Graduation date: 1994

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, 2004. / ENGLISH ABSTRACT: Lactic acid bacteria play an essential role in the majority of fermented foods by producing organoleptic compounds and increasing the shelf life. The best-studied antimicrobial compounds are bacteriocins, i.e. ribosomally synthesized peptides. Most of these peptides have a narrow spectrum of activity and are usually only active against bacteria from the same ecological niche. The fact that all bacteriocins are degraded by proteolytic enzymes enlarges their potential use as natural food preservatives. The ideal would be to replace or reduce chemical preservatives such as sulfur dioxide, nitrates and nitrites. Bacteriocins are classified into four groups according to their structural and functional characteristics. Plantaricin 423, produced by Lactobacillus plantarum 423, is heat stable, plasmid encoded, relatively small (3.5 kDa) and is classified as a class Iia bacteriocin. The peptide is active from pH 1.0 to 10.0 and inhibits Gram-positive bacteria, including Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp. and pathogens such as Bacillus cereus, Clostridium spp. and Listeria monocytogenes. Production of bacteriocins may occur constitutively or may be regulated by a cell-density dependent system called quorum sensing. Plantaricin 423 is produced throughout logarithmic growth, with no apparent change in production levels when the producer strain is cultured in the presence of plantaricin 423 or Listeria innocua and Lactobacillus sakei. This led us to believe that plantaricin 423 may be produced constitutively. A reporter system was constructed which consisted of the plantaricin 423 promoter, P423, fused to the luxAB genes and cloned into a shuttle vector, pTRKH2. The newly constructed plasmid, pTAB4, was transformed to a bacteriocin-negative mutant of L. plantarum (423 B} Despite several repeats, no luciferase activity was recorded and no RNA homologous to the luxAB genes was detected. The region necessary for expression of plantaricin 423 may be located stream-up of the -80 region homologous to the -80 and -40 conserved repeats of regulated class II bacteriocins. Inclusion of the latter region in the reporter construct may result in the successful expression of luxAB. / AFRIKAANSE OPSOMMING: Melksuurbakteriee speel 'n belangrike rol in die meeste gefermenteerde voedselsoorte deur die produksie van organoleptiese komponente en die verlenging van rakleeftyd. Van aile antimikrobiese komponente is bakteriosiene (ribosomaal gesintetiseerde peptiede) die beste bestudeer. Hierdie peptiede het gewoonlik 'n nou spektrum van antimikrobiese werking en is meestal aktief teen bakteriee in dieselfde ekologiese nis. Die feit dat bakteriosiene deur proteolitiese ensieme in die spysverteringskanaal vernietig word, verhoog die potensiele gebruik van bakteriosiene as preserveermiddels. Die ideaal sal wees om die konsentrasie van chemiese preserveermiddels soos swaweldioksied, nitrate en nitriete te verlaag of rnoontlik te vervang met bakteriosiene. Bakteriosiene word in vier groepe op grond van hul strukturele en funksionele karaktereienskappe geklassifiseer. Plantarisien 423, geproduseer deur Lactobacillus plantarum 423, is hitte-stabiel, word deur 'n plasmied gekodeer, is relatief klein (3.5 kDa) en sorteer onder die klas Iia bakteriosiene. Die peptied is aktief oor 'n wye pH-reeks (pH 1.0-10.0) en inhibeer Gram-positiewe bakteriee, insluitend Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp. en patogene soos Bacillus cereus, Clostridium spp. en Listeria monocytogenes. Produksie van bakteriosiene kan konstitutief plaasvind of kan gereguleer word deur 'n seldigtheids- afhanklike sisteem naamlik "quorum sensing". Plantarisien 423 word regdeur logaritmiese groei geproduseer, met geen verandering in produksievlakke wanneer die produserende stam in die teenwoordigheid van plantarisien 423 of Listeria innocua en Lactobacillus sakei gekweek word nie. Dit het gelei tot die hipotese dat plantarisien 423 moontlik konstitutief geproduseer word. 'n Verklikkersisteem bestaande uit 'n fusie van die plantarisien 423 promoter, P423, aan die luxAB gene is gekonstrueer en in die pendelplasmied pTRKH2 gekloneer. Die nuutgekonstrueerde plasmied, pTAB4, is na 'n bakteriosien-negatiewe mutant van L. plantarum (stam 423 B-) getransfonneer. Ten spyte van etlike herhalings kon geen lusiferase-aktiwiteit opgespoor word nie en kon ook geen homologie in die RNA met die luxAB gene opgespoor word nie. Dit is moontlik dat die area nodig vir uitdrukking van plantarisien 423 verder stroom-op van die -80 area, homoloog aan die -80 en -40 gekonserveerde herhalings van reguleerbare klas II bakteriosiene, gesetel is. Insluiting van laasgenoemde area in die verklikker-konstruk mag lei tot die suksesvolle uitdrukking van luxAB.

