The methylotrophic yeast, Pichia past oris, is one of the most respected and widely used systems today. The ability of this yeast to produce large masses of protein and metabolize methanol as a sole source of carbon and energy is attributed to the highly induceable Alcohol Oxidase I promoter (AOXI). Despite of the disperse popularity and use of this promoter over the last 15 years, little is known about the transcription controls at a molecular level. A 5'>3' deletion analysis of the AOXI promoter was perrormed to gain understanding of the promoter's regulation and provided insight to the approximate locations of the important regulatory regions. A total of 10 truncations were made unveiling two areas ofhigh activity located between positions, -257 to-235, and, -235 to -188. In addition, a 14-base pair internal deletion was made between positions, -215 to -201. This region was shown to be necessary for transcriptional activation by deletion analysis. Sufficiency studies suggested that this 14-base pair element could serve as an activator sequence in both glucose and methanol.

Pichia pastoris, a popular protein expression system, is limited in its ability to secrete heterologous proteins. The PMR1 gene, the disruption of which is known to improve the secretion of prochymosin, human prourokinase, and human tissue plasminogen activator in Saccharomyces cerevisiae, was cloned from P. pastoris. The pmr 1 mutant in S. cerevisiae also displayed a slow growth phenotype when grown on low Ca2+ medium. The putative P. pastoris PMR1 gene, encoding for a 924 amino acid P-type Ca2+ ATPase, was disrupted in P. pastoris and the secretion of horseradish peroxidase (HRP) and β-galactosidase (β-gal) analyzed. Secreted HRP activity was determined using 3,3',5,5' tetramethylbenzidine (TMB) colorimetric assay and western analysis. β-gal expression and secretion was determined by western analysis. Secretion in P. pastorius Δpmr1 for both heterologous proteins showed no appreciable difference compared to wild type, nor did P. pastoris Δpmr1 display the slow growth phenotype seen in S. cerevisiae Δpmr1 (Rudolph H. et al., 1989).

Diagnostics serve as the frontline defense for the containment and mitigation of infectious diseases. The emerging synthetic biology field established numerous useful applications of engineered biological systems and networks that led to the development of living biosensors. Significant effort has been made to develop G-coupled protein receptor (GPCR)-based yeast biosensors for applications in drug discovery, environmental monitoring, and clinical diagnostics of small molecules and fungal pathogens. Of the living biosensor chassis, yeast-based biosensors offer exceptional advantages over other in vitro diagnostics, such as long-term storage in a reagent-free and dried dormant state, an engineered colorimetric readout for yes/no result interpretation, and high scalability through industrial fermentation. These advantages could be the next innovation as a low-cost, low-tech, and reliable diagnostic alternative in point-of-care and at-home contexts. Chapter 1 provides background information related to synthetic biology, living biosensors, direction evolution, and point-of-care diagnostics. Chapter 2 covers the development of engineered living yeast as a diagnostic tool for viral infections by tailoring the biosensing recognition element to sense any amino acid-based biomarker of choice via directed evolution. Chapter 3 describes the development of living yeast biosensor for the detection of the pathogenic fungus, Aspergillus fumigatus, in clinical supernatants and patient samples. Chapter 4 describes the progression of a living yeast biosensor for the multi-drug resistant pathogenic fungus, Candida auris, and its detection in clinical culture supernatants and samples.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The conversion of sugar into ethanol and carbon dioxide is a process that has been intertwined with human culture and long as civilized man has existed. This fermentation process has been dominated by the micro-organism Saccharomyces cerevisiae and from providing ancient seafaring explorers of a non perishable beverage to equipping bakers with a raising agent to turn flour into bread; this organism with its fermentative potential, has formed an essential part of most societies. In more recent times, many industries still rely on this basic principle. The complexities and efficiencies of the conversion of sugar into its various fermentative byproducts have been studied and optimised extensively to meet the specific demands of industries. Depending on the raw material used as starting point, the major beneficiaries of the useful characteristics have been alcoholic beverage producers (wine, beer, and whiskey amongst others), bakers (bread leavening) and biofuel producers. One of the obstacles in fermentation optimisation is the sugar consumption preferences displayed by the organism used. S. cerevisiae can consume a wide variety of sugars. Depending on the complexities of its structures, it