Isolation of a novel plasmid, pNi15, from Enterobacter sp. Ni15 containing a nickel resistance gene.

We isolated nickel-resistant bacterium from soil in order to identify a novel nickel resistance determinant. Using 16S rRNA gene sequencing, an isolate was identified as Enterobacter sp. Ni15. This species showed a medium-level (resistant to up to 10 mM) nickel resistance in nutrient-rich media. Enterobacter sp. Ni15 has a novel plasmid, pNi15, and an increased nickel resistance to Escherichia coli DH5alphain trans. To isolate the nickel resistance gene from pNi15, the plasmid was digested with XbaI and its fragments were cloned into pBluescriptIISK(+). The clones were transferred into E. coli DH5alpha. The nickel resistance of the clones was then assayed. From these results, a pNi15100 isolate containing a 5,328 bp XbaI fragment of pNi15 was identified and sequenced. The E. coli DH5alpha harboring the pNi15100 showed a resistance to up to 7 mM nickel. Using a subcloning analysis, we were able to identify the novel nickel resistance determinant: the nrp gene encoding the putative proteins NrpA and NrpB.