Browsing by Subject "Microbiology"

Now showing 1 - 20 of 66
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    The analysis of an enzyme (Ce1A) and a gene system (abg) involved in the utilization of lignocellulose in the rumen
    (1996) Brown, Gordon Douglas; Thomson, Jennifer Ann
    As lignocellulose represents an abundant renewable resource, research is in progress to obtain a better understanding of the natural mechanisms whereby this resource is utilised. Of particular interest is the degradation of forage in the rumen and one research goal is to ultimately increase animal productivity through an improvement in lignocellulose utilisation. However, although the mechanisms behind lignocellulose utilisation are reasonably well understood, relatively little is known about the mechanisms which occur in the rumen. Thus, the aim of this thesis was to gain more insight into the mechanisms of lignocellulose utilisation which occur in the rumen. Initially this research was focused on the poorly characterised exo-acting cellulases from rumen bacteria. Preliminary enzymology studies on one cellulase from Ruminococcus flavefaciens FD-1, previously isolated in this laboratory, indicated that an exo-acting cellodextrinase, CelA, had been isolated. In this report, the enzyme was purified and biochemically characterised and was shown to be an exo-acting cellodextrinase.
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    Bacterial populations and their activity in the Benguela upwelling system
    (1986) Muir, David Gordon
    An investigation of variability in hydrological and bacterial parameters at a fixed coastal station (Oudekraal. Cape Peninsula 33°59'S 17°21'E) showed that bacterial populations varied in numbers and biomass on both a short term (daily) and seasonal basis in response to changes in hydrological conditions which were largely wind induced.
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    The binding of divalent cations to tobacco mosaic virus and to some isometric plant viruses
    (1977) Hendry, Donald Arthur; Durham, Tony
    The binding of divalent cations (particularly calcium and magnesium) to strains of tobacco mosaic virus (TMV) and their isolated proteins was investigated, using equilibrium dialysis and potentiometric titration, in an attempt to elucidate the role of divalent cations in virus stabilisation. It was found that dissociation of bound calcium ions from TMV is apparently a necessary but insufficient condition for in vitro virus disassembly. TMV and the closely related strain, Y-TAMV, possessed three groups per protein subunit which titrated near neutral pH and which showed significant metal ion binding. The tightest of the three calcium binding sites, which was absent on the RNA-free protein, had a computed pKH of 8.3 and pKCa of 5.2 and had a significantly higher affinity for Ca⁺² over Mg⁺² This group thus had some of the characteristics to be expected for a calcium-mediated switch controlling in vivo virus disassembly, and possibly controlled the in vitro alkaline degradation of TMV as well. Both the U2 and cowpea strains of TMV bound one additional metal ion per protein subunit relative to vulgare, this binding site being retained by the polymerised proteins. However, calcium ions stabilised the polymerised forms of the proteins of all four TMV strains at pH values where depolymerisation would normally have occurred. Both bromegrass mosaic virus and turnip crinkle virus bound calcium ions, which stabilised compact forms of these viruses. The phenomenon of cation binding is thus not limited to TMV. In the light of published evidence, it appears that most if not all plant viruses are able to bind divalent cations, which thus represent a hitherto disregarded stabilising element.
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    Characterization and regulation of serine exoproteases and collagenase in vibrio alginolyticus
    (1982) Hare, Patricia; Woods, David R; Robb, Frank T
    The production of an extracellular collagenase and serine proteases by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37°C and by lack of oxygen. V. alginolyticus had identical growth rates at 30 and 37°C. Aeration did not affect the growth rate of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. The regulation of exoprotease production by temperature and oxygen is specific and has implications regarding the ecology of V. alginolyticus. The synthesis of a 100 000 molecular weight protein was induced in V. alginolyticus by either raising the temperature from 30 to 37°C, a lack of oxygen or (NH₄)₂SO₄. Histidine stimulated synthesis of a 52 000 molecular weight protein. The possibility that these proteins have a regulatory role in exoenzyme synthesis is discussed.
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    Characterization of a transposon-induced pleiotropic metronidazole resistant mutant of Clostridium acetobutylicum P262
    (1996) Collett, Helen Jeanne; Reid, Sharon J
    Metronidazole is a pro-drug which must be reduced to elicit a bactericidal effect. In the clostridia, some of the electron transport proteins that provide the source of electrons for the reductive activation of metronidazole play a key role in electron distribution, which in turn regulates the direction of carbon flow in the cell. The aim of this research project was to isolate electron transport gene(s) from the solvent-producing Clostridium acetobutylicum strain P262, using transposon-induced metronidazole resistance as a selection system. In the process, the feasibility of transposon mutagenesis in this strain, which lacks conventional systems for DNA delivery, was assessed, and the nature of metronidazole susceptibility in the C. acetobutylicum wild type was investigated. The metronidazole resistant transconjugant of interest, referred to as mutant 3R, was shown to harbour a single insertion of the Tn925: :Tn917 transposon cointegrate within a structural gene, designated sum (susceptibility to metronidazole).