shows a preference for the simpler saccharides. The fermentation of certain more complex sugars is delayed and runs the risk of being left residually after fermentation. Many of the crops utilised in fermentation-based products contain large amounts of starch. During the starch degradation process many different forms of sugars are made available for fermentation. Improved fermentation of starch and its dextrin products would benefit the brewing, whiskey, and biofuel industries. Most strains of Saccharomyces ferment glucose and maltose, and partially ferment maltotriose, but are unable to utilise the larger dextrin products of starch. This utilisation pattern is partly attributed to the ability of yeast cells to transport the aforementioned mono-, di- and trisaccharides into the cytosol. The inefficiency of maltotriose transport has been identified as the main cause for residual maltotriose. The maltotriose transporting efficiency also varies between different Saccharomyces strains. By advancing the understanding of maltotriose transport in yeast, efforts can be made to minimise incomplete fermentation. This aim can be reached by investigating the existing transporters in the yeast cell membrane that show affinity for maltotriose. This study focuses on optimising maltotriose transport through the comparison of the alpha glucoside transporter obtained from different strains of Saccharomyces. Through specific genetic manipulations the areas important for maltotriose transport could be identified and characterised. This study offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer, whiskey, and biofuel industries. / AFRIKAANSE OPSOMMING: Die transformasie van suiker na etanol en koolstof dioksied is so oud soos die beskawing self, en dit is van die vroegste tye af onlosmaaklik met die mens se kultuur verbind. Hierdie fermentasie-proses word gedomineer deur die Saccharomyces cerevisiae mikroorganisme. Hierdie organisme het antieke seevaarders voorsien van ‘n nie-bederfbare drankie en van ouds af aan bakkers ‘n rysmiddel verskaf waarmee meel in brood verander kon word. As gevolg van hierdie fermenteringspotensiaal het hierdie organisme ‘n onmisbare rol in meeste beskawings gespeel. Baie industrieë is steeds op hierdie basiese beginsel gebou. Die kompleksiteite en effektiwiteit van die transformasie van suiker na sy verskeie gefermeenteerde neweprodukte is breedvoerig bestudeer en geoptimiseer om aan die spesifieke behoeftes van verskeie industrieë te voeldoen. Afhangend van die grondstowwe wat as beginpunt gebruik is, is die primêre begunstigdes van die fermentasie proses die alkoholiese drankprodusente (onder andere die wyn-, bier- en whiskey produsente), bakkers en biobrandstofprodusente. Die suikerverbruik-voorkeur van die organisme wat die fermentering fasiliteer is een van die struikelblokke in die optimisering van die proses. S. cerevisiae kan ‘n wye spektrum van suikers verbruik maar dit toon ‘n voorkeur vir die eenvoudiger suikers. Die fermentasie van sekere van die meer komplekse suikers is vertraag en loop die risiko om agtergelaat te word na fermentasie. Vele van die gewasse wat in die gefermenteerde produkte gebruik word bevat groot hoeveelhede stysel. Vele soorte suikers word gedurende die afbreek van die stysel beskikbaar gestel vir fermentasie. Die brouers-, whiskey- en biobrandstof industrieë sal almal voordeel trek uit die verbeterde fermentasie van stysel en sy gepaardgaande dekstrin produkte. Meeste Saccharomyces gisrasse fermenteer glucose en maltose; maltotriose word gedeeltelik gefermenteer, maar die meer komplekse dekstrien produkte gevind in stysel word nie gefermenteer nie. Hierdie verbruikerspatroon kan gedeeltelik toegeskryf word aan die vermoë van gisselle om die bogenoemde mono-, di- and trisaccharides in die sitosol op te neem. Die oneffektiwiteit van maltotriose transport is identifiseer as die hoofoorsaak van post-fermentatiewe, oortollige maltotriose. Die effektiwiteit van maltotriose transport verskil ook tussen verskillende Saccharomyces rasse. Pogings om onvolledige fermentasie te veminder kan bevorder word deur die kennis rondom maltotriose transport in gis uit te bou. Hierdie oogmerk kan bereik word deur die bestaande transporters in die gissel se membraan wat ‘n affiniteit vir maltotriose toon te ondersoek. Hierdie studie fokus op die optimisering van maltotriose transport deur die vergelyking van die alpha glucoside transporter (AGT1) wat van verskillende Saccharomyces rasse afkomstig is. Die areas wat relevant is tot maltotriose transport kon deur spesifieke genetiese manipulasies identifiseer en gekarakteriseer word. Hierdie studie bevorder die vooruitsig op die ontwikkeling van gisrasse met verbeterde maltose en maltotriose transport vermoëns wat op sy beurt weer kan aanleiding gee tot die verbeterde fermentasie effektiwiteit in die bier, whiskey en biobrandstof industrieë.