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Title: Transposition of a mycobacterial insertion sequence IS6100 in Streptomyces lividans
Author: Smith, B. N.
Awarding Body: University College of Swansea
Current Institution: Swansea University
Date of Award: 1995
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The insertion sequence IS6100 was tested to determine if it could transpose into Streptomyces lividans, with a view to adopting it as tool for the genetic analysis of these biotechnologically important bacteria. IS6100 was originally isolated from Mycobacterium fortuitum, related to S. lividans, as a flank of the compound transposon Tn610 which encodes resistance to sulphonamides. IS6100 was engineered into a temperature-sensitive Streptomyces plasmid, creating pUCS30. On antibiotic containing solid media transformed cells were able to grow normally into colonies at 28°C, permissive for plasmid replication. Upon transfer to the non-permissive temperature, however, colony growth ceased and any subsequent outgrowths were shown to result from transposition of the plasmid into the chromosome. Evidence for transposition in these isolates was obtained by Southern hybridization to restriction ladders. Experiments showed that the majority contained cointegrates with duplicated copies of IS6100 flanking the chromosomally integrated plasmid. Multiple insert sites were observed and pulsed-field gel electrophoresis indicated no regional preference. IS6100 is the first heterologous transposable element shown to transpose in Streptomyces. pUCS30 was constructed in a manner so as to place the thiostrepton inducible promoter, PtipA, upstream of the transposase gene but external to the inverted repeats. Induction of transposition to a high frequency was possible by the addition of thiostrepton to the growth medium. Multiple outgrowths appeared from every colony at 40°C, at a frequency 100-fold greater than in the absence of thiostrepton. Results showed that, in contrast to other bacterial transposons, IS1600 appears to be unable to protect itself from external activation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available