Thesis (MSc (Microbiology))--University of Stellenbosch, 2009. / Biomining organisms are generally found in metal-rich, inorganic environments such as iron and sulfur containing ores; where they play a vital role in mineralization and decomposition of minerals. They are typically obligatory acidophilic, mesophilic or thermophilic, autotrophic, usually aerobic, iron-or sulfur oxidizing chemolithotrophic bacteria. The most prominent biomining organisms used in bioleaching of metal sulfides are Acidithiobacillus ferrooxidans, At. thiooxidans, At. caldus, Sulfobacillus spp. and Leptospirillum spp. Biomining enables us to utilize low grade ores that would not have been utilized by conventional methods of mining. Research has focused on the backbone features of plasmids isolated from bacteria of biomining environments. The aim of this study is to sequence and analyze an 18 kb region of the 66 kb plasmid pTcM1 isolated from At. caldus MNG, focusing on accessory genes carried by this plasmid. Fifteen putative genes / open reading frames were identified with functions relating to metabolism and transport systems. The genes are located in two divergently located operons. The first operon carries features related to general metabolism activities and consists of a transcriptional regulator (ORF 2), a succinate / fumarate dehydrogenase-like subunit (ORF 3), two ferredoxin genes (ORF 4 and ORF 7), a putative HEAT-like repeat (ORF 6) which is interrupted by an insertion sequence (ORF 5) and a GOGAT-like subunit (ORF 8). The second operon contains an ABC-type nitrate / sulfonate bicarbonate-like gene (ORF 9), a binding protein-dependent inner membrane component-like gene, another ABC sulfonate / nitrate-like gene (ORF 12i and 12ii) which is interrupted by an insertion sequence (ORF 13) and two hypothetical proteins with unknown functions (ORF 14 and ORF 15). Southern hybridization analysis have shown that most of the genes from the two operons are found in other At caldus strains #6, “f”, C-SH12 and BC13 from different geographical locations. Expression of the GOGAT-like subunit and the succinate / fumarate-like subunit was demonstrated in At. caldus MNG showing that these genes are functional and actively transcribed. The transcriptional regulator (ORF 2) has been shown to repress the downstream genes of putative operon 1. The persistence of these genes on plasmids together with the fact that they are being expressed, represents a potential metabolic burden, which begs the question why they have been maintained on the plasmid from geographically separated strains (and perhaps also growing under very different nutrient availability conditions) and therefore what possible role they may play.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: It is known that many hyphomycetous fungi are dispersed by wind, water and insects. However, very little is known about how these fungi may differ from each other regarding their ability to be disseminated by different environmental vectors. Consequently, to obtain an indication of the primary means of spore dispersal employed by representatives of the genera Acremonium, Aspergillus and Penicillium, isolated from soil and indoor environments, we monitored spore liberation of cultures representing these genera in an airflow cell. The experimental data obtained, of plate counts conducted of the air at the outlet of the airflow cell, were subjected to an appropriate analysis of variance (ANOVA), using SAS statistical software. Intraspecific differences occurred regarding aerial spore release. Under humid conditions, however, Penicillium species were more successful in releasing their spores than Aspergillus and the Acremonium strain. Under desiccated conditions the Aspergillus took longer to release their spores than representatives of Acremonium and Penicillium. The taxa that were investigated did not differ from each other regarding the release of spores in physiological salt solution (PSS). Although not proven, indications are that water may act as an important dispersion agent for these fungi, because washing of cultures with PSS resulted in all cases in an immediate massive release of colony forming units. Subsequently, using standard plate count techniques, conidial adhesion of the fungi mentioned above to synthetic membranes, leaf cuttings and insect exoskeletons differing in hydrophobicity and electrostatic charge were investigated. We found that the different genera showed different adhesion profiles for the series of test surfaces, indicating differences in physico-chemical characteristics of the fungal spore surfaces. In general, the Penicillium strains showed a greater ability to adhere to the test surfaces, than the aspergilli, while the representative of Acremonium showed the least adherence. No significant difference in the percentage spore adhesion was found between hydrophobic and hydrophilic materials. Furthermore, evidence was uncovered supporting the contention that, under dry conditions, electrostatic surface charges play a role in the adherence of fungal spores to surfaces, because adherence was positively correlated (Correlation coefficient = 0.70898, p = 0.001) to positive electrostatic charges on the lamellar surfaces. In the next part of the study, standard plate count methods were used to determine the relative adhesion of the above mentioned hyphomycetous fungi, as well as a polyphyletic group of yeasts, to the test surfaces submerged in 10 mM sodium phosphate buffer (pH 7.0). As was found with the experiments with the dry surfaces, both intraspecific and intergenus differences were uncovered. Overall, the fungi adhered better to hydrophilic surfaces than to hydrophobic surfaces. This indicated that the fungal surfaces were covered with relatively hydrophilic compounds such as carbohydrates. Subsequently, it was demonstrated that all the fungi adhered to plasma membrane glycoprotein coated polystyrene and the presence of fungal carbohydrates on the surfaces of the fungal propagules was confirmed using epi-fluorescence microscopy. Differences in the strategy of the fungal genera to release their airborne spores, as well as differences in their adhesion profiles for the series of test materials, may be indicative of a unique environmental niche for each genus. In future, this phenomenon should be investigated further. / AFRIKAANSE OPSOMMING: Hifomisete fungi is daarvoor bekend om te versprei deur middel van wind, water, en insek vektore. Maar nietemin, daar is bykans geen kennis m.b.t. hoe hierdie fungi van mekaar verskil t.o.v. hul vermoë om versprei te word deur omgewings vektore nie. Gevolglik was spoorvrystelling van kulture, verteenwoordigend van die genera Acremonium, Aspergillus en Penicillium gemoniteer om ‘n aanduiding te kry van primêre wyse van spoorverspreiding waardeur verteenwoordigers van die onderskeie genera ingespan word. Eksperimentele data ingewin, vanaf plaat tellings wat uitgevoer was op lug afkomstig vanuit die uitlaat-klep van die lugvloei kapsule, was onderwerp aan ‘n toepaslike analise van afwyking (ANOVA), deur gebruik te maak van ‘n SAS statistiese pakket. Intraspesie verskille is waargeneem t.o.v. lug spoorvrystelling. Desnieteenstaande was Penicillium meer suksesvol onder vogtige kondisies t.o.v. spoorvrystelling in vergelyking met Aspergillus en die Acremonium stam. Onder droë kondisies het verteenwoordigers van Aspergillus langer geneem om hul spore vry te stel as verteenwoordigers van onderskeidelik, Penicillium en Acremonium. Geen verskille was waargeneem m.b.t. spoorvrystelling in fisiologiese soutoplossing (FSO) tussen die verskillende filogenetiese stamme nie. Alhoewel dit nie bewys is nie, wil dit voorkom asof water as belangrike verspreidingsagent van die betrokke fungi dien, aangesien die spoel van kulture met FSO tot ‘n oombliklike enorme vrystelling van kolonie-vormende eenhede gelei het. Gevolglik, deur gebruik te maak van standaard plaattellings tegnieke, was spoor aanhegting van bogenoemde fungi aan sintetiese membrane, blaar snitte en insek eksoskelette wat verskil in terme van hidrofobisiteit en elektriese lading, ondersoek. Daar was gevind dat die aanhegtingsprofiele m.b.t. hierdie reeks toetsoppervlaktes van die verskillende genera verskil, wat op sigself ‘n aanduiding was van verskille in fisieschemiese eienskappe van die swamspoor oppervlaktes. Penicillium stamme het ‘n hoër aanhegtings vermoë aan die toetsoppervlaktes getoon as die aspergilli, terwyl die verteenwoordiger van Acremonium die laagste aanhegting getoon het. Geen betekenisvolle verskille i.t.v. persentasie spoor aanhegting was gevind tussen hidrofobiese en hidrofiliese oppervlakte nie. Daarbenewens