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Wine protein haze formation is a major challenge for wine makers, and several wine clarifying agents such as bentonite are used in the industry to protect wine from this occurrence. However, clarifying agents may have an undesirable impact on wine quality. Yeast mannoproteins have been shown to possess haze-protective properties, while also positively impacting on the sensorial properties of the product. However, while such mannoproteins are released into the wine during the wine making process, the amounts are low and therefore of limited oenological significance. However, and although commercial wine yeast strains display significant genotypic and phenotypic diversity, no broader assessment of haze protective activity and of mannoproteins release by different wine yeast strains has been undertaken. In this study, several yeast strains were screened for their impact on wine haze formation in Chardonnay must and in a grape juice model system. The data show that strains of the species Saccharomyces paradoxus possess better haze protective properties than the common Saccharomyces cerevisiae wine yeast strains. Differences in the nature of the proteins released by these two species were investigated, and indicated that several mannoproteins were released at significantly higher levels by S. paradoxus, and that some of these proteins might indeed contribute to the haze-protective activity. A further exploration of yeast cell wall properties indicated that the cell walls of haze-protective S. paradoxus strains contained higher levels of chitin than non-haze protective strains. Grape chitinases are likely to be primarily responsible for wine haze formation, and the data clearly demonstrate that these enzymes are able to bind to the yeast cell walls, and that strains with higher amounts of chitin in the cell wall will bind more chitinases. This finding suggests that the haze-protective nature of the strains is at least in part linked to the chitin levels of the strains. Furthermore, the impact of some genetic modifications in two wine strains (namely S. cerevisiae VIN13 and S. paradoxus RO88) suggests that several proteins contribute to wine haze protection. However, none of the mannoprotein-encoding flocculation genes, FLO1, FLO5, and FLO11 showed any impact on this property. Further studies are required to assess the full impact of the S. paradoxus strains on haze protection. In particular, the possible use of such strains as starter cultures or the use of S. paradoxus yeast hulls as clarifying agent needs to be further explored. / AFRIKAANSE OPSOMMING: Wyn proteïen-waas vorming is 'n groot uitdaging vir wynmakers en verskeie wyn verhelderings agente soos bentoniet word in die wynbedryf gebruik om wyn te beskerm teen die vorming van waas. Hierdie verheldering agente het egter 'n ongewenste impak op wynkwaliteit. Gis mannoproteïene is uitgewys as proteïene met moontlike waas-beskermende eienskappe wat ook 'n positiewe uitwerking op die sensoriese eienskappe van die produk het. Al word hierdie mannoproteïene egter vrygestel in die wyn tydens die wynmaak proses, is die hoeveelhede oor die algemeen te laag om van wynkundige belang te wees. Verder, ten spyte van die beduidende genotipiese en fenotipiese diversiteit van kommersiële wyngisrasse is daar nog geen breër assessering van die waas beskermende aktiwiteit van mannoproteïene, vrygestel deur verskillende rasse, tot dusver onderneem nie. In hierdie studie is verskeie gisrasse gekeur vir hul impak op wyn waas-vorming in Chardonnay mos en ook in 'n model druiwesap. Die data wys dat rasse van die spesie Saccharomyces paradoxus besit beter waas beskermende eienskappe as die algemene Saccharomyces cerevisiae wyngisrasse. Verskille in die aard van die proteïene wat vrygestel is deur hierdie twee spesies is ondersoek, en dit is aangedui aangedui dat verskeie mannoproteins vrygestel aan aansienlik hoër vlakke deur S. Paradoxus. Dit is ook aangedui dat sommige van hierdie proteïene wel bydra tot die waas-beskermende aktiwiteit. 'n Verdere verkenning van gis selwand eienskappe het aangedui dat die selwande van waas-beskermende rasse van S. paradoxus hoër vlakke chitien as nie-waas beskermende stamme bevat. Druiwe chitinases is waarskynlik hoofsaaklik verantwoordelik vir wyn waas vorming, en die data toon duidelik dat hierdie ensieme in staat is om te bind aan die gis selwande, en dat die stamme met hoër vlakke chitien in die selwand meer chitinases sal bind. Hierdie bevinding dui daarop dat die waas-beskermende aard van die stamme ten minste gedeeltelik gekoppel is aan die chitien vlakke van die stamme. Die impak van sekere genetiese modifikasies in twee verskillende gisrasse, naamlik die S. cerevisiae ras VIN13 en die S. paradoxus ras RO88, dui verder daarop dat verskeie proteïene dra by tot die beskerming teen wyn waas. Geeneen van die mannoprotein-koderende flokkulasie gene, FLO1, FLO5 en FLO11 het egter 'n impak op hierdie eienskap nie. Verdere studies is nodig om die volle impak van die S. paradoxus rasse op waas beskerming te assesseer. In die besonder, die moontlike gebruik van sulke rasse as 'n inkolasie kultuur of die gebruik van S. paradoxus gis doppe as verheldering agent moet verder ondersoek word.