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    Clinical Antibiotic Stewardship in South Africa
    (2015) Boyles, Tom
    This course on clinical antibiotic stewardship consists of 22 lectures split into 2 sections. Section 1 covers the background to the problem of antibiotic resistance and teaches principles of antibiotic prescribing and infection control. Section 2 covers specific clinical conditions in turn and explains the clinical approach to managing these problems.
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    The cloning and characterization of an α-amylase and a branching enzyme from Butyrivibrio fibrisolvens H17c and their expression in Escherichia coli
    (1991) Rumbak, Elaine; Woods, David R; Rawlings , Douglas E
    Butyrivibrio fibrisolvens H17c is an important anaerobic bacterium found in the rumen of most ruminants. The aim of this thesis was to establish a genebank of B. fibrisolvens H17c DNA in E.coli and to isolate and characterize genes encoding enzymes involved in the degradation of the major plant polysaccharides. A library of chromosomal DNA fragments from B. fibrisolvens was established in the E. coli-Bacillus subtilis shuttle vector pEBl. The library was screened for the expression of B. fibrisolvens genes in E. coli. E. coli clones expressing glutamine synthetase, carboxymethylcellulase, β-glucosidase and amylolytic-type activities were isolated. A gene (amyA) expressing amylolytic activity and encoding an α-amylase was located on a 5.0 kb DNA fragment and expressed from its own promoter in E. coli. It was shown that more than 86% of the amylolytic actvity was located in the periplasm of the E.coli host and TnphoA mutagenesis indicated the presence of a functional signal peptide. The nucleotide sequence of amyA was determined and encoded a protein of 976 amino acids with a calculated Mr of 106,964. High sequence similarity was demonstrated between the B. fibrisolvens α-amylase and other α-amylases in the three highly conserved regions which constitute the active centre. Conserved regions were all located in the N-terminal half of the B. fibrisolvens amylase and no homology to other amylases was detected for the remainder of the protein. Approximately 40% of the C-terminal region of the protein could be deleted without loss of enzymatic activity. The B. fibrisolvens α-amylase degraded amylose, amylopectin and soluble starch with maltotriose as the major initial hydrolysis product. A gene (glgB) encoding a glycogen branching enzyme, the activity of which produced clearing on starch azure plates, was isolated. The glgB gene was expressed from its own promoter in the host E.coli and encoded a protein of 639 amino acids with a calculated Mr of 73,875. The deduced amino acid sequence of the glgB gene showed high sequence homology (46-50%) to other branching enzymes. The branching enzyme was purified to homogeneity and the properties of the purified enzyme were investigated. Optimal activity of the branching enzyme was at pH 7.2 and 37°C. The branching enzyme was shown to transfer chains of between 5 to 10 glucose units using α-1,4 glucans as substrates, and to stimulate the "de novo" synthesis of a polysaccharide similar to glycogen.
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    The effect of aphid lethal paralysis virus (ALPV) on the biology of grain aphids in the Western Cape
    (1992) Laubscher, Jacobus Martin; Von Wechmar, M Barbara
    Aphid lethal paralysis virus (ALPV) could be detected by indirect immunofluorescent technique in dissected aphids. This technique was found to be more sensitive when compared to DAS-ELISA. The choice of a sensitive, low cost .detection method was of importance to test for low levels of virus in infected aphid body tissues where inapparent infection could cause detection problems. ALPV was visualized in ultrathin sections of diseased aphid body tissues by immunocytochemistry utilizing immunogold label. ALPV antigen was detected in the ovariole tissue, tracheocytes, symbionts of the mycetocytes, fat body cells, brain tissue, nerve tissue and stomach epithelial tissue. Virions were detected predominantly in the cytoplasm but were also found in the nucleus. ALPV antigen was not detected in muscle fibres or mitochondria. ALPV and Rhopalosiphum padi virus (RhPV) are transmitted transovarially. Different incidences of transmissions of ALPV were obtained for R. padi (2996) and Sitobion avenae (1696) and ALPV infections dramatically reduced the longevity and fecundity of these aphids. Infected apterous R. padi aphids were more fecund than alate aphids of the same clone. The percentage of viral infections in different aphid species (R. padi, S. avenae and Diuraphis noxia) was positively associated with temperature; higher temperatures dramatically increased the incidence of ALPV and RhPV and vice versa. The influence of ALPV on a natural R. padi aphid population was found to reduce the population size by 4996. This reduction coincided with a high death factor (70) of aphids per plant. A dramatic decline in R. padi aphid numbers and a high incidence of ALPV present in this aphid population was experienced. Parasitic fungal infections peaked at a later stage than ALPV, and a level of 21 parasitized aphids per plant was reached during this period. This appears to indicate that the presence of ALPV contributes to limit population development in R. padi aphids. Similar results were obtained with S. avenae aphids. Based on this data, ALPV could be considered as a major growth limiting factor in the development of small grain aphid populations in the western Cape. If the presence of the virus is taken into consideration, it could influence pest management strategies directly.