was die argument dat spoorvrystelling onder droë kondisies beïnvloed word deur elektrostatiese oppervlak ladings, bevestig deur ons bevindinge, want aanhegting het positief gekoreleer (Korrelasie koëffisient = 0.70898, p = 0.001) met positiewe ladings op die oppervlaktes. ‘n Standaard plaattellingstegniek was aangewend in die volgende fasset van die studie om die relatiewe aanhegting van bogenoemde hifomisete fungi, sowel as ‘n polifilitiese groep giste aan die toetsoppervlaktes, gedompel in 10 mM natrium fosfaat buffer (pH 7.0) vas te stel. Intraspesie en intragenus verskille was weereens waargeneem, net soos in die geval van die eksperimente met die droë oppervlakte. In die algemeen het die swamme baie beter geheg aan hidrofiliese oppervlaktes in vergelyking met hidrofobiese oppervlakte. Dit was ‘n aanduiding dat die swamspoor oppervlaktes bedek was met relatiewe hidrofiliese verbindings bv. koolhidrate. Verder was daar bewys dat alle swamme ingesluit in hierdie studie die vermoë het om plasmamembraan glikoproteïn bedekte polistireen te bind, en gevolglik was die teenwoordigheid van van koolhidrate op die swamspore bevestig m.b.v epi-fluoresensie mikroskopie. Verskille in die strategie van swamme om spore in die lug vry te stel, sowel as verskille in die aanhegtingsprofiele vir ‘n reeks toetsmateriale, mag net ‘n aanduiding wees van ‘n unieke omgewings nis vir elke genus wat in hierdie studie ondersoek is. Hierdie verskynsel moet dus in die nabye toekoms nagevors word.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: The euascomycetous fungus Aureobasidium pullulans produces xylanolytic accessory enzymes, including an α-L-arabinofuranosidase. The deduced amino acid sequence of the abfA gene encoding α-L-arabinofuranosidase was 69-76% identical to family 54 glycoside hydrolases. The abfA gene encoded a 498 amino acid polypeptide including a signal peptide consisting of 20 amino acids. The mature protein had a calculated molecular weight of 49.9 kDa. One putative N-glycosylation site was found and an iso-electric point of 4.97 was calculated. A. pullulans AbfA was also found to consist of an N-terminal catalytic domain (residues 1-317) and a C-terminal arabinose-binding domain (residues 318-442). The abfA gene from the colour-variant strain of A. pullulans, NRRL Y-2311-1, was recently transferred in Saccharomyces cerevisiae Y294. The yeast culture was grown on synthetic defined medium and α-L-arabinofuranosidase was expressed successfully, secreted from the cells and was purified from the supernatant in a single step using gel filtration. It had an apparent mobility of 52.7 kDa on SDS-PAGE and 36 kDa estimated by gel filtration. The heterologous enzyme was characterized according to pH and temperature dependence and stability, apparent mobility and kinetic properties. The temperature optimum of the recombinant α–L-arabinofuranosidase was 55 °C and it was stable over 3 h at 40 °C. The enzyme displayed optimum activity between pH 4 and 4.5 and was stable at pH 4 over 3 h. Kinetic analysis on p-nitrophenyl-α-arabinofuranoside yielded a Km of 1.43 mM and a Vmax of 23.7 U/mg. Product inhibition was observed and a Ki of 28 ± 3 mM was determined during assaying in the presence of arabinose. A specific activity of 3.85 ± 0.008 U/mg was determined on p-nitrophenyl-α-L-arabinofuranoside and no activity was found on chromogenic substrates which contained a β-linked arabinofuranosyl. The enzyme showed low activity against the 1,5-α-L-arabino-oligosaccharides and cleaved arabinose from corn fibre, oat spelt arabinoxylan and to a lesser degree wheat arabinoxylan. No release of arabinose was observed from larch wood arabinogalactan, α-1,5-debranched arabinan and lignin-arabinose substrates. Linkage preference showed less activity against α-1,5-linked than α-1,2 or α-1,3-linked arabinofuranosyl subunits. Synergism between α–L-arabinofuranosidase and endo-β-1,4-xylanase occurred when measuring the increase in arabinose during wheat arabinoxylan degradation. A. pullulans NRRL Y-2311-1 was grown on synthetic defined medium and native α-L-arabinofuranosidase was expressed and secreted into the culture medium. The native enzyme was partially purified from the supernatant in two steps using gel filtration. The native α–L-arabinofuranosidase had an apparent mobility of 51.5 kDa on SDS-PAGE, displayed optimum activity at 50°C and pH 3. Kinetic analysis on p-nitrophenyl-α-arabinofuranoside gave a Km of 8.33 mM and a Vmax of 1.54 U/mg, and the enzyme showed slight activity against 1,5-α-L-arabinotriose. The properties of the native enzyme were similar to that of the heterologous α–L-arabinofuranosidase. Hydrolysis of sugar cane bagasse by heterologous α–L-arabinofuranosidase and xylanase revealed that pre-treatment with liquid ammonium was more effective in releasing component sugars than a pre-treatment with water at 140º C. A three-dimensional homology model of the heterologous α–L-arabinofuranosidase was constructed using the solved crystal structure of arabinofuranosidase (AkabfB) from Aspergillus kawachii, which was 71 % identical. / AFRIKAANSE OPSOMMING: Die euaskomisetiese swam Aureobasidium pullulans produseer xilanolitiese ensieme, insluitend ‘n α-L-arabinofuranosidase. Die afgeleide aminosuurvolgorde van die abfA geen wat α-L-arabinofuranosidase enkodeer, was 69-76% identies aan familie 54 glikosied hidrolase. Die abfA geen enkodeer ‘n 498 aminosure polipeptied, insluitend ‘n sein peptied bestaande uit 20 aminosure. Die volwasse proteïen het ‘n berekende molekulêre gewig van 49.9 kDa. Een moontlike N-glikosilasie plek is gevind en ‘n iso-elektriese punt van 4.97 is bereken. A. pullulans AbfA bestaan uit ‘n N-terminaal katalitiese gebied (residu 1-317) en ‘n C-terminaal arabinose-bindingsgebied (residu 318-442). Die abfA geen van die kleur-variante ras van A. pullulans, NRRL Y-2311-1, is onlangs na Saccharomyces cerevisiae Y294 oorgedra. Die giskultuur is op ‘n sinteties gedefinieërde medium gegroei en α-L-arabinofuranosidase was suksesvol uitgedruk, uitgedra van af die selle en van uit die supernatant gesuiwer in ‘n enkele stap deur gel filtrasie te gebruik. Dit het ‘n berekende mobiliteit van 52.7 kDa op SDS-PAGE en 36 kDa geskat deur middel van gel filtrasie. Die heteroloë ensiem is gekarakteriseer volgens pH en temperatuurafhanklikheid en stabiliteit, klaarblyklike mobiliteit en kinetiese eienskappe. Die temperatuur optimale van α–L-arabinofuranosidase was 55 °C en dit was stabiel oor 3 ure teen 40 °C. Die ensiem het optimale aktiwiteit tussen pH 4 en 4.5 getoon en was stabiel teen pH4 en oor 3 ure. Kinetiese analiese op p-nitrofeniel-α-L-arabinofuranosied het ‘n Km van 1.43 gelewer en ‘n Vmax van 23.7 U/mg. Produkinhibisie is opgelet en ‘n Ki van 28 ± 3 mM is vasgestel gedurende toetsing in die teenwoordigheid van arabinose. ‘n Spesifieke aktiwiteit van 3.85 ± 0.008 U/mg is vasgestel op p-nitrofeniel-α-L-arabinofuranosied en geen aktiwiteit was gevind op chromogeniese substrate wat ‘n β-verbinding arabinofuranosiel bevat het nie. Die ensiem het ‘n lae aktiwiteit getoon teen die 1,5-α-L-arabino-oligosakkaried en het die arabinose van mielievesel, hawerspelt arabinoxilaan en tot ‘n mindere mate koring arabinoxilaan geskei. Geen vrystelling van arabinose is van af lorkehout arabinogalactan, α-1,5-onvertakte arabinan en lignin-arabinose substrate nie opgemerk. Verbindingsvoorkeure het minder aktiwiteit teen α-1,5-verbindings as α-1,2 of α-1,3- verbindings arabinofuranosiel subeenhede getoon. Sinergisme tussen α–L-arabinofuranosidase en endo-β-1,4-xilanase het plaasgevind met die bepaling van meer arabinose gedurende koring arabinoxilaan degradasie. A. pullulans NRRL Y-2311-1 is op sinteties gedefinieerde medium gegroei en α-L-arabinofuranosidase was uitgedruk en uitgeskei in die kultuur medium. Die inheemse ensiem was gedeeltelik gesuiwer van die supernatant in twee stappe met die gebruik van gel filtrasie. Die inheemse α–L-arabinofuranosidase het ‘n klaarblyklike mobiliteit van 51.5 kDa op SDS-PAGE, het optimum aktiwiteit vertoon by 50°C en pH 3. Kinetiese analiese op p-nitrofeniel-α-arabinofuranosiede het ‘n Km van 8.33 mM en ‘n Vmax van 1.54 U/mg, en die ensiem het effense aktiwiteit teen 1,5-α-L-arabinotrios getoon. Die eienskappe van die inheemse ensiem was soortgelyk aan die van die heteroloë α–L-arabinofuranosidase. Hidroliese van suikerrietbagasse met heteroloë α–L-arabinofuranosidase en xilanase het aan die lig gebring dat vooraf behandeling met vloeistof ammonium meer effektief is in die vrystelling van komponent suikers as ‘n vooraf behandeling met water teen 140º C. ‘n Driedimensionele homologiese model van die heteroloë α–L-arabinofuranosidase is gekonstrueer deur die gebruik van die verklaarde kristal struktuur van arabinofuranosidase (AkabfB) van Aspergillus kawachii, wat 71% identies was.