Functional Characterization of IS 1999 , an IS 4 Family Element Involved in Mobilization and Expression of β-Lactam Resistance Genes

Abstract

ABSTRACT IS 1999 and a point mutant derivative, IS 1999.2 , have been described inserted upstream of emerging antibiotic resistance genes bla VEB-1 and bla OXA-48 . 5′ Rapid amplification of cDNA ends experiments revealed that expression of these β-lactamase genes was driven by the outward-directed promoter, P out , located in the IS 1999 elements. These findings led us to study IS 1999 -mediated gene mobilization. Thus, the transposition properties of IS 1999 and of IS 1999 -based composite transposons, made of two copies of IS 1999 in different orientations, were investigated. IS 1999 or IS 1999 -based composite transposons were capable of transposing onto the conjugative plasmid pOX38-Gen. Sequence analysis of the insertion sites revealed that IS 1999 inserted preferentially into DNA targets containing the consensus sequence NGCNNNGCN. Transposition was more efficient when at least one left inverted repeat end was located at an outside end of the transposon. The transposition frequency of IS 1999.2 was 10-fold lower than that of IS 1999 , and transposition frequencies of the putative natural transposon, Tn 1999 , were below detection limits of our transposition assay. This reduced transposition frequency of IS 1999.2 -based elements may result from a lower transcription of the transposase gene, as revealed by reverse transcription-PCR analyses.