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Cellulose, a ~-1,4-linked polymer of glucose, is the most abundant renewable carbon source on earth. It is well established that efficient degradation of cellulose requires the synergistic action of three categories of enzymes: endoglucanases (EG), cellobiohydrolases (CBH) and ~-glucosidases. ~-Glucosidases are a heterogenous group of enzymes that display broad substrate specificity with respect to hydrolysis of cellobiose and different aryl- and alkyl-ê-u-glucosides. They not only catalyse the final step in the saccharification of cellulose, but also stimulate the extent of cellulose hydrolysis by relieving the cellobiose mediated inhibition of EG and CBH. The ability to utilize cellobiose is widespread among gram-negative, gram-positive, and Archaea bacterial genera. Cellobiose phosphoenolpyruvate- dependent phosphotransferase systems (PTS) have been reported in various bacteria, including: Bacillus species. In this study, we have used a cellobiose chromophore analog, p-nitrophenyl- ~-D-glucopyranoside (pNPG), to screen a Bacillus pumilus genomic library for cellobiose utilization genes that are functionally expressed in Escherichia coli. Cloning and sequencing of the most active clone with subsequent sequence analysis allowed the identification of four adjacent open reading frames. An operon of four genes (celBACH), encoding a cellobiose phosphotransferase system (PTS): enzyme II (encoded by celB, celA and celC) and a ó-phospho-f-glucosidase (encoded by celH) was derived from the sequence data. The amino acid sequence of the celH gene displayed good homology with ~-glucosidases from Bacillus halodurans (74.2%), B. subtilis (72.7%) and Listeria monocytogenes (62.2%). .As implied by sequence alignments, the celH gene product belongs to family 1 of the glycosyl hydrolases, which employ a retaining mechanism of enzymatic bond hydrolysis. In vivo PTS activity assays concluded that the optimal temperature and pH at which the recombinant E. coli strain hydrolysed pNPG were pH 7.5 and 45°C, respectively. Unfortunately, at 45°C the CelBACH-associated activity of the recombinant strain was only stable for 20 minutes. It was also shown that the enzyme complex is very sensitive to glucose. Since active growing cells metabolise glucose very rapidly this feature is not a significant problem. Constitutive expression of the B. pumilus celBACH genes in E. coli enabled the host to efficiently metabolise cellobiose as a carbon source. However, cellobiose utilization was only achievable in the presence ofO.01% glucose. This phenomenon could be explained by the critical role of phosphoenolpyruvate (PEP) as the phosphate donor in PTS-mediated transport. Glucose supplementation induced the glycolytic pathway and subsequently the availability of PEP. Furthermore, it could be concluded that the general PTS components . (enzyme I and HPr) of E. coli must have complemented the CelBACH system from B. pumilus to allow functionality of the celBACH operon, in the recombinant E. coli host. / AFRIKAANSE OPSOMMING: Sellulose (' n polimeer van p-l,4-gekoppelde glukose) is die volopste bron van hernubare koostof in die natuur. Effektiewe afbraak van sellulose word deur die sinnergistiese werking van drie ensiernklasse bewerkstellig: endoglukanases (EG), sellobiohidrolases (CBH) en P-glukosidases. p-Glukosidases behoort tot 'n heterogene groep ensieme met 'n wye