The aims of the project were twofold. The initial objective of the study, based on previous results, was to develop an economically viable methodology for immobilizing yeast cells for the treatment of heavy metal-laden waste water. The non-viable yeast cross-linked by 13% (w/v) formaldehyde/1N HNO₃ exhibited satisfactory mechanical strength and rigidity in a continuous-flow column operation. No apparent disruption of the biomass after repeated use was observed. The cost of immobilizing 1kg dry yeast pellets was estimated at less than US$I. Zn uptake capacity of FA-cross-linked pellets, on batch trials, remained similar to that of raw yeast, reflecting that the immobilizing procedure did not hinder its metal removing capacity. In column studies, cation metals were effectively removed by the yeast pellets from aqueous solution at natural pHs, and then recovered completely by washing the pellets in situ with O.1M HCl. The recovered metals were concentrated in such small volumes that recycling or precipitation of them was facilitated. The metal uptake capacity of the regenerated biomass remained constant in comparison with cycle 1, indicating that reuse of the yeast would be possible. In the case of Cr⁶⁺, a gradual breakthrough curve of Cr in the column profile was noted, with a simultaneous reduction of Cr⁶⁺ to Cr³⁺. However, Cr⁶⁺ in the effluent can be markedly minimised either by accumulation onto the biomass or reduction to its trivalent form. Desorption of bound Cr⁶⁺ with either alkali or salt could not accomplish the regeneration of the biomass. A combination of reduction and desorption with FA/HNO₃ appeared promising in regeneration of the saturated biomass at 4°C. The metal sorption capacities of the yeast pellets, on a batch or a fixed-bed system are relatively lower than that of documented sorbents. Apparently more of the yeast pellets would be required for treating a certain volume of waste effluent, than with other sorbents. Therefore Azolla filiculoides was examined as a suitable sorbent for this purpose. This constitutes the second part of the project. Azolla filiculoides, a naturally-abundant water fern, was screened for its metal sorption and recovering capacities, mechanical stability, flow-permeability and reusability. The azolla biomass appeared to have fulfilled the required mechanical criteria during the repeated sorption-desorption column operations. It is water-insoluble and appears flexible under pressure when rinsed with water. These characters are of crucial importance in a continuous-flow system since a column can be operated at high flow rates without apparent compact of the biomass and pressure loss. Therefore, immobilization of the biomass can be avoided. The sorption isotherm data, obtained from batch removal of Cr⁶⁺, showed that the sorption process was effective, endothermic and highly pH dependent. Considerable amounts of Cr⁶⁺ were accumulated at the optimum pHs of 2-2.5. Column sorption of Cr⁶⁺ at a low flow rate and pH of 2.5 showed optimum performance with a total Cr uptake of 50.4mg/g at 60% saturation of the biomass. Removal of Cr⁶⁺ from an electroplating effluent using an azolla column was deemed reasonably satisfactory, although the uptake declined slightly. Desorption of bound Cr⁶⁺ with various desorbents was incomplete, which resulted in a low regeneration efficiency of about 50%. However, removal and recovery of Cr³⁺ using the azolla column was than that of Cr⁶⁺. Desorption of Cr³⁺ from the spent biomass column was accomplished with the recovery of 80% using O.5N H₂SO₄, The regeneration efficiencies for Cr³⁺ removal were up to 90% and demonstrated that the biomass is reusable. Cation metal uptake capacities of azolla, obtained either from batch or column experiments, are reasonably high in comparison with other sorbents. The uptake of Ni or Zn ions from solution is pH dependent showing the optimum pH of around 6 to 6.5, under the current experimental conditions. The sorption kinetics for cation metals was rapid with about 80% of the bound Ni ions being taken up in the first 10 min. The character of rapid binding is extremely important in a column sorption process, especially on a large scale since it favours an optimum uptake of metals at high flow rates. The Ni or Zn uptakes in column sorption were not markedly affected when the flow rates were increased from 80mllh up to 800ml/h for the 5g biomass used. The cation heavy metals removed from waste effluents were recovered in a concentrated solution of small volume. The desorption of bound Ni and Zn ions from the saturated biomass was accomplished with either O.2N HCl or H₂SO₄ that resulted in recoveries of more than 95%. The metals recovered, in the case of Ni and Zn, are identical to that of plating agents ego nickel sulphate or chloride, so that recycling of the metals is possible. An effluent-free, closed loop of Ni or Zn treatment system was proposed, whereby the Ni or Zn ions can be recycled to the plating bath whilst the purified water is fed back to the rinse tanks. Ca and Mg ions, commonly present in the electroplating effluents, appeared to affect sorption of heavy metals by azolla when metal concentrations were relatively low, presumedly through its competitive binding for the shared sites on surfaces of azolla. The data obtained from column sorption of Ni and Zn follows the BDST model well, enabling the application of the model to predicting design parameters for scale-up of the biosorption column system. It is interesting that the values of metal uptake, expressed in molar quantities, obtained on respective single-metal solutions and the multiple metal system, are similar, implying that the mechanisms involved in the sorption of all metal cations are similar and that the binding sites on surfaces of azolla are probably shared by all cation metals. The surface of the biomass provides sites for metal binding estimated in the range of 0.45-0.57mmol/g, based on the current experiments. The biomass has a surface area of 429 m²/g and water retention of 14.3 ml/g. The functional groups on the surface of azolla were partially identified using chemical modification and metal binding comparison. Among the functional groups examined, carboxyl groups, provided by amino acids and polysaccharides, appeared to play an important role in metal cation binding. The infrared spectra of the samples support this conclusion.

Indiana University-Purdue University Indianapolis (IUPUI) / Growth and division are tightly coordinated with available nutrient conditions. Cells of the budding yeast, Saccharomyces cerevisiae, grow to a larger size prior to budding and DNA replication when preferred carbon sources such as glucose, as opposed to less preferred sources like ethanol and acetate, are available. A culture’s doubling time is also significantly reduced when the available carbon and nitrogen sources are more favorable. These physiological phenomena are well documented but the precise molecular mechanisms relaying nutrient conditions to the growth and division machinery are not well defined. I demonstrate here that Cdc34, the ubiquitin conjugating enzyme that promotes S phase entry, is phosphorylated upon a highly conserved serine residue which is part of a motif that defines the family of Cdc34/Ubc7 ubiquitin conjugating enzymes. This phosphorylation is regulated by multiple, nutrient sensing kinases including Protein Kinase A, Sch9 and TOR. Furthermore, this phosphorylation event is regulated through the cell cycle with the sole induction occurring in the G1 phase which is when nutrients are sensed and cells commit to another round of division. This phosphorylation likely activates Cdc34 and in turn propagates a signal to the cell division cycle machinery that nutrient conditions are favorable for commitment to a new round of division. This phosphorylation is critical for normal cell cycle progression but must be carefully controlled when cells are deprived of nutrients. Crippling the activity of Protein Kinase A, SCH9 or TOR increases the proportion of cells that survive stationary phase conditions, which because of the metabolic conditions that must be maintained and the similarity to post-mitotic mammalian cells, is referred to as a yeast culture’s chronological lifespan. Yeast cells expressing Cdc34 mutants that are no longer subject to this regulation by phosphorylation have a reduced chronological lifespan. A precise molecular mechanism describing the change in Cdc34 activity after phosphorylation of this serine residue is discussed.

