Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Leptospirillum ferriphilum is a moderately thermophilic, iron-oxidizing bacterium that was isolated from a continuous-flow biooxidation plant used for the recovery of gold from arsenopyrite ore concentrates. Over many years of continuous selection, L. ferriphilum and other bacteria associated with this environment developed resistance to high concentrations of arsenic. We investigated the arsenic resistance genes (ars) of Leptospirillum ferriphilum strain Fairview and compared these genes to the ars genes from other Leptospirilli. An arsenic resistance operon (ars operon) was isolated from a L. ferriphilum Fairview genebank. We discovered that this ars operon was situated in between divergently transcribed transposase (tnpA) and resolvase (tnpR) genes related to the Tn21 subfamily of transposons. Sequence analysis of this transposon ars operon indicated the presence of arsRCDAB genes and an additional CBS orf, located in between the arsA and arsB genes. The 8.5 kb L. ferriphilum transposon ars operon (TnLfArs) was shown to be present only in L. ferriphilum strain Fairview and none of the other Leptospirillum strains. The TnLfArs conferred resistance to arsenate and arsenite in an Escherichia coli ars mutant. We also showed that the TnLfArs is capable of transposition in Escherichia coli. / AFRIKAANSE OPSOMMING: Leptospirillum ferriphilum, ‘n matig termofilies, yster-oksiderende bakterium, is een van `n konsortium bakterieë betrokke by die biologiese herwinning van goud uit arsenopiriet erts. Oor vele jare het die selektiewe druk, weens hoë arseen konsentrasies teenwoordig in die erts, veroorsaak dat L. ferriphilum en die ander bakteriee geassosieer met die omgewing, verhoogde vlakke van weerstandbiedendheid teen die metaal opgebou het. Die doel van die studie was om die aard van die aanpassing op die molukulere vlak vas te stel deur die gene wat in L. ferriphilum (Fairview ras) hiervoor verantwoordelik is te identifiseer en te vergelyk met die van ander Leptospirilli. `n Arseen weestandbiedendheids operon (ars operon) is met behulp van `n L. ferriphilum geen-bank geisoleer. DNA-volgorde bepaling het aangedui dat die operon arsRCDAB gene bevat, sowel as `n CBS orf, gelee tussen die arsA en arsB gene. Die hele operon is gelee tussen `n tnpR- (resolvase) en tnpA (transposase) gene wat in teenoorgestelde rigtings getranskribeer word. Hierdie gene behoort aan die Tn21 familie van transposons. Daar is gevind dat die 8.5 kb L. ferriphilum transposon wat die ars operon bevat (TnLfArs) slegs teenwoordig is in die Fairview ras van L. ferriphilum maar in geen van die ander Leptospirillum rasse nie. Die TnLfArs het weerstanbiedendheid verleen, teen beide arsenaat en arseniet, aan `n Escherichia coli arseen-sensitiewe mutant. Die vermoë van die transposon (TnLfArs) om transposisie te ondergaan is ook in E. coli bevestig.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Strain ST4SA, isolated from soya beans, was identified as Enterococcus mundtii. BacST4SA, a bacteriocin produced by strain ST4SA inhibited the growth of Acinetobacter baumannii, Bacillus cereus, Clostridium tyrobutyricum, Enterococcus faecalis, Enterococcus faecium, Lactobacillus sakei, Propionibacterium spp., Streptococcus caprinus, Pediococcus sp., Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae, and unidentified middle ear isolates A, BW, DW, F, G, and H. BacST4SA was active against Pseudomonas aeruginosa G, BG, I, J, B and E, although variable degrees of resistance were observed for some strains. BacST4SA is positively charged, hydrophobic, contains the YGNGV sequence in the N-terminal, a double-glycine processing site and a disulphide bridge, all of which is typical of a class IIa bacteriocin. The operon, which contains a structural-, ATP-dependent transporter- and immunity gene, is located on a 50-kb plasmid. The 58-amino acid prepeptide is homologous to mundticin KS, mundticin AT06 and bacteriocin QU 2, and differs from enterocin CRL35 by only two amino acids. The 674-amino acid ATP-dependent transporter, consisting of a peptidase C39B domain, an ABC-transporter and an ABC-DLP family domain, displayed 98.9% homology to mundticin KS and 99.25% to enterocin CRL35. The 98-amino acid immunity gene of bacST4SA is completely homologous to enterocin CRL35 and 96.9% to mundticin KS. BacST4SA is 3.950 kDa in size, based on electron spray mass spectrometry. The