The object of the present investigation was to determine quantitatively the extent of glucose and d- and l-arabinose utilization in order to separate it from the other two factors influencing the pH change in the medium. The pH of the carbohydrate media employed was determined potentiometrically, using a glass electrode in order that the pH produced could be compared with the extent of utilization of these carbohydrates. Likewise, the pH produced by the same <i>Bacilli</i> in peptone alone was determined so that it could be compared with the pH produced in the carbohydrate-peptone media. The fundamental observations concerned with the metabolism of the genus <i>Bacillus</i> (which may be derived from this study) may be of value in the taxonomic identification of various members of this group of organisms, and might lead to other interesting conclusions regarding the metabolism of these organisms in pure laboratory culture and also in mixed cultures in the natural habitat. / Ph. D.

Twenty-eight different species of the genus Bacillus have been examined both as to morphological characteristics and physiological reactions. The physiological reactions tested include gelatin liquefactions, acetyl-methyl-carbinol production, nitrate reduction and 36 different carbohydrate fermentations. The action of all 78 strains of the 28 species was also determined on peptone and graphs have been prepared comparing this action with that of the organisms of the carbohydrates. Definite explanations are given for many of the inconsistencies existing in the literature for the fermentation reactions of the bacilli. The results obtained are compared with those recorded by three previous workers, namely Fitzgerald (8), Soriano (29) and Bergey’s Manual (1). Reasons have been advanced to explain cases of disagreement. These are concerned mainly with the indicator used and the failure to take into account the action of the organisms under investigation upon the basal medium. Where it was felt that a representative number of strains were examined, definite conclusions as to the ability of the organisms to dissimilate various carbohydrates are drawn. A key for the classification of the B. mesentericus group has been formulated based entirely on carbohydrate fermentations. The procedure followed appears to be applicable to other groups as well as to the entire genus. / Master of Science

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Rep-PCR was used to compare the banding patterns of each sample against B. cereus ATCC14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe, and were capable of causing gastroenteritis in consumers. / Department of Biology

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Esterases play a variety of roles in nearly every aspect of life ranging from cellular metabolism, signal transduction to defence mechanisms in plants. One aspect of esterases that recently is receiving more attention is the role esterases play in the .degradation of plant material. With fossil fuels (coal and oil) estimated to run out in the next 20 to 30 years, renewable sources such as plant biomass are becoming increasingly important. Plant biomass contains hemicellulosic and cellulosic materials that need to be degraded to their different constituents before they can be optimally used for the production of commodities. Although the enzymes needed to hydrolyse the xylan backbone (xylanases and P-xylosidases) are important, enzymes that remove side chains from the polymer are equally important. They facilitate hydrolysis by xylanases and P-xylosidases and will improve the availability of monomeric sugars for utilisation when used in conjunction with other xylanolytic enzymes. Many of these side-chains are esters and they need to be removed through the action of esterases, either directly from the xylan backbone or from shorter xylo-oligomers. An existing genomic DNA library of Bacil/us pumilus in Escherichia coli was screened for the presence of an acetyl esterase encoding gene. Positive clones were identified by the formation of clearing zones on plates containing glucose pentaacetate. Plasmid DNA was isolated from a positive E. coli clone. The DNA insert was sequenced and found to contain two open reading frames, one of which encoded a novel esterase (estA). Using different primers the gene was amplified by polymerase chain reaction and inserted into an inducible expression vector (pKK223- 3) for expression in E. coli. The plasmid