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Infection is one of the major causes of increased morbidity and the escalating costs associated with orthopedic surgery. The areas that are infected are often difficult to reach and thus difficult to treat. In some surgeries antibiotic-loaded bone cements are used to control infection. Polymethylmethacrylate (PMMA) and calcium phosphate-based bone cements (CPC) are usually used as bone fillers. CPC are bioresorbable and biocompatible (unlike PMMA cements), but can only be used in non- or low-load bearing areas and are thus more applicable in cranio-and maxilla-facial surgeries. Several in vitro and in vivo trials have been conducted on the incorporation of antibiotics and other therapeutic agents into CPC and the release of these agents. As with any solid matrix, release is defined by specific parameters, i.e. matrix porosity, solubility of the drug and interaction of the drug with the cement. The increase in antibiotic-resistant pathogens, mainly as a result of overuse of antibiotics, has a major impact on the choice of antibiotics that are used in the treatment of bacterial infections. The search for alternative antimicrobial compounds that are active against resistant pathogens, is thus of utmost importance. Antimicrobial peptides (bacteriocins) produced by lactic acid bacteria may pose a possible alternative to antibiotics. Some of these peptides are active against antibiotic-resistant pathogens. Bacteriocins are small cationic, hydrophobic, or amphiphilic peptides active against a narrow range of target organisms. Most of these peptides are active in the nanomolar range. It may then be advantageous to incorporate bacteriocins into CPC to evaluate if they may be used as an alternative to antibiotics. The aim of the project was to evaluate if bacteriocins could be successfully incorporated into self seting brushite bone cement and remain effective in vivo without altering basic cement characteristics. Incorporation of bacteriocins into CPC is a novel concept. The low setting temperature and pH of CPC renders it the ideal matrix for incorporation of antimicrobial peptides. In this study, peptide ST4SA, a class IIa broad-spectrum bacteriocin, has been incorporated into brushite bone cement and characterized in vitro. Incorporation of the peptide did not have a significant effect on the crystal entanglement or setting reaction of the cement. Peptide ST4SA was rapidly released and inhibited the growth of the target strain effectively. In another experiment, peptide ST4SA was suspended in poly (lactide-co-glycolide) and electrosprayed to form micro particles that were entrapped in brushite cement. Association of the peptide with microparticles resulted in a delayed release from the cement, followed by a constant release. Nisin F, a class Ia bacteriocin was also incorporated into brushite cement and its activity studied in vitro and in vivo. Similar results were observed in vitro as recorded with peptide ST4SA incorporated into brushite cement. Small cylinders of brushite cement loaded with nisin F were implanted into subcutaneous pockets in mice and each pocket infected with a bioluminescent strain of Staphylococcus aureus (Xen 36). Nisin F in the bone cement prevented the growth of S. aureus in the wound and controlled infection. With this study we have shown that antimicrobial peptides that differ in structure (classes I and II) could be incorporated into bone cement and control the growth of S. aureus in vivo and in vitro. The mode of action of these peptides differs from antibiotics in that they form a permanent pore in the cell membrane of the target organism. This minimizes the chance of a strain becoming resistant to the peptide. Incorporation of antimicrobial peptides into bone cement may be a possible alternative to antibiotics in the control of bacterial infections associated with implants. / AFRIKAANSE OPSOMMING: Infeksie is een van die grootste bydraende faktore tot sterftes en verhoogde kostes in ortopediese chirurgie. Geinfekteerde areas is dikwels moeilik bereikbaar en dus ook moeilik om te behandel. In sommige operasies word antibiotika-gelaaide beensement gebruik om infeksie te beheer. Polymetielmetakrilaat (PMMS) en kalsium fosfaat gebaseerde beensement (KFS) word gebruik as been vullers. KFS is bioverenigbaar en bio-absorberend (in teenstelling met PMMS), maar kan slegs in geen- of liggewig-draende areas gebruik word en is dus van groter toepassing in skedel-, kaak- gesig- en mondchirurgie. Verskeie in vitro en in vivo toetse is al gedoen op die inkorporering van antibiotika en ander terapeutiese middels in KFS en die vrystelling daarvan uit die matriks. Soos met enige soliede matriks is vrylating van die geinkorporeerde bestanddeel afhanklik van sekere parameters, onder andere porositeit, oplosbaarheid van die middel, en die interaksie van die middel met beensement. Die toename in antibiotika-weerstandbiedende patogene plaas geweldige druk op die keuse van antibiotika wat gebruik word in die beheer van bakteriese infeksie. Die soeke na alternatiewe antimikrobiese middels aktief teen bestande patogene is dus van kardinale belang. Antimikrobiese peptiede (bakteriosiene) gepproduseer deur melksuur bakteriee mag dalk . alternatief tot antibiotika wees. Sommige van hierdie peptiede is aktief teen verskeie weerstandbiedende patogene. Bakteriosiene is kationiese, hidrofobiese of amfifiliese peptiede wat naverwante bakteriee inhibeer of doodmaak. Die meeste van hierdie peptiede is aktief op nanoskaal vlak. Dit mag dalk dus voordelig wees om bakteriosiene in been sement te evalueer as moontlike alternatiewe tot antibiotika. Die doel van die proejek was om te evaleer of bakteriosiene suksesfol in "brushite" sement geïnkorporeer kan word en steeds effektief in vivo bly sonder om die basiese eienskappe van die sement te verander. Inkorporasie van bakteriosiene in KFS is 'n nuwe konsep. Die lae stollingstemperatuur en pH van KFS maak dit moontlik om bakteriosiene daarin te inkorporeer. In hierdie studie is peptied ST4SA, . klas IIa wye-spektrum bakteriosien, in "brushite" sement geïnkorporeer en in vitro bestudeer. Die toevoeging van die peptied het nie 'n beduidende effek op die stolreaksie of kristal verstrikking van die sement gehad nie. Peptied ST4SA is effektief vrygelaat en het die groei van die teikenorganisme suksesvol onderdruk. In 'n ander eksperiment is peptied ST4SA in poli (D,L-laktied-ko-glikolied) gesuspendeer en met behulp van elektrosproeiing tot mikropartikels omvorm en is in "brushite" sement geïnkorporeer. Assosiasie van die peptied met mikropartikels het die inisiële vrylating van die peptied vertraag, gevolg deur 'n konstante vrylating. Nisien F, . klas Ia lantibiotikum, is ook in "brushite" sement geïnkorporeer en die aktiwiteit daarvan in vitro en in vivo bestudeer. Die in vitro eienskappe is soortgelyk aan die eienskappe wat vir peptied ST4SA-gelaaide sement waargeneem is. Klein stafies "brushite" sement, waarin nisien F geïnkoproreer is, is in onderhuidse sakkies in muise geplaas en die area met 'n bio-liggewende bakterie (S. aureus Xen 36) geïnfekteer. Nisien F in die beensement het die groei van S. aureus in die wond onderdruk en infeksie beheer. Met hierdie studie het ons bewys dat bakteriosiene wat struktureel van mekaar verskil (klasse I en II) in beensement geïnkorporeer kan word en die groei van S. aureus in vitro en in vivo kon beheer. Die wyse waarop hierdie peptiede die groei van sensitiewe organismes inhibeer verskil van die van antibiotika deurdat dit porieë in die selmembraan vorm. Die moontlikheid dat organismes weerstandbiedend raak tot die peptied is dus heelwat skraler. Die insluit van antimikrobiese peptiede in beensement mag dalk 'n alternatief tot antibiotika wees in die voorkoming van bakteriële infeksie geassosieer met ortopediese chirurgie.