peptide was isolated from the cell-free supernatant, precipitated with 80% saturated ammonium sulphate, dialysed and freeze-dried to 1 638 400 AU (arbitrary units) per ml. No change in antimicrobial activity was recorded when bacST4SA was incubated in buffer ranging from pH 2 to 12, heated to 100 °C for 90 min and 121 °C for 20 min, and when incubated in the presence of Tween 20, Tween 80, Triton X-100, SDS, urea, EDTA, middle ear fluid and blood. Optimal levels of bacST4SA production (51 200 AU/ml) was recorded after 14 h of growth in MRS broth at 30°C. Maximum production (102 400 AU/ml) was recorded in modified MRS media supplemented with tryptone, yeast extract, a combination of tryptone and yeast extract, K2HPO4 (10.0 or 20.0 g/l), or with the addition of DL-6,8-thoictic acid, L-ascorbic acid, and thiamine, respectively. BacST4SA is bactericidal towards E. faecium HKLHS and bacteriostatic towards S. pneumoniae 40 and middle ear isolates F, BW and H. The peptide adsorbed maximal (94%) to S. pneumoniae 40, P. aeruginosa 25 and E. faecium HKLHS. BacST4SA forms pores in the cytoplasmic membrane of sensitive cells, leading to dissipation of the cell membrane and leakage of cytoplasmic material. BacST4SA was compared with various other antimicrobial treatment agents, and revealed similar to a higher activity towards a number of these agents. BacST4SA revealed a similar level of activity against E. faecium HKLHS and middle ear pathogens P. aeruginosa J and S. pneumoniae 27 when compared with tetracycline (30μg). However, bacST4SA revealed much higher activity when compared to nasal sprays, aminoglycosides, cephalosporins, fluoroquinolones, lincosamides, macrolides, nitroimidazole, penicillin, quinolones, sulfonamides, chloramphenicol, furanzolidone, fusidic acid, rifampicin, trimethoprim, trimethoprim-sulfamethoxazole and vancomycin when tested in vitro. / AFRIKAANSE OPSOMMING: Stam ST4SA, geïsoleer uit sojabone, is as Enterococcus mundtii geidentifiseer. BacST4SA, ‘n bakteriosien geproduseer deur stam ST4SA het die groei van Acinetobacter baumannii, Bacillus cereus, Clostridium tyrobutyricum, Enterococcus faecalis, Enterococcus faecium, Lactobacillus sakei, Propionibacterium spp., Streptococcus caprinus, Pediococcus sp., Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae en ongeïdentifiseerde middeloor isolate A, BW, DW, F, G, en H geinhibeer. BacST4SA is aktief teen Pseudomonas aeruginosa stamme G, BG, I, J, B en E, alhoewel effense weerstand soms waargeneem is. BacST4SA het ‘n netto positiewe lading, is hidrofobies, bevat die YGNGV-volgorde in die N-terminaal, ‘n dubbel-glisien prosesserings setel en ‘n disulfied brug, kenmerkend van klas IIa bakteriosiene. Die operon, wat bestaan uit ‘n strukturele geen, ‘n ATP-afhanklike transport sisteem geen en ‘n immuniteits-geen, is op ‘n 50 kb plasmied gelokaliseer. Die voorloper peptied (58 aminosure lank), is homoloog aan mundticin KS, mundticin AT06 en bakteriosien QU 2 en verskil van enterocin CRL35 met slegs twee aminosure. Die ATP-afhanklike transporter (674 aminosure lank) bestaan uit ‘n peptidase C39B domein, ‘n ABC-transporter en ‘n ABC-DLP tipe domein en is 98.9% homoloog aan mundticin KS and 99.25% aan enterocin CRL35. Die immuniteits-geen (98 aminosure lank) van bacST4SA is ten volle homoloog aan enterocin CRL35 en 96.9% homoloog aan mundticin KS. BacST4SA is 3.950 kDa groot, gebaseer op elektrosproei-massa spektrometrie. Die peptied is uit selvrye supernatant geïsoleer, met 80% versadigde ammonium sulfaat gepresipiteer, gedialiseer en gevriesdroog tot ’n finale konsentrasie van 1 638 400 AE (arbitrêre eenhede) per ml. Geen verandering in antimikrobiese aktiwiteit is waargeneem tydens inkubasie van bacST4SA in buffer van pH 2 tot 12, tydens verhitting (100 °C vir 90 min en 121 °C vir 20 min) en tydens inkubasie in die teenwoordigheid van Tween 20, Tween 80, Triton X-100, SDS, ureum, EDTA, middeloor vloeistof en bloed. Optimale vlakke van bacST4SA produksie (51 200 AE/ml) is na 14 h groei in MRS media by 30°C waargeneem. Maksimale vlakke van die peptied (102 400 AE/ml) is geproduseer in gemodifiseerde MRS medium, aangevul met triptoon, gisekstrak, ‘n kombinasie van triptoon en gisekstrak, K2HPO4 (10.0 of 20.0 g/l), of met byvoeging van DL-6,8-thioktiensuur, L-askorbiensuur, en tiamien onderskeidelik. BacST4SA is bakteriosidies teenoor E. faecium HKLHS en bakteristaties teenoor S. pneumoniae 40 en middeloor isolate F, BW en