was introduced into E coli and the esterase activity determined, using the chromogenic substrate a-naphthyl acetate. Activity levels decreased shortly after induction with IPTG and therefore plasmid pAP4 was used for enzymatic assays. Cultures containing plasmid pAP4 produced extracellular activity of 2.5 nkatal/ml. The pH and temperature optima as well as temperature stability of the enzyme was determined. The enzyme exhibited optimal activity at pH 6 and 60°C and was stable at 60°C after 2 h. Enzyme assays on different substrates yielded activity on methylumbelliferyl butyrate and methylumbelliferyl acetate in addition to the glucose pentaacetate and a-naphthyl acetate. The estA gene was cloned into a yeast expression vector between the PGK promoter and terminator sequences for expression of the gene in Saccharomyces cerevisiae. The estA open reading frame was also fused to the MFa 1 secretion signal for secretion of the protein from S. cerevisiae. The expression vector was successfully transformed into S. cerevisiae, but no extracellular activity was detected. Only low intracellular activity of 0.260 nkatal/ml was detected in S. cerevisiae. / AFRIKAANSE OPSOMMING: Esterases speel 'n verskeidenheid van rolle in feitlik elke aspek van lewe, van sel metabolisme, sein transduksie tot verdedigingsmeganismes in plante. Een aspek van esterases wat al hoe meer aandag geniet, is die rol wat esterases in die afbraak van plant en plantmateriaal speel. Met olie- en steenkoolbronne wat na beraming oor 20 tot 30 jaar tot niet sal gaan, raak die rol wat hernubare bronne speel al hoe belangriker. Plantbiomassa bevat sellulose en hemisellulose wat tot die verskillende komponente afgebreek moet word voordat dit optimaal vir die vervaardiging van produkte aangewend kan word. Alhoewel die ensieme wat vir die hidrolise van die xilaanruggraat benodig word, (xilanases en ~-xulosidases) belangrik is, is die ensieme wat die sygroepe vanaf die polimeer verwyder ewe belangrik aangesien hulle die hidrolise deur xilanases en ~-xulosidases bevorder. Wanneer hulle saam met die ander xilanolitiese ensieme gebruik word, sal hulle die beskikbaarheid van monomeriese suikers vir fermentasie verhoog. Baie van hierdie sygroepe is esters en hulle word deur die aksie van esterases verwyder, of direk van die ruggraat, ofvanafkorter xilo-oligosakkariede. 'n Bestaande genoom DNA biblioteek van Bacillus pumilus in Escherichia coli is vir die teenwoordigheid van 'n asetielesterase-koderende geen gesif. Positiewe klone is deur die vorming van 'n sone op plate wat glukose pentaasetaat bevat, geïdentifiseer. Die DNA-invoeging van die positiewe E. coli-kloon se DNA-volgorde is bepaal en twee oopleesrame is gevind waarvan een vir 'n unieke esterase (estA) kodeer. Met behulp van verskillende inleiers is die geen met die polimerasekettingreaksie (PKR) geamplifiseer en in 'n induseerbare promotor vir uitdrukking in E. coli gekloneer. Die plasmied is getransformeer in E. coli en aktiwiteit is bepaal deur cc-naftielasetaatte gebruik. Vlakke van aktiwiteit het kort na induksie met IPTG weer gedaal en daarom was plasmied pAP4 vir ensiematiese toetse gebruik. E. coli-transformante met plasmied pAP4 het ekstrasellulêre aktiwiteit van 2.5 nkatal/ml gelewer. Die pH en temperatuur optima sowel as die temperatuurstabiliteit van die ensiem was bepaal. Die ensiem toon optimale aktiwiteit by pH 6 en 'n temperatuur van 60°C. Aktiwiteitstoetse op verskillende substrate het aktiwiteit op metielumbelliferielasetaat en metielumbelliferielbutiraat bo-en-behalwe die glukosepentaasetaat en c-naftielasetaar getoon. Die estA geen is in uitdrukkingskasette bevattende die PGKpromotor en-termineerder vir uitdrukking in Saccharomyces cerevisiae gekloneer. Dit is ook agter die MFal-sekresiesein gekoppel vir sekresie vanuit S. cerevisiae. Geen ekstrasellulêre aktiwiteit is gevind nie. Slegs intrasellulêre aktiwiteit van 0.26 nanokatal per mililiter was bepaal.