Aeromonas spp. isolates, which are fish and opportunistic human pathogens, form biofilms, however the factors involved in and affecting biofilm formation have not been fully elucidated. Biofilm formation is affected by motility, cell surface characteristics, and/or metabolism, thus it is important to identify factors potentially contributing to initial attachment and/or biofilm formation and their correlation with biofilm formation by Aeromonas spp. isolates. With knowledge of the stages of biofilm formation, mechanisms involved in biofilm formation and its physiology, various strategies may be applied to control aeromonad biofilms. Factors potentially involved in initial attachment and/or biofilm formation were investigated for 99 Aeromonas isolates obtained from seawater and cultured fish. Aeromonad biofilm formation was assessed using microtiter plate assays under varying physicochemical conditions. The disk diffusion method was used to determine the antimicrobial susceptibility profiles of isolates, for comparison to clinical and aquaculture isolates reported in other studies. The MICs and MBICs for antimicrobial agents (azithromycin, ceftazidime, ciprofloxacin, gentamicin and tetracycline) of planktonic cells and biofilm cells, respectively, were investigated using the broth microdilution and modified microtiter plate assays. The effect of sub-MIC (0.5 × MIC) and supra-MIC (2 × MIC) exposures on biofilm-forming cells was also determined using microtiter plate assays. The presence of efflux pump-mediated resistance in 45 Aeromonas spp. isolates was determined using the disk diffusion assay incorporating efflux pump inhibitors (EPIs) [carbonyl cyanide 3-chlorophenylhydrazone (CCCP), phenylalanine arginine β-naphthylamide (PAβN) and 1-(1-naphthylmethyl)-piperazine (NMP)]. Modified microtite plate assays were used to determine the effect of EPIs [CCCP, PAβN, and NMP], matrix-degrading DNase I and quorum-sensing inhibitors (QSIs; vanillin, 2(5H)-furanone, S-adenosylhomocysteine and cinnamaldehyde) on initial attachment and mature biofilm. Majority of isolates were motile by swimming and swarming and displayed caseinase, gelatinase, and DNase activities, as well as an A-layer phenotype. Majority of isolates displayed high levels of autoaggregation and were hydrophilic. Isolates showed varying levels of adherence, but majority were strongly adherent in nutrient-rich media at 30 ºC. Motility appeared to be a significant characteristic for biofilm formation. Majority of Aeromonas isolates spp. showed high levels of resistance to β-lactams, trimethoprim and sulphamethoxazole, and were susceptible to augmentin, piperacillin-tazobactam, aztreonam, 2nd and 3rd generation cephalosporins, carbapenems, macrolides, fluoroquinolones and aminoglycosides . High levels of resistance towards ceftazidime (MIC > 32 μg/ml) were observed for isolates, while levels of resistance towards remaining antimicrobial agents tested (tetracycline, azithromycin, ciprofloxacin, and gentamicin) were ≤ 32 μg/ml. There was a ≥16-fold increase in MBICs (4096 μg/ml) compared to the MICs for all the antimicrobial agents. The sub-MIC, MIC, and supra-MIC exposures of all antimicrobial agents had an inhibitory effect on both initial attachment and pre-formed biofilms by Aeromonas spp. isolates. Majority of isolates were more susceptible to tetracycline, norfloxacin, and azithromycin due to CCCP and NMP inhibition of the efflux pumps eliminating these antimicrobial agents. Susceptibility to erythromycin was observed for 51% and 47% of isolates, respectively, due to NMP and PAβN inhibition of the efflux pump/s eliminating erythromycin. In the microtiter plate assays, CCCP, NMP and PABN exposures resulted in significant reduction of biofilm formation by majority of Aeromonas spp. isolates in both initial attachment and mature biofilm assays, with CCCP being more effective. DNase I was more effective in reducing mature biofilm, causing reduction for 60% of isolates, compared to its effect on initial attachment. QSIs were also more effective in reducing mature biofilm compared to inhibiting initial attachment. Although increased biofilm dispersal was observed with all QSIs, vanillin and 2(5H)-furanone were more effective compared to S-adenosylhomocysteine and cinnamaldehyde. Based on data obtained in this study, antimicrobial agents, EPIs and QSIs can be used as potential biofilm-inhibiting compounds in aquaculture to control aeromonad infections and may not only prevent disease outbreaks but they could also increase the effectiveness of existing therapeutic agents. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