Thesis (M. Tech. Environmental health) -- Central University of technology, Free State, 2011 / In nature microorganisms do not exist alone, but in association with one another. These kinds of associations can also be found in food industries, where cells of the same or different species can attach to pipes (biofilm formation) and a variety of surfaces in food processing environments and in food product such as yoghurt which can contain both yeast and bacteria originating from the starter culture as well as fruit. To control food spoilage organisms and food-borne pathogens preventative measures such as good manufacturing processes, the use of sanitizers and preservatives as well as hazard analysis critical control points (HACCP) are crucial in food industries. Sanitation of the working surface, floors, pipes, containers and equipment is a stepwise application of a detergent, acid or alkali rinse, a disinfectant treatment followed by final rinsing. If rinsing of the sanitizer is not done properly it may end up in the product in sub-lethal doses. In this study the influence of Liquid Hypochlorite (LH) and Liquid Iodophore (LI) sanitizers on organism growth and toxicity was evaluated. The organisms investigated included Escherichia coli 0113, Escherichia coli 026 and Zygosaccharomyces bailii Y-1535 in yeast malt broth, which was supplemented with LH and LI at sub-lethal concentrations 0.05% LH, 0.2% LH and 0.075% LI. Subsequently, bacterial and yeast growth responses as pure cultures and in combination (E. coli + Z. bailii) were measured as colony forming units and optical density values. Incorporation of the sanitizers in the growth media resulted in different levels of growth inhibition. Z. bailii proved more robust and the growth rate was not influence significantly by the addition of sanitizers or communal growth with either E. coli strains. The growth rate of both E. coli strains decreased where grown in combination with Z. bailii as well as in the presence of sanitizers, with the most influence exerted by LH. Changes in endotoxicity following the growth of the test samples (stressed cells) and the control (unstressed) were measured by the limulus amoebocyte lysate (LAL) and porcine IL-6 ELISA methods. Where E. coli strains were cultured together with Z. bailii the toxicity of tire mixture showed a decrease over time when measured with the limulus amoebocyte assay method. Interestingly the communal growth of the E. coli strains and Z bailii produced different toxicity profiles when the IL-6 porcine method was used, hi both cases, where E. coli strains were cultured together with Z. bailii the toxicity of the mixture showed an increase over tune when measured by this assay. Other than a similar toxicity profile for E. coli 0113 grown in pure culture, the comparison between results obtained using the LAL or porcine IL-6 methods yielded no correlation in determined toxicity. It was established that LH and LI sanitizers as well as communal growth had an influence in the toxicity of LPS/EPS and the method used to determine such toxicity should be carefully considered.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Numerous workers studied the impact of pollutants and agricultural chemicals, containing heavy metals such as copper (Cu), on soil microbes. It was found that elevated soil Cu levels do have a detrimental effect on soil bacterial populations however the filamentous fungi seemed to be less affected. Most of these studies were conducted in soils containing already relatively high Cu levels and the effect of this heavy metal on the nonfilamentous fungi (i.e. yeasts) was never investigated. The aim of this study was therefore to determine the impact of elevated Cu levels on filamentous fungi and yeasts occurring in soils containing relatively low natural Cu levels. A synthetic selective medium containing glucose as carbon source, thymine as nitrogen source, vitamins, minerals and chloramphenicol as anti-bacterial agent (TMV-agar), was used to enumerate ascomycetous and basidiomycetous Cu resistant yeasts in a sample of virgin soil containing ~ 2ppm Cu. Media that were used to enumerate Cu resistant filamentous fungi were malt extract agar, malt extract agar with streptomycin sulfate, maltyeast- extract-peptone agar with chloramphenicol and streptomycin sulfate, benomyl–dichloran-streptomycin medium for the enumeration of hymenomycetous fungi and two selective media for the isolation of mucoralean fungi. Cu resistant fungi able to grow on all of the above mentioned solid media supplemented with 32 ppm Cu occurred in the soil sample. To obtain an indication of the level of Cu tolerance of fungi present in this soil sample, a number of fungal isolates were screened for the ability to grow on a series of agar plates, prepared from glucose-glutamate-yeast extract agar, containing increasing concentrations of Cu. It was found that filamentous fungi and yeasts that were able to grow on this agar medium containing up to 100 ppm Cu were present in the soil. A series of soil microcosms was subsequently prepared from the soil sample by experimentally contaminating the soil with increasing amounts of copper oxychloride, were after fungal populations in the microcosms, including Cu resistant fungi, were monitored using plate counts. At the end of the incubation period, after 245 days, fungal biomass in the microcosms was compared by determining the concentrations of the fungal sterol, ergosterol, inthe soil. Generally, Cu had little impact on the numbers of filamentous fungal colony forming units on the plates, as well as on the ergosterol content of the