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Thirteen Sulfobacillus strains from different geographical locations and two Alicyclobacillus strains were included in this study. These organisms proved to be moderately thermophillic (two different sets of optimal temperatures of 45°C and 55°C were found), Gram-positive, endospore forming bacteria. The pH optima of the strains tested was pH 2.5 and the pH range lay between pH 1.5 and pH 5.0. It was established that some strains of Sulfobacillus had the capacity for anaerobic growth when using ferric iron as an electron donor. It was determined that S. thermosuljidooxidans was the species found within South African biooxidation plants. Plasm ids were identified within strain 611 (S. thermosuljidooxidans) isolated from a Billiton commercial plant. The sample of Sulfobacillus strains used in this study could clearly be divided into two groups based on the analysis of their 16S rRNA gene sequences as well as the number of ribosomal (rm) operons present as determined by Southern hybridization. A system for the convenient identification of Sulfobacillus species was developed using several of the techniques employed in this study. Preliminary identifications can be obtained by restriction enzyme digestion of the PCR amplified 16S rRNA gene. Confirmation of this placement can be done by comparison of the 16S - 23S rRNA spacer region amplification band sizes. Once the preliminary identification has been completed it is possible to place the isolate in the correct species by making use of the differences in sugar utilization that the species exhibit. The more laborious method of 16S rRNA sequence comparisons can be undertaken if there is still any uncertainty as to which species an isolate belongs to. Phylogenetic results obtained from the 16S rRNA gene sequence indicates that the genus Sulfobacillus should probably be divided into two individual genera. Further information gathered from the phylogenetic comparisons indicates that strain Riv-14 previously assigned to S. ambivalens is more closely related to S. montseratensis. Data obtained from 16S - 23S rRNA spacer region analysis confirms this result. Future work includes the use of DNA-DNA hybridization studies and mol% G+C ratio's in order verify the presence of two distinct genera as well as placing Riv-14 within the correct species. / AFRIKAANSE OPSOMMING: Dertien isolate van die genus Sulfobacillus afkomstig van geografies verskillende areas en twee isolate van die genus Alicyclobacillus is in die studie ingesluit. Hierdie organismes het gewys dat hulle gematigde termofiele (twee verskillende groepe met optimale temperature van 45°C en 50°C elk was waargeneem), Gram-positiewe, endospoorvorrnende organismes is. Die pH optima van die isolate was pH 2.5 en die reeks van pH waar groei moontlik was het tussen pH l.5 en pH 5.0 gelê. Dit was bewys dat sekere van die Sulfobacillus isolate oor die vermoë beskik het om anaerobies te respireer deur ferri yster (Fe3+) as elektron akseptor te gebuik. Dit was bepaal dat S. thermosulfidooxidans die spesies is wat teenwoordig was in die bio-oksidasie reaktors in Suid Afrika. Plasmiede vanuit die isolaat 611 (s. thermosulfidooxidans) afkomstig vanuit 'n Billiton komersieële reaktor, is geidentifiseer. Die toetsmonster van Sulfobacillus isolate gebruik in hierdie studie het duidelik daarop gewys dat daar twee groepe binne Sulfobacillus is. Hierdie stelling is gebaseer op data afkomstig van die analiese van die 16S rRNA volgorde asook die aantal ribosomale (rm) kopieë teenwoordig soos bepaal deur Southern klad eksperimente. 'n Sisteem vir die maklike identifikasie van Sulfobacillus spesies is ontwerp deur van verskeie tegnieke, soos in hierdie studie toegepas, gebruik te maak. Aanvanklike identifikasie kan verkry word deur gebruik te maak van restriksie ensiem vertering van PKR geamplifiseerde 16S rRNA geen. Hierdie plasing van die isolaat kan bevestig word deur die grootte van die 16S - 23S rRNA intergeniese amplifikasie produkte te vergelyk. Sodra die aanvanklike plasing van die isolaat voltrek is, kan daar van die verskille in die vermoëns van die spesies om sekere suikers the benut, gebruik gemaak word om die isolaat binne die regte spesies te plaas. Die meer werksintensiewe metode van 16S rRNA volgorde vergelyking kan gebruik word indien daar enige onsekerheid is oor by watter spesies die isolaat hoort. Filogenetiese resultate verkry van die vergelyking van die 16S rRNA geen volgorde dui daarop aan dat die genus Sulfobacillus waarskynlik uit meer as een genus bestaan. Die filogenetiese data dui verder daarop dat die isolaat Riv-14 wat as 'n S. ambivalens geklassifiseer is, nader verwant is aan die spesies S. montseratensis. Data verkry vanaf die 16S - 23S intergeniese gebied analiese bevestig hierdie resultaat. Toekomstige werk sluit DNA-DNA hibridisasie en mol% Gte ratio eksperimente in om sodoende die teenwoordigheid van meer as een genus sowel as die plasing van Riv-14 in die korrekte spesies te bevestig.