Xylan is the major component of hemicellulose and its degradation can be achieved through the hydrolytic action of microbial xylanases. Xylanases have an array of applications one being bioethanol production. The lack of thermophilic xylanases has prompted the search for new enzymes with increased thermostability. Previous work on the crude enzyme of Phialophora alba has demonstrated optimal activity (39 U/μg) at a pH of 4 and two temperature optima of 50°C and 90°C. These desirable properties highlighted the need for further research on the purified enzyme. In the present study P. alba was identified as a thermophilc Ascomycete that forms conidia and chlamydospores during the asexual and sexual stages of its life cycle, respectively. The various isozymes present in the crude enzyme extract were subsequently detected by zymogram analysis. Up to six xylanase isozymes ranging from 90-210 kDa in size were detected. The crude enzyme was subsequently purified by precipitation and ion exchange chromatography (IEX). Protein precipitation methods, desalting methods, IEX resins, elution buffers and NaCl gradients were optimized. The 31-70% ammonium sulphate precipitate had the highest levels of xylanase activity. Separation of proteins with the anion exchanger, HiTrap Q sepharose fast flow column and a linear gradient of 0-2.5 M NaCl in phosphate buffer (50 mM, pH 7) yielded a partially pure xylanase isozyme with molecular weight of 210 kDa. A final yield of 1.4% and purification fold 10.6 was obtained after ion exchange chromatography. The specific activity of the xylanase was 21 IU/μg. At optimum pH (pH 4) and temperature (50°C) a combined xylanase activity of 32 IU.ml⁻¹ was detected. The partially pure xylanase was stable from pH 4-6 with 86% of xylanase activity retained for 90 minutes. Thermostability was observed from 40-70°C with 95% of activity retained for 90 minutes at optimum temperature. The ability of the partially pure xylanase and crude enzyme to hydrolyze untreated and pretreated (alkali and temperature/pressure) sugarcane bagasse was tested at a constant enzyme loading rate of 15 IU/g. Overall, maximum hydrolysis was achieved with the alkali pretreatment and saccharification with the crude enzyme: approximately, 2.4 g/ml of reducing sugars were liberated over a 48 hours. The partially pure xylanase liberated a maximum amount of 2.3 g/ml reducing sugars after 48 hours. The results obtained highlight the desirable characteristics of the partially pure enzyme and its applicability to bioethanol production. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

No abstract available. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1976.

The production of 1,2-Dichloroethane (1,2-DCA) exceeds 5.44 billion kg per year, and is higher than that of any other industrial halogenated chemical. Improper disposal practices or accidental spills of this compound have made it a common contaminant of soil and groundwater. 1,2-DCA has been classified as a priority pollutant by the Environmental Protection Agency owing to its toxicity, persistence and bioaccumulation in the environment. It has also been shown to have mutagenic and potential carcinogenic effects on animals and humans. Bioremediation is emerging as a promising technology for the clean-up of sites contaminated with chlorinated hydrocarbons. However, sites co-contaminated with heavy metals and 1,2-DCA may pose a greater challenge for bioremediation, as the former pollutant could inhibit the activities of microbes involved in bioremediation. Therefore, this study was undertaken to quantitatively assess the effects of heavy metals on 1,2-DCA biodegradation and to investigate the use of biostimulation, bioaugmentation, dual bioaugmentation, and biosorption