substraatspesifisiteit m.b.t. sellobiose en verskeie ariel- and alkiel-ê-n-glukosidiesc verbindings. Alhoewel hierdie ensieme primêr as kataliste vir die omskakeling van sellulose afbraak-produkte funksioneer, stimuleer hulle ook die mate waartoe sellulose hidroliese plaasvind deur eindprodukinhibisie van EG en CBH op te hef. Sellobiose word algemeen deur verskeie genera van die gram-negatiewe, gram-positiewe en Archae bakterieë gemetaboliseer. Die sellobiose-spesifieke fosfoenolpirovaatfosfotransportsisteem (PTS) is reeds is in verskeie bakterië, insluitende die Bacillus spesies, beskryf. In hierdie studie word die sifting van 'n Bacillus pumilus genoombiblioteek m.b.V. 'n chromofoor analoog van sellobiose, p-nitrofeniel-p-o-glukopiranosied (pNPG), vir die teenwoordigheid van gene wat moontlike sellobiose-benutting in Escherichia coli kan bewerkstellig, beskryf. Die DNA-volgorde van die mees aktiewe kloon is bepaal en daaropvolgende analiese van die DNA-volgorde het vier aangrensende oopleesrame geïdentifiseer. 'n Operon (celBACH), bestaande uit vier gene, wat onderskeidelik vir die ensiem II (gekodeer deur celB, celA en celC) en fosfo-B-glukosidase (gekodeer deur celH) van die sellobiose-spesifieke PTS van B. pumilus kodeer, is vanaf die DNA-volgorde afgelei. Die aminosuuropeenvolging van die celH-geen het goeie homologie met P-glukosidases van Bacillus halodurans (74.2%), B. subtilis (72.7%) en Listeria monocytogenes (62.2%) getoon. Belyning van die DNA-volgordes het aangedui dat die celH geenproduk saam met die familie 1 glikosielhidrolases gegroepeer kan word. Hierdie familie gebruik 'n hidrolitiese meganisme waartydens die stoigiometriese posisie van die anomeriese koolstof behou word. PTS-aktiwiteit van die rekombinante E. coli ras, wat die celBACH gene uitdruk, is in vivo bepaal. Die optimale temperatuur en pH waarby die rekombinante ras pNPG hidroliseer, is onderskeidelik pH 7.5 en 45°C. Alhoewel die ensiernkompleks baie sensitief is vir glukose, is dit nie 'n wesenlike probleem nie, omdat aktief groeiende E. coli selle glukose teen 'n baie vinnige tempo benut. Die celBACH operon het onder beheer van 'n konstitiewe promotor in E coli die rekombinante gasheer in staat gestelom sellobiose as 'n koolstofbron te benut. Die benutting van sellobiose word egter aan die teenwoordigheid van 'n lae konsentrasie glukose (0.01 %) gekoppel. Hierdie verskynsel dui op die kritiese rol van fosfoenolpirovaat (PEP) as die fosfaatdonor gedurende PTS-gebaseerde transport. Glukose speel waarskynlik 'n rol in die indusering van glikoliese, en sodoende die produksie van PEP as tussenproduk. Verder kan afgelei word dat die algemene PTS komponente (ensiem I en HPr) van E. coli die B. pumilis CelBACH-sisteem komplementeer en derhalwe funksionering van die celBACH operon in E. coli toelaat.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The winemaking process involves a complex microbial flora where the interaction of yeasts, lactic acid bacteria and acetic acid bacteria play an important role in the quality and wholesomeness of the final product. Yeasts are primarily responsible for alcoholic fermentation. Malolactic fermentation follows alcoholic fermentation and is conducted by lactic acid bacteria. These bacteria are important in winemaking and can have a positive or negative effect on the wine quality. Biogenic amines are one of the compounds produced by lactic acid bacteria, which affect the hygienic quality