Dissertation (PhD)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The acidophilic, chemolithoautotrophic bacterium, Acidothiobaci/lus ferrooxidans is one of a consortium of bacteria involved in biornining, including the recovery of gold from arsenopyrite ores. The genes conferring arsenic resistance to At. ferrooxidans were cloned and sequenced and shown to be chromosomally located. Homologues to the arsB (membrane located arsenite efflux pump), arsC (arsenate reductase) and arsH (unknown function) genes from known arsenic resistance (ars) operons were identified. A fourth gene was found to have weak homology to the ArsR-family of regulators. The arsenic resistance genes of At. ferrooxidans are arranged in an unusual manner, with the arsRC and arsBH genes divergently transcribed. This divergent arrangement was found to be conserved in all four of the At. ferrooxidans strains we tested. All of the At. ferrooxidans ars genes were expressed in Escherichia coli and the arsB and arsC genes conferred arsenite (and antimonite) and arsenate resistance, respectively, to an E. coli ars mutant (AW311 0). Analysis of the putative amino acid sequences of these ars genes revealed that the ArsB from At. ferrooxidans is closely related to the ArsB proteins from other Gram-negative bacteria. However, the ArsC protein is more closely related to the ArsC proteins from Gram-positive bacteria. Furthermore, a functional thioredoxin (trxA) gene was required for ArsC-mediated arsenate resistance in E. coli. This suggests that reduction of arsenate by At. ferrooxidans has a similar reaction mechanism as that by Gram-positive ArsC proteins. While arsH was expressed in an E. coli-derived in vitro transcription-translation system, the presence of this gene was not required for, nor enhanced, arsenite or arsenate resistance in E. coli. We predict that the function provided by this gene is not required in E. coli. While the putative ArsR from At. ferrooxidans does contain a potential DNA-binding helix-turn-helix (HTH) domain, it does not contain the arsenite binding motif (ELCVCDL), required for response to the presence of inducer. Instead, the ArsR-like protein from At. ferrooxidans is related to a group of unstudied ArsR-like proteins that have been associated with other ars-like genes identified during genome sequencing projects. Using arsB-lacZ, arsC-lacZ, and arsR-lacZ fusions, it has been shown that this atypical ArsR protein from At. ferrooxidans did repress expression from the arsBH and arsRC promoters and that this repression was relieved by the presence of either arsenite or arsenate. Deletion of 19 amino acids from the C-terminus of the ArsR protein did not affect regulation, while deletion of a further 28 amino acids inactivated ArsR. Northern blot hybridization confirmed that expression of the arsRC and arsBH transcripts is increased in the presence of either arsenite or arsenate. This study is the first to show that the ars genes from the acidophilic biorning bacterium At. ferrooxidans are able to be studied in the neutrophilic bacterium, E. coli. We have also shown that the atypical ArsR found in this ars operon is able to regulate expression of these genes in response to arsenic, despite not containing the arsenite binding domain, suggesting that this protein senses arsenic by a different mechanism to that used by the ArsR family members already studied. / AFRIKAANSE OPSOMMING: Acidothiobacillus ferrooxidans, 'n asidofiliese, chemolitotrofiese bakterium, is een van 'n konsortium bakterieë betrokke by