for remediation of water co-contaminated with 1,2-DCA and heavy metals in microcosms. The combined effect of 1,2-DCA and the respective heavy metals on the microbial population and diversity was also investigated. The minimum inhibitory concentrations (MICs) and concentrations of the heavy metals (arsenic, cadmium, mercury and lead) that caused half-life doubling (HLDs) of 1,2-DCA as well as the degradation rate coefficient (k1) and half-life (t1/2) of 1,2-DCA were measured in two different wastewater types. An increase in heavy metal concentration from 0.01–0.3 mM, resulted in a progressive increase in the t1/2 and relative t1/2 and a decrease in k1. The MICs and HLDs of the heavy metals were found to vary, depending on the heavy metals and wastewater type. In addition, the presence of heavy metals was shown to inhibit 1,2-DCA biodegradation in a dose-dependent manner, with the following order of decreasing inhibitory effect: Hg2+ > As3+ > Cd2+ > Pb2+. For the bioremediation experimental set-up, 150 ml wastewater was spiked with 1,2-DCA (2.5 mM) and the respective heavy metal in air-tight serum bottles (Wheaton). The bottles were biostimulated, bioaugmented, dual-bioaugmented or undergoing biosorption. The microcosms were incubated at 25 °C and the 1,2-DCA concentration was measured weekly. Co-contaminated water undergoing biostimulation, bioaugmentation and, in particular, dual bioaugmentation were observed to exhibit higher degradation of 1,2-DCA in the presence of the heavy metal, compared to co-contaminated water receiving none of the treatments. Dual bioaugmentation, proved to be most effective, resulting in up to 60% increase in 1,2-DCA degradation after 4 weeks, followed by bioaugmentation (55%) and biostimulation (51%). In addition, an increase in the total number of 1,2-DCA degrading bacterial population was observed in the bioaugmentated microcosms compared to those biostimulated, which corresponds to an increased 1,2-DCA degradation observed in the bioaugmentated co-contaminated microcosms. Dominant bacterial strains obtained from the co-contaminated microcosms were identified as members of the genera, Burkholderia, Pseudomonas, Bacillus, Enterobacter and Bradyrhizobiaceae, that have been previously reported to degrade 1,2-DCA and other chlorinated compounds. Some of these isolates also belong to genera that have been previously shown to be resistant to heavy metals. PCR-DGGE analysis revealed variations in microbial diversity over time in the different co-contaminated microcosms, whereby the number of bands was reduced, the intensity of certain bands increased, and new bands appeared. Agricultural biosorbents (AB) were found to adsorb heavy metals effectively when utilized at a concentration of 2.5%, with the level of biosorption found to be dependent on the type of AB as well on the type of heavy metal present. OP proved to be the most efficient biosorbent for the heavy metals tested, followed by CNF, and corn cobs (CC) least efficient; therefore CC was not used in further bioremediation experiments. Both orange peel (OP) and coconut fibre (CNF) were found to be excellent at removing heavy metals from co-contaminated microcosms, with OP removing 14.59, 74.79, 60.79 and 87.53% of As3+, Cd 2+, Hg2+ and Pb2+, respectively, while 10.03, 40.29, 68.47 and 70.00% of As3+, Cd2+, Hg2+ and Pb2+, respectively, was adsorbed by CNF. Consequently, a higher degradation of 1,2-DCA was observed in the presence of OP and CNF, compared to the untreated control. It can be concluded that the remediation approaches utilized in this study proved effective in the bioremediation of water co-contaminated with 1,2-DCA and heavy metals and may provide the foundation for new and innovative treatment strategies for co-contaminated sites. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2011.