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    Effects of far ultra-violet radiation and oxygen on macromolecular synthesis and protein induction in Bacteroides fragilis Bf-2
    (1984) Schumann, Jacoba Petronella; Woods, David R; Jones, David T
    This thesis deals with a study of the effects of far-UV radiation, oxygen and hydrogen peroxide on macromolecular synthesis and viability in the obligate anaerobe, Bacteroides fragilis, as well as the specific proteins induced in this organism by these different DNA damaging agents.
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    Fermentation studies on Clostridium acetabutylicum
    (1982) Van der Westhuizen, André; Woods, David R; Jones, David T
    The initial aim of this work was to develop a laboratory system for the study of the ABE fermentation under laboratory conditions. The development of defined and simple laboratory inoculation and build-up procedures for the ABE process was investigated. A defined spore preparation in distilled water gave solvent yields comparable to the yields obtained in the commercial ABE process. A laboratory inoculation procedure was developed which avoided the lengthy culture build-up procedures presently utilised. An investigation into solvent production by Clostridium acetobutylicum in clostridial basal medium (CBM) , reinforced clostridial medium (RCM) , Leung and Robson media was undertaken with the aim of developing a partially defined laboratory medium which produced solvent yields comparable to the molasses fermentation medium (MFM). The solvent yields obtained in the partially defined laboratory media were substantially lower than those obtained in MFM. It became apparent that the initial aim of trying to identify and manipulate a few key factors to give better solvent yields would not be easily attained. Both the solvent levels and the overall pattern of cell development were markedly different in the various laboratory systems. In view of these differences, a more detailed investigation of the growth patterns, morphological and physiological changes were undertaken.
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    Gene expression associated with drought tolerance in Xerophyta viscosa Baker
    (2000) Ndima, Tozama Beauty; Mundree, Sagadevan G; Farrant, Jill M; Thomson, Jennifer Ann
    Herophyta viscosa (Baker) is a monocytyledonous resurrection plant that can tolerate extremes of dessication. Upon rewatering, it rehydrates completely and assumes its full physiological activities. Studies on changes in gene expression associated with dehydration stress tolerance were conducted. A cDNA library constructed from m RNA isolated from dehydrated (85%, 37% and 5% relative water content) X. viscosa leaves, was differently screened. Of the 192 randomly selected cDNAs screened, 30 showed higher expression levels when X. viscosa was dehydrated while 20 showed lower expession. XVLEA, XVDH and XVLEC represent three cDNAs that were upregulated during dehydration stress. XVLEA showed the highest identity at the amino acid level with a late embryogenesis abundant protein, LEA29G, from Gossipium hirsutum (30%) and LEA D-29 from cotton (50%). XVDH exhibited significant identity to dehydrin proteins from Arabidopsis thaliana (45%) and Pisum sativum (43%) at the amino acid level. It encodes a glycine-rich protein (27kDa) which is largely hydrophilic and contains a hydrophobic segment at the C-terminus. XVLEC showed 28% identity and 50% similarity to a lectin-like protein from Arabidopsis thaliana. Southern blot analysis confirmed the presence of the three cDNAs in the X.viscosa genome. Both XVLEA and XVDH transcripts were highly expressed during dehydration- (37% RWC) and rehydration (4%, 32%, 72% RWC) treatment of the plant ͌ 1.0kb was observed. However, with XVDH a transcript of ͌ 1.0 kb and 1.09 kb were observed. XVDH transcripts accumulated in X. viscosa plants in response to low temperature, heat and dehydration stresses, as well as to exogenous supply of abscisic acid, ethylene and methyl jasmonate. Localization studies of the XVDH encoded protein showed that XVDH is located in the plasma membrane-cell wall region.