biologiese ontgunnig ("biomining") asook by die herwinning van goud uit arsenopiriet erts. Die gene wat aan At. ferrooxidans weerstandbiedendheid teen arseen verleen, is gekloneer. Die DNA-volgorde van hierdie gene is bepaal en daar is bewys dat die gene op die chromosoom geleë is. Homoloë van die arsB (membraan geleë pomp wat arseniet uitpomp), arsC (arsenaat reduktase) en die arsH (funksie onbekend) gene is in bekende arseenweerstanbiedheidsoperons (arsoperons) geïdentifiseer. Verder is daar 'n vierde geen geïdentifiseer wat lae homologie met die ArsR-familie van reguleerders toon. At. ferrooxidans se ars gene is op 'n ongewone manier gerangskik met twee van die gene, arsRC en arsBH wat lil teenoorgestelde rigtings getranskribeer word. Hierdie rangskikking van gene IS waargeneem in al vier die At. ferrooxidans rasse wat getoets is. Al die At. ferrooxidans ars gene is in Escherichia coli uitgedruk. Die arsB en arsC gene het aan 'n E. coli ars mutant (AW311 0) weerstandbiedendheid teen aseniet, antimoniet en arseen verleen. Analiese van die afgeleide aminosuurvolgorde van die ars proteïene het getoon dat die At. ferrooxidans ArsB naby verwant aan die ArsB-proteïene van ander Gram negatiewe bakterieë is. In teenstelling hiermee, is gevind dat die ArsC-proteïene nader verwant aan die ArsC-proteïene van Gram positiewe bakterieë is. Daar is ook gevind dat 'n funksionele tioredoksien (trxA) geen vir ArsC-bemiddelde arsenaat weerstandbiedendheid in E.coli benodig word. Dit dui daarop dat die meganisme van arsenaatreduksie deur At. ferrooxidans soortgelyk is aan die ArsC-proteïen-meganisme van Gram positiewe bakteriee. In vitro studies met behulp van 'n E. coli gebaseerde transkripsie-translasie sisteem het getoon dat arsH nie nodig is vir arsenaat of aseniet weerstanbiedendheid in sensitiewe E.coli rasse nie en ook nie help om weerstand in hierdie rasse te verhoog nie. Daarom kan daar aangeneem word dat die funskie van die arsH geen nie deur E. coli benodig word nie. Die vermeende ArsR van At. ferrooxidans bevat 'n potensiële DNA-binding heliks-draaiheliks motief, maar nie die arsiniet binding motief (ELCVCDL) wat nodig is vir reaksie in die teenwoordigheid van 'n induseerder nie. Die ArsA-proteïen van At. ferrooxidans is soortgelyk aan 'n groep ArsA-proteïene wat tydens genoom DNA- volgordebepalingsprojekte geïdentifiseer is. Hierdie groep gene is egter nog nie verder bestudeer nie. Deur gebruik te maak van 'n stel fusie gene, arsB-IacZ, arsC-IacZ en arsRlacZ kon daar bewys word dat die ongewone ArsH-proteïen van At. ferrooxidans uitdrukking van arsBH en arsRC onderdruk en dat die onderdrukking deur arseniet of arsenaat opgehef kan word. Delesie van die eerste 19 aminosure vanaf die C-terminus van die ArsA-proteïen het geen uitwerking op die regulering van die proteïen nie, maar delesie van 'n vedere 28 aminosure het ArsR geïnaktiveer. Verhoogde vlakke van transkripsie van arsRC en arsBH in die teenwoordigheid van arseniet en arsenaat is met behulp van Noordelike kladanalise bewys. Hierdie is die eerste studie waarin daar bewys word dat die ars gene van die asidofiliese bakterium Atferrooxidans in die neutrofiliese bacterium E. coli bestudeer kan word. Daar is ook bewys dat ten spyte daarvan dat die ArsR in die ars operon nie 'n arseniet bindingsdomein het nie, dit die uitdrukking van die gene in hierdie operon reguleer in reaksie op arseen. Dit dui dus daarop dat hierdie proteïen op arseen in die omgewing reageer met behulp van 'n meganisme wat verskil van die ArsR-proteïene wat tot dusver bestudeer is.