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Saccharomyces cerevisiae is commonly associated with the wine industry. However, this yeast was also isolated from soils not associated with vines. Despite the fact that S. cerevisiae is not perceived as an autochthonous soil yeast, its interaction with other soil microbiota suggests the contrary. Aside from a few in vitro studies, the fate of S. cerevisiae in soil is largely unknown. This may partly be ascribed to the lack of reliable methods to enumerate fermentative yeasts in soil. Consequently, we evaluated an enumeration procedure for fermentative yeasts in soil, whereby yeast malt extract (YM) agar plates containing selective agents, were incubated in anaerobic jars before the colonies were enumerated. This procedure proved to be selective for fermentative yeasts, such as industrial strains of S. cerevisiae. We then commenced studying the growth and survival of S. cerevisiae in soil differing in moisture content and nutrient levels, using S. cerevisiae strain S92 and the genetically modified strain S. cerevisiae ML01, as well as two autochthonous soil yeasts, Cryptococcus laurentii and Cryptococcus podzolicus. The yeast strains were each inoculated into three series of microcosms containing sterile soil with a moisture content of ca. 30% (v/w), a moisture content of ca. 15% (v/w), or a moisture content of ca. 30% supplemented with nutrients used in agriculture. Growth of each strain was monitored for a period of 48 days and all the yeasts were found to grow or survive under these conditions, up until the end of the incubation period. Generally, the cryptococci reached larger population sizes in the soil than the Saccharomyces strains, which may be due to their ability to utilize a wider range of carbon sources and to survive in semi-arid soils. Aside from cell numbers observed in nutrient supplemented soil, in which S. cerevisiae ML01 reached higher numbers than S92, there was no significant difference between the growth and survival of the Saccharomyces strains. In all the microcosms, metabolic rates, as determined by measuring CO2 emissions from soil, reached a maximum within the first day and then declined over the remainder of the trial, possibly due to depletion of nutrients. Differences in CO2 emissions from the different series of microcosms were attributed to different metabolic rates and energy expenditure needed to maintain yeast populations under different conditions. Each of the above-mentioned yeasts was subsequently inoculated in a microcosm prepared from non-sterile soil and monitored using selective enumeration procedures. The Saccharomyces strains were enumerated using the above-mentioned soil dilution plates incubated in anaerobic jars. The presence of natural soil biota caused a decrease in viable yeast numbers for all strains and this was ascribed to competition with and predation by other soil borne organisms. Further evidence for competition and/or amensalism impacting on Saccharomyces populations in soil was obtained when monitoring co-cultures of Saccharomyces with C. laurentii 1f and C. podzolicus 3f in soil microcosms, revealed a significant reduction in Saccharomyces numbers during a 28 day incubation period. However, when the two Saccharomyces strains were cultured in soil microcosms inoculated with a protistan predator, populations of both strains increased and remained at these high levels for the duration of the trial. These findings point to a possible symbiosis between Saccharomyces and the protista whereby the predators ensure continuous nutrient cycling within the soil microcosms. In the final part of the study, epifluorescence microscopy revealed that, similar to known soil cryptococci, the two Saccharomyces strains were able to form biofilms in oligotrophic conditions. The results of this study showed that in the presence of natural soil microbes, no differences exist between the growth and survival of S. cerevisiae S92 and S. cerevisiae ML01. Also, the findings point to a natural niche for this species somewhere in the soil habitat. / AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae word algemeen met die wynindustrie geassosieer. Hierdie gis is egter ook uit grond geïsoleer wat nie met wingerd geassosieer word nie. Ten spyte van die feit dat S. cerevisiae nie as ‘n outogtoniese grondgis beskou word nie, dui sy interaksie met ander grondmikrobiota op die teendeel. Behalwe vir ‘n paar in vitro studies, is die lot van S. cerevisiae in grond grootliks onbekend. Dit mag gedeeltelik aan die gebrek aan betroubare