The use of transposon mutagenesis for the identification of genes involved in <I>Streptomcyes </I>development has been frustrated in part by the high target site specificity of many transposable elements. This investigation reports the low insertion site specificity of the IS<I>6100</I>-based mini-transposon Tn/<I>792</I> from a temperature-sensitive delivery plasmid to the genome of the avermectin producer <I>Streptomyces avermitilis</I>. Sequencing of Tn/<I>792</I> insertion sites in <I>S. avermitilis</I> transposants revealed no meaningful shared homology of the target sites or predilection of Tn/<I>792</I> for GC or AT rich regions of the genome. Furthermore, the cointegrate structure normally formed as the end product of the IS<I>6100</I> family of transposable elements was found to undergo resolution resulting in the precise excision of one of the duplicated copies of the transposon and the delivery vector. Several insertions revealed previously uncharacterized genes, of which one termed <I>orf219</I> was implicated in morphological differentiation. Disruption of wild-type <I>S. avermitilis</I> with the cloned insertion site carrying Tn<I>1792</I> generated colonies unable to sporulate (<I>whi</I>), suggesting <I>orf219</I> was involved in development of the aerial hyphae. Complementation of the disruptants with a functional copy of the gene confirmed this involvement in development, producing reversion of the <I>whi</I> phenotype to that of wild-type. Microscopic analysis revealed that in strains in which <I>orf219</I> was disrupted the aerial hyphae are grossly elongated and unable to develop spore compartments. This suggests that <I>orf219</I> is involved in differentiation at or prior to septation of aerial hyphae.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:638541 |
| Date | January 1997 |
| Creators | Pitman, A. B. |
| Publisher | Swansea University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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