remain to be answered pertaining to them and it is my hope that this study will provide a foundation and stimulus for further work.Item Open Access Evaluation of the growth and survival of probiotic microorganisms in commercial bio-yogurt(University of the Free State, 2000-11) Hattingh, Analie; Viljoen, B. C.A review of the literature highlighting the importance of the 'therapeutic minimum' and the survival of the probiotic bacteria in fermented milk bioproducts is given in Chapter 2. Special reference is made to the historical background of probiotics, its therapeutic value and the survival through passage in the gastrointestinal tract. In addition, technology of bio-yogurt, factors affecting the survival of probiotic bacteria in yogurt, and the media for the differential enumeration of these microorganisms in dairy products are discussed. In Chapter 3 existing media proposed for the selective enumeration of starter cultures employed in the manufacture of bio-yogurt are compared and evaluated. It is essential for comparison reasons to standardize enumeration methods for microbial analyses in order to study the incidence of the probiotic bacteria in the presence of the conventional starter cultures. The media proposed by Chr. Hansen's laboratory proved to be the most suitable for the enumeration of the different cultures. It is essential that bio-yogurts meet the criteria of a minimum of 106 cfu/rnl of probiotic bacteria until the expiry date to induce any potential therapeutic advantages for the consumer. Consequently, in Chapter 4 we evaluated samples of AB-yogurt obtained from supermarket outlets statistically based on the enumeration of viable probiotic cultures, Lactobacillus acidophilus and Bifidobacterium bifidum; as well as conventional yogurt starter cultures, Streptococcus thermophilus and Lactobacillus bulgaricus and the maintenance with respect to the 'therapeutic minimum'. Based on the data obtained, the AB-yogurts examined comply with the criteria regarding the number of viable cells of L. acidophilus, but the consumer would not have received sufficient numbers of B. bifidum cells at the time of consumption. In Chapter 5, we monitored the survival of viable cells of the probiotic cultures and starter cultures present in bio-yogurt at frequent intervals from day 1 until the expiry date at day 31 stored at 4eC and loec. B.bifidum never exceeded counts of 106 cfuZgin any of the samples and a constant decline in its numbers was observed. L. acidophilus, despite maintaining counts higher than 106 cfu/ g in the yogurt samples, also exhibited a substantial decrease in its numbers during storage. Due to the poor survival of probiotic cultures in yogurt, we incorporated a probiotic yeast species, S. boulardii as part of the starter culture in Chapter 6 and monitored its progression and survival in yogurt and milk products. Despite good growth and the survival of the yeast species until the expiry date, excessive gas and alcohol production proved, however, to be major constraints. In order to further study the effect of yeast growth on the survival of probiotic bacteria in bio-yogurt, pure cultures of Kluyveromyces marxianus, Issatchenkia orienialis, Debaryomyces harisenii and Yarrotoia lipolytica were inoculated into commercial AB-yogurt, sterile milk and pasteurised sweetened yogurt in Chapter 7. The yeast species were able to progress in the bio-yogurt reaching maximum counts exceeding 107 cfu/ g. Despite the inability of some species to utilise lactose, the yeast species utilised available organic acids, galactose and glucose derived from bacterial metabolism of the milk lactose, as well as possible free fatty acids or free amino acids present in the dairy products and thereby sufficiently contributed to the retention or enhancing of the pH values. The production of excessive gas and alcohol was major constraints in implementing Kluyveromyces marxianus, Issatchenkia orienialis. The inclusion of Y. lipolytica and D.hansenii in AB-yogurts, therefore, seemed the most promising in controlling the pH to assure the viability of the pro-biotic microorganisms. In Chapter 8, Y. lipolytica and D.hansenii cultures were inoculated into commercial plain and fruit AB-yogurt at moderate (105 -106 cfujml) and low level (10² - 10³ cfujml), directly after manufacture and with the ABT-starter before fermentation, to compare the effects that the yeast will have on viability of probiotic bacteria. Viable bifidobacteria counts remained virtually the same in the bio-yogurt inoculated with the yeast cultures during the refrigerated storage period. A rapid decrease in L. acidophilus occurred after 2 weeks storage (2-4 log cycles) in the yeast-inoculated bio-yogurt suggesting possible antagonistic action of the yeast against L. acidophilus. Addition of the yeast primarily encouraged the growth of streptococci, which had an influence on the pH of the yogurt environment. A gradual decrease in the pH of all the bioyogurt products was observed. pH was affected by enhanced growth of streptococci, utilization of organic acids by the yeasts and the fact that L. bulgaricus was excluded form the yogurt starter culture. The yogurt inoculated with D. hanserui was still acceptable and had a pleasant taste compared to the control yogurt after 30 days storage. Inclusion of yeast as part of the starter culture for bio-yogurts seems promising. In Chapter 9, possible enhancement of the growth and survival of Bifidobacteria in bio-yogurt by the addition of a prebiotic was investigated. Commercial AB-yogurt was fortified with 1, 2 and 3% of the fructooligosaccharide, neokestose, and growth and survival of bifidobacteria as well as L.acidophilus and S. thermophilus were monitored during storage at SoC over 30 days. With the addition of neokestose the viable bifidobacteria count remained significantly higher in all the yogurts when compared to traditional yogurts without the addition of neokestose. L. acidophilus reduction in viable counts did not exceed a 30% reduction; therefore neokestose also had a better survival effect on L. acidophilus. The addition of neokestose had no affect on the survival of S. thermophilus.Item Open Access Utilisation of edible oils and GLA production by Mucor in the presence of acetate(University of the Free State, 1998-10) Badenhorst, Jacqueline; Kock, J. L. F.English: Surveys launched across South Africa indicate that many frying establishments abuse their frying oils and fats during the frying process, resulting in degradation and concomitant production of potentially toxic oxidation products. Some of these compounds have been shown to be toxic to animals and in human in vitro studies. Consequently, strict regulations under the Foodstuffs, Cosmetics and Disinfectants Act, 1972 (Act 54 of 1972) were published on 16 August 1996. It is now an offense to use or sell used cooking oil or fat for human consumption containing high levels of these degradation products. Since frying establishments are not allowed to discard their used oils and fats by selling to the public for consumption or dumping into municipal drainage systems, it is important that these oils and fats are collected for re-use in another form. Consequently, the aim of this study was the biotransformation of used oil wastes (containing no toxic substances) to high value lipids containing gamma-linolenic acid (GLA). This polyunsaturated fatty acid is prescribed for the treatment of eczema. In order to achieve this, Mucor circinelloides f. circinelloides CBS 108.16 was first grown on 40 gil unused sunflower oil and, as expected, produced neutral lipids (NL) similar in fatty acyl composition to the original oil. The apparent repression of the Á6 fatty acid desaturation was partially reversed when cells were grown on oil (30 g/l) and sodium acetate (10 gil) as mixed substrates resulting in an increase in GLA content. Furthermore, a three-fold increase in oil substrate utilisation and doubling of biomass production to 19.1 gil occurred when sodium acetate was added to the oil substrate. When sodium acetate (10 gil) was added to a growth medium containing used cooking oil (UCO) similar results were obtained. This experimental procedure was repeated for seven additional Mucor strains and again the stimulatory effect of sodium acetate in combination with UCO was obvious. Next, the effect of different UCO concentrations in the presence of 10 g sodium acetatell on biomass and lipid production was investigated in Mucor circinelloides CBS 108.16. According to our results, a maximum biomass concentration of 48 gIl consisting of 82 % oil yielding about 35 g NUl and up to 900 mg GLNI was achieved. The addition of 30 g UCO/I in combination with 10 g sodium acetate/l proved to be the optimum UCO concentration in order to obtain maximum GLA yield. Similar results with this strain were obtained when UCO was replaced with fresh unused cooking oil. When these experiments were repeated with linseed oil and sodium acetate as sole carbon sources, much less GLA was produced (351 mg GLNI). According to bioreactor studies, the effects of sodium acetate addition can be attributed to the change in pH of the medium during cell growth in the presence and absence of acetate. In the absence of sodium acetate the pH decreased to 2.2, whereas in its presence it increased to about pH 8.0. During metabolism of sunflower oil in the presence of sodium acetate, the percentage of saturated fatty acids in the extracellular lipids increased, suggesting a higher specificity of the fungal lipase for unsaturated fatty acids. When the sodium acetate was omitted from the medium and the pH gradually increased according to a pH profile mimicking the natural increase in pH found in the medium containing sodium acetate, similar results as in the presence of sodium acetate were obtained. This observation indicated that the pH increase alone during cultivation was responsible for the increased sunflower oil utilisation, biomass and GLA production.Item Open Access The repurposing of chemical compounds as anti-Cryptococcus drugs(University of the Free State, 2017-01) Ogundeji, Adepemi Olawunmi; Sebolai, O.M.; Pohl, C. H.English: The manifestation of disseminated cryptococcal infection in HIV-infected individual is a life-threatening infection, with 70% mortality rate in sub-Saharan Africa. People are dying because of the complications around the management. Thus, there is a need for alternative drugs for better management of HIV-associated cryptococcal infection. This thesis successfully demonstrated in vitro anti-cryptococcus activity of: 1) anti-inflammatory drugs (aspirin and ibuprofen), 2) aspirinate-metal complex (CAS), and 3) anti-psychotic drugs (quetiapine and olanzapine). In this thesis, EUCAST guidelines were followed closely and this provides a sense of effectiveness, which is very important for improving patient outcomes. Chapter 2 focused on repurposing aspirin and ibuprofen as alternative anti-cryptococcal drugs. The major findings from this part of the thesis show that, all the tested fungal strains revealed a dose dependent response profile towards aspirin and ibuprofen. Compared to aspirin, ibuprofen exerts greater antimicrobial action. More importantly, the MICs of both drugs did not negatively affect the functioning of macrophages - rather they enhanced the phagocytic capability of macrophages to internalize more cryptococcal cells. Ibuprofen was also shown to act in synergy with fluconazole and amphotericin B at lower concentrations than individual concentrations tested. Our findings revealed the mode of action employed by aspirin and ibuprofen which is via oxidative damage. Chapter 3 focused on a derivative of aspirin viz. copper acyl salicylate (CAS). CAS possess anti-fungal activity against cryptococcal cells and acted in synergy with fluconazole and amphotericin B at lower concentrations than their individual concentrations tested. CAS also kills cells via reactive oxygen species (ROS)-mediated membrane damage. The effect of CAS did not negatively affect macrophages but rather enhance their phagocytic function. Comparing with aspirin, CAS led to more growth reduction and showed less toxicity. Today, one of the main challenges in the management of disseminated cryptococcal infections is the secondary complications such as psychosis. Therefore, chapter 4 considers repurposing two anti-psychotic drugs i.e. quetiapine and olanzapine as suitable candidate anti-Cryptococcus drugs. The in vitro susceptibility results revealed that quetiapine and olanzapine have anti-cryptococcus activity and kill cells by compromising their membrane integrity. Importantly, the concentrations of drug tested were within the recommended dosage in the blood. Additionally, they acted in synergy with conventional drugs at concentrations that were lower than their defined MICs. It was also interesting to find that these two drugs chemosensitised macrophages, just like cytokines, which in turn, increase the appetite of macrophages against cryptococcal cells. The presented data from this thesis has highlighted the potential clinical application of aspirin, ibuprofen, CAS, quetiapine and olanzapine as candidate anti-cryptococcus drugs. More encouragingly, all the drugs were able to effect synergism at reduced concentrations, which in turn can minimize the issues of side effects. These compounds employed a killing mechanism that was efficient against cryptococcal cells, although of lower eukaryotic origin. Therefore, it is important to demonstrate the effectiveness of these drugs in higher eukaryotic host cells. Towards this end, animal studies should be used as model to establish their therapeutic benefits in higher eukaryotic hosts. The duality of these compounds should also be assessed, which may provide additional beneficial therapeutic outcomes such as to manage pathogen-emergent psychosis and out-of-control inflammatory responses. Lastly, it is also important to determine if the antimicrobial activities of these compounds also occurs in other medically important pathogens.Item Open Access Elucidation of African elephant beta casein phosphorylation state and casein micelle structure(University of the Free State, 2017-01) Madende, Moses; Osthoff, G.; Kemp, G.English: The exact structure of casein micelles still remains a debated subject. While most of the experimental work on cow caseins and casein micelles has provided a wealth of data, data of caseins and casein micelles of non-bovine origin provide a new insight into the structure of casein micelles. Microscopic examination of cow, sheep, horse, human and African elephant milk casein micelles show that the respective casein micelles are all spherical in shape but differ in size as well as surface appearances. Human casein micelles were the largest of the casein micelles whereas sheep casein micelles were the smallest. Apart from their smaller size, sheep micelles also had a smooth surface compared to a rough surface observed on the rest of the casein micelles. African elephant casein micelles were the second largest of the five casein micelles compared. It may be derived that, although casein micelle shape and size seem to be species specific, the differences observed