soil. The numbers of filamentous fungi in the soil, including the Mucorales and hymenomycetes, seemed to be less affected by the addition of copper oxychloride than the numbers of soil yeasts able to grow on TMV-agar. The focus of the next chapter was on the response of yeasts in different soils to elevated levels of Cu in the soil. TMV-agar was used to enumerate yeasts in soil microcosms prepared from four different soil samples, which were experimentally treated with copper oxychloride resulting in Cu concentrations of up to 1000 ppm. The selective medium supplemented with 32 ppm Cu was used to enumerate Cu resistant yeasts in the microcosms. The results showed that the addition of Cu at concentrations ≥ ~1000 ppm did not have a significant effect on total yeast numbers in the soil. Furthermore, it was found that Cu resistant yeasts were present in all the soil samples regardless of the amount of Cu that the soil was challenged with. At the end of the incubation period, yeasts in the microcosms with zero and ~1000 ppm additional Cu were enumerated, isolated and identified using sequence analyses of the D1/D2 600-650bp region of the large subunit of ribosomal DNA. Hymenomycetous species dominated in the control soil, while higher numbers of the urediniomycetous species were found in the soil that received Cu. These observations suggest that urediniomycetous yeasts may play an important role in re-establishing overall microbial activity in soils following perturbations such as the addition of Cu-based fungicides. / AFRIKAANSE OPSOMMING: Vele navorsers het al die impak van besoedelingstowwe en landbouchemikalieë wat swaarmetale soos koper (Cu) bevat, op grond-mikrobes bestudeer. Dit is gevind dat verhoogde Cu vlakke ‘n nadelige effek het op grond-bakteriese populasies, maar dat die filamentagtige fungi geneig is om minder geaffekteer te word. Meeste van hierdie studies is gedoen met gronde wat alreeds relatief hoë Cu vlakke bevat het en die effek van hierdie swaarmetaal op die nie-filamentagtige fungi (d.i. giste) is nooit ondersoek nie. Die doel van hierdie studie was dus om die impak van verhoogde Cu vlakke op filamentagtige fungi en giste in gronde, wat natuurlike lae vlakke van Cu bevat, te bepaal. ‘n Sintetiese selektiewe medium wat glukose as koolstofbron, timien as stikstofbron, vitamiene, minerale asook chloramfenikol as anti-bakteriese agent bevat (TMV-agar), is gebruik om askomisete en basidiomisete Cu weerstandbiedende giste in ‘n monster ongeskonde grond, bevattende ~ 2dpm Cu, te tel. Media wat gebruik is om Cu weerstandbiedende filamentagtige fungi te tel, was mout-ekstrak agar, moutekstrak agar met streptomisiensulfaat, benomiel-dichloran-streptomisien medium vir die tel van hiemenomiseetagtige fungi en twee media vir die isolasie van mukoraliese fungi. Cu-weerstandbiedende fungi wat op al die bogenoemde media, aangevul met 32 dpm Cu, kon groei, het in die grondmonster voorgekom. Om die mate van Cu-weerstandbiedendheid van fungi wat in die grondmonster voorkom, te bepaal, is ‘n getal fungus-isolate op agarplate, voorberei met glukose-glutamaat-gis ekstrak agar, bevattende verhoogde konsentrasies Cu, nagegaan. Daar is gevind dat daar filamentagtige fungi en giste in die grond voorkom wat die vermoë het om op media bevattende 100 dpm Cu te groei. ‘n Reeks grond mikrokosmosse is dus voorberei vanaf die grondmonster deur om dit eksperimenteel te kontamineer met verhoogde hoeveelhede koper oksichloried, waarna die fungus-populasies asook die Cu-weerstandbiedende fungi in die mikrokosmos gemoniteer is deur middel van plaattellings. Aan die einde van die inkubasie periode, 245 dae, is die fungus biomassa in al die mikrokosmosse bereken deur die konsentrasie van die fungus sterool ergosterool te bepaal en dit met mekaar te vergelyk. Oor die algemeen het Cu min impak ten opsigte van diegetal filamentagtige fungi kolonie vormende eenhede die plate, asook op die ergosterool inhoud van die grond gehad. Dit wil voorkom of die getal filamentagtige fungi in die grond, insluitende die Mucorales en die hymenomisete, minder geaffekteer is deur die toediening van koperoksichloried as die aantal grondgiste wat op die TMV-agar kan groei. Die fokus van die volgende hoofstuk was dus op die reaksie wat giste in verskillende grondtipes gehad het op verhoogde Cu in die grond. TMV-agar is gebruik om die getal giste te bepaal in die grond mikrokosmosse van die vier verskillende grondmonsters, wat voorberei is deur om dit eksperimenteel met koper oksikloried te kontamineer tot en met Cu konsentrasies van 1000 dpm. Die selektiewe medium wat gesupplementeer is met 32 dpm Cu, is gebruik om Cu weerstandbiedende giste in die mikrokosmosse te bepaal. Die resultate toon dat die toevoeging van Cu by konsentrasies ≥ ~1000 dpm nie enige beduidende effek op die totale gis getalle gehad het nie. Daar is ook gevind dat daar Cu weerstandbiedende giste in die grond monsters voorkom gekom het ten spyte van die hoeveelheid Cu wat tot die grond toegevoeg is. Aan die einde van die inkubasie periode is die giste wat die die mikrokosmosse bevattende nul en ~1000 dpm Cu getel, geïsoleer en geïdentifiseer deur gebruik te maak van DNA volgorde bepaling van die D1/D2 600-650 bp areas geleë in die groter subeenheid van die ribosonale DNA. Hymenomisete spesies het in die grond kontrole gedomineer, terwyl hoër getalle uredinomisete spesies in die grond met addisionele Cu gevind is. Die resultate dui daarop dat uredinomisete giste dalk ‘n belangrike rol kan speel in die hervestiging van die oorwegende mikrobiese aktiwiteit in grond na skoktoestande soos die aanwending van Cu-gebaseerde fungisiede.