and wholesomeness of the wine negatively and directly pose a health risk to the consumer. The demand of consumers for higher quality and healthier foods has led to renewed interest in studies on biogenic amines. Biogenic amines occur in a wide variety of food products, such as cheese, dried sausage, sauerkraut, fishery products, chocolates, wine and beer. This thesis focussed on the presence of biogenic amines in wine. The first objective of the study was to determine the ability of lactic acid bacteria isolated from South African wine to produce biogenic amines, using a decarboxylase screening plate method. The potential to produce the biogenic amines histamine, tyramine, putrescine and cadaverine was investigated. The results obtained showed that Lactobacillus species (Lactobacillus brevis and Lactobacillus hilgardil) might be the lactic acid bacteria responsible for tyramine and putrescine production and that it can contribute significantly to the overall biogenic amine content in wines. The results also suggest that amine production is strain dependent and not species specific. None of the lactic acid bacteria tested had the ability to produce histamine or cadaverine. It is important to remember that the ability of the lactic acid bacteria to produce biogenic amines has only been investigated in synthetic media and that it does not necessarily imply similar behaviour in wine. Wine represents a complex environment with a wide number of factors influencing microbial growth and decarboxylase activity and, thus, further investigation is necessary to determine if these amine-producing bacteria behave similarly in wine conditions. In addition, the polymerase chain reaction (PCR) amplification method was used for the identification of the tyrosine decarboxylase (TOe) gene in some of the tyramine-producing lactic acid bacteria. This was followed by the sequencing of the amplified products, which are partial TOe gene sequences, of two L. brevis strains and of a L. hilgardii strain. Only one tdc gene sequence has been described for bacteria (Enterococcus faecalis), while a partial TOC gene sequence from L. brevis lOEB 9809 was described. An amino acid sequence alignment of the three TOe gene fragments, obtained in this study, with the known TOe gene fragment of L. brevis lOEB 9809 and the tdc gene of E. faecalis showed a high degree of relatedness and conserved regions. To meet consumer demands, procedures are necessary to prevent the formation of amines in food products. One way of preventing the formation of biogenic amines is to relate amine production with certain lactic acid bacteria species involved in the winemaking process. Another possible way would be to develop a rapid detection method for bacteria carrying amino acid decarboxylase genes. The results of this study provide knowledge about which lactic acid bacteria in the winemaking process could contribute to the production of biogenic amines and the sequencing of additional partial TOe genes could possibly assist in the development of a rapid detection method for tyramine-producing lactic acid bacteria in food products. / AFRIKAANSE OPSOMMING: Die wynmaakproses behels 'n komplekse mikrobiese flora waar die interaksie van giste, melksuurbakterieë en asynsuurbakterieë 'n belangrike rol speel in die kwaliteit en heilsaamheid van die finale produk. Giste is primêr verantwoordelik vir alkoholiese fermentasie. Appelmelksuurgisting volg op