H. Die peptied adsorbeer optimaal (94%) aan S. pneumoniae 40, P. aeruginosa 25 en E. faecium HKLHS. BacST4SA vorm porieë in die selmembraan van sensitiewe selle en lei tot vernietiging van die selmembraan en lekkasie van die sitoplasma inhoud. In vergelykende studies het bacST4SA ‘n soortgelyke en selfs hoër antimikrobiese aktiwiteit teenoor ‘n aantal bekende antimikrobiese middels getoon. Die aktiwiteit van bacST4SA is soortgelyk aan dié van tetrasiklien (30μg) in toetse teen E. faecium HKLHS en middeloor patogene P. aeruginosa J en S. pneumoniae 27. BacST4SA het egter in ’n in vitro vergelyking met neussproeie, aminoglisiedes, cephalosporiene, fluoroquinolone, lincosamides, makroliede, nitroimidazole, penisilien, quinolone, sulfonamide, chloramphenicol, furanzolidone, fusiensuur, rifampisien, trimethoprim, trimethoprim-sulfamethoxazool en vankomisien ‘n baie hoër aktiwiteit teen patogene getoon.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: In this study the chromosomal arsenic resistance (ars) genes shown to be present in all Acidithiobacillus. caldus isolates were cloned and sequenced from At. caldus #6. Ten open reading frames (ORFs) were identified on a clone conferring arsenic resistance, with three homologs to arsenic genes, arsC (arsenate reductase), arsR (regulator) and arsB (arsenite export). This ars operon is divergent, with the arsRC and arsB genes transcribed in opposite directions. Analysis of the putative amino acid sequences of these arsRC and arsB genes revealed that they are the most closely related to the ars genes of Acidithiobacillus ferrooxidans. These ars genes were functional when transformed into an Escherichia coli ars deletion mutant ACSH50Iq, and conferred increased levels of resistance to arsenate and arsenite. ArsC was required for resistance to arsenate, but not for resistance to arsenite. None of the other ORFs enhanced arsenic resistance in E. coli. A transposon located arsenic resistance system (TnAtcArs) has been described for highly arsenic resistant strains of the moderately thermophilic, sulfur-oxidizing, biomining bacterium At .caldus #6. In the latter study it was shown that TnAtcArs confers higher levels of resistance to arsenate and arsenite than the chromosomal operon. TnAtcArs was conjugated into a weakly ars resistant At. caldus strain (C-SH12) and resulted in greatly increased arsenite resistance. RT-PCR analysis revealed that arsR and arsC are co-transcribed. Despite ORF1 (cadmium inducible-like protein) and ORF5 (putative integrase for prophage CP-933R) not being involved in resistance to arsenic, ORF1 was co-transcribed with arsRC and ORF5 with arsB. Using arsR-lacZ and arsB-lacZ fusions it was shown that the chromosomal ArsR-like regulator of At. caldus acts as a repressor of the arsR and arsB promoter expression. Induction of gene expression took place when either arsenate or arsenite was added. The chromosomal located ArsR was also able to repress TnAtcArs, but the transposon-located ArsR was unable to regulate the chromosomal system. / AFRIKAANSE OPSOMMING: In hierdie studie is die chromosomale arseen weerstandbiedendheidsgene (ars gene), teenwoordig in alle Acidithiobacillus caldus isolate, gekloon en die DNA volgorde daarvan vanaf At. caldus #6 bepaal. Tien oopleesrame (ORFs) is geïdentifiseer op ‘n kloon wat arseen weerstandbiedend is, met drie homoloog aan ars gene, nl. arsC (arsenaat reduktase), arsR (reguleerder) en arsB (membraan-geleë pomp wat arseniet uitpomp). Die ars operon is gerangskik met die arsRC en arsB gene wat in teenoorgestelde rigtings getranskribeer word. Analise van die afgeleide aminosuurvolgorde van dié ars gene het getoon hulle is naverwant aan die ars gene van Acidithiobacillus ferrooxidans. Die ars gene was funksioneel na transformasie na ‘n E. coli ars mutant (ACSH50Iq), en het ‘n hoër vlak van weerstand teen arsenaat en arseniet gebied. ArsC was nodig vir weerstand teen arsenaat, maar nie vir weerstand teen arseniet nie. Geen van die ander ORFs het arseen weerstandbiedendheid in E. coli bevorder nie. Voorheen is ‘n ars operon, geleë op ‘n transposon (TnAtcArs), in ‘n hoogs arseen-weerstandbiedende stam van die middelmatige termofiliese, swawel-oksiderende, bio-ontgunning (“biomining”) bakterie Acidithiobacillus caldus #6 beskryf. In laasgenoemde studie is gevind dat TnAtcArs hoër vlakke van weerstand bied teen arsenaat en arseniet as die chromosomale operon. TnAtcArs is na ‘n lae