metodes om fermenterende giste in grond te tel, toegeskryf word. Ons het gevolglik ‘n tellingsmetode vir fermenterende giste in grond geëvalueer waarin gis-mout ekstrak (GM) agar plate, bevattende selektiewe agente, in anaërobiese flesse geïnkubeer is voordat die kolonies getel is. Hierdie metode was selektief vir fermenterende giste, soos die industriële stamme van S. cerevisiae. Hierna is die groei en oorlewing van S. cerevisiae bestudeer in gronde met verskillende vog- en nutriëntvlakke deur gebruik te maak van S. cerevisiae stam S92 en die geneties gemodifiseerde stam S. cerevisiae ML01, asook twee outogtoniese grondgiste, Cryptococcus laurentii en Cryptococcus podzolicus. Die gisstamme is elk geïnokuleer in drie reekse van mikrokosmosse bestaande uit steriele grond met ‘n vogvlak van ca. 30% (v/w), ‘n vogvlak van ca. 15% (v/w), of ‘n vogvlak van ca. 30% aangevul met landbounutriënte. Die groei van elke stam is waargeneem vir ‘n tydperk van 48 dae en al die giste het onder hierdie omstandighede tot aan die einde van die inkubasietydperk gegroei of oorleef. Oor die algemeen het die cryptococci groter populasies in die grond gevorm as die Saccharomyces stamme, wat toegereken kan word aan hul vermoë om ‘n wyer reeks koolstofbronne te benut en om in droë gronde te oorleef. Behalwe dat S. cerevisiae ML01 ‘n hoër aantal selle in nutriënt aangevulde grond behaal het as S92, was daar geen beduidende verskil tussen die groei en oorlewing van die Saccharomyces stamme nie. In al hierdie mikrokosmosse het die metaboliese tempo, soos bepaal deur CO2 vrystellings vanuit grond te meet, ‘n maksimum bereik binne die eerste dag en dan het dit afgeneem oor die res van die toetsperiode, waarskynlik as gevolg van die uitputting van die nutriënte. Verskille in die CO2 vrystellings wat vir die verskillende reekse van mikrokosmosse aangeteken is, is te wyte aan die verskillende metaboliese tempo’s en energiegebruik benodig om gispopulasies onder verskillende omstandighede in stand te hou. Elk van bogenoemde giste is vervolgens geïnokuleer in ‘n mikrokosmos wat voorberei is van nie-steriele grond, en waargeneem deur selektiewe enumerasie prosedures toe te pas. Die Saccharomyces stamme is getel deur gebruik te maak van bogenoemde grondverdunningsplate wat in anaërobiese flesse geïnkubeer is. Die teenwoordigheid van natuurlike grondbiota het in alle stamme ‘n afname in lewensvatbare gisgetalle veroorsaak en is toegeskryf aan die kompetisie met en predasie deur ander grondorganismes. Verdere bewys van die impak van kompetisie en/of amensalisme op Saccharomyces populasies in die grond, is die beduidende afname in Saccharomyces getalle tydens ‘n 28 dag inkubasie tydperk, waartydens ko-kulture van Saccharomyces stamme met C. laurentii 1f en C. podzolicus 3f in grond mikrokosmosse ondersoek is. Toe die twee Saccharomyces stamme egter in grond mikrokosmosse opgekweek is wat met ‘n protistiese predator geïnokuleer is, het populasies van albei stamme gegroei en om hierdie hoë vlakke gebly tot aan die einde van die toets. Hierdie bevindings dui ‘n moontlike simbiose tussen Saccharomyces en die protista aan waardeur die predatore deurlopende nutriëntsiklering binne die grondmikrokosmos verseker. In die laaste deel van die studie toon epifluoressensie mikroskopie aan dat, net soos bekende grond cryptococci, die twee Saccharomyces stamme in staat is om biofilms in oligotrofiese omstandighede te vorm. Die resultaat van die studie toon aan dat in die teenwoordigheid van natuurlike grondmikrobe daar geen verskil tussen die groei en oorlewing van S. cerevisiae S92 en S. cerevisiae ML01 is nie. Die bevindings dui ook aan dat daar ‘n natuurlike nis vir hierdie spesie iewers in die grondhabitat is.