Nic1p, a relative of bacterial transition metal permeases in Schizosaccharomyces pombe, provides nickel ion for urease biosynthesis.

The Schizosaccharomyces pombe genome sequencing project identified an open reading frame (O74869 and O74912, named Nic1p in the present study) with significant similarity to members of a family of bacterial transition metal permeases. These uptake systems transport Ni(2+) ion with extremely high affinity across the bacterial cytoplasmic membrane, but they differ in selectivity toward divalent transition metal cations. An S. pombe mutant harboring an interrupted nic1 allele (nic1-1) was strongly impaired in (63)Ni(2+) uptake in the presence of a high molar ratio of Mg(2+) relative to Ni(2+), conditions that reflect the natural situation. Under these conditions, the nic1-1 mutant contained only background activities of the nickel-dependent cytoplasmic enzyme urease and could not catabolize urea. Among a series of divalent transition metal cations tested (Cd(2+), Co(2+), Cu(2+), Mn(2+), and Zn(2+)), only Co(2+) caused considerable inhibition of Nic1p-mediated Ni(2+) uptake. On the other hand, experiments with (57)Co(2+) (at nm concentrations) did not show significant differences in Co(2+) uptake between the nic1-1 mutant and the parental strain. Our data suggest that Nic1p acts as a plasma-membrane nickel transporter in fission yeast, a finding that invites searches for isologous counterparts in higher eukaryotes.

Nickel transport systems in microorganisms.

The transition metal Ni is an essential cofactor for a number of enzymatic reactions in both prokaryotes and eukaryotes. Molecular analyses have revealed the existence of two major types of high-affinity Ni2+ transporters in bacteria. The Nik system of Escherichia coli is a member of the ABC transporter family and provides Ni2+ ion for the anaerobic biosynthesis of hydrogenases. The periplasmic binding protein of the transporter, NikA, is likely to play a dual role. It acts as the primary binder in the uptake process and is also involved in negative chemotaxis to escape Ni overload. Expression of the nik operon is controlled by the Ni-responsive repressor NikR, which shows functional similarity to the ferric ion uptake regulator Fur. The second type of Ni2+ transporter is represented by HoxN of Ralstonia eutropha, the prototype of a novel family of transition metal permeases. Members of this family have been identified in gram-negative and gram-positive bacteria and recently also in a fission yeast. They transport Ni2+ with very high affinity, but differ with regard to specificity. Site-directed mutagenesis experiments have identified residues that are essential for transport. Besides these uptake systems, different types of metal export systems, which prevent microorganisms from the toxic effects of Ni2+ at elevated intracellular concentrations, have also been described.

Selective transport of divalent cations by transition metal permeases: the Alcaligenes eutrophus HoxN and the Rhodococcus rhodochrous NhlF.

nhlF and hoxN, the genes encoding a cobalt transporter of Rhodococcus rhodochrous J1 and a nickel permease of Alcaligenes eutrophus H16, respectively, were expressed in Escherichia coli. 57CO2+ and 63Ni2+ transport of the recombinants was examined by means of a previously described physiological assay. Although the transporters are highly similar, different preferences for divalent transition metal cations were observed. HoxN was unable to transport 57CO2+, but mediated 63Ni2+ uptake. The latter activity was unaffected by a tenfold excess of other divalent cations, showing the specificity of HoxN for Ni2+. In contrast, NhlF transported both 57CO2+ and 63Ni2+ ion. NhlF-mediated 63Ni2+ uptake was markedly reduced in the presence of CO2+, while 57CO2+ uptake was only slightly lower in the presence of Ni2+. These results indicate different affinities of NhlF for CO2+ and Ni2+ and identified CO2+ ion as the preferred substrate.

A Ni2+ binding motif is the basis of high affinity transport of the Alcaligenes eutrophus nickel permease.

