Transposition of Cyanobacterium Insertion Element ISY 100 in Escherichia coli

Abstract

ABSTRACT The genome of the cyanobacterium Synechocystis sp. strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY 100 in 22 copies is the most abundant. A typical ISY 100 member is 947 bp long and has imperfect terminal inverted repeat sequences. It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS 630 , indicating that ISY 100 belongs to the IS 630 family. To determine whether ISY 100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-β- d -thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY 100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY 100 , at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid. Mini-ISY 100 transposed to the target plasmid in the presence of IPTG at a very high frequency. Mini-ISY 100 was inserted into the TA sequence and duplicated it upon transposition, as do IS 630 family elements. Moreover, the mini-ISY 100 -carrying plasmid produced linear molecules of mini-ISY 100 with the exact 3′ ends of ISY 100 and 5′ ends lacking two nucleotides of the ISY 100 sequence. No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc 1 / mariner family elements, Tc 1 and Tc 3 , which transpose to the TA sequence, have. These findings suggest that ISY 100 transposes to a new site through the formation of linear molecules, such as Tc 1 and Tc 3 , by excision. Some Tc 1 / mariner family elements leave a footprint with an extra sequence at the site of excision. No footprints, however, were detected in the case of ISY 100 , suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system. ISY 100 transposes in E. coli , indicating that it transposes without any host factor other than the transposase encoded by itself. Therefore, it may be able to transpose in other biological systems.