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Plant biomass is potentially an inexhaustible source of bioenergy. To be more useful in an industrialised context, conversion to liquid biofuel is necessary, which could provide the motor vehicle market with energy. To enable fermentation of both hexose and pentose sugars present in plant biomass, many researchers have introduced eukaryotic D-xylose utilisation metabolic pathways into S. cerevisiae as these yeasts cannot utilise D-xylose. The aim of this study was to increase D-xylose utilisation and lower the xylitol production found with the eukaryotic pathway, thus redirecting carbon to the increased production of ethanol. In order to reduce xylitol yield a two-fold approach was followed. Firstly S. cerevisiae transformed with eukaryotic XR and XDH genes were subjected to random mutagenesis and selection for improved D-xylose utilisation. Unfortunately no mutant superior to the parental strain with respect to D-xylose utilisation, lowered xylitol production and improved ethanol production was obtained. Subsequently a bacterial xylose isomerase (XI) gene was introduced into S. cerevisiae. Bacterial xylose isomerase converts D-xylose to xylulose in a single step, while eukaryotic pathways produce the intermediate xylitol. The chosen gene encodes for a putative xylose isomerase gene (xylA) from the bacterium Bacteroides thetaiotaomicron, which has not previously been transformed into yeast. When the native xylA was expressed in E. coli and S. cerevisiae no XI activity was found, nor growth on D-xylose sustained. Lack of activity was surmised to be due to an amino acid modification, or possibly due to a vastly different codon bias in yeast compared to the Bacteroides strain. Northern analysis revealed that no D-xylose transcript was formed. A synthetic D-xylose isomerase gene (SXI) based on the B. thetaiotaomicron XI amino acid sequence, but optimised for S. cerevisiae codon bias, was designed and manufactured. S. cerevisiae transformed with the synthetic gene showed sustained, non-pseudohyphal growth on D-xylose as sole carbon source, both on solid and liquid medium. This ability to utilise D-xylose represents a significant step for recombinant S. cerevisiae to potentially ferment D-xylose for bioethanol. / AFRIKAANSE OPSOMMING: Plant biomassa is potensieel ‘n onuitputlike bron van bio-energie. Om in die huidige industriële konteks van groter nut te wees, en die motor-industrie met energie te voorsien, is omskakeling na ‘n vloeistof-energievorm nodig. Om die fermentasie van beide heksoses en pentoses teenwoordig in plantbiomassa te bewerkstellig, het verskillende navorsingspanne eukariotiese D-xilose-afbraak metabolise weë na S. cerevisiae oorgedra om dié gis die vermoë te gee om D-xilose af te breek. Die doel van hierdie studie was om D-xilose-verbruik in geneties gemodifiseerde S. cerevisiae te verhoog en die hoeveelheid xilitol wat met die eukariotiese sisteem verkry word, te verminder om ‘n hoë etanol opbrengs te handhaaf. Twee moontlikhede is ondersoek om die xilitol opbrengs te verminder. Eerstens is ‘n rekombinante S. cerevisiae met die xilose reduktase (XR) en xilitol dehidrogenase (XDH) gene aan nie-spesifieke mutagenese onderwerp en vir verbeterde D-xilose verbruik geselekteer. Ongelukkig kon geen mutante wat beter as die oorspronklike ras D-xilose kon gebruik, en etanol produseer met relatief min xilitol opbrengs, gevind word nie. Daarna is ‘n bakteriese D-xilose-afbraak geen na S. cerevisiae oorgedra. Bakteriese xilose isomerases skakel D-xilose om na xilulose in ‘n enkele stap, terwyl die eukariotiese paaie die tussenganger xilitol produseer. Die gekose xylA geen wat vir xilose isomerase (XI) van die bakterium Bacteriodes thetaotaomicron kodeer, is vir die eerste keer in gis getransformeer. Toe die natuurlike xylA geen In E. coli en S. cerevisiae uitgedruk is, is geen XI-aktiwiteit of volhoubare groei op D-xilose waargeneem nie. Die tekort aan aktiwiteit is aan 'n aminosuurverandering, of aan die groot verskil tussen kodonkeuse (“codon bias”) in gis teenoor die Bacteroides ras toegeskryf. Noordkladanaliese het bepaal dat geen mRNA spesifiek tot die XI-geen geproduseer is nie. Die xilose isomerase geen van B. thetaiomicron is toe sinteties ontwerp, met die DNA-volgorde vir die S. cerevisiae kodonkeuse geoptimiseer. S. cerevisiae wat met die sintetiese geen (SXI) getransformeer is, het aanhoudende, nie-pseudohife groei op D-xilose as enigste koolstofbron op beide soliede en in vloeibare medium getoon. Die vermoë om D-xilose te verbruik verteenwoordig ‘n betekenisvolle stap tot die fermentasie van D-xilose na etanol met geneties gemodifiseerde S. cerevisiae.