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    Generic studies on the oxytetracycline producing organism, Streptomyces alboflavus
    (1981) Botha, Christine Leone; Kirby, Ralph
    The Actinomycete organism, Streptomyces alboflavus, produces the antibiotic oxytetracycline. Treatment with curing agents resulted in S. alboflavus losing the ability to produce oxytetracycline. However, the loss of oxytetracyc1ine production was reversible, and non-producing colonies regained the ability to produce oxytetracycline when the curing agent was removed. Therefore the loss of oxytetracyc1ine production was not due to the irreversible loss of genetic material specifying oxytetracycline production, but was possibly due to genetic instability. Two extrachromosomal DNA species were isolated from S. alboflavus, (SAP1 and SAP2). SAP1 was approximately 8 - 10 x 10â ¶ da1tons, and appeared to be linear and heterogenous. SAP2 was 20 - 25 x 10â ¶ daltons and appeared to be covalently closed circular. The functions of SAP1 and SAP2 are unknown, although transformation experiments with SAP1 suggested that it may play a role in the production of an antibiotic-like substance, possibly by acting as a promotor of the genes coding for the antibiotic-like substance.
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    Genetic studies of Streptomyces cattleya and Streptomyces olivaceus
    (1985) Coyne, Vernon Errol; Kirby, Ralph
    Actinophage VCll is able to virulently infect 11 of the 20 Streptomyces strains tested. Examination of VCll infection of Streptomyces cattleya, Streptomyces olivaceus and Streptomyces lividans TC10 indicated the absence of restriction-modification systems which affect VCll infectivity of these Streptomyces strains.
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    Genetic studies on Clostridium acetobutylicum
    (1982) Allcock, Errol Ralph; Woods, David; Reid, Sharon J
    The aim of this study involved the characterisation of the cellulolytic properties of Clostridium acetobutylicum and the development of a genetic transfer system for this organism. The production of a carboxymethyl cellulose and a cellobiase by C acetobutylicum was demonstrated. In liquid medium the carboxymethyl cellulase was induced by molasses, and it was not repressed by glucose. Optimum carboxymethyl cellulase activity occurred at pH 4.6 and 37°C. Optimum conditions for autolysis and autoplast formation in C. acetobutylicum were defined. Autolysis-deficient mutants which produced less autolysin than the parent strain were isolated. Growth of the P262 strain and the lyt-1 mutant was inhibited by the same concentrations of wall-inhibiting antibiotics.
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    Genetic studies on the region downstream of the unc operon of Thiobacillus ferroxidans
    (1996) Oppon, Joseph Cruick-Shank; Rawlings, Doug
    A Tn7-like element was found in a region downstream of a cosmid (p818.1) isolated from a genomic library of Thiobacillus ferrooxidans ATCC 33020. A probe made from the Tn7-like element hybridized to restriction fragments of identical size from both cosmid p818.1 and T ferrooxidans chromosomal DNA. The same probe hybridized to restricted chromosomal DNA from two other T ferrooxidans strains (ATCC 23270 and 19859). There were no positive signals when an attempt was made to hybridize the probe to chromosomal DNA from two Thiobacillus thiooxidans strains (ATCC 19733 and DSM504) and a Leptospirillum ferrooxidans strain DSM 2705. A 3.5 kb BamHI-BamHI fragment was subcloned from p818.1 downstream the T ferrooxidans unc operon and sequenced in both directions. One partial open reading frame (ORFl) and two complete open reading frames (ORF2 and ORF3) were found. On the basis of high homology to previously published sequences, ORFI was found to be the C-terminus of the T ferrooxidans glmU gene encoding the enzyme GlcNAc I-P uridyltransferase (EC 1.7.7.23). The ORF2 was identified as the T ferrooxidans glmS gene encoding the amidotransferase, glucosamine synthetase (EC 2.6.1.16). The third open reading frame (ORF3) was found to have very good amino acid sequence homology to TnsA of transposon Tn7. Inverted repeats very similar to the imperfect inverted repeat sequences of Tn7 were found upstream of ORF3. The cloned T ferrooxidans glmS gene was successfully used to complement an E.coli glmS mutant CGSC 5392 when placed behind a vector promoter, but was otherwise not expressed in E.coli.
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    Identification and characterisation of South African strains of cucumber mosaic virus
    (1985) Lupuwana, Pumezo; Von Wechmar, M Barbara
    This project was then aimed at finding naturally occurring isolates of CMV, characterising them, producing much needed antisera and to use such antisera in a comparison with other well characterised strains by the use of new contemporary sensitive serological techniques.