Amino acid exchanges in the Alcaligenes eutrophus nickel permease (HoxN) were constructed by site-directed mutagenesis, and their effects on nickel ion uptake were investigated. Mutant hoxN alleles were expressed in Escherichia coli, and activity of the altered permeases was examined via a recently described physiological assay (Wolfram, L., Friedrich, B., and Eitinger, T. (1995) J. Bacteriol. 177, 1840-1843). Replacement of Cys-37, Cys-256, or Cys-318 by alanine did not severely affect nickel ion uptake. This activity of a C331A mutant was diminished by 60%, and a similar phenotype was obtained with a cysteine-less mutant harboring four Cys to Ala exchanges. Alterations in a histidine-containing sequence motif (His-62, Asp-67, His-68), which is conserved in microbial nickel transport proteins, strongly affected or completely abolished transport activity in the E. coli system. The analysis of HoxN alkaline phosphatase fusion proteins implied that His-62, Asp-67, and His-68 exchanges did not interfere with overall membrane topology or stability of the nickel permease. These mutations were reintroduced into the A. eutrophus wild-type strain. Analyses of the resulting HoxN mutants indicated that exchanges in the histidine motif led to a clearly decreased affinity of the permease for nickel ion.

hyp gene products in Alcaligenes eutrophus are part of a hydrogenase-maturation system.

In Alcaligenes eutrophus H16 the hyp gene complex consists of six open reading frames hypA1, B1, F1, C, D and E whose products are involved in maturation of the two NiFe hydrogenases: an NAD-reducing cytoplasmic enzyme (SH) and a membrane-bound electron-transport-coupled protein (MBH). hypB1 and hypF1 were originally considered to form a single open reading frame designated hypB [Dernedde, J., Eitinger, M. & Friedrich, B. (1993) Arch. Microbiol. 159, 545-553]. Re-examination of the relevant sequence identified hypB1 and hypF1 as two distinct genes. Non-polar in-frame deletions in the individual hyp genes were constructed in vitro and transferred via gene replacement to the wild-type strain. The resulting mutants fall into two classes. Deletions in hypC, D and E (class I) gave a clear negative phenotype, while hypA1, B1 and F1 deletion mutants (class II) were not impaired in hydrogen metabolism. Class I mutants were unable to grow on hydrogen under autotrophic conditions. The enzymatic activities of SH and MBH were disrupted in all three class I mutants. Immunoblot analysis showed the presence of the H2-activating SH subunit (HoxH) at levels comparable to those observed in the wild-type strain whereas the other three subunits (HoxF, U and Y) were only detectable in trace amounts, probably due to proteolytic degradation. Likewise, MBH was less stable in hypC, D and E deletion mutants and was not attached to the cytoplasmic membrane. In the wild-type strain, HoxH and the MBH large subunit (HoxG) undergo C-terminal proteolytic processing before attaining enzymatic activity. In class I mutants this maturation was blocked. 63Ni-incorporation experiments identified both hydrogenases as nickel-free apoproteins in these mutants. Although class II mutants bearing deletions in hypA1, B1 and F1 showed no alteration of the wild-type phenotype, a role for these genes in the incorporation of nickel and hence hydrogenase maturation cannot be excluded, since there is experimental evidence that this set of genes is duplicated in A. eutrophus.

The Alcaligenes eutrophus protein HoxN mediates nickel transport in Escherichia coli.

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 63NiCl2 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.

A topological model for the high-affinity nickel transporter of Alcaligenes eutrophus.

The gene hoxN of Alcaligenes eutrophus encodes a membrane protein with a molecular mass of 33.1 kDa that mediates energy-dependent uptake of nickel ions. Based on the hydrophobicity of the HoxN protein five, six, or seven transmembrane segments were predicted, depending on the algorithm used for computer analysis. To distinguish between these possibilities varying segments of the amino-terminal end of the transporter were fused to the Escherichia coli enzymes alkaline phosphatase (PhoA) or beta-galactosidase (LacZ). The enzymatic activity of 16 HoxN-PhoA and 15 HoxN-LacZ fusions was determined. On the assumption that PhoA fusions only exhibit high activity when fused to periplasmic domains of the target, while LacZ fusions are only active when oriented towards the cytoplasm, a two-dimensional model for the nickel transporter was developed. This model proposes that HoxN contains four periplasmic and four cytoplasmic regions, and seven transmembrane helices. The amino terminus is located in the cytoplasm, and the carboxyl terminus faces the periplasm.

