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.
de Pina
K
K
Laboratoire de Génétique Moléculaire des Microorganismes et des Interactions Cellulaires, CNRS URA 1486, Villeurbanne, France.
Navarro
C
C
McWalter
L
L
Boxer
D H
DH
Price
N C
NC
Kelly
S M
SM
Mandrand-Berthelot
M A
MA
Wu
L F
LF
D001426:Bacterial Proteins; D001483:Base Sequence; D001693:Biological Transport, Active; D002633:Chemotaxis; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009532:Nickel / Q000378:metabolism; D009876:Operon
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008670:Metals; C068025:nikA protein, Plasmid R64; D009532:Nickel; D004264:DNA Topoisomerases, Type I