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: In the aquaculture setting, opportunistic pathogens are present as part of the normal aquatic microflora, colonizing surfaces in fish tanks as part of biofilm communities, and often causing severe economic losses to the aquacultural industry. Isolates belonging to the genera Chryseobacterium, Elizabethkingia, Myroides and Empedobacter have been isolated from diseased fish, and are responsible for causing secondary fish infections, fish- and food-product spoilage, and have been described as etiological agents of various human diseases. Thirty-four Chryseobacterium and Elizabethkingia spp. and five Myroides and Empedobacter spp. isolates, obtained from various diseased fish species and biofilm growth in South African aquaculture systems, were characterised genetically using 16S rRNA gene PCR restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD) PCR, whole cell protein (WCP) and outer membrane protein (OMP) analyses. Genetic heterogeneity was displayed by the Myroides and Empedobacter spp. study isolates following OMP analysis, although 16S rRNA gene RFLP, RAPD-PCR and WCP analysis did not allow for differentiation of these isolates. A high degree of genetic heterogeneity was displayed by the Chryseobacterium and Elizabethkingia spp. study isolates following OMP analysis, 16S rRNA gene RFLP with MspI, and RAPD-PCR with primer P2. However, based on the results obtained by WCP analysis, 16S rRNA gene RFLP with CfoI and TaqI, and RAPD-PCR with primer P1 the isolates appeared genetically very homogeneous. High MAR indices and potential multi-drug resistance phenotypes were obtained for the Myroides and Empedobacter spp. and some of the Chryseobacterium and Elizabethkingia spp. isolates by antimicrobial susceptibility testing. Primary adherence and the influence of environmental changes on adherence was investigated by a modified microtitre-plate adherence assay. Nutrient composition, temperature and hydrodynamic incubation conditions were observed to influence adherence abilities of all study isolates. In addition, adherence varied greatly among isolates of the genera Chryseobacterium and Elizabethkingia, as opposed to a consistent strong adherence profile observed for the Myroides and Empedobacter spp. isolates. The influence of cell surface properties such as capsule presence and cell surface hydrophobicity, on primary adherence of the isolates was also investigated. Quantitative analysis of capsular material revealed the presence of thick capsular material surrounding the Myroides and Empedobacter spp. and some of the Chryseobacterium and Elizabethkingia spp. isolates, but could not be directly associated with adherence. Hydrophobicity were investigated using the salt aggregation assay (SAT) and bacterial adherence to hydrocarbon test (BATH). A very hydrophilic cell surface was observed for all of the Myroides and Empedobacter spp. isolates, and majority (74%) of the Chryseobacterium and Elizabethkingia spp. isolates. Cell surface hydrophobicity could not be correlated to the adherence of the Myroides and Empedobacter spp. isolates, and only SAT-determined hydrophobicity could be positively correlated to adherence of Chryseobacterium and Elizabethkingia spp. isolates under certain conditions. Coaggregation studies were performed between the study isolates and various important clinical and aquacultural microorganisms. High coaggregation indices were observed between the Myroides and Empedobacter spp. isolates and E. faecalis and S. aureus, and between E. faecalis, S. enterica serovar Arizonae, S. aureus and Listeria spp. and the Chryseobacterium and Elizabethkingia spp. isolates. Biofilm-forming capacity of the study isolates in an environment simulating their natural environment was investigated microscopically using a flow cell system. Typical ‘cone-like’ biofilm structures were observed for selected strains of both Myroides and Empedobacter spp. and Chryseobacterium and Elizabethkingia spp. isolates. The effect of increased hydrodynamics on biofilm architecture was seen through the narrowing of the biofilm structures and the formation of single cell chains towards the increased hydrodynamic area of the flow chambers. Chryseobacterium and Elizabethkingia spp. and Myroides and Empedobacter spp. appear to be potential primary biofilm-formers associating with a variety of microbes thus perpetuating their survival in a variety of aquatic habitats. / AFRIKAANSE OPSOMMING: Opportunistiese patogene kom gereeld in akwakultuur sisteme voor as deel van die akwatiese mikroflora wat dikwels biofilms vorm op oppervlaktes in hierdie sisteme. Visinfeksies veroorsaak deur hierdie patogene lei tot ernstige ekonomiese verliese vir akwakultuur industrieë. Chryseobacterium, Elizabethkingia, Myroides en Empedobacter spp. is reeds voorheen van verskeie geïnfekteerde visspesies geïsoleer hierdie bakterieë is verantwoordelik vir sekondere visinfeksies, die bederf van vis- en kosprodukte, asook menslike siektes. Vier-en-dertig Chryseobacterium en Elizabethkingia spp. en 5 Myroides en Empedobacter spp. isolate, geïsoleer vanaf verskeie geïnfekteerde visspesies en biofilm-groei in Suid Afrikaanse akwakultuur-sisteme, is geneties met behulp van 16S rRNS geen PKR restriksie fragment lengte polimorfisme (RFLP), toevallig geamplifiseerde polimorfiese DNS (TGPD) PKR, heel-sel protein (HSP) en buitemembraan protein (BMP) analise gekarakteriseer. BMP analise het getoon dat die Myroides en Empedobacter spp. isolate geneties heterogeen is, alhoewel 16S rRNS TGPD-PKR, TGPD-PKR en HSP analise nie tussen die isolate kon onderskei nie. BMP analise, 16S rRNS TGPD-PKR met MspI en TGPD-PKR met inleier P2 was meer suksesvol as HSP analise, 16S rRNS TGPD-PKR met CfoI en MspI, en TGPD-PKR met inleier P1, om onderskeid te tref tussen die Chryseobacterium en Elizabethkingia spp. isolate en het gedui op ‘n hoë vlak van genetiese heterogeniteit tussen hierdie isolate. Beide die Chryseobacterium en Elizabethkingia spp. en Myroides en Empedobacter spp. isolate het ‘n hoë vlak van antibiotika weerstand getoon wat dui op ‘n menigvuldigde antibiotika weerstands-fenotiepe. Primêre vashegting vermoëns en die invloed van omgewingsfaktore op vashegting is met behulp van ‘n gemodifiseerde mikrotiterplaat vashegtings toets ondersoek. Vashegting van die isolate is beïnvloed deur variasies in die samestelling van die medium, temperatuurveranderings en verskillende hidrodinamiese inkubasie kondisies. Inteenstelling met die sterk vashegtingsvermoë van die Myroides en Empedobacter spp. isolate, het die vermoë om vas te heg grootliks tussen die Chryseobacterium en Elizabethkingia spp. isolate gevarieer. Verder is ondersoek ingestel op die invloed van seloppervlak eienskappe soos die teenwoordigheid van kapsules en hidrofobisiteit op die isolate se vermoë om aan oppervlaktes te heg. Die Myroides en Empedobacter spp. isolate en verskeie Chryseobacterium en Elizabethkingia spp. isolate is omring deur dik kapsules, maar geen verband tussen vashegting en die teenwoordigheid van kapsules kon bepaal word nie. Die sout aggregasie toets (SAT) en bakteriële vashegting aan koolwaterstowwe (BVAK) toets was gebruik om die hidrofobisiteit van die isolate se seloppervlaktes te bepaal. Die Myroides en Empedobacter spp. isolate en 74% van die Chryseobacterium en Elizabethkingia spp. isolate het ‘n baie hidrofiliese seloppervlak getoon. Slegs die hidrofobisiteit bepaal deur die SAT toets het ‘n positiewe verwantskap met die aanhegtingsvermoë van die Chryseobacterium en Elizabethkingia spp. isolate getoon. Mede-aggregasie tussen die isolate en verskeie belangrike mediese en akwakultuur mikroörganismes is ook ondersoek. Die Myroides en Empedobacter spp. isolate het ‘n sterk assosiasie met E. faecalis en S. aureus getoon Die Chryseobacterium en Elizabethkingia spp. isolate het sterk met E. faecalis, S. aureus, S. enterica serovar Arizonae en Listeria spp. geassosieer. Vloei-sel studies is uitgevoer om die biofilm-vormingsvermoë van die isolate te ondersoek. Vir beide die Myroides en Empedobacter spp. en Chryseobacterium en Elizabethkingia spp. isolate is tipiese kegelagtige biofilm stukture waargeneem. Die invloed van verhoogde hidrodinamiese kondisies in die vloei-sel het vernouing van die biofilm strukture en die vorming van enkel-sel kettings tot gevolg gehad. Vanuit hierdie studie is afgelei dat die Myroides en Empedobacter spp. en Chryseobacterium en Elizabethkingia spp. isolate onder verskeie kondisies aan oppervlaktes kan vasheg en dus potensiële primêre biofilm-vormings organismses is. Hierdie organismes besit ook die vermoë om met ‘n verskeidenheid ander organismes te assosieer, wat waarskynlik hulle suksesvolle oorlewing in akwakultuursisteme verseker.