Isolation and characterization of the nikR gene encoding a nickel-responsive regulator in Escherichia coli

Abstract

Expression of the nickel-specific transport system encoded by the Escherichia coli nikABCDE operon is repressed by a high concentration of nickel. By using random transposon Tn10 insertion, we isolated mutants in which expression of the nik operon became constitutive with respect to nickel. We have identified the corresponding nikR gene which encodes a nickel-responsive regulator. Expression of nikR was partially controlled by Fnr through transcription from the nikA promoter region. In addition, a specific transcription start site for the constitutive expression of nikR was found 51 bp upstream of the nikR gene.

FIG. 1

Complementation analysis of Tn10 insertion mutants with plasmids carrying the nik region. KS01 and KS02 are Tn10 insertion derivatives of HYD723 (nikA-lacZ). Cells which carried the indicated plasmid were grown microaerobically at 37°C in LB medium supplemented with 2 μM ammonium molybdate, 2 μM sodium selenite, and kanamycin (25 μg/ml) when required (21). Plasmids pLW22, pLW25, and pLW26 were described previously (24). β-Galactosidase activity was measured for cells treated by addition of 0.0025% sodium dodecyl sulfate–5% chloroform, and the specific activity is expressed as nanomoles of o-nitrophenol produced per minute per milligram bacterial dry weight. Values quoted are the averages of three separate experiments. Symbols: E, EcoRI; H, HincII; M, MluI; N, NsiI; S, SspI; V, EcoRV.

FIG. 2

Specific expression of the nikR, nikD, and nikE gene products, under control of the T7 φ10 promoter in E. coli K38/pGP1-2. Cells containing vector pT7-6 (lane 1), its derivative pHD4, harboring nikDER (lane 2), vector pKSM710 (lane 4), and its derivative p8611, harboring nikR (lane 3), were labeled with [³⁵S]methionine and [³⁵S]cysteine and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 17% denaturing polyacrylamide gel. Molecular mass standards (in kilodaltons) and NikE, NikD, and NikR proteins are indicated on the right and on the left, respectively.

FIG. 3

Determination of the constitutive nikR transcription start site. Total RNAs (50 μg) isolated from E. coli NM522/pLW22 grown anaerobically in Luria broth in the absence (lane 1) or presence (lane 2) of 0.5 mM NiCl₂ were analyzed by primer extension with primer NikR (see text). The DNA sequence ladder (lanes TGCA) was obtained with the same primer and plasmid pLW22 as a template. Part of the sequence and the nikR transcription start site are indicated on the right.