Expression of the nickel-specific transport system encoded by theEscherichia 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 nikRwas partially controlled by Fnr through transcription from thenikA promoter region. In addition, a specific transcription start site for the constitutive expression of nikR was found 51 bp upstream of the nikR gene.

The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system specific for nickel. In this report, we describe the overproduction of the periplasmic nickel-binding protein NikA by cloning the nikA gene into an overexpression vector, pRE1. NikA was purified free of nickel to near homogeneity from the periplasm by hydrophobic and ion-exchange chromatography. N-terminal amino acid sequencing confirmed that the leader peptide of NikA had been removed. The nickel-binding properties of the protein has been studied by monitoring the quenching of intrinsic protein fluorescence. NikA binds one atom of nickel/molecule of protein with a dissociation constant (Kd) of less than 0.1 microM. Other metals (cobalt, copper, iron) are bound at least 10-fold less tightly. The high specificity for Ni2+ is also demonstrated by high-performance immobilized-metal-ion affinity chromatography. Biosynthesis of NikA occurred only under anaerobic conditions and was dependent on the general anaerobic regulator FNR. It was repressed by the presence of 250 microM Ni2+ in the growth medium and was not affected by either 30 mM formate or 100 mM nitrate. Anaerobically grown wild-type strain MC4100 contains about 23,000 molecules of NikA/cell. In addition to the effect on nickel transport, nikA mutation affects also the nickel sensing in Tar-dependent repellent chemotaxis.

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The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system specific for nickel. In this report, we describe the overproduction of the periplasmic nickel-binding protein NikA by cloning the nikA gene into an overexpression vector, pREl. NikA was purified free of nickel to near homogeneity from the periplasm by hydrophobic and ion-exchange chromatography. N-terminal amino acid sequencing confirmed that the leader peptide of NikA had been removed. The nickel-binding properties of the protein has been studied by monitoring the quenching of intrinsic protein fluorescence.NikA binds one atom of nickellmolecule of protein with a dissociation constant (Kd) of less than 0.1 pM.Other metals (cobalt, copper, iron) are bound at least 10-fold less tightly. The high specificity for Ni2+ is also demonstrated by high-performance immobilized-metal-ion affinity chromatography. Biosynthesis of NikA occurred only under anaerobic conditions and was dependent on the general anaerobic regulator FNR. It was repressed by the presence of 250 pM NiZ+ in the growth medium and was not affected by either 30 mM formate or 100 mM nitrate. Anaerobically grown wild-type strain MC4100 contains about 23000 molecules of NiWcell. In addition to the effect on nickel transport, nikA mutation affects also the nickel sensing in Tar-dependent repellent chemotaxis.Keywords. nikA gene ; nickel binding ; periplasmic protein ; transport; overproduction.Bacterial cold-osmotic-shock-sensitive transport systems are complex permeases composed of a periplasmic substrate-binding receptor and a membrane-bound complex containing 2 -4 proteins (Ames, 1986). These permeases are energized by the hydrolysis of ATP, mediated by one or two of the proteins in the complex. The periplasmic substrate-binding proteins are the primary receptors for the transport systems specific for different small molecules such as sugars, amino acids, peptides and inorganic ions. In some cases, they also serve as the primary receptors for bacterial chemotaxis (Bourret et al., 1991;Furlong, 1987;Macnab, 1987). For example, in addition to its role in transport, the periplasmic maltose-binding protein, MBP, encoded by ma1E functions as the primary maltose chemoreceptor (Koiwai and Hayashi, 1979;Richarme, 1982). Generation of the chemotactic response to maltose requires a second protein, the chemotactic signal transducer Tar, which is also required for the chemotactic response to aspartate and to two repellents Ni2+ and Co2+ (Springer et al., 1977;Tso and Adler, 1974 Nickel has long been known as a heavy metal toxic to both eukaryotic and prokaryotic organisms (Barbich and Stotzky, 1983 ;Brown and Sunderman, 1988). Epidemiological studies have identified nickel as potentially carcinogenic and allergenic to humans (Dool et al., 1977; MennC et al., 1989). We demonstrated recently that nickel has an antagonistic effect on the fermentative growth of Escherichia coli . In E.coli the only known nickel-containing enzymes are the three hydrogenase isoenzymes whic...