may be a result of the differences in total casein content, the proportions of the individual casein types and the presence and or absence of some of the casein types. The elucidation of African elephant β-casein phosphorylation state by LC MS/MS, showed the presence of a single phosphorylation site at Ser9. In contrast, electrophoresis analysis showed that there are up to five phosphoforms of African elephant β-casein. The LC MS/MS also showed that the presence of a short length African elephant β-casein that is 200 amino acids long and that the gene sequences coded for by exons 4 and 5 have been truncated. Homology modeling of cow, sheep, horse, human and African elephant caseins showed that the secondary structure of α-caseins predominantly consist of α-helices, whereas the secondary structure of β- and κ-caseins is dominated by random coils. Alpha caseins give micelles a slightly compact structure whereas random coils result in a more open and larger size of micelles. These structural differences of caseins could possibly explain the varied size of casein micelles in milk. Comparative genomics of casein genes across mammalian species shows that several mammalian species are devoid of CNS1S1 and CSN1S2 genes. Considering the evolution of the casein gene locus organization, it appears that the CNS1S1 gene has been lost whereas the CSN1S2 gene has not been gained or developed in these species. In contrast, the CSN2 and CSN3 genes have been preserved and gained respectively, in most mammalian species. This suggests that these genes have a more important role in casein micelle formation and consequently the sequestration of large amounts of calcium and phosphate. Evidence from this study suggests that studying of non-cow caseins may shed more light on the casein micelle structure.Item Open Access Utilization of wood-decay fungi for biokraft pulping of softwood(University of the Free State, 1999-05) Wolfaardt, Jacobus Francois; Rabie, C. J.; Wingfield, M. J.English: The forest products industry is one of the most important earners of foreign exchange for South Africa. The major focus of the industry is the production of pulp with an annual capacity of 2,4 million tons. Wood from plantations of exotic trees is the most important source of fibre, but other fibre sources are also used. Biotechnology can play a significant role in the industry to produce high value products at lower cost and could reduce the environmental impact of conventional processes. Biopulping is potentially the most important of these biotechnological processes, because it can influence all downstream operations. The aim of this study was, therefore, to develop a biopulping process for the treatment of softwood at the Sappi Ngodwana kraft mill in Mpumalanga. Initially, 278 strains of wood-decay fungi were collected from various natural habitats. This collection represents a diversity of fungal families and included species that have not previously been recorded from South Africa. The first step in selecting suitable fungal strains for biopulping was to characterize different groups on the basis of the enzymes that they produce and their oxidase reactions. The suitability of these strains for the pre-treatment of softwood chips prior to kraft pulping was subsequently assessed by evaluating their influence on kappa number, yield and strength properties of pulp. Seven of the strains tested were able to reduce the kappa number of pulp significantly, without having a significant influence on the pulp yield. These strains were more efficient than strains of Phanerochaete chrysosporium and Ceriporiopsis subvermispora that have been patented for other biopulping applications. Treatment of wood with strains of Peniophora sp., an unidentified specie as well as two strains of Stereum hirsutum resulted in pulp with improved strength properties. The envisaged biopulping process aimed at treating wood chips in outside chip storage with a biopulping fungus. The aim of one study was to investigate conditions such as temperature, moisture, CO2 and microbial populations that develop in a chip pile, and to determine the suitability of the chip pile for colonization by biopulping fungi. High temperatures and high moisture levels were observed in some areas of the chip pile, which suggested that part of the pile was unsuitable for colonization by mesophilic fungi. It will, therefore, be necessary to manage the chip pile to maintain a suitable environment for biopulping. Problems were experienced with poor colonization of freshly chipped softwood by biopulping fungi. The effect of contaminating microbes and inhibitory compounds present in wood was, therefore, studied. It was found that the inhibition of biopulping fungi by α-pinene and by contaminating microbes were both very important. The inhibition by microbes, as well as by extractives, was mitigated by a short steam treatment of wood chips. Steaming for ten minutes under atmospheric pressure could be an economical method to improve colonization by biopulping fungi. Alternatively biopulping fungi with good competitive ability and tolerance to monoterpenes could be selected. Pinus patula wood chips were pre-treated with a selected strain of Stereum hirsutum to determine the optimal conditions for the kraft pulping of pre-treated softwood and to do an economic evaluation of the process. Chips were pulped on a small scale under various pulping conditions. Lignin content, yield, and viscosity of the pulp as well as alkali consumption were evaluated. The results were used to develop models for biokraft pulping. This study showed that biopulping can reduce the kappa number of pulp or reduce the pulping time. Pulp yield losses were relatively small when pulps with low kappa number were produced. Increased alkali consumption was, however, an important factor in the economic evaluation.Item Open Access Diseases of Acacia mearnsii in South Africa, with particular reference to ceratocystis wilt(University of the Free State, 1998-12) Roux, Jolanda; Wingfield, M. J.English: The Acacia mearnsii industry is a relatively small, though very profitable industry in South Africa. Wood derived from A. mearnsii is currently in greater demand than that of either pine or eucalyptus in South Africa. Despite the importance of this industry, very little attention has been given to the genetic improvement, disease tolerance or general improvement of A. meamsii as a forestry species. The result has been that, during the last few decades, pathogens have become adapted to, and spread through plantations of this tree. Although relatively little research has been conducted on the impact of pathogens on A. mearnsii, this situation has changed during the past nine years, and particularly since the identification of Ceratocystis wilt. The planting of exotics has many advantages over native plants. In South Africa, exotic forestry species, such as Eucalyptus spp., Pinus spp. and A. mearnsii were introduced to halt the uncontrolled logging of native forests. These native forests were logged mainly for furniture and building material, but also for fuel wood, resulting in the near complete destruction of South Africa's native forests. The introduced exotics prevented the further destruction of these forests and soon became a large industry. This was particularly due to the fact that it was found that they also had a superior growth rate when compared to native species. This accelerated growth rate brought rapid results from breeding trials and, thus, a relatively rapid improvement of the material planted. Because they had been separated from