Thesis (MScAgric)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defence reactions comprising both constitutive and inducible barriers. While disease is the exception, such exceptions can be costly and even devastating. In particular, fungal diseases remain one of the major factors limiting crop productivity worldwide, with huge losses that need to be weighed up against massive cash inputs for pesticide treatments. Part of the defence reactions of plants is the synthesis of pathogenesis-related proteins, such as the plant hydrolases, glucanases and chitinases. In recent years, attention has been paid to the implementation of these proteins in plant transformation schemes. The rationale for this approach was that these antimicrobial agents not only degrade the main cell wall components of fungi, but also produce glucosidic fragments that act as elicitors of the biosynthesis of defence metabolites by the host. Furthermore, since these active antimicrobial agents are individually encoded by single genes, these defence systems should and have been shown to be highly amenable to manipulation by gene transfer. In this study, yeast glucanases from Saccharomyces cerevisiae were evaluated for their potential as antifungal proteins. The glucanases tested for their antifungal activity against Botrytis cinerea were the yeast EXG1 and BGL2 genes, encoding an exoglucanase and an endoglucanase respectively. An in vitro assay performed on these glucanases indicated that exoglucanase had a more detrimental effect on B. cinerea hyphal development and growth than the endoglucanase; the former caused typical disruption of the cells and leakage of cell material. The yeast exoglucanase was subsequently subcloned into a plant expression cassette containing the strong constitutive 358 promoter, yielding plasm ids pEXG1 and pMJ-EXG1. The pMJ-EXG1 construct targeted the exoglucanase to the apoplastic region with a signal peptide from an antimicrobial peptide from Mirabilis jalapa, Mj-AMP2. The pEXG1 and pMJ-EXG1 constructs were mobilised into Agrobacterium tumefaciens to facilitate the subsequent tobacco transformation, which yielded transgenic tobacco lines designated E and MJE respectively. Transgene integration was confirmed with southern blot and PCR analyses for both the E and MJE lines. The expression and heterologous production of the EXG1-encoded exoglucanase in the E-transgenic lines was shown with northern blots and activity assays respectively. Moreover, the high level of expression of the yeast exoglucanase led to a decrease in susceptibility of the E lines to B. cinerea infection in comparison to the untransformed tobacco controls. An average decrease in disease susceptibility of 40% was observed in an in planta detached leaf assay. Crude protein extracts from the E lines were also analysed in an in vitro quantitive fungal growth assay, inhibiting in vitro fungal growth by average 20%, thus further confirming the antifungal nature of the yeast exoglucanase. Although integration of the MJ-EXG1 expression cassette was confirmed, no mRNA levels could be detected with northern blot or RT-PCR analysis of the MJE lines. These lines also did not show any in vitro antifungal activities or a decrease in susceptibility to B. cinerea infection in the detached leaf assay. It is suspected that this result is possibly linked to gene silencing, a phenomenon quite frequently associated with heterologous and/or overexpression of glucanases in plant hosts. It appears as if the targeted overexpression to the apoplastic space triggered the gene silencing response, since the intracellularly overexpressed product was produced and shown to display activity. The yeast exoglucanase thus joins the list of silenced glucanases in overexpression studies in plants. Overall, this study confirmed the antifungal characteristics of the Saccharomyces exoglucanase and provides valuable information of the possibility of utilising yeast glucanases in a transgenic environment. A decrease in the susceptibility of tobacco to B. cinerea infection, as shown by the overexpressed EXG1-encoded exoglucanases, merits further investigation into the use of this gene in the engineering of disease-resistant crops. / AFRIKAANSE OPSOMMING: Die weerstand van plante teen infeksie deur fitopatogeniese mikroórganismes is die resultaat van verskeie meervoudige verdedigingsreaksies