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Journal of Bacteriology, September 2008, p. 6111-6118, Vol. 190, No. 18
0021-9193/08/$08.00+0     doi:10.1128/JB.00452-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Bias between the Left and Right Inverted Repeats during IS911 Targeted Insertion {triangledown}

P. Rousseau,{ddagger}* C. Loot,{dagger},{ddagger} C. Turlan, S. Nolivos, and M. Chandler

Laboratoire de Microbiologie et Génétique Moléculaires, CNRS-UMR5100, Université de Toulouse III, Toulouse 31062, France

Received 2 April 2008/ Accepted 22 June 2008

IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3'-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction.

* Corresponding author. Mailing address: Laboratoire de Microbiologie et Génétique Moléculaires, CNRS-UMR5100, Université de Toulouse III, Toulouse 31062, France. Phone: (33) 0561335916. (33) 0561335886. E-mail: philippe.rousseau{at}ibcg.biotoul.fr

{triangledown} Published ahead of print on 27 June 2008.

{ddagger} P.R. and C.L. contributed equally to this work.

{dagger} Present address: Unité Plasticité du Génome Bactérien—CNRS URA 2171, Institut Pasteur, Paris, France.

Journal of Bacteriology, September 2008, p. 6111-6118, Vol. 190, No. 18
0021-9193/08/$08.00+0     doi:10.1128/JB.00452-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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