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Cellulose, a glucose polymer, is considered the most abundant fermentable polymer on earth. Agricultural waste is rich in cellulose and exploiting these renewable sources as a substrate for ethanol production can assist in producing enough bioethanol as a cost-effective replacement for currently used decreasing fossil fuels. Saccharomyces cerevisiae is an excellent fermentative organism of hexoses; however the inability of the yeast to utilize cellulose as a carbon source is a major obstruction to overcome for its use in the production of bio-ethanol. Cellobiose, the major-end product of cellulose hydrolysis, is hydrolyzed by -glucosidase or cellobiose phosphorylase, the latter having a possible metabolic advantage over -glucosidase. Recently, it has been showed that S. cerevisiae is able to transport cellobiose. The construction of a cellulolytic yeast that can transport cellobiose has the advantage that end-product inhibition of the extracellular cellulases by glucose and cellobiose is relieved. Furthermore, the extracellular glucose concentration remains low and the possibility of contamination is decreased. In this study the cellobiose phosphorylase gene, cepA, of Clostridium stercorarium was cloned and expressed under transcriptional control of the constitutive PGK1 promoter and terminator of S. cerevisiae on a multicopy episomal plasmid. The enzyme was expressed intracellulary and thus required the transport of cellobiose into the cell. The fur1 gene was disrupted for growth of the recombinant strain on complex media without the loss of the plasmid. The recombinant strain, S. cerevisiae[yCEPA], was able to sustain aerobic growth on cellobiose as sole carbon source at 30°C with Vmax = 0.07 h-1 and yielded 0.05 g biomass per gram cellobiose consumed. The recombinant enzyme had activity optima of 60°C and pH 6-7. Using Michaelis-Menten kinetics, the Km values for the colorimetric substrate p-nitrophenyl-b-D-glucopyranoside (pNPG) and cellobiose was estimated to be 1.69 and 92.85 mM respectively. Enzyme activity assays revealed that the recombinant protein was localized in the membrane fraction and no activity was present in the intracellular fraction. Due to an unfavourable codon bias in S. cerevisiae, CepA activity was very low. Permeabilized S. cerevisiae[yCEPA] cells had much higher CepA activity than whole cells indicating that the transport of cellobiose was inadequate even after one year of selection. Low activity and insufficient cellobiose transport led to an inadequate glucose supply for the yeast resulting in low biomass formation. Cellobiose utilization increased when combined with other sugars (glucose, galactose, raffinose, maltose), as compared to using cellobiose alone. This is possibly due to more ATP being available for the cell for cellobiose transport. However, no cellobiose was utilized when grown with fructose indicating catabolite repression by this sugar. To our knowledge this is the first report of a heterologously expressed cellobiose phosphorylase in yeast that conferred growth on cellobiose. Furthermore, this report also reaffirms previous data that cellobiose can be utilized intracellularly in S. cerevisiae. / AFRIKAANSE OPSOMMING: Sellulose, ‘n homopolimeer van glukose eenhede, word beskou as die volopste suiker polimeer op aarde. Landbou afval produkte het ‘n hoë sellulose inhoud en benutting van diè substraat vir bio-etanol produksie kan dien as ‘n koste-effektiewe aanvulling en/of vervanging van dalende fossielbrandstof wat tans gebruik word. Die gis, Saccharomyces cerevisiae, is ‘n uitmuntende organisme vir die fermentasie van heksose suikers, maar die onvermoë van die gis om sellulose as koolstofbron te benut is ‘n groot struikelblok in sy gebruik vir die produksie van bio-etanol. Sellobiose, die hoof eindproduk van ensiematiese hidrolise van sellulose, word afgebreek deur -glukosidase of sellobiose fosforilase. Laasgenoemde het ‘n moontlike metaboliese voordeel bo die gebruik van -glukosidase vir sellobiose hidrolise. Daar was onlangs gevind dat S. cerevisiae in staat is om sellobiose op te neem. Die konstruksie van ‘n sellulolitiese gis wat sellobiose intrasellulêr kan benut, het die voordeel dat eindproduk inhibisie van die ekstrasellulêre sellulases deur sellobiose en glukose verlig word. Verder, wanneer die omsetting van glukose vanaf sellobiose intrasellulêr plaasvind, word die ekstrasellulêre glukose konsentrasie laag gehou en die moontlikheid van kontaminasie beperk. In hierdie studie was die sellobiose fosforilase geen, cepA, van Clostridium stercorarium gekloneer en uitgedruk onder transkripsionele beheer van die konstitutiewe PGK1 promoter en termineerder van S. cerevisiae op ‘n multikopie episomale plasmied. Die ensiem is as ‘n intrasellulêre proteïen uitgedruk en het dus die opneem van die sellobiose molekuul benodig. Die disrupsie van die fur1 geen het toegelaat dat die rekombinante ras op komplekse media kon groei sonder die verlies van die plasmied. Die rekombinante ras, S. cerevisiae[yCEPA], het aërobiese groei by 30°C op sellobiose as enigste koolstofbron onderhou met mmax = 0.07 h-1 en ‘n opbrengs van 0.05 gram selle droë gewig per gram sellobiose. Die rekombinante ensiem het optima van 60°C en pH 6-7 gehad. Die K m waardes vir die kolorimetriese substraat pNPG en sellobiose was 1.69 en 92.85 mM onderskeidelik. Ondersoek van die ensiem aktiwiteit het getoon dat die rekombinante proteïen gelokaliseer was in die membraan fraksie en geen aktiwiteit was teenwoordig in die intrasellulêre fraksie nie. CepA aktiwiteit was laag as gevolg van ‘n lae kodon voorkeur in S. cerevisiae. Verder het geperforeerde S. cerevisiae[yCEPA] selle aansienlik beter CepA aktiwiteit getoon as intakte selle. Hierdie aanduiding van onvoldoende transport van sellobiose na binne in die sel tesame met die lae aktiwiteit van die CepA ensiem het gelei tot onvoldoende glukose voorraad vir die sel en min biomassa vorming. Sellobiose verbruik het toegeneem wanneer dit tesame met ander suikers (glukose, galaktose, raffinose, maltose) gemeng was, heelwaarskynlik deur die vorming van ekstra ATP’s vir die sel wat ‘n toename in sellobiose transport teweeg gebring het. Fruktose het egter kataboliet onderdrukking veroorsaak en sellobiose was nie benut nie. Sover ons kennis strek, is hierdie die eerste verslag van ‘n heteroloë sellobiose fosforilase wat in S. cerevisiae uitgedruk is en groei op sellobiose toegelaat het. Verder, bewys die studie weereens dat S. cerevisiae wel sellobiose kan opneem.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: It is commonly known that a diversity of fungi, including yeasts, may occur on plant surfaces. Similarly, on fallen trees an ecological succession of different fungal species is known to occur during wood degradation. Some of these fungi may be pioneer fungi contributing to the initial degradation process, while others may be yeasts associated with the fruiting bodies of macro-fungi which in turn are able to utilize the more recalcitrant polymers in wood. Previously, it was revealed that an increase occurs in the wood degradation rate of certain white-rot fungi when co-cultured with selected yeast species. A well known inhabitant of decomposing trees is the white rot fungus Pycnoporus sanguineus. It was found by some that this fungus is capable of selective delignification while growing on the wood of poplar trees, while other authors found a simultaneous delignification pattern on Eucalyptus grandis trees. In the latter case cellulose and lignin are degraded simultaneously. We were interested in how yeasts occurring on the surface of P. sanguineus fruiting bodies, and the pioneer fungus Aspergillus flavipes, impact on wood degradation by this white-rot fungus. Restriction Fragment Length Polymorphisms (RFLP) analyses were used to obtain an indication of the species composition of the culturable yeast community associated with fruiting bodies of P. sanguineus. The impact of the most dominant of these yeasts species, i.e. Pichia guilliermondii and Rhodotorula glutinis, as well as A. flavipes, on wood degradation by P. sanguineus was then determined by analyzing the major wood components after growth of co-cultures on hot water washed E. grandis wood chips. Co-cultures of P. sanguineus with the other fungi were prepared by inoculating the wood chips, contained in solid state bioreactors and supplemented with molasses and urea, with the an appropriate volume of fungal inoculum, resulting in an initial moisture content of 60%. After two weeks of incubation at 30°C with constant aeration, the chips were harvested. Standard protocol (TAPPI Standard Methods), commonly used by the paper and pulp industry, were then employed to determine the percentage cellulose, Klason Lignin, as well as polar and solvent-borne extractives in the chips. The resulting data were analyzed using box plots, as well as biplots. No degradation of Klason lignin was observed, while the percentage cellulose did decrease during fungal degradation. Taking into account the inherent shortcomings of the Klason Lignin determination, the results supported the findings of others that P. sanguineus shows a simultaneous delignification pattern while