arseen-weerstandbiedende At. caldus stam (C-SH12) gekonjugeer. Die resultaat was ‘n groot verhoging in arseen weerstandbiedendheid. RT-PCR analise het onthul dat arsR en arsC saam getranskribeer word. Benewens die feit dat ORF1 (kadmium induseerbare protein) en ORF5 (afgeleide integrase vir profaag CP-933R) nie betrokke is in weerstand teen arseniet and arsenaat nie, is ORF1 saam met arsRC getranskribeer en ORF5 saam met arsB. Deur gebruik te maak van die fusie-gene arsR-lacZ en arsB-lacZ is bewys dat die chromosomale ArsR reguleerder van At. caldus as ‘n inhibeerder van die arsR en arsB promoter uitdrukking funksioneer. Indusering van geen uitdrukking vind plaas wanneer arseniet of arsenaat bygevoeg word. Die chromosomaal-geleë ArsR is ook in staat om TnAtcArs te inhibeer, terwyl die transposon geleë ArsR nie daartoe in staat is om die chromosomale ars sisteem te reguleer nie.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / Mastitis is considered to be the most costly disease affecting the dairy industry. Management strategies involve the extensive use of antibiotics to treat and prevent this disease. Prophylactic dosages of antibiotics used in mastitis control programmes could select for strains with resistance to antibiotics. In addition, a strong drive towards reducing antibiotic residues in animal food products has lead to research in finding alternative antimicrobial agents. Streptococcus macedonicus ST91KM, isolated from bulgarian goat yoghurt, produces the bacteriocin macedocin ST91KM with a narrow spectrum of activity against Grampositive bacteria. These include mastitis pathogens Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus epidermidis as well as Lactobacillus sakei and Micrococcus varians. Macedocin ST91KM is, according to tricine-SDS PAGE, between 2.0 and 2.5 kDa in size. The activity of macedocin ST91KM remained unchanged after 2 h of incubation at pH 2.0 to 10.0 and 100 min at 100 °C. The peptide was inactivated after 20 min at 121 °C and when treated with pronase, pepsin and trypsin. Treatment with α-amylase had no effect on activity, suggesting that the mode of action does not depend on glycosylation. Precipitation with 60 % saturated ammonium sulphate, followed by Sep-Pak C18 separation recovered 43 % of macedocin ST91KM. Amplification of the genome of strain ST91KM with primers designed from the sequence of the macedocin prescursor gene (mcdA) produced two fragments (approximately 375 and 220 bp) instead of one fragment of 150 bp recorded for macedocin produced by S. macedonicus ACA-DC 198. Strain ACA-DC 198 was not available. However, the DNA fragment amplified from strain LMG 18488 (ACA-DC 206), genetically closely related to strain ACADC 198, revealed 99 % homology to the mcdA of S. macedonicus ACA-DC 198 (accession number DQ835394). Macedocin ST91KM may thus be a related bacteriocin described for S. macedonicus. The peptide adsorbed equally well (66 %) to L. sakei LMG13558 and insensitive cells, e.g. Enterococcus faecalis BFE 1071 and FAIR E92, and Streptococcus caprinus ATCC 700066. Optimal adsorption of macedocin ST91KM was recorded at 37 °C and 45 °C and at pH of 8 - 10. Addition of solvents decreased adsorption by 50%, suggesting that the receptors to which the bacteriocin binds have lipid moieties. The addition of MgCl2, KI and Na2CO3 completely prevented adsorption of macedocin ST91KM to the target cells, possibly due to competitive ion adsorption on the bacterial cell surface. The peptide has a bacteriocidal mode of action, resulting in lysis and the release of DNA and β-galactosidase. Atomic force microscopy of sensitive cells treated with macedocin ST91KM have shown deformation of the cell structure and developing of irregular surface areas. Antimicrobial susceptibility patterns were evaluated against eighteen mastitis pathogens. All isolates tested were resistant to methicillin and oxacillin, but had minimum inhibitory concentrations (MICs) falling in the intermediate and susceptible range against erythromycin. S. agalactiae and S. epidermidis had the highest sensitivity to macedocin ST91KM. A teat seal preparation containing macedocin ST91KM effectively released bacteriocin inhibiting the growth of the bacterial pathogen. Macedocin ST91KM could form the basis for an alternative dry cow therapy to prevent mastitis infections in dairy cows, as it is effective against pathogens that display resistance to conventional antibiotic therapy.

