Browsing by Author "Woods, David R"
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- ItemOpen AccessAnalysis of genes and enzymes involved in the degradation of cellulose and proteins by Butyrivibrio fibrisolvens H17c(1990) Berger, Eldie; Woods, David RButyrivibrio fibrisolvens H17c is a gram-negative obligate anaerobic bacterium found in the rumen of most ruminants. The aim of this thesis was to investigate the enzymes produced by B. fibrisolvens H17c involved in the degradation of cellulose, xylan, and protein. A library of chromosomal DNA fragments from B. fibrisolvens H17c was established in the plasmid pEcoR251, an Escherichia coli positive selection vector. The library was screened for genes expressing cellulase, xylanase, and protease activity. Two genes expressing endo-β-1,4-glucanase and cellodextrinase activity were cloned in E. coli as host. The gene expressing endo-β-1,4-glucanase activity (end1) was cloned on a recombinant plasmid pES400. The end1 gene was located on a 6.8 kb DNA fragment and expressed from its own promoter in the E. coli host. It was shown that 64% of the endoglucanase activity was located in the periplasm of the E. coli host. TnphoA mutagenesis indicated the presence of a functional E. coli-like signal peptide. The nucleotide sequence of end1 was determined and the amino acid sequence (547 amino acids) deduced. The catalytic domain of End1 showed very good similarity to the catalytic domain of the Clostridium thermoceiium EGE endoglucanase. End1 also has a non-catalytic domain similar to the binding domains of the CenA and Cex cellulases from Ceilulomonas fimi The gene expressing cellodextrinase activity (ced1) was cloned on a recombinant plasmid pES500. This gene was located on a 3.55 kb fragment and was also expressed from its own promoter in the E. coli host. The Ced1 enzyme was also exported to the periplasm of the E. coli host, but did not contain a functional E. coli-like signal peptide. The nucleotide sequence was determined and the deduced amino acid sequence (547 residues) showed high similarity to the catalytic domain of the C. thermocellum EGD endoglucanase. The proteins of End1 and Ced1 showed no similarity. The End1 and Ced1 enzymes were characterized using a range of different substrates. The End1 enzyme showed optimal activity at pH 5.6 and 45°C. Optimal activity for the Ced1 enzyme was obtained at pH 6.6 and 50°C. The proteolytic activity of B. fibrisolvens H17c was characterized using gelatin-SD5-PAGE. Ten bands of protease activity with apparent molecular weights ranging between 42 000 and 101 000 were detected at different stages during the growth cycle. The effect of protease inhibitors indicated that all ten protease bands were serine proteases. Optimal activity was observed between pH 6.0 to 7.5 and at a temperature of 50°C. The proteolytic activity of B. fibrisolvens H17c varied depending on the type of carbohydrate substrate in the medium, and was positively correlated with the growth rate.
- ItemOpen AccessCharacterization and regulation of serine exoproteases and collagenase in vibrio alginolyticus(1982) Hare, Patricia; Woods, David R; Robb, Frank TThe 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.
- ItemOpen AccessCharacterization of pigment production by Escherichia Coli containing a cloned Rhodococcus gene(1991) Hart, Stephen Lewis; Woods, David RThe screening of a Rhodococcus gene bank in Escherichia coli lead to the discovery of a pigment-producing clone. Initial studies revealed a blue component and a pink component in bacterial pigment preparations and suggested that they were different from the prodigiosins and actinorhodins produced by other actinomycetes. This dissertation represents a further analysis of the genetics and biochemistry of pigment production, and the development of an insertional-inactivation cloning vector utilizing the Rhodococcus pigment gene. The DNA of the pigment-producing clone was analysed by restriction mapping and sequenced. A 'single Rhodococcus gene of 1.1 kbp was found to be responsible for pigment production in E. coli. This gene had a putative ribosome binding site and coded for an enzyme of Mr 42,560. Deletion analysis and in vitro transcription-translation experiments supported the hypothesis that pigment production in E. coli was due to a single enzyme. The gene and gene product did not show any similarity when compared with nucleotide and protein sequences in computer data bases. Evidence was obtained from bacterial studies that the pigment pathway involved the conversion of tryptophan to indole by tryptophanase of E. coli and then to the pigment by the action of the cloned Rhodococcus gene. The solubility properties of the pigment, TLC analysis and NMR and visible spectrophotometry supported the hypothesis that the blue pigment was indigo and that the red pigment was indirubin, an isomer of indigo. An insertional-inactivation cloning vector, pSLH8, was developed containing the pigment gene as a marker. The advantages of this vector were that it produced pigmented colonies on LB agar plates without any further requirement for expensive substrates such as X-Gal ,which is required by the pUC series of vectors, and it is capable of being used in any E. coli strain whereas the pUC plasmids require special mutant strains for the detection of recombinants.