growing on E. grandis wood. In addition, it was found that the yeasts played no significant role in the degradation ability of P. sanguineus, while A. flavipes showed an antagonistic effect on P. sanguineus with respect to cellulose degradation. However, it was clear that the analytical methods used in this study were inadequate to accurately determine fungal degradation of wood. In addition, it was obvious that the methods used did not distinguish between fungal biomass and wood components. Nevertheless, the methods provided us with a fingerprint of each culture growing on E. grandis wood, allowing us to compare the chemical composition of the different cultures and the un-inoculated hot water washed wood chips. The question, therefore, arose whether the effect of a particular coculture, on the chemical composition of wood, differs between tree species. Consequently, chemical alterations in different tree species, induced by a P. sanguineus / A. flavipes co-culture, were investigated in the next part of the study. Wood chips originating from four tree species, i.e. Acacia mearnsii, Eucalyptus dunnii, E. grandis, and Eucalyptus macarthurii, were inoculated with this co-culture. The culture conditions and subsequent analyses of the wood components were the same as in the first part of the study. From the box- and biplots constructed from the resulting data, it was clear that the chemical composition of each tree species were altered in a different manner by the coculture. Lignin content showed an apparent increase in A. mearnsii, while E. dunnii showed a decrease in cellulose content. The results indicate that wood of different tree species are degraded in a different manner and this phenomenon should be taken into account in selecting fungi for biopulping. / AFRIKAANSE OPSOMMING: Dit is algemeen bekend dat 'n verskeidenheid fungi, insluitend giste, op plantoppervlaktes mag voorkom. Dit is ook bekend dat 'n ekologiese opeenvolging van verskillende fungusspesies tydens hout-afbraak op omgevalle bome voorkom. Van hierdie fungi mag pionierfungi wees wat bydra tot die aanvanklike afbraakproses, terwyl ander giste mag wees wat geassosieer word met die vrugliggame van makro-fungi, wat op hul beurt weer in staat is om die meer weerstandbiedende polimere in hout te benut. Dit is voorheen bekendgemaak dat daar 'n toename plaasvind in die tempo van houtafbraak deur sekere witvrot-fungi wanneer dit in ko-kulture met geselekteerde gisspesies voorkom. 'n Bekende bewoner van verrottende bome is die wit-vrotfungus Pycnoporus sanguineus. Dit is gevind dat hierdie fungus tot selektiewe delignifikasie in staat is terwyl dit op die hout van populierbome groei, terwyl ander outeurs 'n gelyktydige patroon van delignifisering op Eucalyptus grandis bome gevind het. In laasgenoemde geval is sellulose en lignien gelyktydig afgebreek. Ons was geïnteresseerd in die effek van giste op die oppervlak van vrugliggame van P. sanguineus, en die pionierfungus Aspergillus flavipes, op die houtafbraak deur hierdie wit-vrotfungus. Restriction Fragment Length Polymorphisms (RFLP) analises is gevolglik gebruik om 'n aanduiding te kry van die spesiesamestelling van die kweekbare gisgemeenskap wat met die vrugliggame van P. sanguineus geassosieer word. Die impak van die mees dominante van hierdie gisspesies, naamlik Pichia guilliermondii en Rhodotorula glutinis, asook A. flavipes, op houtafbraak deur P. sanguineus is voorts bepaal deur die analise van die belangrikste houtkomponente na die kweek van ko-kulture op E. grandis houtskyfies wat met warm water gewas is. Ko-kulture van P. sanguineus met die ander fungi is voorberei deur die houtskyfies in vaste fase bioreaktore, aangevul met melasse en ureum, te inokuleer met 'n toepaslike volume van die fungus inokulum om 'n aanvanklike voginhoud van 60% te verkry. Na twee weke se inkubasie by 30°C met konstante belugting is die skyfies ge-oes. Standaard protokol (TAPPI Standard Methods), algemeen deur die papier en pulpindustrie gebruik, is ingespan om die persentasie sellulose, Klason Lignien, asook polêre en oplosmiddel-gedraagde ekstrakte in die skyfies te bepaal. Die gevolglike data is geanaliseer deur gebruik te maak van box plots en biplots. Daar is geen afbraak van Klason Lignien bespeur nie, terwyl die persentasie sellulose wel toegeneem het tydens fungus degradasie. Met die inherente tekortkominge van die Klason Lignien bepaling inaggenome, het die resultate die bevindings ondersteun van andere wat getoon het dat P. sanguineus 'n gelyktydige delignifikasiepatroon openbaar terwyl dit op E. grandis hout groei. Daarby is dit gevind dat die giste geen beduidende rol in die afbraakvermoeë van P. sanguineus gespeel het nie, terwyl A. flavipes 'n antagonisiese effek ten opsigte van die sellulose degradering van P. sanguineus getoon het. Dit was egter duidelik dat die analitiese metodes wat in hierdie studie gebruik is, onvoldoende was om die degradering van hout akkuraat te bepaal. Daarby was dit duidelik dat die metodes nie tussen fungus biomassa en houtkomponente kon onderskei nie. Nogtans het die metodes 'n vingerafdruk verskaf van elke kultuur wat op E. grandis hout groei, wat ons toegelaat het om die chemiese samestelling van die verskillende kulture en die ongeïnokuleerde, met warm water gewasde houtskyfies te vergelyk. Die vraag het gevolglik ontstaan of die effek van 'n bepaalde ko-kultuur op die chemiese samestelling van hout van boomspesie tot boomspesie verskil. Gevolglik is die chemiese wisselinge in verskillende boomspesies, geïnduseer deur 'n P. sanguineus / A. flavipes ko-kultuur, in die volgende gedeelte van die studie ondersoek. Houtskyfies van vier boomspesies, naamlik Acacia mearnsii, Eucalyptus dunnii, E. grandis, en Eucalyptus macarthurii, is met hierdie ko-kultuur geïnokuleer. Die kultuurkondisies en daaropvolgende analises van die houtkomponente was dieselfde as in die eerste deel van die studie. Van die box- en biplots wat van die resultate getrek is, is dit duidelik dat die chemiese samestelling van elke boomspesie op 'n verskillende manier deur die ko-kulture verander is. Lignien-inhoud het ’n waarskynlike toename getoon in A. mearnsii, terwyl E. dunnii 'n afname in sellulose-inhoud getoon het. Die resultate toon dat hout van verskillende boomspesies op verskillende maniere afgebreek word en dat hierdie fenomeen in aanmerking geneem moet word wanneer fungi vir bioverpulping geselekteer word.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The arsenic resistance operon (ars operon) of the Gram-positive, iron-oxidizing, acidophilic, moderately thermophilic bacterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t. VKM B-1269T), was isolated and characterised. The ars operon was chromosomally located and consisted of an arsR (codes for a transcriptional regulator) and an arsB (codes for a membrane located arsenic/antimony efflux pump). The arsRB genes were transcribed in the same direction. An arsC (codes for an arsenate reductase), usually associated with ars operons, was absent from this ars operon. PCR and Southern-hybridization experiments revealed that no arsC, representative of either the Grx/GSH or Trx ArsC families was present in the genome of Sb. t. VKM B-1269T. An interesting feature of the ars operon was the presence of a gene encoding a 525 amino acid (60.83 kDa) kumamolisin-As precursor located upstream of the arsRB operon. The intergenic region between the termination end of the kumamolisin-As precursor gene and the transcriptional start of the arsR gene was only 77 bp, suggesting that this ars operon might consist of three genes. RT-PCR analysis showed that the ars operon of Sb. t. VKM B-1269T, was not co-transcribed with the kumamolisin-As precursor gene in its native Sulfobacillus host. The ars operon of Sb. t. VKM B-1269T did not complement an Escherichia coli arsenic sensitive mutant. mRNA transcript analysis and promoter expression studies confirmed that processes involved in the production of functional proteins from the ars operon transcript were likely to be responsible for the inability of the arsRB operon of Sb. t. VKM B-1269T to confer resistance to arsenic in the heterologous E. coli host. Eight Sulfobacillus strains isolated from different geographical areas were subjected to amplified ribosomal DNA restriction enzyme analysis (ARDREA) using the restriction endonuclease Eco1015 (SnaBI) and revealed that they could be divided into the proposed Sulfobacillus spp. subgroup I and subgroup II, respectively (Johnson et al., 2005). The presence, distribution and relatedness of the ars genes among members of genus Sulfobacillus was determined. Phylogenetic sequence comparisons revealed two clearly