their natural enemies, these trees were also initially disease free. The A. mearnsii industry has, and will continue, to face many problems and challenges from pests and diseases. After the initial phase in which the tree was removed from the pathogens affecting it in it's native range, it faced attacks by native South African pests and diseases. These can spread from native Acacia species, or from any other native plants in the same, or even different families. Exotic, mono culture industries are also constantly under threat from the introduction of pathogens from other countries, including the country of origin. This can be done by the introduction of new germ plasm or on any other plant species or plant material brought into a country. Because A. mearnsii is now planted as a monoculture, in contrast to it's native situation, diseases and pests can potentially be much more severe and will spread more rapidly and widely throughout even aged and genetically uniform stands. Propagation of A. mearnsii has, recently, advanced considerably and this is concurrent with increased demand for this wood on world markets. Lessons learned from eucalypt and pine forestry need, however, to be heeded to save unnecessary losses and time. With the advent of vegetative propagation of A. mearnsii in South Africa, it is important to include disease screening trials at the early stages of the development of clones. In order to do this, a knowledge of all possible pathogens of A. mearnsii is needed. This includes pathogens known in South Africa and those that occur beyond the borders of the country. It is also necessary to have a detailed knowledge of the biology and population structure of these pathogens in order to gain an impression of the possible success of control measures. This thesis is a compilation of work conducted on some of the known pathogens of A. mearnsii in South Africa. It also includes a large component dealing with the identification and clarification of previously unknown pathogens of A. mearnsii. It, therefore, does not focus only on diseases of A. mearnsii, but includes a chapter on a disease of Eucalyptus. The causal agent of this disease has, however, also recently been found on A. mearnsii in South Africa and this chapter aims at elucidating the possible origin of the isolates from South Africa. It also illustrates the potential threat of this pathogen to the A. mearnsii industry. South Africa is a semi-arid country that regularly suffers from severe drought. Forestry activities in the country are also mainly restricted to areas with poorer soil and where agriculture cannot be pursued on a profitable basis. Factors such as drought, hail, frost and sub-optimal soil conditions can all contribute to increased stress on trees. Under these conditions, many fungi can act as opportunistic pathogens, causing large scale losses. They often live as endophytes within their hosts, not causing any negative affect until the onset of stress. At this stage, they spread throughout trees, preventing them from recovering from the stress condition and leading to cankers and tree death. Careful management, particularly site/species matching, is required to minimise losses caused by these pathogens. This thesis provides a basis for future research on the development of management strategies to control diseases of A. mearnsii in South Africa. Information, however, also provide valuable knowledge for forestry industries outside South Africa by highlighting the threat of exotic pathogens and the importance of strict quarantine measures to prevent the spread of pathogens. This is true for the movement of not only A. mearnsii material, but as was seen here, the movement of any forestry products, since many pathogens have a wide host range. Although the thesis is comprised of a series of individual entities, these all provide information regarding the hygiene of A. mearnsii plantations. This thesis thus aims at identifying future focus points for intensive research, while at the same time focusing on those pathogens that have been known to the South African industry for a longer period of time. Chapter one provides a review of the available literature on diseases affecting not only A. mearnsii, but also other Acacia spp. important to the forestry industry, world wide. It also highlights some of the uses of these species in the countries where they are planted. The multi-purpose use of Acacia spp. is an important aspect emerging from this review. In many countries, Acacia spp. are not only planted as forestry species but are also used for soil reclamation, nitrogen fixation and fodder. The main focus of the chapter, however, is on the A. mearnsii industry in South Africa, with a brief discussion on all the diseases currently known to occur in the country. It is concluded that much research is still needed to reduce the impact of these diseases and to ensure that the Industry functions optimally. Ceratocystis albofundus must be considered as one of the most important pathogens of Acacia spp., world-wide. Currently this pathogen occurs only in South Africa, but if it is to spread to other countries, large scale losses will be incurred. It may also affect, not only A. mearnsii, but most likely many other plant species. Breeding programmes for A. mearnsii in South Africa focus strongly on this pathogen. In Chapter two, the population diversity of C. albofundus was investigated and compared with data for other Ceratocystis spp., using nuclear and mitochondrial DNA fingerprinting. It was found that the C. albofundus population has a greater genetic diversity than any of the species with which it was compared. This will thus mean that intensive breeding programmes will be necessary to ensure durability of disease tolerance. It also supports previous hypotheses that C. albofundus is native to South Africa and may be a temperate species, not found in tropical areas where its close relative, C. fimbriata, commonly occurs. The first unequivocal report of C. fimbriata and Ch. elegans from A. mearnsii is presented in Chapter three. Both these fungi were isolated from dying trees with typical symptoms of Ceratocystis wilt caused by C. albofundus. Both were shown to be capable of causing disease to seedlings under green house conditions. It was, however, found that C. albofundus is more virulent than either Ch. elegans or C. fimbriata. Both isolates were identified using molecular and morphological approaches. Unfortunately only one isolate of each exists and surveys to obtain additional samples continue to be a priority. The first report of a wilt disease of Eucalyptus, caused by Ceratocystis fimbriata in the Republic of the Congo in West Africa is recorded in Chapter four. This is not only the first report of C. fimbriata as a pathogen of Eucalyptus in Africa but is also one of the few unequivocal reports of this fungus from the continent. Pathogenicity of C. fimbriata on Eucalyptus spp. was confirmed in glass house tests. In this Chapter, C. fimbriata and C. albofundus from A. mearnsii, and C. fimbriata from Eucalyptus in Brazil were also compared to the C. fimbriata from the Congo. Comparison of the lTS region of the rRNA operon showed that isolates from all three areas grouped together in a clade of C. fimbriata, separate from European isolates. Sequence data showed that C. fimbriata from A. mearnsii in South Africa is nearly identical to the fungi from Eucalyptus in Brazil and Congo, suggesting that they may have a common origin. These findings stress the importance of sound quarantine measures to prevent the introduction of potentially