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 (PhD (Microbiology))--University of Stellenbosch, 2009. / Multidrug resistant strains of Staphylococcus aureus is presenting an increasing threat, especially immune compromised individuals. Many of these strains have developed resistance to newly approved drugs such as quinupristin-dalfopristin, linezolid and daptomycin. The search for alternative treatment, including bacteriocins (ribosomally synthesized antimicrobial peptides) of lactic acid bacteria is increasing . Lactococcus lactis subsp. lactis F10, isolated from freshwater catfish, produced a new nisin variant active against clinical strains of S. aureus. The operon encoding nisin F is located on a plasmid and the structural gene has been sequenced. The lantibiotic is closely related to nisin Z, except at position 30 where valine replaced isoleucine. The antimicrobial activity of nisin F against S. aureus was tested in the respiratory tract of Wistar rats. Non-immunosuppressed and immunosuppressed rats were intranasally infected with S. aureus K and then treated with either nisin F or sterile physiological saline. Nisin F protected immunosuppressed rats against S. aureus, as symptoms of an infection were only detected in the trachea and lungs of immunosuppressed rats treated with saline. The safety of intranasally administered nisin F was also evaluated and proved to have no adverse side effects. The potential of nisin F as an antimicrobial agent to treat subcutaneous skin infections was evaluated by infecting C57BL/6 mice with a bioluminescent strain of S. aureus (Xen 36). Immunosuppressed mice were treated with either nisin F or sterile physiological saline 24 h and 48 h after infection with subcutaneously injected S. aureus Xen 36. Histology and bioluminescence flux measurements revealed that nisin F was ineffective in the treatment of deep dermal staphylococcal infections. Non-infected and infected mice treated with nisin F had an influx of polymorphonuclear cells in the deep stroma of the skin tissue. This suggested that nisin F, when injected subcutaneously, may have modulated the immune system. Nisin F proved an effective antimicrobial agent against S. aureus-related infections in the respiratory tract, but not against subcutaneous infections. The outcome of nisin F treatment thus depends on the route of administration and site of infection.

Dissertation (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: To manipulate recombinant microorganisms for industrial processes, controllable genetic systems are needed that can coordinate expression of recombinant metabolic pathways. All components are sensitive to change and thus putative targets for modification and genetic elements and regulatory systems need to be understood and determined. Central in gene regulation is the transcription activators that mediate gene transcription mechanisms by binding to promoters in response to environmental signals. Promoter engineering entails the modification of transcription factors and their target promoters. In this study, a metabolic control system in Saccharomyces cerevisiae was constructed that would allow induction in response to physiological environment, specifically hypoxia and low temperature conditions. Two approaches were undertaken to find such a system. Firstly, a bi-directional reporter gene cloning vector was designed to search for novel hypoxiainducible promoters. Secondly, a transcription regulatory circuit was built, consisting of an inducible transcription regulator and promoter with a reporter gene through which it mediates transcription. Advantage was taken of the modular nature of proteins and functional domains originating from different transcriptional proteins were combined. A search for promoter elements sensitive to hypoxia from a S. cerevisiae genomic DNA (gDNA) library, using a bi-directional cloning vector, did not yield highly inducible promoters. It was concluded that a multitude of signals overlap, rendering genetic induction difficult to control. A synthetic regulatory system would minimize the impact of these multiple interactions. Such a genetic circuit was constructed, consisting of a chimeric transcription activator and a target fusion promoter. The chimeric transcription activator consisted of the GAL4 DNA binding domain, ADR1 TADIII transactivation domain and three domains of the MGA2 regulatory protein. The functional domains of Mga2p responsible for unregulated expression (at high basal levels) under both aerobic and hypoxia conditions were located, as well as a further upregulation under low temperature, and were mapped to the Nterminal and mid-Mga2p regions. A target fusion promoter consisting of a partial GAL10/1 promoter sequence and a Trichoderma reesei core xyn2 promoter were constructed as target for this chimeric transactivator. This synthetic promoter was fused to the T. reesei xyn2 open reading frame encoding for a readily assayable β-xylanase activity. Both the chimeric transactivator and fusion promoter-reporter gene cassettes were expressed from the same episomal plasmid, named pAR. Transformed into S. cerevisiae Y294, this regulatory system induced transcription under aerobic and hypoxia conditions. Furthermore, the reporter gene expression was upregulated by the chimeric transactivator at low temperatures. The chimeric transactivator mediated