defined arsB clusters within genus Sulfobacillus and showed that the arsB of a specific Sulfobacillus sub specie is distinctive of that specific Sulfobacillus sub specie. Futhermore, sequence analysis of the isolated arsB homologue fragments from the respective Sulfobacillus spp. showed that four distinctive profiles could be identified based on differences in the location of restriction endonuclease recognition sites. / AFRIKAANSE OPSOMMING: Die arseen weerstandbiedendheidsoperon (ars operon) van die Gram-positiewe, ysteroksiderende, asidofiliese, matige termofiliese bakterium, Sulfobacillus thermosulfidooxidans VKM B-1269T (Sb. t. VKM B-1269T), was geïsoleer en gekarakteriseer. Die ars operon was op die chromosoom geleë en het uit ‘n arsR (kodeer vir ‘n transkripsionele reguleerder) en ‘n arsB (kodeer vir ‘n membraan geleë arseen/timien uitskeidings pomp) bestaan. Die arsRB gene word in dieselfde rigting getranskribeer. ‘n arsC (kodeer vir ‘n arsenaat reductase), wat gewoontlik geassosïeer word met ars operons, was afwesig van hierdie ars operon. PKR en Southern-hibridisasie eksperimente het aangedui dat geen arsC, verteenwoordigend van beide die Grx/GSH of Trx ArsC families, nie teenwoordig was in die genoom van Sb. t. VKM B-1269T, nie. ‘n Interressante eienskap van hierdie ars operon was die teenwoordigheid van ‘n geen wat stroom-op van die arsRB operon geleë is en ‘n 525 amino suur (60.83 kDa) kumamolisin-As voorloper kodeer. Die intergeniese gedeelte tussen die terminerings einde van die kumamolisin-As voorloper en die transkriptionele begin van die arsR geen was slegs 77 bp, wat voorgestel het dat die ars operon moontlik uit drie gene bestaan. RT-PKR analiese het bewys dat die ars operon van Sb. t. VKM B-1269T, nie geko-getranskribeer word met die kumamolisin-As voorloper in sy oorspronklike Sulfobacillus gasheer nie. Die ars operon van Sb. t. VKM B-1269T, het nie ‘n Escherichia coli arseen sensitiewe mutant gekomplimenteer nie. mRNA transkrip-analiese en promoter uitdrukkings eksperimente het bevestig dat prosesse wat betrokke is in die produksie van funksionele proteïene vanaf die ars operon transkrip, moontlik vir die onvermoë van die arsRB operon van Sb t. VKM B-1269T verantwoordelik was om weerstandbiedendheid teen arseen in die heteroloë E. coli gasheer te verleen. Agt Sulfobacillus stamme wat geïsoleer is vanuit verskillende geografiese areas, was onderhewig aan geamplifiseerde ribosomale DNA restriksie-ensiem-analiese (ARDREA) deur gebruik te maak van restriksie endonuklease Eco1015 (SnaBI) en het aangedui dat hulle in die voorgestelde Sulfobacillus spp. subgroup I en subgroup II ingedeel kan word (Johnson et al., 2005). Die aanwesigheid, verspreiding en verwantskappe van die ars gene tussen lede van genus Sulfobacillus was bepaal. Filogenetiese DNA volgorde vergelykings het aangedui dat twee duidelik definïeerbare arsB groepe van mekaar onderskei kan word en dat die arsB van ‘n spesifieke Sulfobacillus sub spesie uniek tot daardie spesifieke Sulfobacillus subspesie is. Bykomend, DNA volgorde analiese van die geïsoleerde arsB homoloog fragmente van die Sulfobacillus spp. het gewys dat vier unieke profiele, op grond van verskille in die ligging van restriksie ensiem herkenning setels, geïdentifiseer kan word.

Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Grapevine trunk diseases are caused by invasive pathogens that are responsible for the slow decline of vines. In particular, Eutypa dieback of grapevine has had a devastating impact on vineyards worldwide, reducing growth and yield, eventually killing the grapevine. The causal organism of Eutypa dieback was first described as Eutypa armeniacae Hansf. & Carter, the pathogen that causes dieback of apricots, but since 1987 this species has been considered a synonym of Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Recently, it was proposed that at least two species that are capable of infecting grapevines are responsible for Eutypa dieback. Consequently, the molecular identification and characterisation of Eutypa dieback was used to delineate the species occurring on infected grapevines in South Africa. This involved the molecular analyses of three molecular markers, namely, the internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA operon, and the -tubulin gene. The results obtained revealed the presence of a second species, namely, Eutypa leptoplaca (Mont.) Rappaz, that occurred together with E. lata on infected grapevines. Also co-habiting with these pathogens were related fungi form the Diatrypaceae family, Cryptovalsa ampelina (Nitschke) Fuckel and Eutypella vitis (Schwein.) Ellis & Everhart. Pathogenicity tests conducted on isolates representing C. ampelina, E. lata, E. leptoplaca, and E. vitis revealed that all were pathogenic to grapevine. Several species of Botryosphaeriaceae that commonly invade the woody tissue of grapevines are also pathogenic to grapevine. The symptoms in grapevine commonly associated with Botryosphaeriaceae are easily confused with the symptoms produced by Eutypa dieback which prompted the need for the development of a detection method that can correctly identify the presence of multiple pathogens. A reverse dot blot hybridisation (RDBH) method was subsequently applied to provide a rapid, accurate and reliable means of detecting the Eutypa species involved in the Eutypa disease complex, as well as those species of Botryosphaeriaceae known to cause disease in grapevines. The method involved the use of multiplex PCR to simultaneously amplify and label the regions of DNA that are used as pathogen specific probes. Consequently, membrane immobilised species-specific oligonucleotides synthesised from the ITS, - tubulin and LSU molecular data were evaluated during the application of this diagnostic method to detect Eutypa species. It was found that the species-specific oligonucleotides, designed from ITS sequence data, could consistently detect E. lata and E. leptoplaca. The application of the RDBH method for the detection of these Eutypa species, based on -tubulin and LSU sequence data, however, proved to be unsuccessful. Subsequently, a RDBH method, utilising species-specific oligonucleotides designed from elongation factor-1α sequence data, was successfully applied for the detection of Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips. The method, however, was unsuccessful for the detection of Diplodia seriata De Not. In addition to the above-mentioned shortcomings, the RDBH was not amenable to the detection of pathogens directly from field or environmental samples, but required preparation of DNA from pure cultures. The method, however, allows for the identification of multiple pathogens in a single assay. As DNA extraction methods are amended, improved and honed to obtain DNA from environmental samples, so would it increase the usefulness of RDBH. / AFRIKAANSE OPSOMMING: Wingerd stamsiektes word veroorsaak deur patogene wat die vermoë het om wingerdplante te infekteer en dan stadige agteruitgang van dié wingerde te veroorsaak. Veral Eutypa terugsterwing het ‘n vernietigende effek op wingerde wêreldwyd deurdat dit groeikrag en oesmassa verlaag, maar ook omdat dit uiteindelik wingerdstokke kan dood. Die veroorsakende organisme is aanvanklik as Eutypa armeniacae Hansf. & Carter beskryf, die patogeen wat terugsterf by appelkose veroorsaak, maar sedert 1987 word hierdie spesies beskou as ‘n sinoniem van Eutypa lata (Pers.:Fr.) Tul & C. Tul (anamorph Libertella blepharis A. L. Smith). Dit is egter onlangs voorgestel dat ten minste twee spesies die vermoë het om wingerd te infekteer om Eutypa terugsterwing te veroorsaak. Gevolglik is molekulêre identifikasie- en karakteriseringstudies geloods om te bepaal watter spesies Eutypa terugsterwing in Suid-Afrikaanse wingerde veroorsaak. Dit het die molekulêre analise van drie molekulêre merkers behels, naamlik die interne getranskribeerde spasiëerderarea (“ITS”), die groot ribosomale subeenheid (“LSU rDNA”) en β-tubilien geen. Resultate van die filogenetiese analise dui daarop dat ’n tweede spesies, naamlik Eutypa leptoplaca (Mont.) Rappaz, saam met E. lata in geïnfekteerde plante voorkom. Saam met bogenoemde twee spesies het daar ook verwante spesies van die Diatrypaceae familie voorgekom, naamlik Cryptovalsa ampelina (Nitschke) Fuckel en Eutypella vitis (Schwein.) Ellis & Everhart. Patogenisiteitstudies wat uitgevoer is met verteenwoordigende isolate van C. ampelina, E. lata, E. leptoplaca, en E. vitis dui daarop dat almal patogene van wingerd is. Verskeie Botryosphaeriaceae spesies wat gereeld in houtagtige wingerdweefsel aangetref word, is ook patogene van wingerd. Interne simptome wat algemeen