Thesis (MSc (Microbiology))--University of Stellenbosch, 2005. / Intraspecific diversity among yeasts, including basidiomycetous yeasts has mostly been studied from a taxonomic point of view. The heterobasidiomycetous genus Cryptococcus is no exception and it was found to contain species that display heterogeneity both on a genetic and physiological level, i.e. diversity among strains originating from different geographical areas. It was stated that this diversity within yeast species is possibly caused by intrinsic attributes of the different habitats the strains of a particular species originate from. However, little is known about the diversity of a species within a specific habitat. Thus, in this study intraspecific diversity among selected cryptoccoci isolated from a single soil sample originating from pristine Fynbos vegetation , was investigated.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Members of the genus Sulfobacillus form an important part of the microbial consortia that are active in the biooxidation of sulphide ores in biomining processes, yet very little is known about these industrially important organisms. The study of sulfobacilli, and other biomining organisms, is hampered by the absence of effective gene cloning and inactivation systems. During this study, the groundwork was laid for the development of a genetic system for the genus Sulfobacillus. The plasmid diversity present in industrial and environmental isolates of sulfobacilli was assayed. Plasmids were plentiful in the assayed strains, providing the basis for development of cloning vectors for sulfobacilli. Plasmid DNA isolated from Sulfobacillus thermosulfidooxidans strain DSM 9293T was methylated at dam and dcm sites. Whether the methylase enzymes responsible for this methylation pattern form part of restriction-methylation systems or only play a regulatory role is unknown, but it does indicate the appropriate methylation state of DNA for the transformation of this strain. The DNA sequences of three plasmids originating from sulfobacilli were analysed and compared. There was no significant similarity between the three plasmid sequences, indicating diversity in plasmid genetic load and replication mechanisms. Plasmid pSulfBC1 was predicted to replicate via the rolling circle mechanism, while the replication mechanisms of pKara and pTHWX could not be predicted from sequence data. Two antibiotics, chloramphenicol and tetracyline, were found to be suitable for selection of Sulfobacillus transformants. E. coli – Sulfobacillus shuttle vectors were constructed using the Sulfobacillus plasmid, pKara, as the backbone with a Gram-positive chloramphenicol resistance marker and appropriate elements allowing replication in, and mobilization from, E. coli. These shuttle vectors were used in the evaluation of electroporation and conjugation as methods for the delivery of DNA to Sulfobacillus. Transformants of sulfobacilli could not be obtained by either transformation method, although some progress was made towards determining the optimal conditions for both methods. The most promising finding was that cells of E. coli and Sulfobacillus could be maintained on the same medium for a theoretically sufficient time to allow mating. It is likely that Sulfobacillus transconjugants can be obtained with the right combination of donor, mobilizable vector, selectable marker and treatment to neutralize restriction systems.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Lactobacillus plantarum JW3BZ and Lactobacillus fermentum JW15BZ isolated from boza, a Bulgarian cereal based fermented beverage, produce bacteriocins JW3BZ and JW15BZ active against a wide range of food spoilage and pathogenic bacteria. Strains JW3BZ and JW15BZ are resistant to low pH (pH 2.0–4.0). Both strains grow well in MRS broth with an initial pH ranging from 5.0 to 10.0. Strain JW3BZ displayed intrinsic resistance to bile salts. Strain JW15BZ, on the other hand, is sensitive to bile salts exceeding concentrations of 0.3% (w/v). Both strains are weakly hydrophobic and are resistant to a wide range of antibiotics, antiinflammatory drugs and painkillers. Strains JW3BZ and JW15BZ adhered at 4% to Caco-2 cells and they did not compete with Listeria monocytogenes Scott A for adhesion. A homologue of MapA, a gene known to play a role in adhesion, was detected in L. plantarum JW3BZ. Both strains have high auto- and co-aggregation properties. Bacteriocin JW15BZ was partially purified with ammonium sulfate, followed by separation on Sep-Pak C18 and reverse phase High Pressure Liquid Chromatography (HPLC). Two separate peaks with antimicrobial activity were recorded for bacteriocin JW15BZ, suggesting that it consists of at least two antimicrobial peptides. Lactobacillus plantarum JW3BZ contains genes homologous to plnE, plnF and plnI of the plnEFI operon that encode for two small cationic bacteriocin-like peptides with double-glycine-type leader peptides and its respective immunity proteins. The antimicrobial activity displayed by strain JW3BZ may thus be ascribed to the production of plantaricins E and F. Bacteriocin JW3BZ and JW15BZ displayed activity against herpes simplex virus (HSV-1) (EC50=200 μg/ml). Both strains were identified in boza after 7 days at storage at 4 oC and repressed the growth of Lactobacillus sakei DSM 20017, indicating that the bacteriocins are produced in situ. The sensory attributes of boza prepared with different starter cultures did not vary considerably, although statistical differences were observed