a seven-fold induction of the reporter gene under aerobic conditions in S. cerevisiae Y294 when transformed with plasmid AR. A two- to three-fold induction at 23ºC was reported under anaerobic conditions, relative to a reference strain expressing a transcription activator without the Mga2p domains. At 30ºC, a two- to three-fold induction under aerobic conditions and similar induction under oxygen-limited conditions were observed. Replacing the reporter gene with your favorite gene (for example a recombinant enzyme) and incorporating such a pAR system into a recombinant yeast should induce expression of the chosen gene under low temperatures, both aerobic and anaerobically (thus creating a controllable system). The system also has wider application in identifying other transcription factors’ signal-sensitive domains. The design of this system provides the ability to add a linker to a transactivator and to either create specific signal sensitivity or relieve the regulator of its signal dependence. It creates an easy system for assessing other transactivators and their domains with unknown functions and thus provides a ”workhorse and prospector in one”. / AFRIKAANSE OPSOMMING: Vir die manipulering van rekombinante mikroörganismes vir industriële prosesse word beheerbare genetiese stelsels benodig om gekoördineerde uitdrukking van rekombinante metaboliese weë teweeg te bring. Alle komponente van sulke stelsels is sensitief vir verandering en genetiese elemente en reguleerbare sisteme moet dus deeglik verstaan of bepaal word. Sentraal tot geenregulering is die transkripsie-aktiveerders wat geentranskripsie beheer deur aan promoters te bind in reaksie op eksterne omgewingsfaktore. Promotoringenieurswese behels wysigings van transkripsiefaktore en hul teikenpromotors. In hierdie studie is 'n genetiese beheerstelsel vir Saccaromyces cerevisiae ontwikkel wat induksie in reaksie tot spesifieke fisiologiese omgewingreaksies, naamlik hipoksie- en lae temperatuur, toelaat. Twee benaderings is gevolg: eerstens is ‘n tweerigting verklikker-geen vektor ontwikkel en gebruik om vir unieke induseerbare hipoksie-promoters te soek. Tweedens is ‘n transkripsie reguleringstelsel gebou wat uit ‘n induseerbare transkripsiereguleerder and promotor met ‘n verklikkergeen bestaan, waardeur transkripsie bemiddel kan word. Hierdie benadering benut die modulêre onderbou van proteïene en funksionele domeine afkomstig vanaf verskillende transkripsiefaktore is gekombineer. 'n Soektog na hipoksie-sensitiewe promotors vanuit 'n Saccharomyces cerevisiae-genoom- DNA (gDNA), deur van ‘n tweerigting verklikker-vektor gebruik te maak, het ongelukkig nie hoogs-induseerbare promotors opgelewer nie. Die gevolgtrekking was dat ‘n veelvoud van seine met mekaar oorvleuel en die beheer van genetiese induksie dus bemoeilik. Die ontwikkeling van ‘n sintetiese regulering-sisteem kan die impak van die veelvuldige interaksies verminder. Vir dié doel is ‘n sintetiese reguleringstelsel ontwerp, bestaande uit ‘n chimeriese transkripsie-aktiveerder met ‘n teiken fusie-promotor. Die chimeriese transaktiveerder bestaan uit die GAL4 DNA bindingsdomein, die ADR1 TAD III transaktiveringsdomein en drie domeine van die Mga2 reguleringsproteïen. In die studie is die funksionele domeins van Mga2p betrokke by lae temperatuur-respons en ongereguleerde uitdrukking (teen hoë basale vlakke) onder beide aërobiese en anaërobiese toestande aangedui en is tot die N-terminaal en middel-Mga2p areas gekarteer. ‘n Teiken-fusie-promoter, bestaande uit 'n gedeeltelike GAL1/10 DNA promotoropeenvolging en ‘n Trichoderma reesei kern xyn2-promoter, is as teiken vir hierdie chimeriese transaktiveerder saamgestel. Hierdie sintetiese promotor is aan die T. reesei xyn2 oopleesraam, wat vir ‘n maklik meetbare β-xylanase aktiwiteit kodeer, gekoppel. Beide die chimeriese transaktiveerder and fusie-promoter-verklikker-geenkaset word vanaf dieselfde episomale plasmied, bekend as pAR, uitgedruk. Hierdie reguleringsisteem induseer transkripsie onder aërobiese en hipoksie toestande in S. cerevisiae Y294. Verder word die verklikkergeen se uitdrukking deur die chimeriese transaktiveerder by lae temperature verhoog. Die chimeriese transaktiveerder induseer ‘n sewe-voudige induksie van die verklikkergeen onder aërobiese toestande by 23ºC vanaf die pAR-stelsel in S. cerevisiae Y294. ‘n Twee- tot drie-voudige induksie teen 23ºC is onder hipoksie toestande gevind, relatief tot induksievlakke van ‘n verwysingstam met ‘n transaktiveerder sonder die Mga2 domeine. By 30ºC is ‘n twee- tot drie-voudige induksie onder aërobiese en lae suurstofvlakke waargeneem. Deur die verklikker geen met ‘n jou-gunsteling-geen te vervang (bv. ‘n rekombinante ensiem) en so 'n pAR-sisteem in ‘n rekombinante gis te inkorporeer, word uitdrukking onder lae temperature onder beide aërobiese- en anaërobiese toestande geïnduseer (en sodoende word ‘n reguleerbare sisteem geskep). Die sisteem het wyer toepassing om sein-sensitiewe domeine van ander transkripsiefaktore te identifiseer. Die ontwerp van die stelsel maak dit moontlik om 'n skakel tot die transaktiveerder by te voeg wat óf sensitiwiteit tot 'n spesifieke sein skep, óf die reguleerder vanaf seinafhanklikheid verlos. So word ‘n bruikbare stelsel vir die bestudering van ander transaktivators en hul domeine met onbekende funksie geskep – ‘n “werksesel en prospekteerder in een”.