met Botryosphaeriaceae infeksies geassosieer word, kan baie maklik met dié van Eutypa terugsterwing verwar word en dit het die nood laat ontstaan om ‘n opsporingsmetode te ontwikkel wat akkuraat genoeg is om tussen veelvoudige infeksies te onderskei. ’n Omgekeerde-stippelklad-hibridisasie (OSH) metode is gevolglik aangewend om Eutypa spesies betrokke in die Eutypa-siektekompleks op ‘n vinnige, akkurate en betroubare manier op te spoor, sowel as die Botryosphaeriaceae species wat bekend is as patogene van wingerd. Die metode behels ’n saamgestelde PKR vir die vermeerdering en merk van DNS areas wat gebruik word as patogeen spesifieke peilers. Spesies-spesifieke oligonukleotiede ontwikkel vanaf die ITS, -tubilien en LSU molekulêre data is op ‘n membraan vasgeheg en gebruik om ’n diagnostiese toets te ontwikkel vir Eutypa species. Merkers ontwikkel vanaf die ITS kon E. lata and E. leptoplaca konsekwent opspoor. Die opspoor van Eutypa spesies met merkers vanaf die -tubulien en LSU gene met OSH was onsuksesvol. Die OSH metode met merkers vanaf die verlengingsfaktor-1α kon susksesvol gebruik word om Botyrosphaeria dothidea (Moug.:Fr.) Ces. & De Not., Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, A.J.L. Phillips and Neofusicoccum ribis (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips op te spoor. Dié metode kon egter nie Diplodia seriata De Not. opspoor nie. Bykomend tot bogenoemde tekortkominge, kon die omgekeerde-stippelklad-hibridisasie metode ook nie aangepas word om patogene direk vanuit plantmateriaal op te spoor nie en word DNS afkomstig vanaf suiwer kulture benodig. Dié metode laat egter identifikasie van verskeie patogene in ‘n enkele toets toe. Soos DNS ekstraksie metodes aangepas, verbeter en verfyn word om DNS vanuit plantmateriaal te verkry, sal die bruikbaarheid van die omgekeerde stippelklad hibridisasie metode ook verbeter.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The search for a cost-effective, environmentally friendly replacement for fossil fuels resulted in bio-ethanol production receiving a lot of attention. Lignocellulose, is considered to be the most abundant renewable source on earth, and consists of cellulose, hemicellulose and lignin. Exploitation thereof as a substrate for ethanol production, can serve as solution in producing bio-ethanol as an adequate replacement for fossil fuels. Hemicelluloses, contributing up to a third of the lignocellulosic substrate, consists mainly of xylan and mannan and can be degraded by hemicellulolytic enzymes that are produced by plant cell wall degrading organisms. Galactoglucomannan is the most complex form of mannan and requires a consortium of enzymes for complete hydrolysis. These enzymes include β-mannanase, β-mannosidase, α-galactosidase, β-glucosidase and galactomannan acetylesterases. Saccharomyces cerevisiae is a well-known fermentative organism that has been used in various industrial processes and is able to produce ethanol from hexose sugars. Although this organism is unable to utilize complex lignocellulosic structures, DNA manipulation techniques and recombinant technology can be implemented to overcome this obstacle. Strains of S. cerevisiae pose other shortcomings like hyperglycosylation and therefore other non-conventional yeasts (such as Kluyveromyces lactis) are now also being considered for heterologous protein production. The mannanase gene (manI) of Aspergillus aculeatus was expressed in K. lactis GG799 and S. cerevisiae Y294. K. lactis transformants were stable for two weeks in consecutive subcultures and secreted a Man1 of 55 kDa. The recombinant Man1 displayed an optimum temperature of 70°C and a pH optimum of 5 when produced by K. lactis. Activity levels of about 160 – 180 nkat/ml was obtained after 86 hours of cultivation, which was similar to the activity observed with S. cerevisiae under the same conditions. Disruption of the ku80 gene did not contribute to the stability of the cultures and a heterogeneous culture developed for 10 days of consecutive subculturing. The mannosidase gene (man1) from A. niger and mannanase gene (manI) from A. aculeatus were constitutively expressed in S. cerevisiae Y294 and S. cerevisiae NI-C-D4. The MndA and Man1 proteins appeared as a 140 kDa and 58 kDa species on the SDS-PAGE analysis when expressed in S. cerevisiae Y294, respectively. MndA had an optimum temperature of 50°C and optimum pH 5. Man1 produced by S. cerevisiae Y294 indicated a pH optimum of 6 and temperature optimum of 70°C. The MndA displayed low levels of endomannanase activity and no β-mannosidase activity could be detected. Co-expression of man1 and mndA in either S. cerevisiae Y294 and S. cerevisiae NI-C-D4, resulted in less hydrolysis of galactoglucomannan. An increase in the size of the plasmid generally results in a decrease in the copy number, leading to a decrease in the amount of ManI protein being produced. The co-expression of ManI and MndA could also have resulted in a higher metabolic burden on the cell, hence the amount of ManI are produced. This study confirms that more research should be done on the evaluation of alternative hosts for expression of foreign proteins. Furthermore, producing enzymes cocktails for industrial application should be considered rather than co-expression of various enzymes in one host. / AFRIKAANSE OPSOMMING: ‘n Behoefte na ‘n koste-effektiewe en omgewingsvriendelike vervoer brandstof is besig om toe te neem. Lignosellulose word beskou as die volopste hernubare bron vir biobrandstof en lignosellulose bestaan uit sellulose, hemisellulose en lignien. Die gebruik daarvan vir die produksie van bio-etanol kan ’n voldoende alternatief vir fossielbrandstowwe bied. Verbruik van lignosellulose as bron vir die produksie van biobrandstof bied ’n oplossing vir die energie krises. Hemisellulose vorm ’n derde van lignosellulose substraat en bestaan uit xilaan en mannaan en word deur hemisellolitiese ensieme afgebreek wat algemeen by plantselwand-verterende organismes voorkom. Galaktoglukomannaan is die mees komplekse vorm van mannaan en benodig verskeie ensieme vir volkome hidroliese. Hierdie ensieme sluit in β-mannanase, β-mannosidase, α-galaktosidase, β-glukosidase en galaktomanaan asetielesterases. Saccharomyces cerevisiae is ‘n bekende fermenterende organisme wat gereeld in verskeie industriële prosesse gebruik word en kan etanol van heksose suikers produseer. Die organisme beskik nie oor die vermoë om komplekse polisakkarides wat in lignosellulose voorkom te hidroliseer nie maar. DNS-manipuleringstegnieke en rekombinante tegnologie maak dit egter moontlik die probellm te oorbrug. S. cerevisiae het nogtans tekortkominge soos hiperglikosilering en daarom word ander nie-konvensionele giste (soos Kluyveromyces lactis) tans ook vir die produksie van rekombinante proteine ondersoek. Die mannanase geen (manI) vanaf Aspergillus aculeatus is in K. lactis GG799 en S. cerevisiae Y294 uitgedruk. K. lactis transformante was stabiel vir twee weke in opeenvolgende subkluture en het ‘n Man1 van 55 kDa geproduseer. Die rekombinante Man1 ensiem het ‘n temperatuur optimum van 70°C en pH optimum van 5.0 getoon in K. Lactis. Aktiwiteitsvlakke van 160 – 180 nkat/ml was bereik na 86 uur klutivering, In vergelyking met S. cerevisiae was aktiwiteitsvlakke eenders oor ‘n periode Die disrupsie van die ku80 geen het geen effek op die stabiliteit van die transformante in 10 dae opeenvolgende sub-kulture getoon nie. Die mannosidase geen (mndA) vanaf Aspergillus niger en die mannanase geen (man1) van Aspergillus aculeatus is konstitutief in S. cerevisiae Y294 en S. cerevisiae NI-C-D4 uitgedruk. Uitdrukking van die MndA en Man1 proteïen in S. cerevisiae Y294 het onderskeidelik ‘n 140 kDa en 58 kDa spesie getoon met SDS-PAGE analisering. Die MndA ensiem het ‘n temperatuur optimum van 50°C and pH optimum van 5.0 getoon. Man1 het ‘n pH optimum van 6.0 en ‘n temperatuur optimum van 70°C getoon. MndA het lae hidrolitiese aktiwiteit op galaktoglukomannaan, maar geen β-mannosidase aktiwiteit getoon nie. Wanneer man1 and mndA saam in S. cerevisiae Y294 en S. cerevisiae NI-C-D4 uitgedruk is, het die hidroliese van galaktoglukomannan dramaties afgeneem. ‘n Toename in die grootte van ‘n plasmied veroorsaak dikwels ‘n afname in kopiegetal wat die produksie van ManI verlaag. Die ko-uitdrukking van ManI en MndA kan ook tot ’n hoër metaboliese las lei en dus die laer produksie van ManI. Resultate in hierdie studie wys daarop dat meer navorsing benodig word in die soeke na alternatiewe gashere vir uitdrukking van mannanases. Ensiem mengsels vir industriële toepassings behoort eerder gebruik te word as die ko-ekspressie van verskeie ensieme in ’n enkel gasheer.