alkoholiese fermentasie en word deur melksuurbakterieë uitgevoer. Hierdie bakterieë is belangrik in die maak van wyn en kan 'n positiewe of negatiewe uitwerking op die kwaliteit van wyn hê. Biogeniese amiene is een van die komponente wat deur melksuurbakterieë geproduseer kan word en wat die higiëniese kwaliteit en heilsaamheid van die wyn benadeel. Dit hou ook 'n gesondheidsrisiko vir die verbruiker in. Die vereiste van verbruikers vir hoër kwaliteit en gesonder voedselprodukte het nuwe belangstelling in studies op biogeniese amiene ontlok. Biogeniese amiene kom in 'n wye verskeidenheid voedselprodukte voor, soos kaas, droëwors, suurkool, vis, sjokolade, wyn en bier. Hierdie tesis fokus op die teenwoordigheid van biogeniese amiene in wyn. Die eerste doelwit van die studie was om melksuurbakterieë, wat uit Suid- Afrikaanse wyn geïsoleer is, se vermoë te bepaal om biogeniese amiene op dekarboksilase-agarplate te produseer. Die potensiaal om die biogeniese amiene histamien, tiramien, putresien en kadawerien te produseer, is bestudeer. Die resultate wat verkry is, toon dat Lactobacillus-spesies (Lactobacillus brevis en Lactobacillus hilgardit) vir tiramien- en putresienproduksie verantwoordelik is en dat hulle 'n belangrike bydrae kan lewer tot die totale biogeniese amienkonsentrasie in wyn. Die resultate dui ook daarop dat die produksie van amiene afhanklik is van die ras, en nié 'n spesifieke spesie nie. Geen melksuurbakterieë wat getoets is, het die vermoë getoon om histamien of kadawerien te produseer nie. Dit is belangrik om in ag te neem dat die vermoë van die melksuurbakterieë om amiene te produseer slegs in sintetiese media bestudeer is en dat dit nie noodwendig dieselfde gedrag in wyn sal toon nie. Wyn is 'n komplekse omgewing met 'n wye verskeidenheid faktore wat die mikrobiese groei en dekarboksilase-aktiwiteit kan beïnvloed, daarom is verdere studie nodig om vas te stelof hierdie amien-produserende bakterieë dieselfde gedrag in wyn sal toon. Die polimerase-kettingreaksie (PKR) amplifikasie-metode is vir die identifikasie van die tirosiendekarboksilase-geen (TDK) in sommige van die tiramienproduserende melksuurbakterieë gebruik. Dit is gevolg deur die volgordebepaling van die geamplifiseerde produkte, wat gedeeltelike TDK-geenvolgordes is, van twee L. brevis- en van een L. hilgardii-ras. Slegs een tdk-geenvolgorde is al voorheen vir bakterieë beskryf, nl. Enterococcus faecalis, asook 'n gedeeltelike TDK-geenvolgorde vir L. brevis lOEB 9809. 'n Vergelyking van die aminosuurvolgordes van die drie TDK-geenfragmente wat in die studie verkry is, het 'n hoë graad van ooreenkoms en gekonserveerde areas met die bekende TDK-geenfragment van L. brevis lOEB 9809 en die tdk-geen van E. faecalis getoon. Om verbruikers se behoeftes te bevredig, is dit noodsaaklik dat die vorming van amiene in voedselprodukte voorkom word. Een manier van voorkoming is om amienproduksie aan sekere melksuurbakterieë wat in die wynmaakproses betrokke is, te koppel. 'n Ander manier sal wees om 'n vinnige metode te ontwikkel vir die opsporing van bakterieë wat aminosuurdekarboksilase-gene dra. Die resultate van die studie verskaf kennis van watter melksuurbakterieë in die wynmaakproses tot die produksie van biogeniese amiene kan bydra. Die volgordebepaling van addisionele gedeeltelike TDK-gene kan moontlik tot die ontwikkeling van 'n vinnige opsporingsmetode van tiramien-produserende melksuurbakterieë in voedselprodukte bydra.