devastating pathogens to South Africa. It is not yet known why C. fimbriata has not caused more diseases on A. mearnsii or Eucalyptus spp. in the country, but the situation will need to be monitored closely. Apart from C. albofundus, there are many other fungi that cause disease of A. mearnsii in South Africa. Chapter five reports on a species of Seiridium that was isolated from stem cankers on A. mearnsii. Morphological and molecular comparisons, as well as pathogenicity studies have shown that the species from A. mearnsii is similar to those species responsible for Cypress canker in many parts of the world. It also confirms previous reports that the taxonomy of the three Seiridium spp. causing cypress canker needs re-evaluation, since molecular data support the view that the three species, represent a single taxon. Pathogenicity trials on mature Cuppressus lusitanica and on A. mearnsii trees showed that both the cypress and A. mearnsii isolates are capable of causing lesions on both hosts. Many of the fungi isolated from diseased A. mearnsii during the current and previous studies of diseases resulted in the isolation of fungi, commonly found as latent pathogens on other forest trees. Chapter six encompassed a survey of the endophytic fungi of A. mearnsii, with the specific aim of identifying possible pathogens. Thirty different fungal taxa were found as endophytes of the xylem and rachi. These included F. graminearum and Botryosphaeria dothidea, which are known pathogens. During periods of environmental stress, these fungi can apparently cause disease. This is especially true because A. mearnsii is often planted on marginal sites in South Africa. Chapter seven represents the first report of Fusarium graminearum from A. mearnsii and presents evidence for the fungus being involved in disease of A. mearnsii. This pathogen was first isolated during 1994-95 disease surveys, but was not identified due to the fact that cultures on artificial media did not sporulate. In the current study, additional isolates were obtained from stem cankers and die-back symptoms and the fungus was identified based on β-tubulin gene sequences. Field inoculations using F. graminearum showed extensive lesion formation in the xylem. Previously, this Fusarium sp. was known only as a pathogen of maize and wheat in various parts of the world. Results of this study are, therefore, enigmatic and intriguing.Item Open Access Mycotoxin contamination of maize in relation to insect infestation, agricultural practices and agroecology in the Republic of Cameroon(University of the Free State, 1999-09) Ngoko, Zachee; Marasas, W. F. O.; Wingfield, M. J.; Cardwell, K. F.English: Maize (Zea mays L.), the staple food crop of the majority of the population of Cameroon, is damaged by insects and diseases from the fields to the stores. As a result, the quantity and the quality of harvested grain is reduced. This study was undertaken to identify constraints associated with the production and post-harvest losses of this commodity in two ecological zones ofCameroon from 1995 to 1997. Farmers' perceptions of diseases and pests play an important role in their acceptance of new pest management technologies. From the survey conducted to assess their perceptions, farmers reported that borers (Busseola fusca) were the main constraint to maize production in the Humid Forest and Western Highlands. Locusts (Zonecerus variegatus) and rodents were the second most important limiting factor in the Humid Forest. The storage weevil (Sitophilus zeamais) was the most damaging storage insect. Diseases were not generally known by farmers who could only recognize smuts and ear rots by the visible damage caused by them. While the period of the outbreaks of insect infestation was not reported with precision, most farmers reported that diseases occurred at the mid-season. Control practices were not well established. Disease surveys conducted from 1995 to 1997, revealed that lowland blight (Bipolaris maydis, Diplodia leaf spot (Stenocarpella macrospora) and sheath blight (Rhizoctonia solani) were the most important maize diseases in the Humid Forest, while highland blight (Exserohilum turcicum) and grey leaf spot (Cercospora zeae-maydis) prevailed in the Western Highlands. Phaeosphearia leaf spot (Phaeosphaeria maydis) was specific to the Western Highlands with a negative relationship with grey leaf spot. Busseola fusca infested maize plants at all stages of growth with high prevalence in the Humid Forest. The identification of factors -affecting maize yield demonstrated that diseases, insects and their interactions with soil infertility, soil texture, weeds, and maize varieties were responsible for the reduction of maize production. Yield reductions were 30% and 33.6% respectively, in the Humid Forest in 1995 and 1996 due to Stenocarpella macrospora, Puccinia polysora and Rhizoctonia solani. In the Western Highlands, Cercospora zeaemaydis, Busseola fusca, stem diseases, and physiological spot caused yield reductions of 51.2%, and 37.9% in 1996 and 1997, respectively. Mycological and chemical analyses of maize grain collected from 72 farmers' stores showed that several pathogens were associated with grain quality deterioration. Nigrospora spp. were the most frequently isolated fungi on kernels, followed by Fusarium moniliforme and Fusarium graminearum. Aspergillus spp. were rare in both zones. Fumonisin B1, deoxynivalenol and zearalenone were detected in maize samples at levels ranging from 300 to 26,000ng/g, 100 to 1300 ng/g, and 50 to 110 ng/g, respectively. This is the first report on the natural occurrence of these Fusarium mycotoxins in maize in Cameroon. Surveys conducted to identify the biological and physical factors that enhanced the infection of maize kernels by fungi and the contamination with fumonisin , identified several agricultural techniques related to grain quality in the Western Highlands. Harvesting in June (11.1 %) or July (23.6%), sorting right from the field (16.7%), drying over the fireplace with husk (19.4) or without husk (33.3%) and storing shelled grain in bags (19.4%) or boxes (9.7%) reduced fumonisin contamination. Continuous production of maize on the same field, harvesting in August, and the infestation by the weevil Sitophilus zeamais were factors that increased fumonisin contamination. Crop rotation, sorting maize during all the post-harvest processes and the treatment of maize grain with appropriate insecticides should decrease the risk of contamination by fumonisin. Continuing collaborative research should aim at understanding farmers' needs and priorities, investigating the epidemiology of maize diseases, screening for resistance to the most important maize diseases and improving harvesting, sorting, drying and storing methods in Cameroon.Item Open Access Factors associated with coniothyrium canker of Eucalyptus in South Africa(University of the Free State, 1999-05) Van Zyl, Leonel Merwe; Wingfield, M. J.; Coutinho, T. A.; Wingfield, B. D.English: English: In chapter one of this thesis, the literature pertaining to the genus Coniothyrium and its importance in plant pathology, is reviewed. Special attention is given to Coniothyrium species associated with Eucalyptus but the focus is on Eucalyptus stem canker pathogen, C. zuluense. Coniothyrium zuluense is an important pathogen in South Africa and has, since its discovery, become widespread throughout plantation areas of KwaZulu-Natal. The current means for reducing the impact of this disease is to plant disease resistant species and clones of Eucalyptus. It is evident from this review that very little