Browsing by Author "Ras, Tertius Alwyn"

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    Bioinformatic characterisation of genes associated with coenzyme A biosynthesis in mycoplasmas and expression and isolation of dephospho-coenzyme A kinase from Mycoplasma sp. Ms02
    (Stellenbosch : Stellenbosch University, 2018-03) Ras, Tertius Alwyn; Botes, Annelise; Strauss, Erick; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The South African ostrich industry is internationally the leading provider of ostrich products. The increasing popularity of ostrich products has resulted in the adjustment of production strategies, which includes intensifying rearing conditions by using feedlot systems. However, this intensive rearing strategy creates an ideal environment for pathogens, such as mycoplasmas, to spread. There are three Mycoplasma species that infect ostriches, which are associated with respiratory diseases. These mycoplasma infections can result in production losses, which not only have an economic impact on the ostrich industry but also significant socio-economic implications. Hence, there is a need for specific and cost-effective treatment against these ostrich-infecting mycoplasmas. The enzymes involved in the biosynthesis pathway of coenzyme A have long been regarded as potential targets for drug development. These enzymes could, therefore, offer a solution to the control of mycoplasma infections in ostriches. Since the coenzyme A biosynthetic pathway is relatively unexplored in mycoplasmas, the first aim of this study was to determine the presence or absence of enzyme-encoding genes involved in this pathway in Mycoplasma species. This was done using a bioinformatics approach. Of the 62 Mycoplasma species investigated, there were eight species (13%) found to have none of the enzyme-encoding genes, while the remaining species had at least one. Additionally, twelve enzyme-encoding gene homologues were identified and their predicted identities confirmed by evaluating the conserved and functional motifs and domains. The enzyme-encoding gene found to be most common amongst the investigated species was that of dephosphocoenzyme A kinase (DPCK), the final enzyme in the biosynthesis pathway. Furthermore, there was no correlation between the number of identified coenzyme A biosynthetic pathway enzyme-encoding genes in a species and the phylogeny of the respective proteins. There was also no correlation with the 16S rRNA phylogenetic groupings. Given the common presence of the DPCK-encoding gene, the second aim of this study was to recombinantly express the DPCK of the ostrich-infecting Mycoplasma sp. Ms02 (Ms02) and isolate the protein using a His-tag. The Ms02 DPCK-encoding gene was successfully amplified, cloned and mutated by site-directed mutagenesis to allow for expression in a nonmycoplasma host. However, the soluble expression and isolation of the Ms02 DPCK protein proved to be challenging. Using a variation of methods, the protein was eventually solubilised using a sarkosyl treatment method. A pure isolate of the Ms02 DPCK protein could, however, not be attained when using immobilised metal affinity chromatography (IMAC) purification. Subsequent activity testing of the isolated DPCK enzyme, using an HPLC-based method, also showed no activity.