The thiostrepton inducible P-<I>tipA</I> promoter of <I>Streptomyces</I> is widely used as a tool for gene cloning and over expression studies. This thesis describes the experiments carried out to investigate the regulation of P-<I>tipA</I> in <I>S. lividans</I> 1326 in order to improve expression of the promoter. Regulation of the promoter was studied using three different reporter genes, <I>aph</I>II, <I>luxAB</I> and <I>egfp</I> in both plasmid borne and chromosomally integrative forms. The promoter was found to be induced by soya flour as well as thiostrepton (Tsr). Expression of the promoter was determined to be growth-phase development and sensitive to extracellular osmolarity. The expression of the promoter was increased and prolonged in high osmolarity media in the presence and absence of Tsr. The transcriptional activator protein TipA<SUB>L</SUB>, which is required for thiostrepton induction was also shown to be required for induction by soya flour and increased extracellular osmolarity. P<I>-tipA</I> has several features, which suggest that it may be responsive to DNA superhelicity. These include a sub-optimally spaced promoter, which is contained within a palindromic sequence with the potential to form a cruciform secondary structure <I>in vivo</I>. The possible role of DNA superhelicity in the regulation of P-<I>tipA</I> was analysed using the gyrase inhibitors, novobiocin and coumermycin. These had little effect on P-<I>tipA</I> expression in the presence of high extracellular osmolarity. The promoter was also exploited for the production of transposon Tn<I>1798</I>, which carries an inducible outward reading P-<I>tipA</I>. The purpose of this transposon is to produce conditional lethal mutations for the study of essential genes. The construction and application of this transposon is described.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:635755 |
| Date | January 2000 |
| Creators | Ali, N. A. |
| Publisher | Swansea University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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