information is available pertaining to the biology, reproductive system, or the population structure of C. zuluense. Such information is essential for managing the disease successfully in the future. The strategy currently used to reduce the impact of Coniothyrium canker in plantations is to deploy Eucalyptus species or clones that are resistant to the disease. Considerable success has already been achieved in this regard, but the long-term durability of resistance is of concern. Results of the study represented in chapter two showed that there is considerable variation in colony colour and pathogenicity of a large collection (344) of C. zuluense isolates. Conidial morphology and growth requirements are, however, similar for all isolates tested. The considerable variation in pathogenicity indicates that C. zuluense has been present in South Africa for an extended period of time, or that virulence is changing rapidly due to strong directional selection pressure. Results of the taxonomic and pathogenicity studies in chapter two, suggest that the C. zuluense population is well established. In chapter three, the population diversity of 108 C. zuluense isolates, differing in their pathogenicity to a susceptible Eucalyptus clone, was investigated using Amplified Fragment Length Polymorphism (AFLP) technology. Results indicated that the level of genetic diversity is relatively low, but higher than expected for an asexually reproducing pathogen. Genetic similarity values also indicated a significant population differentiation between different plantation regions (subpopulations), suggesting that gene flow, together with selection, might be responsible for most of the gene diversity. New epidemics would, therefore, not be as a result of the emergence of new aggressive strains, but would rather be due to the introduction of susceptible Eucalyptus species, together with environmental conditions favouring disease development. A Coniothyrium species associated with similar symptoms to those associated with C. zuluense in South Africa was observed on E. camaldulensis in Thailand in 1996. It was previously thought that C. zuluense was restricted to South Africa. In chapter four, I show using morphological and molecular comparisons, as well as pathogenicity studies, that C. zuluense and the Coniothyrium sp. from Thailand are the same organism. This is, thus, the first record of this important Eucalyptus stem canker pathogen, C. zuluense, outside South Africa. Bacteria commonly exude from necrotic cankers on severely infected Eucalyptus clones in plantations. In chapter five, it was shown that bacteria associated with Coniothyrium canker in the field are species of the genus Pantoea. These species were identified based on 16S rDNA sequence data as P. ananatis pv. ananatis and a species closely related to P. stewartii subsp. stewartii. It was also shown that a synergistic interaction between C. zuluense and both Pantoea species exists. Inoculation studies, using both Pantoea species together with C. zuluense isolates, resulted in a significant increase in pathogenicity as opposed to inoculations where the bacterial and fungal isolates were used alone. Future studies should consider the presence or absence of both bacteria species in disease development in Thailand. During plant-pathogen interactions, pathogens are known to produce cell wall degrading enzymes, in particular pectin degrading enzymes. Polygalacturonase (PG) is the first enzyme produced during such interactions and is known to be a determining factor in pathogenicity. Chapter six showed that C. zuluense isolates and both Pantoea species, P. ananatis pv. ananatis and an unknown Pantoea sp., produce PG. Experimental assays show that levels of PG activity for both Pantoea spp. are significantly higher than those obtained for C. zuluense isolates. As PG is the first enzyme produced during disease development it is hypothesised that the two Pantoea species might play a significant role in the development of Coniothyrium canker. Production of PG could also be used as an assay to evaluate pathogenicity in different isolates of C. zuluense. Pathogen-produced cell wall-degrading enzymes play a key role in activating plant defence responses. Most inducible defence responses are the result of transcriptional activation of genes. Various plant resistance (R) genes, as well as pathogenesis-related proteins, such as polygalacturonase inhibiting proteins (PGIPs), have been linked with resistance to various fungal and bacterial pathogens. In chapter seven, a partially sequenced resistance gene from disease resistant E. grandis clone, TAG 5, was shown to be similar to a gene associated with a disease resistance gene in Arabidopsis thaliana. The most exciting aspect of this study was, however, the discovery of a shift in reading frame of this gene for the susceptible Eucalyptus clone, ZG 14. The complete sequence of this gene should provide a more complete view of its importance in disease resistance. Screening for similar interruptions in the open reading frame of various commercially available Eucalyptus clones could significantly speed up breeding programmes aimed at producing improved disease resistant clones.Item Open Access Biocatalytic resolution of epoxides: epoxide hydrolases as chiral catalysts for the synthesis of enantiomerically pure epoxides and vic diols from α-olefins(University of the Free State, 1999-06) Botes, Adriana Leonora; Smit, M. S.; Litthauer, D.English: The synthesis of chiral pharmaceuticals in an enantiopure form had become increasingly important in the last few years. This same trend is now found in the synthesis of agrochemicals. Epoxides, due to their high reactivity with a large number of reagents, and vie diols, employed as their corresponding cyclic sulfates or sulfites as reactive intermediates, are versatile chiral synthons in the synthesis of many bioactive compounds. Extensive research efforts have thus been directed towards the synthesis of optically active epoxides and viel diols. Kinetic resolution of racemic epoxides by epoxide hydrolases has recently emerged as a very attractive strategy for the synthesis of enantiopure epoxides. Both chemical and biological catalysts that may be employed to obtain enantiopure epoxides from relatively inexpensive racemic substrates had been reviewed (Chapter 1). The potential use of microbial epoxide hydrolases, including those from yeasts as elucidated during this study, was emphasised in this review. At the onset of this study, epoxide hydrolase activity had been identified in only one yeast, Rhodotorula glutinis. The broad range of substrates that were hydrolyzed with excellent enantioselectivity by this yeast, indicated that yeast epoxide hydrolases might be very interesting catalysts. This had indeed been found to be true during the course of this study. Enantioselective hydrolysis of a homologous range of aliphatic 1,2- epoxyalkanes was accomplished in collaboration with the group of Jan de Bont (Division Industrial Microbiology, Wageningen AU, The Netherlands). No other microbial epoxide hydrolases have been found that display this unique enantioselectivity for epoxides lacking other substituents (Chapter 2). Extensive screening of yeasts from the renowned UOFS Yeast Culture Collection revealed that epoxide hydrolase activity was constitutively present in about 20% of the yeasts screened, and that other basidiomycetous yeasts from the genera Rhodotorula, Rhodosporidium and Trichosporon shared this unique enantioselectivity for 1,2- epoxyoctane with Rhodotorula glutinis (Chapter 3). he apparent association between carotinoid production and epoxide hydrolase activity in bacteria as well as the red yeasts Rhodotoru/a and Rhodosporidium, prompted us to investigate the epoxide hydrolase activity of the yellow pigmented bacterium Chryseomonas /uteo/a in our collection. Indeed, this bacterium displayed epoxide hydrolase activity, and moderate enantioselectivity for 1,2-epoxyalkanes (E =20) by a bacterial epoxide hydrolase was found for the first time (Chapter 4). A survey of the enantioselectivities of yeasts for a homologous range of 1,2- epoxyalkanes, 1,2-epoxyalkenes as well as the 2,2-disubstituted 2-methyl-1,2- epoxyheptane and benzyl glycidyl ether was conducted. Excellent biocatalysts for C-5 to C-8 epoxyalkanes and the C-8 epoxyalkene were found. The epoxide hydrolases from all the enantioselective yeasts were found to be membrane-associated (Chapter 5). The epoxide hydrolase from the yeast Rhodosporidium toru/oides was purified in an elegant one-step protocol from the microsomal fraction, using affinity chromatography (Chapter 6). However, initial attempts to obtain amino-acid sequences failed. In lieu of information about the primary structure of yeast epoxide hydrolases, inactivation of the enzyme by modification of specific amino acids was studied. Asp/Glu and His residues were found to be essential for catalytic activity. In addition, it was found that one or more Ser residues in the catalytic site are indispensible for catalytic activity. These results indicate that yeast epoxide hydrolases probably belong to the same subfamily of a,l3- hydrolase fold enzymes as the microsomal epoxide hydrolases from other eukaryotes. Unusual kinetic behaviour was observed during the hydrolysis of 1,2-epoxyalkanes by purified epoxide hydrolase. Hydrolysis was characterised by a strong dependence of enantioselectivity on the presence of the substrate as a second (Iypophilic) phase. The purified epoxide hydrolase was not very stable, with a half-life time at 35°C of 18 hours (Chapter 7).Item Open Access Molecular taxonomy and mating type genes in Ceratocystis sensu stricto(University of the Free State, 1999-01) Witthuhn (née Strydom), Regina Cornelia; Wingfield, B. D.; Wingfield, M. J.; Harrington, T. C.English: Most species of Ceratocystis sensu stricto are virulent pathogens of a wide variety of plants. In the research presented in this thesis, I have developed a rapid and reliable PCR-based RFLP identification method for species of Ceratocystis. A 1.6 kb fragment within the ribosomal DNA operon was directly amplified from living fungal tissue, without extracting DNA. The amplified fragment included part of the small and large sub-unit rRNA genes, the 5.8S rRNA gene and the internal transcribed spaeers 1 and 2. The PCR fragments were digested with eighteen restriction enzymes. Four of these (AluI, DraI, HaeIII and RsaI) produced RFLPs that separated the species of Ceratocystis. The 1.6 kb PCR products, from the better known species of Ceratocystis, were sequenced and phylogenetically analyzed. The delimitation of taxa was consistent with results of previous studies using isozymes and rDNA sequence analysis. Ceratocystis coerulescens is a well-known cause of blue stain in spruce and pine. It was shown that C. coerulescens encompasses at least five morphological types. I compared isolates of C. coerulescens sensu lato and morphologically similar species on the basis of lTS DNA sequences. A 600 bp fragment within the ribosomal DNA operon, including the 5.8S rRNA gene and ITS 1 and 2, was amplified, sequenced and analyzed using PAUP. The five morphological types previously known as C. coerulescens, and the two other taxa from conifers, formed a strongly supported monophyletic group that includes all the Ceratocystis species occurring primarily on conifers. The species from hardwood trees, C. eucalypti, Ch. australis and Ch. neocaledoniae, also formed a monophyletic group, sister to the conifer group. The fourth species from hardwoods, C. virescens, formed a group basal to the two sister groups. The phylogeny of species in the C. coerulescens complex based on ITS DNA sequences were compared to the phylogeny based on the MAT-2 HMG box DNA sequences. A 210 bp PCR fragment of part of the MA T-2 HMG box of species in the C. coerulescens complex was amplified. C.fimbriata was used as the outgroup taxon and was distinct from the other Ceratocystis species studied. The species from conifers formed a single clade, sister to the clade formed by the species from hardwoods. Phylogenetic analysis of the MAT-2 HMG box DNA sequences differed slightly from the phylogenetic analysis based on ITS DNA sequences. Based on ITS DNA sequences C. vireseens was basal to the other species in the C. coerulescens complex, while C. laricicola and C. polonica could not be separated from each other. Differences in the MAT-2 HMG box DNA sequences for the latter two species clearly showed them to be as distinct from each other. Recent mating studies on C. coerulescens have prompted a study of the expression of mating type genes in species of Ceratocystis sensu stricto. C. eucalypti is strictly heterothallic. Most other Ceratocystis species, including C. virescens, C. coerulescens and C. pinicola are homothallic. The MAT-2 strains are self-fertile, while MAT-1 strains are self-sterile and grow more slowly than MAT-2 strains. Part of the MAT-2 idiomorph in C. eucalypti, C. vireseens and C. pinicola was amplified using degenerate primers designed from the conserved MAT-2 HMG DNA binding motif. The expected ~300 bp PCR products were cloned and sequenced. Specific primers were designed that amplified a 210 bp fragment only in MAT-2 isolates of C. eucalypti, C. vireseens and C. pinicola. This fragment was absent from the self-sterile (MAT -1) progeny of C. vireseens and C. pinicola, confirming the deletion of MAT-2 during uni-directional mating type switching. The known DNA sequence data for the C. eucalypti MAT-2 mating type idiomorph was increased from 280 bp to 1 371 bp, using TAIL-peR and uneven PCR.Item Open Access Genome sequencing of the extremophile Thermus scotoductus SA-01 and expression of selected genes(University of the Free State, 2009-05) Gounder, Kamini; Litthauer, D.; Van Heerden, E.; Piater, L. A.Thermus scotoductus SA-01 is an extremophile that was isolated from groundwater samples at 3.2 km depth in a South African gold mine and has previously been shown to grow using nitrate, Fe(III), Mn(IV) or Sº as terminal electron acceptors and to be capable of reducing Cr(VI), U(VI), Co(III) and the quinone-containing compound anthraquinone-2,6-disulfonate. This study reports the sequencing of the T. scotoductus SA-01 genome using a strategy involving the GS20 and FLX pyrosequencing, which is a novel, rapid method of high-throughput sequencing, as well as Sanger technology. The GS20 and FLX pyrosequencing data was assembly into 35 contigs using the Newbler Assembly software. Mapping attempts using various software against the reference strain T. thermophilus, proved unsuccessful due to low levels of synteny and extensive rearrangement noticed between the two organisms. After using various strategies to close the gaps between the 35 contigs with Sanger sequencing, the complete chromosome sequence was obtained. The genome consists of a 2 346 803 bp chromosome and a plasmid, which could not be closed with all sequencing attempts. The draft plasmid sequence consists of 8 383 bp with about 65% in agreement with the chromosome sequence. Automatic annotation of the complete chromosome and draft plasmid sequence performed by TIGR (J. Craig Venter