The gltF gene of Klebsiella pneumoniae: cloning and initial characterization.

From a gene bank of Klebsiella pneumoniae M5a1, a 1.7 kb gene fragment was isolated which was able to restore the Ntr+ phenotype and ammonium (methylammonium) transport, but not glutamate synthase in an Escherichia coli glt mutant (glutamate synthase deficiency). The fragment strongly hybridized with the gltF regulatory gene from E. coli. After subcloning the fragment into an overexpression vector, a protein with a molecular weight of 27,000 dalton was identified as the gene product. The results indicate that the fragment cloned contains the gltF gene from K.pneumoniae.

Construction and properties of a triprotein containing the high-affinity nickel transporter of Alcaligenes eutrophus.

The high-affinity nickel transporter of Alcaligenes eutrophus H16 is encoded by gene hoxN, which maps within the hydrogenase gene cluster of megaplasmid pHG1. A tripartite gene fusion was constructed, consisting of (i) the Escherichia coli lacZ gene for beta-galactosidase, (ii) a segment encoding an endoproteolytically cleavable peptide, and (iii) the A. eutrophus gene hoxN. An E. coli strain harboring this construct (plasmid pCH307) efficiently produced the corresponding triprotein upon induction. A broad-host-range derivative of pCH307 was shown to complement an A. eutrophus HoxN- mutant.

Cloning, nucleotide sequence, and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus.

High-affinity nickel transport in Alcaligenes eutrophus H16 is mediated by a function designated hoxN. hoxN lies within the hydrogenase gene cluster of megaplasmid pHG1. An insertional mutation at the hoxN locus led to an increased nickel requirement. In this mutant (strain HF260) both autotrophic growth on hydrogen and wild-type level of urease, a nickel-containing enzyme, were dependent on high concentration of nickel in the medium. Studies with a heterologous in vivo expression system revealed that the hoxN locus encodes two proteins with Mr = 30,000 and 28,000. Only the larger polypeptide was essential for nickel transport. The hoxN locus was cloned on a 2.2-kilobase pair fragment. Nucleotide sequence analysis of the hoxN locus revealed an open reading frame with a coding capacity for a protein of 33.1 kDa. The insertion leading to the Nic- phenotype of strain HF260 maps within this open reading frame indicating that it does in fact have coding function. The deduced amino acid sequence of the hoxN gene has several features typical of a hydrophobic integral membrane protein. Alkaline phosphatase fusion proteins produced by insertion of the transposon TnphoA into hoxN gave significant levels of alkaline phosphatase activity indicating that protein HoxN contains periplasmic domains. Taken together, our results suggest that gene hoxN encodes the high-affinity nickel transporter of A. eutrophus.

Genetic determinants of a nickel-specific transport system are part of the plasmid-encoded hydrogenase gene cluster in Alcaligenes eutrophus.

Nickel-deficient (Nic-) mutants of Alcaligenes eutrophus requiring high levels of nickel ions for autotrophic growth with hydrogen were characterized. The Nic- mutants carried defined deletions in the hydrogenase gene cluster of the indigenous pHG megaplasmid. Nickel deficiency correlated with a low level of the nickel-containing hydrogenase activity, a slow rate of nickel transport, and reduced activity of urease. The Nic+ phenotype was restored by a cloned DNA sequence (hoxN) of a megaplasmid pHG1 DNA library of A. eutrophus H16. hoxN is part of the hydrogenase gene cluster. The nickel requirement of Nic- mutants was enhanced by increasing the concentration of magnesium. This suggests that the Nic- mutants are impaired in the nickel-specific transport system and thus depend on the second transport activity which normally mediates the uptake of magnesium.