Characterization of the P IB -Type ATPases Present in Thermus thermophilus

Abstract

ABSTRACT P IB -type ATPases transport heavy metals (Cu 2+ , Cu + , Ag + , Zn 2+ , Cd 2+ , Co 2+ ) across biomembranes, playing a key role in homeostasis and in the mechanisms of biotolerance of these metals. Three genes coding for putative P IB -type ATPases are present in the genome of Thermus thermophilus (HB8 and HB27): the TTC1358, TTC1371, and TTC0354 genes; these genes are annotated, respectively, as two copper transporter (CopA and CopB) genes and a zinc-cadmium transporter (Zn 2+ /Cd 2+ -ATPase) gene. We cloned and expressed the three proteins with 8His tags using a T. thermophilus expression system. After purification, each of the proteins was shown to have phosphodiesterase activity at 65°C with ATP and p -nitrophenyl phosphate ( p NPP) as substrates. CopA was found to have greater activity in the presence of Cu + , while CopB was found to have greater activity in the presence of Cu 2+ . The putative Zn 2+ /Cd 2+ -ATPase was truncated at the N terminus and was, surprisingly, activated in vitro by copper but not by zinc or cadmium. When expressed in Escherichia coli , however, the putative Zn 2+ /Cd 2+ -ATPase could be isolated as a full-length protein and the ATPase activity was increased by the addition of Zn 2+ and Cd 2+ as well as by Cu + . Mutant strains in which each of the three P-type ATPases was deleted singly were constructed. In each case, the deletion increased the sensitivity of the strain to growth in the presence of copper in the medium, indicating that each of the three can pump copper out of the cells and play a role in copper detoxification.