El “biol” es un abono líquido rico en fitohormonas, como las auxinas o mejor conocido como Ácido Indol Acético (AIA). Éste ácido estimula el desarrollo de las plantas y en general, la germinación de semillas. El uso del biol, en la agricultura peruana ha cobrado mucha importancia, pues asegura mayor rendimiento de la producción, incrementando a la vez la calidad de los cultivos y sobretodo ofreciendo alimentos libres de productos químicos. Para esta investigación se prepararon 22 muestras de biol artesanal con composiciones variables instalados en Lima y Huancayo. Se realizaron 10 muestreos durante un periodo de 150 días de biodigestión para seleccionar Pseudomonas sp., Bacillus sp. y actinomicetos productores de AIA, los cuales fueron identificados por sus características culturales y pruebas preliminares. Posteriormente con todas las cepas seleccionadas se realizó la prueba cualitativa y cuantitativa del AIA. El 20% de Pseudomonas sp., 17,07% de Bacillus sp. y 21,35% de actinomicetos aislados resultaron positivos a la prueba de AIA y fue posible cuantificar su producción. Asimismo, se realizó una prueba de efectividad del AIA producido por nueve bacterias para evaluar su efecto promotor de crecimiento sobre el hipocotilo y la radícula en semillas de Lactuca sativa L. (lechuga). El análisis estadístico de las pruebas señaló que los resultados son altamente significativos, mostrando los mejores resultados la cepa PSIV-1-L para el hipocótilo y la cepa PSII-7-L para la radícula. Se comprobó así la presencia de bacterias productoras de AIA en las muestras de biol de preparación artesanal y a la vez fue posible evidenciar un efecto positivo sobre la germinación in vitro de semillas de Lactuca sativa L. (lechuga). / Tesis

Graduation date: 1994

The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are well known for their ability to produce a diverse array of antimicrobial compounds. Ofparticular interest is the ability of certain strains to produce antifungal compounds. Such organisms have the potential for application in agriculture where they can be used as biocontrol agents against selected plant pathogenic fungi. A study was undertaken to further characterize selected Bacillus isolates that exhibit broad spectrum antifungal activity. Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying degrees. Two isolates, R29 and B81, were selected for further testing and characterization. Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media composition showed significant influences on the antifungal activities of the isolates tested, but that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found to give rise to the largest inhibition zones. Both isolates were tentatively identified using standard biochemical and morphological tests. Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for homology testing against the GeneBank database. Homology testing confirmed that both isolates were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment used for sequencing proved to be too small to give conclusive identification of the isolates. Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve studies using a defined synthetic medium showed that antifungal activity arose during the stationary phase and appeared to be closely linked to sporulation. The antifungal component of cell free culture supematant was extracted using various methods including thin layer chromatography, acid precipitation, hydrophobic interaction chromatography and methanol extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating that at least two antifungal compounds were produced. Methanol extracted samples produced the cleanest sample extract but only revealed one active peak from the HPLC fraction . Nuclear magnetic resonance analysis of purified samples indicated that the antifungal compound/s have aromatic complex and peptide structures. The extracted antifungal compounds were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and, were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar properties to known antifungallipopeptides i.e. iturin A and fengycin A and B. Further characterization and identification of the active compounds is recommended usmg methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption ionisation time-of- flight. The results presented in this dissertation provide a basis from which antifungal compounds produced by strains ofBacillus can be further characterized. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

Several powdered food products were screened using repetitive element palindrome PCR (rep-PCR) for the presence of enterotoxin producing species of Bacillus. Samples from these products were screened by being placed into a tryptone-peptoneglucose-yeast enrichment medium (TPGY), heat-treated, and shake-incubated. DNA was extracted using a modification of established protocol, leading to the development of an optimized method for each food system. Purified DNA was amplified through rep-PCR using extragenic sequence-targeting primers and optimized for each food product. Amplified PCR products were analyzed electrophoretically and viewed using an ultraviolet photodocumentation system. Bacillus cereus positive control DNA fingerprints were compared to banding patterns from enriched food samples, revealing the presence of the typical diagnostic 1,230 bp band in non-fat dry milk (NFDM). Restriction Fragment Length Polymorphism (RFLP) with Alu I restriction enzyme was performed on the 1230 bp diagnostic band from NFDM and displayed a profile consistent with Bacillus cereus positive control. RPLA (Reverse Passive Latex Agglutination) and BDE ELISA (Bacillus Diarrhoeal Enterotoxin Enzyme Linked Immunosorbent Assay - Tecra Diagnostics) confirmed the presence of HBL and NHE enterotoxin production in NFDM, Coffee creamer, infant milk formula, and two lecithin samples. / Department of Biology