A search of various domestic isolates of Thiobacillus ferrooxidans revealed that some were fairly resistant to mercury ion. A proportion of mercury-resistant clones were able to volatilize mercury, and their corresponding gene was localized not in the plasmid DNA but in chromosomal DNA. This mercury ion resistance gene was cloned in Escherichia coli. E. coli carrying the recombinant plasmid was able to grow in the presence of more than 40 ,ug of HgCl2 per ml. Deletion analysis of the recombinant plasmid showed that the entire coding sequence of the mercury ion resistance gene was located within a 2.3-kilobase fragment of the chromosomal DNA from strain E-15. At least two polypeptides (molecular mass, 56 and 16 kDa, respectively) were coded by this fragment.Thiobacillus ferrooxidans is an acidophilic chemoautotroph that utilizes energy generated by the oxidation of inorganic ferrous ion to ferric ion. This organism has been a focus of interest because of its industrial utility in so-called bacterial leaching. However, the slow growth of this organism has limited its further use. To fully exploit the properties of this bacterium by molecular breeding, an understanding of both its gene structure and its expression is essential. However, knowledge about the genetic background of this organism is scarce. Recently, Rawlings and co-workers (24,25) reported that the oriV, oriT, and mob functions of plasmids from T. ferrooxidans are expressed in Escherichia coli. Furthermore, this group (1, 20, 22) has cloned and determined the nucleotide sequences of the glutamine synthetase gene and nitrogenase iron protein gene from T. ferrooxidans. The Thiobacillus ginA gene is able to complement the E. coli glnA strain (1). These findings indicated that genetic information from T. ferrooxidans is functional in the heterotroph E. coli.It has been reported that T. ferrooxidans is quite resistant to iron, acidity, and some heavy metals (Cu2+, Zn2+, Ni2+, Co2+, etc.) but exceedingly sensitive to uranium, silver, and mercury ions (26). So far, two groups (3, 19) have reported the presence of the enzyme mercuric reductase in mercury ion-resistant T. ferrooxidans strains.In the present study, we showed that (i) the mercury ion resistance gene of T. ferrooxidans is present in the chromosomal DNA but not in plasmid DNAs, (ii) it was possible to clone the corresponding gene into E. coli, (iii) this gene was expressed in E. coli, and (iv) two polypeptides were coded by the minimum essential region. (28) and purified on colloidal silica plates (M. Kawarazaki, personal communication). Each silica plate was composed of 900 ml of colloidal silica no. 30 (Nissan Kagaku Co., Ltd., Tokyo, Japan), 70 ml of 10-fold 9K basal salts, and 30 ml of saturated FeSO4 7H20 solution. Each solution was separately autoclaved, and then mixed and adjusted to pH 3.6 with sulfuric acid. A 20-ml portion was poured into each petri dish and incubated at 60°C for 16 h. After the incubation, the pH of each silica plate became 2.8. A purified T. ferrooxidans clone was grow...