- ItemOpen AccessThe cloning and characterisation of an endoglucanase and an endoxylanase from Clostridium acetobutylicum in Escherichia coli(1988) Zappe, Harold; Woods, David R; Jones, David TClostridium acetobutylicum P262 is an endospore forming Gram-positive obligate anaerobe which has been used for the industrial production of acetone and butanol. Strains of C. acetobutylicum have been reported to exhibit some activity towards cellulosic and hemicellulosic substrates. The aim of this thesis was to establish a genebank of C. acetobutylicum P262 DNA in Escherichia coli and to isolate and characterise genes encoding enzymes which show activity towards hemicellulose and cellulose.
- ItemOpen AccessThe 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 EButyrivibrio 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.
- ItemOpen AccessA cluster of two serine transfer RNA genes from Clostridium acetobutylicum P262(1994) Sealy, Victoria Rosamond; Woods, David R; Reid, Sharon JA cloning system, using metronidazole as a screening tool and E. coli FI9 as a selection host, was previously established to clone C. acetobutylicum P262 electron transport genes which may play a role in solvent metabolism in this bacterium. In theory, metronidazole would be reduced under anaerobic conditions to a cytotoxic intermediate by C. acetobutylicum P262 electron transport genes or reductive enzymes cloned into a recombinant plasmid. This intermediate would kill the host E. coli FI9. One C. acetobutylicum P262 clone, pMETIOB, was found to render the E. coli strain FI9 sensitive to metronidazole, under anaerobic conditions. A number of subclones of the 2.56kb C. acetobutylicum P262 insert DNA were generated in Bluescript pKS and pSK. A range of exonuclease III deletions of this C. acetobutylicum P262 insert DNA were also generated which were shown to lose the metronidazole sensitivity phenotype on progressive deletion of the insert DNA. In vitro and in vivo protein transcription/translation experiments failed to reveal a protein product that was related to the metronidazole sensitivity phenotype. DNA hybridization confrrmed that the insert DNA of pMETIOB hybridized to C. acetobutylicum P262 chromosomal DNA, but not to E. coli chromosomal DNA. The nucleotide sequence of a 933-bp fragment of the C. acetobutylicum P262 insert DNA was determined.
