The Alcaligenes eutrophus protein HoxN mediates nickel transport in Escherichia coli

Abstract

HoxN, an integral membrane protein with seven transmembrane helices and a molecular mass of 33.1 kDa, is involved in high-affinity nickel transport in Alcaligenes eutrophus H16. From genetic analyses, it has been concluded that HoxN is a single-component ion carrier. To investigate this assumption, hoxN was introduced into Escherichia coli. The recombinant strain showed significantly enhanced nickel uptake in a short-interval assay. Likewise, growth in the presence of 63 NiCl 2 yielded a more than 15-fold-increased cellular nickel content. The HoxN-based nickel transport activity could also be demonstrated in a physiological assay: an E. coli strain coexpressing hoxN and the urease operon of Klebsiella aerogenes exhibited urease activity 10-fold greater than that in the strain lacking a functional hoxN. These results strongly suggest that HoxN is sufficient to operate as a nickel permease. Multiple sequence alignment of HoxN and four other bacterial membrane proteins implicated in nickel metabolism revealed two conserved signatures which may play a role in the nickel translocation process.Nickel is an essential trace element for at least four biological processes: (i) oxidation and evolution of molecular hydrogen, (ii) hydrolysis of urea, (iii) carbon monoxide dehydrogenase-mediated acetate metabolism in methanogens and in homoacetogenic bacteria, and (iv) reduction of methyl coenzyme M to methane in methanogenic archaea (6). Uptake of nickel ions is a prerequisite for those organisms catalyzing nickel-dependent reactions. The microbial transport of the divalent Ni 2ϩ cation, the most commonly occurring oxidation state of nickel, has been investigated with regard to physiology, ion specificity, and very recently, the molecular structure of the transport proteins (3, 18). Nickel uptake is mediated by nonspecific Mg 2ϩ transport systems and by high-affinity systems specific for the transport of nickel (for a review, see reference 7).In Alcaligenes eutrophus, a gram-negative aquatic and soil bacterium which can utilize molecular hydrogen as an energy source, nickel uptake occurs by Mg 2ϩ transporters and by a high-affinity nickel transporter, the product of gene hoxN (2, 11). The phenotype of a HoxN-negative mutant manifested itself in the inability to grow on hydrogen as the energy source under nickel limitation (below 100 nM) in the presence of 0.8 mM Mg 2ϩ . As expected for a mutant lacking the specific nickel transporter, this nickel deficiency was physiologically compensated for by either increasing the Ni 2ϩ concentration to 1 M or decreasing the Mg 2ϩ concentration (1, 2). The nucleotide sequence of hoxN has been determined, and the two-dimensional membrane topology of the HoxN nickel transport protein has been investigated. While computer analyses of potential membrane-spanning segments gave ambiguous results, the construction of a set of fusions to alkaline phosphatase and ␤-galactosidase clearly allowed the identification of segments located in the periplasm and in the cytoplasm, respectively. Using these...

Crystal Structures of the Liganded and Unliganded Nickel-binding Protein NikA from Escherichia coli

“…Nickel incorporation in Escherichia coli hydrogenases also requires a GTP-binding accessory protein, HypB (11). The protein sequences of a number of microbial nickel uptake systems have been described (12)(13)(14), but to date no structural information has been obtained. Nickel uptake by bacteria is of interest for bioremediation and water purification as well as its biological function and involvement in pathogenesis.…”

hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

“…The hox gene cluster harbours additional genes encoding a high-affinity nickel permease Wolfram et al, 1995) and regulatory proteins required for hydrogenase gene expression (Zimmer et al, 1995;Lenz et al, 1994).…”

Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake

“…Members of the NiCoT family are grouped into three classes depending on whether they are highly specific for nickel (Class I), prefer cobalt over nickel (Class II) or prefer nickel over cobalt (Class III). 7 The best-studied proteins within this family are the Class I nickel transporters Cupriavidus necator HoxN 8 and Helicobacter pylori NixA. 9 Both proteins are high-affinity nickel transporters with an apparent K m in the low nanomolar range.…”