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

Novel pairs of Bet/Exo recombineering proteins were identified in the beta-proteobacterium Laribacter hongkongensis (LHK) and in the SXT genetic element isolated from Vibrio cholerae. In this research, these new recombineering proteins were functionally characterized using a variety of in vivo and in vitro techniques. The SXT-Exo and LHK-Exo proteins were both found to be alkaline exonucleases, with activities similar to those of Lambda-Exo. Both the SXT-Bet/Exo and LHK-Bet/Exo protein pairs had dsDNA recombination activity within E. coli. / Recombineering is a powerful tool used to manipulate or engineer DNA in vivo, which enables chromosomes and plasmids to be modified precisely and efficiently. It is of critical importance for research into genome and proteome function, and greatly facilitates metabolic engineering applications. The Lambda-Red (Bet and Exo) and RecET proteins constitute the most efficient bacterial recombineering systems characterized to date. However, they work only in E. coli or closely-related bacteria (e.g. Salmonella spp.), which limits their widespread application. / The Lambda-Red and RecET recombineering systems can use PCR products (double stranded DNA, dsDNA) or single stranded DNA (ssDNA, oligonucleotides) to create precise point mutations (substitutions), gene deletions and insertions in chromosomal or episomal DNA. The Exo/RecE exonuclease proteins digest dsDNA and produce long 3'-ssDNA tails. The Bet/RecT ssDNA annealing proteins bind to these 3'-ssDNA tails and promote their homologous recombination with complementary ssDNA regions on the chromosome or episome. / The results described in this thesis will be very useful in assisting the future development of novel recombineering systems that can be used for genetic engineering applications across a wide range of bacterial organisms. Such tools will greatly promote functional genomic and proteomic studies within these organisms, and may also be used for microbial engineering and biotechnological applications. / The ssDNA recombination activities of five different Bet/RecT recombinases were directly compared using an E. coli reporter system. The comparison revealed that Bet protein from LHK had a higher efficiency than Lambda-Bet or RecT. Based on their predicted secondary structure, a set of rationally-designed lambda-Bet protein truncations were prepared and their biological activity was examined, to investigate structure-function relationships within this recombinase. / Chen, Wenyang. / Adviser: Ho W.S. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 113-128). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (PhD (Microbiology))--Stellenbosch University, 2008. / One of the key selection criteria for potential probiotics is the ability to adhere and colonise the host gastrointestinal tract (GIT). Probiotics compete for receptor sites at the host intestinal surface, preventing the colonisation of pathogens, thereby protecting the host from infection. In addition, several important intestinal functions are mediated by the binding of probiotics to host tissue. However, the molecular mechanisms and genotypic characterization of adhesive elements have not received as much attention as other aspects of probiotic research. The present study aims to contribute to this area of research. The first part of the study focused on monitoring the expression of mucus adhesion genes mub, mapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of Lactobacillus plantarum 423, as well as mub, surface layer protein (slp) and EF-Tu of Lactobacillus acidophilus ATCC 4356 when grown in the presence of mucin, bile, pancreatin and at low pH. Real time PCR was used. mub, mapA and EF-Tu of strain 423 were up-regulated in the presence of mucus and expression increased under increasing concentrations of mucus. Expression of mapA was up-regulated under normal gut conditions (0.3%, w/v, bile; 0.3%, w/v, pancreatin; pH 6.5) and at higher levels of bile (1.0%, w/v) and pancreatin (1.0%, w/v). Expression of mub was downregulated in the presence of bile and pancreatin at pH 6.5, whilst the expression of EFTu and plaA remained unchanged. At pH 4.0, the expression of mub and mapA remained unchanged, whilst EF-Tu and plaA were up-regulated. Expression of mapA was down-regulated in the presence of 0.1% (w/v) cysteine, suggesting that the gene is regulated by a mechanism of transcription attenuation that involves cysteine. In the case of L. acidophilus ATCC 4356, none of the genes were up-regulated under increasing concentrations of mucin, whilst only slp and EF-Tu were up-regulated under normal and stressful gut conditions in vitro. In the second part of the study, male Wistar rats were used to evaluate which section of the gastrointestinal tract are colonised by L. plantarum 423 and Enterococcus mundtii ST4SA and determine the effect of adhesion. Fluorescent in situ hybridization (FISH) incorporating strain specific oilgonucleotide probes indicated strong fluorescent signals for L. plantarum 423 along the intestinal lining of the ileum and the cecum. L. plantarum 423 did not colonise the colon as indicated by real timePCR. Fluorescent signals were recorded for E. mundtii ST4SA across the epithelial barrier of cecum and colonic tissue, suggesting that translocation took place. Real time PCR revealed highest cell numbers of strain ST4SA in the cecum and the colon. Haemotoxylin eosin staining of rat tissue revealed no change in morphology or any toxic effects induced upon adhesion of the strains. 