- ItemOpen AccessCyanide degradation by Bacillus pumilus C1 : cellular and molecular characterization(1993) Meyers, Paul Robert; Woods, David R; Rawlings , Douglas EA cyanide-degrading, Gram-positive, aerobic, endospore-forming bacterium was isolated from a cyanide wastewater dam by an enrichment technique and was identified as a strain of Bacillus pumilus. The bacterium was routinely cultured in Oxoid nutrient broth and rapidly degraded 100 mg/1 of free cyanide in the absence of added inorganic and organic substances. The ability to degrade cyanide was linked to the growth phase and was not exhibited before late-exponential/ early-stationary phase. Cyanide-degrading activity could not be induced in early exponential phase by the addition of cyanide or acetonitrile to 20 mg/1. Production of the cyanide-degrading activity required at least 0.01 mg Mn2+ /1 in the growth medium (lower concentrations prevented the development of strong cyanide-degrading activity and also resulted in poor growth of the organism). No induction of cyanide-degrading activity occurred when Mn2+ ions were added to late-exponential-phase cells (or a cell-free extract from these cells) which had been grown with a low endogenous concentration of Mn2+. However, Mn2+ ions could be added to cultures growing in low-Mn2+ broth as late as the mid-exponential phase of growth with no apparent reduction of the cyanidedegrading activity exhibited in stationary phase. Culturing the organism in Difeo nutrient broth resulted in poor growth and very low levels of cyanide-degrading activity; addition of Mn2+ to this medium did not significantly increase the levels of activity. Production of the cyanide-degrading activity required de novo transcription and translation. Cyanide-degrading activity was located intracellularly and cell-free extracts rapidly degraded cyanide (0.27 ± 0.08 μmole cyanide/min/mg protein at 30 °C in pH 7.4 phosphate buffer). Eight strains of cyanide-utilizing fluorescent pseudomonads were also isolated from activated sewage sludge by an enrichment technique and tentatively identified as strains of P. fluorescens and P. putida. These isolates could not degrade cyanide rapidly.
- ItemOpen AccessThe development of a neonatal vital signs database(1992) Berelowitz, Jonathan; Poluta, Mladen; Woods, David R; Van der Elst, Clive; Mann, Michael DModern intelligent monitoring systems use digital computer technology to analyze and evaluate physiological vital signs. This analytical and evaluative process is performed by algorithms developed for this purpose. The degree of 'intelligence' of the monitoring system is dependent on the 'sensitivity' and 'specificity' of these algorithms. In order to develop robust and clinically valid algorithms, a database of representative waveforms is required. The aim of this thesis was to create a neonatal vital signs database to be used for this purpose, by means of a computer-based central station. The computer was interfaced to a number of neonatal monitors (Neonatal ICU, Groote Schuur Hospital). The monitors were interrogated to obtain patient condition, ECG waveforms and respiration waveforms using the impedance technique. When possible, percentage oxygen saturation was also captured. The database contains 509 documented clinical records obtained from 35 patients and 20 records containing examples of technical alarm conditions and high frequency noise. Additional patient record data is included. Clinical events recorded include apnoea, bradycardia, periodic breathing tachycardia, tachypnoea and normal traces. These events were recorded against a variety of signal quality conditions that have been characterized in Appendix C. A prototype rate detection algorithm was checked using samples from the database.
- ItemOpen AccessThe development of an industrial process to produce AC γ-linolenic acid using Choanephora cucurbitarum(1990) Ziniades, Catherine; Woods, David RThe objective of this work was to produce γ-linolenic acid (γLA) using a fungus in submerged fermentation. Selection work was aimed at identifying a fungal strain capable of yielding a high level of γLA in an industrial fermentation. Thirty-nine fungal strains were screened under shake flask conditions. The major criteria used in evaluating these strains were, the yield of γLA per unit volume (g/l) and γLA as a percentage of fatty acids, which is important in the downstream processing of γLA . Other parameters of industrial importance such as strain handling and the fatty acid profile were also considered. Eleven fungi in the order Phycomycetes were identified after initial screening. From these fungi, a strain of Choanephora cucurbitarum was found to give superior γLA yields. c. cucurbitarum produced γLA yields of 331mg/l and 674mg/l in shake flask and laboratory fermenters respectively. This strain had other industrially beneficial qualities such as good sporulation, a good biomass of 22, 5g/l and a relatively high yield of γLA of 2,99g/100g dry matter. Subsequently a Zygorhynchus heterogamus strain was found to give similar yields of γLA to c. cucurbitarum. z. heterogamus also had a high γLA : linoleic acid ratio which aids the purification of γLA . This is the first known report of a high level of γLA in the genus Zygorhynchus. The industrial development of γLA production by Zygorhynchus is not reported.
- ItemOpen AccessThe development, initial implementation and support of a primary health care training programme in rational drug use(1998) Orrell, Catherine Jane; Folb, Peter I; Woods, David RThe Rational Drug Use Training Project is a district-oriented programme designed to improve rational drug use among primary health care prescribers in the South African public sector. This thesis describes the development of the project and details the initial implementation study in 3 facilities in Region B of KwaZulu-Natal. This was a small before-after study, with no control. There were three components: 1. A series of easily collectable drug use indicators, adapted from those developed by WHO/INRUD. These allow primary health care staff to monitor their prescribing patterns in a district or facility. Ninety sets of prescribing indicators were collected as a baseline at 3 facilities in KwaZulu-Natal in December 1996, by the district trainers and the Rational Drug Use Training Project staff. The process was repeated in March 1997, after the training intervention, by the district trainers alone. 2. The intervention was a 2-day training workshop in rational drug use. This is problem-based and trained on-site in primary health facilities. Training was done by 8 district trainers from Region B who were taught to present the workshop by the Rational Drug Use Training Project staff. The workshop covers principles of prescribing, use of standard treatment guidelines, principles of clinic stock management and principles of good dispensing. Staff are encouraged to develop their self-learning skills through questioning, and seeking answers to clinical and drug related queries. 3. A set of resources, including texts, treatment guidelines and information centres, to provide quality, safe and unbiased drug information, are made accessible to staff at primary care level. These are available by post, telephone or e-mail. The Primary Care Medicines Resource Centre at the University of Durban-Westville was developed as a result of this study. Significant improvements in prescribing habits were noticed after the study. There was an increase in the percentage of drugs prescribed by their generic names (p=0.000); an increase in the number of medications adequately labelled (p=0.0132); a decrease in the cost of prescriptions (p=0.0134); and a decrease in the number of prescriptions that did not follow standard treatment guidelines at all for that diagnosis (p=0.0109). The Mann-Whitney U- test was used for statistical analysis. There were no significant changes in the average number of drugs per prescription; the percentage of drugs from the Essential Drugs List; and the number of prescriptions that completely followed standard treatment guidelines. Qualitative feedback was favourable too. This was a difficult study to undertake. The staff and funding organisation, Health Systems Trust, fell outside of the provincial health structure and met resistance at that level. Regional politics shaped the programme's design. District trainers needed for the cascade approach were not available. District staff remained entrenched in a traditional health hierarchy and found it difficult to function as a team. The will of district prescribing staff to learn was low. Rational drug use training is only one of a number of essential elements of in-service training urgently needed by these staff. Despite these problems, quantitative and qualitative success was shown. The Training Manual, developed in support of the training, has been in demand. The Primary Care Medicines Resource Centre is growing. Primary care prescribers have been motivated to monitor their own practices and manage their own clinic stock. The project is a successful example of multi-disciplinary and institutional collaboration. The Rational Drug Use Training Project has expanded to eight other health districts in 1997. A list of criteria, such as the need for a district trainer, have been set. These must be met by the district before training will commence. The project is a resource for Initiative for Sub-District Support, a joint district development programme of Health System Trust and the Department of Health. Most expansion in 1998 will be through this initiative. The difficulties encountered and achievements made during this small study will be used to support, and hopefully strengthen, the development of the primary health care oriented district health system, so urgently needed by the South African population.
- ItemOpen AccessDevelopmental genetic studies on Thiobacillus ferrooxidans(1988) Ramesar, Rajkumar Sewcharan; Rawlings , Douglas E; Woods, David RThiobacillus ferrooxidans is an industrially important bacterium which is used in bioleaching operations. The work reported in this investigation extends current knowledge of the genetics of this organism. Conjugation was attempted as a means for plasmid DNA transfer to T. ferrooxidans. Recombinant T. ferrooxidans plasmids, pDER401 and pDER405, were shown to code for mobilization and replication functions in Escherichia coli and Thiobacillus novellus strains. The plasmids were mobilizable at high frequency by the IncP plasmid, R68.45. Attempts to transfer the T. ferrooxidans recombinant plasmids directly from E. coli to T. ferrooxidans were unsuccessful. In multistage mating experiments, plasmid DNA was transferred from E. coli to T. novellus, and from T. novellus to Thiobacillus intermedius. However, in subsequent matings, plasmid transfer from these thiobacilli to T. ferrooxidans could not be shown. A genomic library of T. ferrooxidans ATCC 33020 was constructed in the plasmid vector, pEcoR251, for the purpose of cloning a recA-like gene from this organism. The library consisted of approximately 1,78 X 10⁴ clones carrying chromosomal DNA fragments of about 3-12 kilobases (kb). The library was successfully screened for functional complementation of E. coli auxotrophic mutants. Clones that conferred resistance to methyl methane sulfonate (MMS), a DNA-damaging agent, were isolated in an E. coli recA⁻ mutant. In an attempt to clone a homologous marker, T. ferrooxidans ATCC 33020 was mutated to rifampicin resistance (Rifʳ) and DNA from the mutant strain was used in the construction of plasmid- and cosmid-based libraries. The plasmid library contained approximately 1,35 X 10⁴ clones with inserts of about 1-13 kb. The cosmid library consisted of approximately 8.2 X 10³ colonies, 4.0 X 10⁴ in vitro packaged cosmids, and an amplified in vivo-packaged cosmid lysate containing approximately 1.82 X 10¹¹ infectious particles, carrying inserts of about 35-55 kb. Complementation of E. coli auxotrophic mutants was observed with the plasmid and cosmid library of the T. ferrooxidans Rifʳ strain. Screening both libraries for a Rifʳ marker was unsuccessful. Three recombinant plasmids, pRSR100, pRSR101, and pRSR102, each containing the functional analogue of the E. coli recA gene, were isolated from the plasmid-based genomic library of T. ferrooxidans ATCC 33020. The plasmid, pRSR100, was used for further characterization of the cloned recA-like gene. pRSR100 complemented defects in DNA repair and homologous recombination in an E. coli recA- strain. Antiserum raised against E. coli RecA⁻ protein reacted with two protein bands with an apparent Mᵣ of approximately 40 000 and 38 000 in extracts of the recA deletion mutant, E. coli JK696, containing pRSR100. A single band with an apparent Mᵣ of approximately 40 000 was detected in T. ferrooxidans cell extracts with the E. coli RecA antiserum. The nucleotide sequence of the T. ferrooxidans recA gene has been determined. No SOS box characteristic of LexA- regulated promoters could be identified in the 196-bp region upstream of the coding region. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and P. aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with ATPase and constitutive protease activity have been substituted, the cloned protein has retained these activities. The cloned recA gene was expressed in E. coli from both the λ Pᵣ and lac promoters. However, no expression from the 2.2 kb T. ferrooxidans DNA preceding the gene was evident.
- ItemOpen AccessDNA repair in Bacteroides fragilis Bf-2(1987) Abratt, Valerie Rose; Woods, David R; Jones, David TRepair deficient mutants of Bacteroides fragilis have been isolated in order to study the responses of this organism to various DNA damaging agents at the physiological and molecular levels. Two types of mutants were isolated by ethyl methane sulphonate mutagenesis of B.fragilis followed by selection for sensitivity to mitomycin C. One mutant (UVS9) showed sensitivity to both mitomycin C and far-UV irradiation. The other (MTC25) was more sensitive to mitomycin C than UVS9, but showed wild-type resistance to UV radiation. Both mutant strains had wild-type resistance to methyl methane sulphonate.
- ItemOpen AccessThe effect of metronidazole on Bacteroides fragilis and Escherichia coli(1992) Dachs, Gabriele Ursula; Abratt, Valerie Rose; Woods, David RThe antibiotic metronidazole is used extensively in the clinical treatment of anaerobic infections, including those caused by the anaerobic pathogen Bacteroides fragilis. Metronidazole is an inert substance that requires reductive activation to become cytotoxic. In its activated form metronidazole induces DNA damage. Relatively little is known about the cytotoxic effects of this drug in vivo. The aim of the work reported in this thesis was to analyze the mode of action of metronidazole in living systems. Furthermore, the potential for bacterial cells to develop resistance mechanisms to metronidazole is largely unknown, and therefore the role played by B. fragilis genes in influencing the potency of metronidazole was investigated. Bibliography: pages 172-201.
- ItemOpen AccessEffects 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 TThis 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.
- ItemOpen AccessExpression of a clostridium acetobutylicum endoglucanase and an endoxylanase in saccharomyces cerevisiae(1989) Lewis, Elizabeth A; Woods, David R; Jones, David TS. cerevisiae is widely used in industrial processes, in particular ethanol production. The aim of this study was to examine the feasability of cloning lignocellulase genes into yeast. These enzymes could possibly extend the substrate range of S. cerevisiae, thereby possibly making the process of ethanol production more cost effective.
- ItemOpen AccessFermentation studies on Clostridium acetabutylicum(1982) Van der Westhuizen, André; Woods, David R; Jones, David TThe 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.
- ItemOpen AccessGene cloning studies in two nocardioform bacteria(1988) Hill, Russell; Woods, David RNocardioforms are Gram-positive, aerobic actinomycetes and are a metabolically diverse group which produce antibiotics, useful enzymes, are important in the biotransformation of organic compounds and the decomposition of organic wastes and are important medically. A gene cloning vector designated pLR591 was constructed from the broad host range, multicopy Streptomyces plasmid pIJ702 and the positive selection Escherichia coli plasmid pEcoR251. This plasmid has useful features for the construction of actinomycete genomic libraries. Cloning of DNA into the unique Bg1II endonuclease site of pLR591 inactivated the lethal EcoRI gene derived from pEcoR251, thereby selecting for recombinant plasmids containing inserted DNA. The thiostrepton resistance gene derived from pIJ702 was shown to be functional in Streptomyces lividans enabling selection of recombinant pLR591 plasmids containing foreign DNA in S. lividans. The vector pLR591 therefore functions as a positive selection Streptomyces-E. coli shuttle vector facilitating construction of actinomycete genomic libraries in E. coli and subsequent transfer of recombinant plasmids into S. lividans.
- ItemOpen AccessGenetic and biochemical studies of Vibrio alginolyticus glutamine synthetase(1988) Maharaj, Romilla; Woods, David R; Robb, Frank TA genomic library of the collagenolytic Vibrio alginolyticus strain was established in Escherichia coli HB101 employing the positive selection vector pEcoR251. A glutamine synthetase (GS) gene, glnA was identified by complementation of the glnA deletion in E. coli ET8051 glnA, glnL, glnG deletion strain. The glnA region of V. alginolyticus was cloned on a 5.7 kb insert in pRM210.
- ItemOpen AccessAn Investigation into the bacterial leaching of a gold-bearing pyrite/arsenopyrite ore(1986) Norman, Philippa Fernandes; Rawlings , Douglas E; Woods, David RThe 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.
- ItemOpen AccessAn investigation of the molecular biology and genetics of Witches' Broom disease of Protea cynaroides(1987) Dorrington, Rosemary Ann; Woods, David RAn investigation was undertaken into the biology and genetics of Witches' Broom disease on Protea cynaroides. The investigation was approached in two ways: firstly, from a physiological and pathological angle and secondly at the genetic level. As very little is known about the causes of Witches' Broom disease on P. cynaroides, an attempt was made to identify a pathogen which could be held responsible for the disease. A number of plants were studied in the field and from these samples were taken and cultured on culture medium. Healthy P. cynaroides tissue was not established in tissue culture, while more success was obtained with teratoma tissue. Attempts were made to transmit the disease but these were unsuccessful. Four strains of Agrobacterium tumefaciens were unable to induce tumours on P. cynaroides seedlings. Sections of vascular tissue from teratoma and healthy tissue were viewed under the electron microscope but revealed no pathogen.