Cloning and characterization of a high-copy-number novel insertion sequence from chemolithotrophic Thiobacillus ferrooxidans

Abstract

Two distinct families of repetitive DNA elements (1.4 and 1.2 kb) were identified from S1 nuclease-treated genomic DNA of four strains ofThiobacillus ferrooxidans.The 1.4-kb fragment hybridized with IST2,an insertion sequence ofT. ferrooxidans.The 1.2-kb fragment was cloned and sequenced. The sequence (IST445), 1219 bp in length, with features characteristic of an insertion element, has a terminal inverted repeat of 8 bp, which can be further extended to 23 or 48 bp with 9 and 26 mismatches, respectively. It displays 54.4% identity in 967 nucleotides of overlap with ISAE1ofAlcaligenes eutrophus.The IST445contains three open reading frames which have codon usage almost similar to 56 different coding genes ofT. ferrooxidans.In Southern blots of restricted genomic DNAs probed with IST445,each of the several strains ofT. ferrooxidansgives a distinctive fingerprint. IST445is present in the range of 10-20 copies per genome in the four strains studied.