for acidity and yeasty aroma. Encapsulation of strain JW3BZ and JW15BZ in 2% sodium alginate protected the cells from low pH (1.6) and 2.0% (w/v) bile. The rate at which cells were released from the matrix varied, depending on the conditions. Better survival of strains JW3BZ and JW15BZ encapsulated in 2% (w/v) alginate was observed during 9 h in a gastro-intestinal model. Highest release of cells was observed at conditions simulating colonic pH (pH 7.4), starting from 56-65% during the first 30 min, followed by 87%. Complete (100%) release was recorded after 2.5 h at these conditions. Strains JW3BZ and JW15BZ could be used as starter cultures in boza. The broad spectrum of antimicrobial activity of bacteriocins JW3BZ and JW15BZ is an added advantage, rendering the cells additional probiotic properties. Encapsulation of the cells in alginate gel increased their resistance to harsh environmental conditions and may be the ideal method to deliver viable cells in vivo.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / The genus Fusarium contains members that are phytopathogens of a number of agricultural commodities causing severe diseases such as wilts and rots. Fusarium species also secrete mycotoxins that have devastating effects on humans and animals. The ability of Fusarium species to change their genetic makeup in response to their immediate environment allows these fungi to exist in diverse habitats. Due to the ubiquitous nature of Fusarium, it forms part of the fungal communities in both agricultural and native soils. Fynbos is the major vegetation type of the Cape Floristic Region (CFR), which is a region that is renowned for its high plant species diversity and endemism. In this study, the occurrence and distribution of Fusarium species in indigenous fynbos soils and associated plant debris is investigated. In addition, the phylogenetic relationships between Fusarium species occurring in this particular habitat are evaluated. Fusarium isolates were recovered from soils and associated plant debris, and identified based on morphological characteristics. The morphological identification of isolates was confirmed using Polymerase Chain Reaction (PCR) based restriction fragment length polymorphism (RFLP) analyses of the translation elongation factor 1 alpha (TEF-1α) and internal transcribed spacer (ITS) regions. Furthermore, phylogenetic relationships between Fusarium species were based on the TEF-1α, ITS and β-tubulin gene regions. One-hundred-and-twenty-two (122) Fusarium strains were isolated from the fynbos soils in the Cape Peninsula area (Western Cape). Based on both morphological and molecular identification, the most prevalent Fusarium species in the fynbos soils were F. oxysporum Schlecht. emend. Snyd. and Hans., F. solani (Martius) Appel and Wollenw. emend. Snyd. and Hans., F. equiseti (Corda) Sacc. and an undescribed Fusarium species. Fusarium oxysporum was the dominant species in fynbos soils and strains of this species displayed significant genetic variability. Some strains of both F. oxysporum and F. solani showed close phylogenetic affinities to formae speciales (strains pathogenic to specific plant hosts) in the phylogenetic analyses. However, no diseased plants were observed in and within the vicinity of our sampling sites. In the third chapter, the undescribed Fusarium strains are described as Fusarium peninsulae prov. nom. Morphologically these strains are characterized by falcate macroconidia produced from brown sporodochia. The macroconidia are pedicellate, falcate to curved with hooked apical cells. Also, this fungus produces apedicellate mesoconidia on polyphialides in the aerial mycelium and forms microconidia sparsely. Chlamydospores are formed abundantly on aerial mycelium and submerged hyphae. All these morphological characteristics closely relate this fungus to F. camptoceras species complex in Fusarium section Arthrosporiella. However, phylogenetic analysis based on the ITS sequences differentiate these strains from F. camptoceras and other related species in section Arthrosporiella. Considering the fact that both as phytopathogens and saprophytic fungi, Fusarium species secrete a variety of cell wall degrading enzymes such as cellulases and xylanases. These enzymes allow the fungi to degrade the plant cell wall components to obtain nutrients. In Fusarium, notably endoxylanases play a role in phytopathogenesis of these fungi. Endoxylanase enzymes from F. oxysporum f. sp. lycopersici, F. verticillioides and F. graminearum have been characterized. In this final chapter, the use of the endoxylanase encoding gene, as a molecular marker in phylogenetic analysis was evaluated using F. graminearum (Fg) clade species as model. Degenerated primers were designed and the endoxylanase region amplified by PCR, cloned and sequenced. PAUPgenerated neighbour-joining analysis of the endoxylanase (XYL) region enabled all species to be distinguished and was as informative as the analysis generated with UTPammonia ligase (URA), phosphate permase (PHO), reductase (RED) and trichothecene 3- О-acetyltransferase (TRI101). Furthermore, the results of the phylogenetic analysis of XYL showed better species resolution in comparison to the analysis of the structural genes (TEF-1α and histone H3). Overall, the results demonstrated that phylogenetic analysis of XYL combined with other functional genes (URA, PHO, RED and TRI101) clearly distinguished between the Fg clade species far better than the analysis of structural genes (TEF-1α and histone H3).