Characterization of the Variants, Flanking Genes, and Promoter Activity of the Leifsonia xyli subsp. cynodontis Insertion Sequence IS 1237

Abstract

ABSTRACT We performed a comprehensive study of the distribution and function of an insertion sequence (IS) element, IS 1237 , in the genome of Leifsonia xyli subsp. cynodontis , a useful genetic carrier for expressing beneficial foreign genes in plants. Two shorter IS 1237 isoforms, IS 1237 d1 and IS 1237 d2 resulting from precise deletion between two nonperfect repeats, were found in the bacterial genome at a level that was one-fifth the level of wild-type IS 1237 . Both the genome and native plasmid pCXC100 harbor a truncated toxin-antitoxin cassette that is precisely fused with a 5′-truncated IS 1237 sequence at one nonperfect repeat, indicating that it is a hot site for DNA rearrangement. Nevertheless, no transposition activity was detected when the putative transposase of IS 1237 was overexpressed in Escherichia coli . Using thermal asymmetric interlaced PCR, we identified 13 upstream and 10 downstream unique flanking sequences, and two pairs of these sequences were from the same loci, suggesting that IS 1237 has up to 65 unique loci in the L. xyli subsp. cynodontis chromosome. The presence of TAA or TTA direct repeat sequences at most insertion sites indicated that IS 1237 inserts into the loci by active transposition. IS 1237 showed a high propensity for insertion into other IS elements, such as IS Lxc1 and IS Lxc2 , which could offer IS 1237 a nonautonomous transposition pathway through the host IS elements. Interestingly, we showed that IS 1237 has a strong promoter at the 3′ end and a weak promoter at the 5′ end, and both promoters promote the transcription of adjacent genes in different gram-positive bacteria. The high-copy-number nature of IS 1237 and its promoter activity may contribute to bacterial fitness.