Transposition insertional mutagenesis of the insertion sequences (IS elements) was discovered for the first time in Acidithiobacillus caldus (A. caldus), when A. caldus MTH-04 hsdM (type I restriction-modification system M-subunit) mutant was constructed by electroporation of a suicide plasmid. The IS element, specifically inserting into hsdM gene, was analyzed, identified, and named ISAtc2. The transposition frequency of ISAtc2 was ranged from 4% to 7%, and no reverse mutation occurred in the mutants after 50 generations of proliferation without selective pressure. These results revealed that transposition of IS elements on A. caldus chromosome could regulate the gene expression and metabolic pathways by gene inactivation, gene loss and gene acquisition. Therefore, the transposition of IS elements in A. caldus may be an important and unique regulation mechanism for adaptation to the living condition. (C) 2013 Elsevier Inc. All rights reserved.