The complete nucleotide sequence of the Escherichia coli nik locus, which has been suggested to encode the specific transport system for nickel, has been determined. It was found to contain five overlapping open reading frames that form a single transcription unit. Deduced amino acid sequence of the nik operon shows that its five gene products, NikA to NikE, are highly homologous to components of oligopeptide- and dipeptide-binding protein-dependent transport systems from several Gram-negative and Gram-positive species. NikA represents the periplasmic binding protein, NikB and NikC are similar to integral membrane components of periplasmic permeases, and NikD and NikE possess typical ATP-binding domains that suggest their energy coupling role to the transport process. Insertion mutations in nik genes totally abolished the nickel-containing hydrogenase activity under nickel limitation and markedly altered the rate of nickel transport. Taken together, these data support the notion that the nik operon encodes a typical periplasmic binding-protein-dependent transport system for nickel.

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.

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...

The facultative anaerobic enterobacterium Escherichia coli requires the activity of nickel-containing hydrogenase for its anaerobic growth. Deficiency of the specific nickel transport system led to a hydrogenase-minus phenotype and slowed down the fermentative growth in the nik mutant. Addition of 300 microM nickel to the growth medium could restore the hydrogenase activity. This restoration resulted in the recovery of anaerobic growth. A further increase of nickel concentration inhibited growth. Thus nickel shows an antagonistic effect on the anaerobic growth of E. coli. To study the mechanism of nickel toxicity, two classes of nickel-resistant mutants were isolated. The nkr mutant was obtained by selecting colonies grown on nickel-containing minimal plate. It acquired simultaneously the resistance to cobalt. A nonspecific magnesium transport mutant corA was isolated on cobalt-containing plate. The corA mutant was also resistant to nickel. When analyzing the influence of nickel and cobalt on the bacterial growth, we obtained two interesting observations. First, anaerobic growth was less sensitive than aerobic growth to cobalt toxicity. In contrast, nickel toxicity did not vary from the growth conditions. Second, cobalt seems to abolish the growth, while nickel appears to slow down the growth rate under the condition used.

The facultative anaerobic enterobacterium Escherichia coli requires the activity of nickel-containing hydrogenase for its anaerobic growth. Deficiency of the specific nickel transport system led to a hydrogenase-minus phenotype and slowed down the fermentative growth in the nik mutant. Addition of 300 pM nickel to the growth medium could restore the hydrogenase activity. This restoration resulted in the recovery of anaerobic growth. A further increase of nickel concentration inhibited growth. Thus nickel shows an antagonistic effect on the anaerobic growth of E. coli. To study the mechanism of nickel toxicity, two classes of nickel-resistant mutants were isolated. The nkr mutant was obtained by selecting colonies grown on nickel-containing minimal plate. It acquired simultaneously the resistance to cobalt. A nonspecific magnesium transport mutant corA was isolated on cobalt-containing plate. The corA mutant was also resistant to nickel. When analyzing the influence of nickel and cobalt on the bacterial growth, we obtained two interesting observations. First, anaerobic growth was less sensitive than aerobic growth to cobalt toxicity. In contrast, nickel toxicity did not vary from the growth conditions. Second, cobalt seems to abolish the growth, while nickel appears to slow down the growth rate under the condition used. -Environ Health Perspect 1 02(Suppl 3):297-300 (1994).

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