Cloning and expression of Thiobacillus ferrooxidans mercury ion resistance genes in Escherichia coli

Abstract

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...

Expression of bacterial mercuric ion reductase in Saccharomyces cerevisiae

“…Nevertheless, this enzyme activity resulted in a fivefold increase in resistance to Hg2+ when cells were grown on minimal medium. Mercuric ion tolerance of both the sensitive and the resistant yeast cells is much higher on complex medium because of the presence of metal-chelating components, e.g., amino acids, and is much higher than that of sensitive E. coli cells (21).…”

“…Expression of the Thiobacillus merA gene in E. coli resulted in a twofold increase in mercury resistance, which was enhanced to fivefold by the additional expression of the merC gene (21). Therefore, enhancement of a specific Hg2+ transport may result in enhancement of resistance to mercuric ion in yeast cells containing the merA gene.…”

“…However, mer determinants may encode up to six gene products in addition to MerA in gram-negative bacteria: the activator/repressor protein MerR, the specific Hg2+ carrier MerT, the periplasmic Hg2+-binding protein MerP, the second Hg2+ transport system MerC, the organomercurial lyase MerB, and the MerD protein, which is involved in the regulation of mer (9,12,15,17,22). The mer determinant cloned from Thiobacillus ferrooxidans, however, encodes only two proteins, MerA and MerC, which confer mercuric ion resistance to Escherichia coli (21). Therefore, along with MerA, at least one additional component appears to be involved in full expression of mercuric ion resistance in prokaryotes.…”

Functional Dissection of a Mercuric Ion Transporter, MerC, fromAcidithiobacillus ferrooxidans

“…0 , and pTM314 20) were used as templates. In the primers used, the restriction enzyme sites used for cloning are marked by underlining, and distinguished from the original enzyme sites marked by double-underlining (see below).…”

Electrotransformation of Thiobacillus ferrooxidans with plasmids containing a mer determinant

“…Plasmid pTM314 (37) contained the 4.6-kb Sall fragment (which includes an operator-promoter region followed by merC and merA genes) from T. ferrooxidans E-15 in the SalI site of pUC18. pTM315 (37) contained the same Sall fragment in reverse orientation on pUC18. pTMR25 (15) generated by using electroporation apparatus (Gene-pulser;…”