Dissertation (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. The fission yeast Schizosaccharomyces pombe effectively degrades L-malate, but only in the presence of an assimilable carbon source. In contrast, the yeast Saccharomyces cerevisiae is unable to effectively degrade L-malate, which is ascribed to the slow uptake of L-malate by diffusion. In contrast, the yeast Candida utilis can utilise L-malate as the only source of carbon and energy, but this is subject to substrate induction and catabolite repression. Very little research has been done on a molecular level in C. utilis and only a few of its genes have been studied. In this study, we have shown that the yeast C. utilis effectively degraded extracellular L-malate and fumarate, but in the presence of glucose or other assimilable carbon sources, the transport and degradation of these dicarboxylic acids was repressed. The transport of both dicarboxylic acids was shown to be strongly inducible by either L-malate or fumarate and kinetic studies suggest that the same transporter protein transports the two dicarboxylic acids. In contrast, S. pombe effectively degraded extracellular L-malate, but not fumarate, only in the presence of glucose or other assimilable carbon sources. The S. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of L-malate. In order to clone the C. utilis dicarboxylic acid transporter, a cDNA library from C. utilis was constructed using a number of strategies to ensure representativeness and high transformation frequencies. The cDNA library was transformed in a S. cerevisiae strain carrying a plasmid containing the S. pombe malic enzyme gene (mae2) to allow screening for a malate-degrading S. cerevisiae clone. However, no positive clones that would indicate the successful cloning of the C. utilis malate transporter were obtained. The C. utilis malic enzyme gene, CuME, was subsequently isolated from the cDNA library based on conserved sequence homologies with the genes of S. cerevisiae and S. pombe, and characterised on a molecular and biochemical level. Sequence analysis revealed an open reading frame of 1926 bp, encoding a 641 amino acid polypeptide with a predicted molecular weight of 70.2 kDa. The optimum temperature for the C. utilis malic enzyme was 52°C and the enzyme was stable at 50°C for 2 hours. The inferred amino acid sequence showed significant homology with the malic enzymes of S. pombe and S. cerevisiae. Expression of the CuME gene is subject to glucose repression and substrate induction, as was observed for the dicarboxylic acid transporter from C. utilis. The CuME gene was successfully coexpressed with the S. pombe malate permease gene (mae1), resulting in a recombinant strain of S. cerevisiae able to effectively degrade L-malate. / AFRIKAANSE OPSOMMING: Daar is ’n merkwaardige verskil in die vermoë van verskillende gisspesies om ektrasellulêre dikarboksielsure af te breek en dit as enigste bron van koolstof te benut. Die splitsingsgis Schizosaccharomyces pombe kan L-malaat effektief afbreek, maar slegs in die teenwoordigheid van ’n ander benutbare koolstofbron. In teenstelling hiermee is dit vir die gis Saccharomyces cerevisiae onmoontlik om L-malaat effektief af te breek en te benut, wat hoofsaaklik toegeskryf kan word aan die stadige opname van L-malaat deur middel van diffusie. Die gis Candida utilis kan egter L-malaat as die enigste bron van koolstof en energie benut, maar dit is onderhewig aan substraat-induksie en kataboliet onderdrukking. Baie min navorsing op molekulêre vlak is tot hede in C. utilis uitgevoer en slegs ’n paar gene in hierdie gis is al bestudeer. In hierdie studie het ons aangetoon dat die gis C. utilis L-malaat en fumaraat effektief afbreek, maar dat glukose of ander benutbare koolstofbronne die opname en afbraak van hierdie dikarboksielsure onderdruk. Die opname van beide dikarboksielsure is sterk induseerbaar deur L-malaat óf fumaraat, terwyl kinetiese studies toon dat beide dikarboksielsure deur dieselfde transporter-proteïen vervoer word. In teenstelling hiermee kan S. pombe ekstrasellulêre L-malaat, maar nie fumaraat nie, in die teenwoordigheid van glukose of ’n ander benutbare koolstofbron effektief afbreek. Die S. pombe L-malaat transporter was nie in staat om fumaraat te vervoer nie, alhoewel fumaraat die opname van L-malaat onderdruk het. Ten einde die dikarboksielsuur transporter van C. utilis te kloneer, is verskeie strategieë gevolg ten einde ’n cDNA-biblioteek van C. utilis te konstrueer wat verteenwoordiging en hoë transformasie-frekwensies kan verseker. Die cDNA-biblioteek is getransformeer in ’n S. cerevisiae ras wat die S. pombe malaatensiem geen (mae2) bevat om die sifting van ’n S. cerevisiae kloon wat malaat effektief kan afbreek, moontlik te maak. Geen positiewe klone wat dui op die klonering van die C. utilis malaat transporter kon egter gevind word nie. Die C. utilis malaatensiem geen, CuME, is vervolgens van uit die cDNA biblioteek geïsoleer deur van gekonserveerde DNA-homologie met S. cerevisiae en S. pombe gebruik te maak, en op molekulêre en biochemiese vlak gekarakteriseer. DNA-volgordebepaling het ’n oopleesraam van 1926 bp onthul, wat kodeer vir ’n 641 aminosuur polipeptied met ’n verwagte molekulêre gewig van 70.2 kDa. Die optimale temperatuur van die C. utilis malaatensiem was 52°C en die ensiem was vir 2 ure stabiel by 50°C. Die afgeleide aminosuurvolgorde het beduidende homologie met die malaatensieme van S. pombe en S. cerevisiae getoon. Die CuME geen is suksesvol saam met die S. pombe malaat permease geen (mae1) uitgedruk om ’n rekombinante S. cerevisiae ras te genereer wat in staat is om L-malaat effektief af te breek.