wat beide konstitutiewe en induseerbare versperrings behels. Terwyl siekte die uitsondering eerder as die reël is, kan sulke uitsonderinge duur en selfs verwoestend wees. In die besonder is swamsiektes een van die vernaamste faktore wat gewasproduksie wêreldwyd beperk, met enorme verliese wat teen kontantinsette vir plaagdoders opgeweeg moet word. Deel van die verdedigingsreaksie van plante is die sintese van patogeen-verwante proteïene, soos die planthidrolases, -glukanases en -chitinases. In die onlangse tyd is aandag geskenk aan die implementering van hierdie proteïene in plant transformasieskemas. Die grondrede hiervoor was dat hierdie antimikrobiese agente nie net die hoof selwandkomponente van swamme kan afbreek nie, maar ook glukosidiese fragmente produseer wat as ontlokkers van metabolietbiosintese vir die verdediging van die gasheer kan optree. Aangesien hierdie aktiewe antimikrobiese agente individueel deur enkele gene enkodeer word, blyk hierdie verdedigingsisteme om hoogs ontvanklik vir manipulasie deur geenoordrag te wees. In hierdie studie is die gisglukanase van Saccharomyces cerevisiae vir hul potensiaal as antifungiese proteïene geëvalueer. Die glukanases wat vir hul antifungiese aktiwiteit teen Botrytis cinerea getoets is, was die gis EXG1- en -BGL2-gene, wat onderskeidelik vir "n eksoglukanase en 'n endoglukanase enkodeer. "n In vitro toets wat op hierdie glukanases uitgevoer is, het aangedui dat die eksoglukanase 'n meer skadelike effek op die hife-groei en -ontwikkeling van B. cinerea as die endoglukanase gehad het; eersgenoemde het die tipiese ontwrigting van die selle en die uitlek van selmateriaal tot gevolg gehad. Die gis-eksoglukanase is gevolglik in 'n plant uitdrukkingskasset wat die sterk konstitutiewe 35S promotor bevat, gesubkloneer, wat plamiede pEXG1 en pMJ-EXG1 opgelewer het. Die pMJ-EXG1-konstruk het die eksoglukanase na die apoplastiese gebied geteiken deur 'n seinpeptied vanaf "n antimikrobiese peptied van Mirabilisjalaba, Mj-AMP2. Die pEXG1- en pMJ-EXG1-konstrukte is in Agrobacterium tumefaciens gemobiliseer, wat die gevolglike tabaktransformasies gefasiliteer het wat die E en MJE transgeniese tabaklyne onderskeikelik gelewer het. Transgeen-integrasie is deur suidelike klad- en PKR-analises vir beide die E en MJE lyne bevestig. Die uitdrukking en heteroloë produksie van die EXG1-enkodeerde eksoglukanase is in die transgeniese E lyne deur noordelike klad en aktiwiteitstoetse onderskeidelik aangetoon. Verder het die hoë uitdrukkingsvlak van die gis-eksoglukanase tot 'n vermindering in die vatbaarheid van die E lyne vir B. cinerea-infeksie relatief tot die ongetransformeerde tabakkontroles gelei. 'n Gemiddelde vermindering in siektevatbaarheid van 40% is in 'n in planta verwyderde-blaartoets waargeneem. Ru proteïen-ekstrakte van die E lyne is ook in 'n in vitro kwantitatiewe swamgroeitoets geanaliseer en het in vitro swamgroei met tot gemiddeld 20% geïnhibeer, wat dus verder die antifungiese aard van die gis-eksoglukanase bevestig het. Alhoewel die integrasie van die pMJ-EXG1 uitdrukkingskasset bevestig is, kon geen mRNA-vlakke met die noordelike klad- of RT-peR-analises van die MJE-Iyne waargeneem word nie. Hierdie lyne het ook geen in vitro antifungiese aktiwiteite of 'n vermindering in die vatbaarheid vir B. cinerea-infeksie getoon nie, soos in die verwyderde-blaartoets uitgevoer is nie. Dit word vermoed dat hierdie resultaat moontlik aan geenstilmaking gekoppel is, 'n verskynsel wat gereeld met heteroloë- en/of ooruitdrukking van glukanases in plantgashere gekoppel word. Dit blyk dat die ooruitdrukking wat tot die apoplastiese ruimte geteiken is, tot die geenstilmaking-respons aanleiding gegee het, aangesien die intrasellulêre ooruitgedrukte produk gemaak is en aktiwiteit getoon het. Die gis-eksoglukanase word dus deel van die lys van stilgemaakte glukanases in die ooruitdrukkingstudies van plante. In die algemeen het hierdie studie dus die antifungiese kenmerke van die Saccharomyces eksoglukanase bevestig en waardevolle inligting oor die moontlike gebruik van gis-glukanases in 'n transgeniese omgewing verskaf. 'n Afname in die vatbaarheid van tabak vir infeksie deur B. cinerea, soos deur die ooruitdrukking van EXG1-enkodeerde eksoglukanase getoon is, verdien dus verdere ondersoek van die gebruik van hierdie geen in die skepping van siekteweerstandbiedende gewasse.