Institute formerly known as The Institute for Genome Research) revealed the presence of 2482 and 12 ORFs, respectively. ORF correction was performed using the Artemis software package. Manual annotation was performed using the ERGO Tool software on half of the genome using various public databases and criteria. BLAST results of the plasmid sequence against the chromosome show that four ORFs present on the draft plasmid are also present in an identical copy (one or more than one copy) on the T. scotoductus SA-01 chromosome, providing evidence of genetic exchange between the chromosome and the extrachromosomal element. Comparative genome analysis was done using strains that are related (3 genomes) to T. scotoductus, isolated from a South African goldmine (1 genome) and metal reducing organisms (2 genomes). Using this data, analysis of metabolism and thermophily of T. scotoductus SA-01 could be comparatively elucidated as well as determining the orthologous gene content. The complete chromosome and draft sequence of T. scotoductus SA-01 not only provides valuable basic data in terms of the organism’s lifestyle and capabilities but is also consists of many genes of potential interest for biotechnological applications. Due to its thermophilic nature, T. scotoductus SA-01 would contain many thermostable enzymes, which possess qualities that make them more robust and better suited for use in molecular applications. The DNA polymerase I and single stranded DNA binding (SSB) protein was chosen for expression studies for their potential use in a PCR reaction. A partially purified DNA polymerase I protein was obtained; however the protein was found to be non-functional in a PCR. Expression of the SSB was performed, but the protein could not be purified for further analysis. Obtaining expression at higher levels and complete protein characterization would be required in order to understand the capabilities of these proteins.Item Open Access Development of Yarrowia lipolytica for enhanced production of heterologous proteins(University of the Free State, 2011-03) Ramagoma, Faranani; Smit, M. S.; Nthangeni, M. B.English: Yarrowia lipolytica is a non-conventional yeast which is considered to be suitable for production of industrially important proteins at commercial scale. The yeast is non pathogenic and is generally regarded as safe and as such can be used to produce biotechnological products for human consumption and applications. The genetic tools for the manipulation of the yeast have been developed over the years. This includes chemical and genetic based mutagenesis techniques to develop and isolate Y. lipolytica strains with enhanced properties in the production of endogenous and heterologous proteins. Protein expression systems comprised of a variety of inducible and constitutive promoters are available; secretion signals for extracellular production of proteins and a choice of replicative or integrative expression systems have also been developed. Auxotrophic and antibiotic based selectable markers are available some of which result in single or multiple copies of the integrative expression cassettes containing the target gene. The Y. lipolytica yeast can utilise a wide variety of carbon sources and its fermentation processes in bioreactors are well established. The property of Y. lipolytica as a prolific producer of the endogenous extracellular Lip2p was exploited for the development of an expression system for production of therapeutic peptides. To this end, the 38 amino acid long RANTES I peptide with therapeutic applications in the treatment of HIV/AIDS was selected as a model peptide for co-expression with Lip2p. The sequence encoding the peptide was cloned downstream of the complete LIP2 gene. The therapeutic peptide was separated from the LIP2 gene by the sequences encoding for 6X His and the DDDDK sequence recognized by the enterokinase proteolytic enzyme. The expression cassette was under the hp4d quasi constitutive promoter with the rDNA as a target for multi copy integration using the defective Ura3d4 integration cassette. The amount of RANTES I produced by the expression system was estimated to be 0.274 μg.L-1 as determined by the CCL5/RANTES immunoassay method. The RANTES I peptide produced in this study was reactive to polyclonal antibodies raised against human RANTES and functionality assays showed that the peptide inhibited the binding of the pseudoviral particles to the TZM-bl cells through the CD4/CCR5 receptors. Another section of the study sought to identify genetic elements responsible for enhanced secretion of the extracellular Lip2p in Y. lipoyltica. Lipase hyperproducing mutants were generated using the zeta based mutagenesis system which integrates randomly within the Y. lipolytica genome. One mutant denoted Y. lipolytica Y12 which showed lipase hyperproducing phenotype was found to be disrupted in the function of the GPI7 gene, a protein encoding a polypeptide of 830 amino acids. The GPI7 protein is 33% identical to the S. cerevisiae GPI7 known to play a role in the maintenance of the yeast cell wall integrity. Consistent with the disruption of cell wall integrity through deletion of GPI7, the Y. lipolytica strain was more sensitive to cell wall hydrolysing enzymes, had defects in cell separation and a daughter cell-specific growth defect at the non-permissive temperature and exhibited hypersensitivity to Calcofluor white and Congo red which are known to interfere with yeast cell wall composition. While the Y. lipolytica Yl12 and the ”wild-type” Po1d strains grew similarly in shake flask cultures, the Y. lipolytica Yl12 strain produced 7 times more extracellular lipase activity in liquid shake flask cultures than the control Y. lipolytica Po1d strain. To investigate the mode of GPI7 action in the enhancement of protein production, a Y. lipolytica YlHmA25 strain expressing intracellular epoxide hydrolase under control of the hp4d promoter was deleted for the GPI7 gene to create a Y. lipolytica YlHmA25ΔGPI7 strain. The YlHmA25ΔGPI7 strain contained epoxide hydrolase activity in the extracellular in contrast to the Y. lipolytica YlHmA25. In addition, the Y. lipolytica YlHmA25ΔGPI7 accumulated more total extracellular protein than Y. lipolytica YlHmA25 an indication that the strain accumulates more proteins extracellulary in comparison with the Y. lipolytica YlHmA25 strain. Taken together, the results suggested that the disruption of GPI7 affects the integrity of the yeast cell wall which in turn results in leakage or “enhanced” secretion of proteins to the extracellular. The challenge in developing a host system with broad applications in heterologous production of proteins including therapeutics lies in the elimination of the endogenous hyper-mannosylated yeast glycans. The OCH1 gene encoding a protein with α-1,6- mannosyltransferase activity was deleted from the GPI7 deleted mutant of Y. lipolytica. The OCH1 and GPI7 double deletion mutant of Y. lipolytica showed similar growth patterns to the GPI7 null mutant. Glycan analyses revealed that the most abundant glycans are the Man8GlcNAc2 and Man9GlcNAc2 which are acceptable substrates of the Trichoderma reesei α-1,2-mannosidase I the enzyme widely used in the engineering of yeast glycosylation pathway to produce Man5GlcNAc2, a prerequisite intermediate for the synthesis of complex glycans in human cells. The OCH1 and GPI7 double deletion mutant of Y. lipolytica is a good candidate for further glycoengineering to enable production of extracellular therapeutic proteins with human-like glycans.
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