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    Immunoassays with chemically modified bacteriophage
    (1977) Du Plessis, Dion Henri
    The immunospecific inactivation of bacteriophage is one of the most sensitive methods available for the detection of very low concentrations of antibody. By chemically modifying the phage coat-protein, this sensitivity can be extended to antibodies against a wide variety of haptens and proteins. Phage particles that have been modified by attaching some molecule onto their surface are sensitive to antibodies directed against the coupled chemical moiety. Furthermore, the inactivation of the modified phage by antibody can be inhibited by free antigen, and this provides a sensitive assay for small quantities of antigen. Antibody and antigens have been detected at the nanogram level by this technique. The modified phage technique can also be used to distinguish antibodies of different specificities and to discriminate between closely related antigens. This technique has not yet been applied to the immunochemical study of viral components and the present work represents such an attempt. Tobacco mosaic virus (TMV) was chosen as a model system since it permits the study of numerous inununological phenomena (Rappaport, 1965; van Regenmortel, 1966). A series of preliminary experiments were performed to obtain experience in the methodology of the technique. These included the inactivation of native T4 phage by phage antiserum and anti phage IgG, the chemical modification of phage with DNP and the attachment of lysozyme to phage under a variety of conditions. The success of the covalent binding of protein to phage particles depends on finding conditions under which a proportion of the phage remains viable and, at the same time, can still be neutralized by anti-protein sera. To this end, different proportions of reactants and three different bifunctional reagents were tested. To prevent aggregation of tobacco mosaic virus protein (TMVP) at the high concentration used, the protein was treated with N-bromosuccinimide. TMVP-phage conjugates which were sensitive to antiserum were prepared using bis-diazobenzidine as the bifunctional reagent.
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    Interactions between selected metal compounds and marine heterotrophic bacteria
    (1984) Thompson, Gillian Ann; Watling, R J
    A method was developed to test the toxicity of several metal compounds to selected bacteria. The technique was based on the agar diffusion method. Reliability and reproducibility were enhanced by careful standardisation of experimental parameters. By quantification of the concentration of metal compounds in the media using sequential strips of agar it was .possible to prepare standard graphs (metal concentration vs. distance from disc). These graphs demonstrated the exponential rate of diffusion of metals. The diffusion rates were metal specific. In the case of lead compounds the diffusion rate was poor. A media was developed which allowed lead chloride to diffuse relatively well.
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    An Investigation into the bacterial leaching of a gold-bearing pyrite/arsenopyrite ore
    (1986) Norman, Philippa Fernandes; Rawlings , Douglas E; Woods, David R
    The main aim of this study was to develop an economically viable bacterial leaching process for a gold-containing pyrite/arsenopyrite ore. The effect of various parameters on, and the mechanism of, bacterial leaching were investigated. Initially milled run-of-mine ore was examined. Batch tests and a continuous bacterial leach were carried out. Bacterial leaching was successful and 91-93% gold dissolution was attained in four days. The process was not economically feasible when compared to the standard flotation-roasting process.
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    An investigation into the high level production of proteins in tobacco using transgenic plants or viral vectors
    (1996) Gehringer, Michelle Martine; Rybicki, Edward P
    The aim of this project was to construct a high level plant expression vector from the RNA 3 of cucumber mosaic virus strain Y (CMV Y). The 5'- and 3'-untranslated regions (UTRs) of this genome segment were reverse transcribed, cloned and sequenced. The chloramphenicol acetyl transferase gene (CAT) was inserted between the two UTRs. This artificial viral cDNA (5'cat3') was cloned immediately downstream of the cauliflower mosaic virus 35 S promoter at the transcription initiation site to make a DNA vector. An RNA vector construct was made by placing the 5'cat3' segment under the control of a T7 RNA promoter sequence. In vitro transcripts, as well as linearised DNA vector constructs were inoculated onto CMV infected plants. Inoculated plants were monitored for CAT expression. No CAT could be detected in total protein extracts of inoculated plants. No CAT mRNA could be detected in northern blots of total RNA extracted from inoculated plants. The vector constructed from the 5' - and 3' -UTR of the RNA 3 of CMV Y did not appear to contain all the necessary attributes for a viral expression vector. To study the expression of a foreign antigen in tobacco, the L 1 capsid protein of human papillomavirus type 16 was cloned into Agrobacterium tumefaciens and used to make transgenic Nicotiana tabacum. Kanamycin resistant tobacco plants were shown to carry the L 1 capsid gene using PCR screening, but western blots on total protein extracts of the transformed plants were indeterminate. Further studies are needed to determine whether the antigen is produced and if it is correctly processed.