Thesis (PhD(Microbiology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Pathogenic Flavobacterium spp. cause serious disease outbreaks in a variety of farmed fish, which lead to large economic losses in the aquaculture industry on an annual basis. The ability of Flavobacterium johnsoniae-like isolates to grow as surface-associated communities (biofilms) in aquaculture systems poses a threat to fish health over extended periods of time. The biofilmforming ability of 28 F. johnsoniae-like isolates obtained from diseased fish were correlated with their chitin-degrading abilities and extracellular carbohydrate complexes (ECC) and their pulsed-field gel electrophoresis (PFGE) genotypes. Physiological changes in the proteome of 5 day planktonic, biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were analyzed by two-dimensional (2-D) gel electrophoresis and 17 differentially expressed and 14 uniquely expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Thirty-two differentially expressed genes in 5 day biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were identified using suppression subtractive hybridization (SSH). Significant negative correlations were observed between the chitin-degrading abilities and ECC and the biofilmforming capacity of 24 h biofilm cultures of F. johnsoniae-like isolates. Genetic heterogeneity was displayed by the F. johnsoniae-like isolates following PFGE. A significant positive correlation was observed between PFGE types and fish host species. Differentially and uniquely expressed proteins identified in planktonic, biofilm and agar surface-associated phases by 2-D/MS as well as differentially expressed genes identified in the biofilm and agar surface-associated phases by SSH were categorized as being involved in adaptation/protection, metabolic processes, membrane/transport/ motility and transcription/ translation. As far as we know, this is the first report on the characterization of differentially expressed genes and gene products of F. johnsoniae-like isolates obtained from diseased fish in South Africa. / AFRIKAANSE OPSOMMING: Patogene Flavobacterium spp. veroorsaak ernstige infeksie uitbrake in ’n verskeidenheid gekweekte vissoorte, wat jaarliks tot groot ekonomiese verliese in die akwakultuur bedryf lei. Die vermoë van Flavobacterium johnsoniae-tipe isolate om as oppervlak-gehegde gemeenskappe (biofilms) in akwakultuur sisteme te groei bedreig visgesondheid oor verlengde periodes. Die vermoë van 28 F. johnsoniae-tipe isolate om biofilms te vorm is vergelyk met hul vermoë om chitien te degradeer, die profiel van hul ekstrasellulêre koolhidraat komplekse (EKK) en bandpatrone verkry met puls-veld jel elektroforese (PVJE). Fisiologiese veranderinge in die proteoom van 5-dagoue planktoniese-, biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 is met twee-dimensionele (2-D) jel elektroforese geanaliseer. Sewentien differensieël uitgedrukte en 14 uniek uitgedrukte proteïene is deur middel van matriks-geassisteerde laser desorpsie ioniserings-tyd van vlug-massa spektrometrie (MGLDI-TVV MS) geïdentifiseer. Twee-en-dertig differensieël uitgedrukte gene in 5-dag-oue biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 was deur middel van suppressie afgetrokke hibridisasie (SAH) geïdentifiseer. Beduidende negatiewe korrelasies is tussen die chitin-degraderings vermoë en EKK en die biofilm-vormings kapasiteit van 24-uur-oue biofilm kulture van F. johnsoniae-tipe isolate waargeneem. Resultate verkry met PVJE het die heterogene samestelling van F. johnsoniae-tipe isolate uitgewys. ‘n Beduidende positiewe korrelasie is tussen PVJE groeperings en vis gasheer spesie waargeneem. Differensieël en uniek uitgedrukte gene geidentifiseer in die planktoniese-, biofilm- en agar oppervlak-geassosieerde fases is deur middel van 2-D/MS asook differensieël uitgedrukte gene geïdentifiseer in die biofilm en agar oppervlakgeassosieerde fases deur middel van SAH was as betrokke by aanpassing/beskerming, metaboliese prosesse, membraan/vervoer/ beweeglikheid en transkripsie/translasie gekategoriseer. Sover bekend is hierdie die eerste beskrywing van differensieël uitgedrukte gene en geenprodukte van F. johnsoniae-tipe isolate afkomstig van geinfekteerde vis in Suid Afrika.

Thesis (DSc)--Stellenbosch University, 1961. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming