Browsing by Author "Rawlings , Douglas E"
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- 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 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 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 AccessEcology and physiology of bacterial activity in a temperate saltmarsh lagoon, with an emphasis on nitrogen fixation(1994) Tibbles, Brian Jonathan; Lucas, Michael I; Rawlings , Douglas E; Branch, George MHeterotrophic bacterial activity and nitrogen fixation are fundamental to nutrient regeneration and nitrogen cycling in saltmarsh ecosystems. Ecological and physiological aspects of bacterial production and nitrogenase activity in marine sediments and water were examined in Langebaan Lagoon, a temperate saltmarsh ecosystem. Emphasis was placed on factors modulating rates and patterns of nitrogen fixation. Nitrogen fixation appeared to be dominated by heterotrophic bacteria. Rates of nitrogen fixation (estimated by the acetylene reduction technique), and bacterial production (estimated by tritiated thymidine incorporation, Tri) were higher in fine, muddy sediments near the head of the lagoon (Geelbek) than in coarser, sandy sediments near the mouth of the lagoon (Oesterwal). These comparisons (between sites) reflected the higher bacterial abundance and organic content of sediments from Geelbek. Examinations of five sedimentary microhabitats at each site (including those associated with beds of the seagrass Zostera capensis, burrows of the sandprawn Callianassa kraussi at Oesterwal, and burrows of the mudprawn Upogebia africana at Geelbek) showed that bacterial activity was higher in surface sediments than in subsurface sediments. Highest rates of nitrogen fixation (annual mean, 0.28 + 0.07 nmol C2H4 g-1 dry sediment h-1) were measured in Zostera bed sediments at Geelbek. Thymidine incorporation activity and nitrogenase activity were higher in burrow linings than in adjacent subsurface sediments, suggesting that burrow linings provided an improved subsurface environment for bacterial activity. Burrow linings also had a higher organic content than subsurface sediments away from burrows. Nitrogenase activity was not detected in lagoon water.
- 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 replication of the broad-host-range plasmid pTF-FC2(1990) Dorrington, Rosemary_Ann; Rawlings , Douglas EPlasmid pTF-FC2, a 12.4 kilobase pairs (kb) cryptic plasmid, was originally isolated from the acidophilic chemoautotroph Thiobacillus ferrooxidans and subsequently cloned into the pMB1-based vector, pBR325. Deletion of the pBR325 origin of replication revealed that pTF-FC2 was able to replicate autonomously in a number of Gram-negative bacteria besides T. ferrooxidans. Constructs carrying the pTF-FC2 origin were able to replicate independently of DNA polymerase I (Pol I) in Escherichia coli and this feature was used to distinguish between replication from the T. ferrooxidans origin and the pMB1-derived origins of the vectors. A 3.2 kb Sau3A partial fragment was obtained which had retained the ability to replicate in a E. coli polA- mutant and also in Pseudomonas aeruginosa. A series of deletions of this fragment was used to identify the minimal replicon, the vegetative origin of replication (orzV) and the areas determining plasmid incompatibility. The copy number of pTF-FC2 in E. coli was estimated at 12 - 15 copies per chromosome and a deletion plasmid was identi.fied which replicated at a reduced copy number. An area which affected the ability of the replicon to replicate in P. aeruginosa, was identified. The nucleotide sequence of the 3.2 kb minimal replicon of pTF-FC2 was determined from overlapping DNA sequence obtained from a series of sequential deletions from each end of the fragment. Analysis of the orzV sequence revealed three, tandemly repeated 22 base pairs (bp) DNA sequences and two sets of complementary inverted repeats. The 22 bp direct repeats appeared to be essential for replication and also for incompatibility. The role of one of the two sets of complementary inverted repeats was unclear. The deletion plasmid previously shown to replicate at reduced copy number was found to have half of the second set of repeats deleted.
- ItemOpen AccessThe microbiology of mining leach liquor regeneration(1983) Barros, Maria Eugenia Carvalho; Rawlings , Douglas E; Woods, David RA method for monitoring the growth of Thiobacillus ferrooxidans independently of iron oxidation was developed. This method involved the analysis of protein using the Cobmassie Blue protein-binding assay. It was found to be free from interference by the inorganic ions present in T. ferrooxidans culture medium. The effect of ferrous iron concentrations and mixtures of ferrous and ferric irons on the rate of iron oxidation by T. ferrooxidans in batch culture was determined. The exponential iron oxidation rate was not affected by the ferric iron concentration per se but rather by the ferrousferric ratio.
- ItemOpen AccessStudies of the cloning and expression of Thiobacillus Ferrooxidans Plasmid and Nitrogenase genes(1986) Pretorius, Inge-Martine; Rawlings , Douglas EThis dissertation forms part of a fundamental investigation into the molecular biology of the industrially important bacterium, Thiobacillus ferrooxidans. The expression of T. ferrooxidans plasmid encoded functions, as well as the identification, cloning, sequencing and expression in a variety of heterotrophic bacteria and in vitro systems of the T. ferrooxidans nitrogenase structural genes were studied.