It is the purpose of this investigation to test for serological characteristics of six Streptomyces species which exhibit identical and/or very closely similar morphological and biochemical characteristics.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014. / During the last few decades, Streptomycetes have shown to be an important and adaptable group of bacteria for the production of various beneficial secondary metabolites. One such secondary metabolite, epsilon polylysine (ε-PL), produced by Streptomyces albulus is of particular interest due to its antimicrobial activity. This work aimed to study different facets surrounding ε-PL and its production. Firstly, to grow S. albulus CCRC 11814, using economically viable crude glycerol as a carbon source and subsequently measure ε-PL production using an anionic dye, trypan blue. Secondly, to evaluate the antimicrobial activity of ε-PL against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger and Penicillium simplissium. Thirdly, to determine whether there is economic feasibility of ε-PL as a food preservative in South Africa. Lastly, to develop and optimise tools for metabolic engineering such as recombineering and group II introns to improve ε-PL production. The results obtained in this study fall into three different areas: In terms of growth studies, S. albulus grew in the presence of crude glycerol, although the growth was suboptimal, 0.48 g/L as compared to 1.04 g/L produced using pure glycerol or glucose. This is due to the pressures on the bacteria from the impurities of crude glycerol such as methanol and salts. ε-PL antimicrobial activity was effective at a concentration of 100 μg/ml against S. aureus, E. coli and A. niger. It was however, ineffective against P. aeruginosa owing to the low outer membrane permeability of the bacteria. Due to the ability of S. albulus to grow in crude glycerol, it could be used as a financially viable option to produce ε-PL as a natural food preservative in South Africa. The economic feasibility of ε-PL as a food preservative in South Africa showed potential in terms of market research as well as the financial evaluations. However; the production volumes are low due to the use of the crude glycerol and may not cater for the large food industry in the country. For these reasons, metabolic engineering could be employed to improve these production volumes. The first step to metabolic engineering was to develop novel tools which can be used for genetic modifications in S. albulus. The group II intron tools for gene knockouts were developed by the construction of a vector which subsequently requires sequencing and testing to perform gene knockouts. Based on current knowledge, this is the first experiment of its kind. In terms of introduction of genetic material post gene knockouts, iii transformation was shown to be a more effective gene transfer technique as opposed to electroporation, producing 7.75 transformants/μg and 0.038 transformants/μg of DNA, respectively. Future work would involve the use of biocatalysis for metabolic engineering of S. albulus by either removing genes inhibiting ε-PL or overexpressing the enzyme responsible for its production. This research has developed the groundwork for future ε-PL production improvement using biocatalysis and economically viable crude glycerol as a carbon source for applications of the secondary metabolite as a food preservative.

Arsenic is commonly known as poison and contaminant from both natural and anthropogenic sources. However, it is mostly unknown to the general public that there is a wide variety of arsenic species, which can either be toxic or benign. Currently, public interest and legislation focuses on inorganic species of arsenic. However, there is increasing evidence for the toxic capabilities of organic arsenic containing compounds, especially arsenolipids. These compounds are commonly found in oils derived from marine animals, such as fish oil or krill oil, which are increasingly popular as food supplements due to their high omega-3 fatty acid content. Other samples of (mostly) marine origin have also been shown to contain arsenolipids. In this thesis, organ samples from a stranded pod of long-finned pilot whales (Globicephala melas) were investigated for their arsenolipid content. This was achieved by coupling reversed phase high performance liquid chromatography (RP-HPLC) with a combination of inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionisation mass spectrometry (ESI-MS). Using these techniques, it is shown that arsenic can accumulate with age in mammalian brains in the form of arsenic containing phospholipids (AsPL). In contrast to inorganic arsenic and due to their lipophilic nature, lipophilic arsenic containing compounds are thus able to pass the blood-brain barrier. In a second part of the thesis, a previously unknown type of lipophilic arsenic containing compounds has been identified in strains of Streptomyces bacteria. This compound was shown to feature a non-methylated arsenic moiety, which is a structural feature that has not been reported for any of the previously known arsenolipids.

Graduation date: 1993

Graduation date: 1994

Univ., Diss.--Göttingen, 1984.

Thèse de doctorat : Sciences et Techniques du Déchet : Villeurbanne, INSA : 2002. / Titre provenant de l'écran-titre. Bibliogr. p.216-225.

Thèse de doctorat : Procédés biotechnologiques et alimentaires : Vandoeuvre-les-Nancy, INPL : 2006. / Titre provenant de l'écran-titre. Bibliogr.

Thèse doctorat : Génétique Moléculaire : Nancy 1 : 2006. / Titre provenant de l'écran-titre.

La gale commune est une des principales maladies affectant la pomme de terre. Cette maladie est causée par Streptomyces scabiei (S. scabies ), une bactérie du sol qui produit de la thaxtomine A (TA), une toxine essentielle à l'apparition des symptômes de la gale commune. En effet, la TA empêche la synthèse ou l'intégration de la cellulose dans la paroi cellulaire des cellules végétales et entraîne la mort des cellules du périderme de la pomme de terre. Comme cette maladie ne peut être contrôlée par aucun pesticide présentement homologué, il est important de développer des stratégies de lutte alternatives. Notre laboratoire étudie le mode d'action et les effets de la TA sur les cellules végétales. Nous avons réussi à augmenter la résistance à la TA de suspensions cellulaires de peuplier en augmentant progressivement la concentration de la toxine qui était ajoutée à leur milieu. Ces cellules ont conservé leur plus forte tolérance à la TA, même après avoir été cultivées en l'absence de la toxine durant plusieurs années. C'est pourquoi nous avons voulu étudier la possibilité d'utiliser cette technique pour produire des plants de pomme de terre plus résistants à la TA et éventuellement à la gale commune. Nous avons donc produit des cals à partir de segments de tiges de différents cultivars de pomme de terre, déposés sur un milieu contenant des concentrations de plus en plus élevées de TA. Une fois les cals développés, nous avons régénéré des embryons somatiques pour obtenir des plantules potentiellement plus résistantes à la phytotoxine. Pour tester cette nouvelle résistance, nous avons produit des microtubercules à partir des plantules, afin de les mettre en présence de fortes concentrations de TA et d'observer l'intensité de la nécrose résultante. Les résultats démontre que quelques lignées habituées du cultivar Russet Burbank ont semblé être plus résistantes que le cultivar parent à la TA et probablement même à la gale commune, ce qui est très encourageant. Nos expériences démontrent qu'il est possible d'habituer des cellules indifférenciées de pomme de terre à la TA, pour ensuite régénérer des lignées de pomme de terre ayant une plus grande résistance à la TA que le cultivar parent. Il serait intéressant de poursuivre l'étude afin de tester les caractéristiques agronomiques des lignées obtenues, pour s'assurer qu'elles restent intéressantes pour l'industrie et de faire une étude protéomique pour mieux comprendre les processus impliqués dans la mise en place de la résistance à la TA.