Crystallization and preliminary X-ray diffraction study of the nickel-binding protein NikA of Escherichia coli.

NikA, a periplasmic nickel-binding protein involved in nickel-repellent chemotaxis has been crystallized. The crystals are hexagonal with space group P6(2) (or its enantiomorph) with a = 160.3 A and c = 138.4 A and they diffract to at least 3.0 A resolution in the laboratory. NikA presents sequence homology with several periplasmic solute-binding proteins from Gram-negative bacteria.

Antagonistic effect of nickel on the fermentative growth of Escherichia coli K-12 and comparison of nickel and cobalt toxicity on the aerobic and anaerobic growth.

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.

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

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.

Purification and characterization of the periplasmic nickel-binding protein NikA of Escherichia coli K12.

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.