IS911-mediated intramolecular transposition is naturally temperature sensitive.
It is shown here that the bacterial insertion sequence IS911 exhibits a temperature-sensitive transposition phenotype. Previous results have demonstrated that elevated levels of the IS911 transposase OrfAB generate significant quantities of a figure-eight form, created by cleavage and circularization of one of the transposon strands, and of an excised circular form, in which both transposon strands have been circularized. We show here that the level of both types of molecule observed in vivo was greatly reduced at 42 degrees C compared with 37 degrees C. On the other hand, reducing the temperature to 30 degrees C resulted in a significant increase in production. Transposition activity at this temperature was sufficiently high to permit detection in vivo of an excised circular form of a defective single IS911 chromosomal copy when OrfAB is supplied in trans. A similar temperature-activity profile is observed for a cell-free reaction that uses partially purified OrfAB and generates the figure-eight form uniquely. Moreover, two point mutants of OrfAB were obtained, which render the reactions partially temperature resistant both in vivo and in vitro. These results suggest that some property of transposase itself is sensitive to elevated temperatures.
Haren
L
L
Laboratoire de Microbiologie et Génétique Moléculaires, CNRS UPR9007,Toulouse, France.
Bétermier
M
M
Polard
P
P
Chandler
M
M
D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004270:DNA, Circular / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007763:Lac Operon; D009690:Nucleic Acid Conformation; D010641:Phenotype; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013696:Temperature; D019895:Transposases / Q000378:metabolism
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004270:DNA, Circular; D029968:Escherichia coli Proteins; C085896:OrfB protein, E coli; D011993:Recombinant Fusion Proteins; D019895:Transposases