16S rDNA PCR and denaturing gradient gel electrophoresis (DGGE) revealed a decrease in enterobacterial species whilst the lactic acid bacterial content remained unchanged. Strains 423 and ST4SA agglutinated yeast cells in vitro, indicating the possible presence of mannose receptors. It is well known that these receptors play a crucial role in the elimination of type 1 fimbriated strains of E. coli. It is thus safe to speculate that mannose receptors may have played a role in diminishing the enterobacterial content in the gut. The third part of the study encompassed characterization of cell surface proteins of L. plantarum 423 and their role in adhesion to Caco-2 cell lines. The strain lacks the typical surface layer protein whilst a multifunctional “intracellular” protein, elongation factor Tu (EF-Tu) and glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI) were detected. Removal of surface proteins reduced adherence of strain 423 to Caco-2 cell lines by 40%, suggesting that these proteins play a role in adhesion. The ability of strain 423 to competitively adhere, exclude and displace Clostridium sporogenes LMG 13570 and Enterococcus faecalis LMG 13566 from Caco-2 cell lines, was studied. Adhesion of C. sporogenes LMG 13570 and E. faecalis LMG 13566 was inhibited by 70% and 90%, respectively. Strain 423 excluded C. sporogenes LMG 13570 from Caco-2 cells by 73% and displaced the pathogen by 80%. E. faecalis LMG 13566 was excluded by 60% and displaced from Caco-2 cells by 90%. Despite removal of the surface proteins, L. plantarum 423 was still capable of competitively adhering to Caco-2 cells and reduced adherence of C. sporogenes LMG 13570 by 50% and E. faecalis LMG 13566 by 70%.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2007. / Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria and are active against other bacteria, either in the same species (narrow spectrum) or across genera (broad spectrum). The application of bacteriocins during the vinification process might help to prevent the production of undesired compounds by inhibiting the indigenous bacterial microflora and allowing malolactic fermentation to be conducted by a selected bacterial strain. Furthermore, the use of bacteriocins might allow reducing the total sulphur dioxide amount in wine. The purpose of this study was the selection of lactic acid bacteria (LAB) belonging to the genera Oenococcus, Lactobacillus and Pediococcus with the ability to produce bacteriocins, with respective biological activity against undesired indigenous wine LAB and the capability to complete malolactic fermentation. The first objective of this study was the screening of LAB isolated from South African red wines for the production of bacteriocins. Only 27 strains out of 330 wine isolates, belonging to the species Lb. plantarum, Lb. paracasei, Lb. hilgardii and O. oeni, showed activity towards various wine-related and non wine-related indicator strains with the colony-overlay method. It is the first time that bacteriocin activity is reported in O. oeni. The second objective was the detection and identification of known structural bacteriocin genes of Lb. plantarum wine strains. Furthermore, the web server BAGEL was used to in silico analyse putative bacteriocin-encoding genes in the genome of O. oeni and primers were designed to amplify four possible bacteriocin-encoding genes. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Moreover, PCR analysis rendered positive results with all four chosen putative bacteriocin-encoding genes in the eight tested O. oeni strains with antimicrobial activity. The latter genes of O. oeni were heterologously expressed in different Escherichia coli host strains, but no antimicrobial activity could be detected. The third objective of this study was the transformation and expression of the heterologous bacteriocin genes nisin A and pediocin PA-1 in two selected Lb. plantarum strains. To enhance their antimicrobial activity a plasmid containing the nisin A gene was successfully cloned into the two strains. Indeed, an enhanced antimicrobial activity could be detected, but the transformed plasmid was not stable. The fourth objective in this project was the evaluation of bacteriocin production in liquid media. A co-culture experiment with a plantaricin producing Lb. plantarum strain and an Enterococcus faecalis strain as indicator was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 hours. The last objective was the evaluation of the impacts of phenolic compounds on the activity of nisin and pediocin. The short term influence of two phenolic acids, two flavan-3-ols, grape tannins and oak tannins on the activity of nisin and pediocin PA-1 was investigated. No influence on the activity was detected. Furthermore, synergistic effects on bacterial growth inhibition were observed. This study confirms the potential use of either bacteriocin additives or bacteriocin-producing LAB in order to control the bacterial microflora during the vinification process.