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J. Biol. Chem., Vol. 275, Issue 24, 18029-18033, June 16, 2000

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Nic1p, a Relative of Bacterial Transition Metal Permeases in Schizosaccharomyces pombe, Provides Nickel Ion for Urease Biosynthesis^*

Thomas Eitinger, Olaf Degen, Ute Böhnke, and Marion Müller

From the Institut für Biologie, Humboldt-Universität zu Berlin, 10115 Berlin, Germany

Received for publication, March 9, 2000, and in revised form, March 30, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

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 Ni²⁺ 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 ⁶³Ni²⁺ uptake in the presence of a high molar ratio of Mg²⁺ relative to Ni²⁺, 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 (Cd²⁺, Co²⁺, Cu²⁺, Mn²⁺, and Zn²⁺), only Co²⁺ caused considerable inhibition of Nic1p-mediated Ni²⁺ uptake. On the other hand, experiments with ⁵⁷Co²⁺ (at nM concentrations) did not show significant differences in Co²⁺ 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Nickel-dependent enzymes catalyze key reactions in energy and nitrogen metabolism in both prokaryotes and eukaryotes (for reviews, see Refs. 1 and 2). The most widespread nickel metalloenzyme is urease (EC 3.5.1.5), which has been identified in prokaryotes, fungi, algae, plants, and invertebrates. Ureases allow their hosts to utilize urea as a source of nitrogen. They are also important virulence factors in bacteria and fungi (reviewed in Refs. 3 and 4). Urease-mediated hydrolysis of urea yielding ammonium ion and carbamate is the common mechanism of biological urea degradation, although an alternative mechanism is known. In baker's yeast Saccharomyces cerevisiae, other yeasts, and certain green algae, the breakdown of urea is mediated by a biotin- and ATP-dependent carboxylation to give 1-carboxyurea (allophanate) and subsequent hydrolysis to ammonia and carbon dioxide (5).

The fission yeast Schizosaccharomyces pombe contains a urease and is able to grow on urea as the sole source of nitrogen. The soluble, cytoplasmic urease has been purified to homogeneity, and its kinetic properties have been determined (6). Its structural gene (ure1⁺) has been cloned and sequenced, and a knockout mutation was shown to inhibit the growth of S. pombe on agar plates containing 10 mM urea as the nitrogen source (7). S. pombe urease is neither controlled by nitrogen repression nor by urea induction (6).

High affinity nickel transport, a prerequisite for the biosynthesis of nickel-containing metalloenzymes, and the underlying uptake mechanisms have been investigated in a number of prokaryotes (reviewed in Refs. 8 and 9). Comparable transporters, however, have not yet been reported for eukaryotes. Trace amounts of Ni²⁺ ion are sufficient for maximal urease activity of S. pombe, and this activity was not stimulated by the addition of Ni²⁺ to the medium (6). This result suggested that an uptake system with a very high affinity for Ni²⁺ operates in fission yeast. The S. pombe genome sequencing project identified an open reading frame (Nic1p) that displays similarity to a family of bacterial transition metal permeases (9). In the present report we show that interruption of the respective gene strongly affected urease activity under conditions of nickel limitation and prevented the growth of S. pombe on urea. We demonstrate that Nic1p is a high affinity nickel permease. This is the first example of this kind of transporter in a eukaryotic organism. We also present evidence that nonspecific metal uptake systems allow S. pombe to transport Ni²⁺ ion under certain conditions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Organisms, Media, and Growth Conditions-- S. pombe var. pombe 972 h^s Lindner (wild type, DSM 70576) was obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). S. pombe strain FY254 (ATCC 201402) (h can1-1 leu1-32 ade6-M210 ura4-D18) was a gift of Susan Forsburg (The Salk Institute, La Jolla, CA) and was used as the parental strain for gene interruption. S. pombe strains were grown in YES (0.5% w/v yeast extract, 3% w/v D-glucose) medium or in Edinburgh minimal medium (EMM) (10) at 30 °C. Under standard conditions, adenine, L-leucine, and uracil were added as supplements to both media to give final concentrations of 100 µg/ml. Cell densities of cultures in YES medium were calculated upon measuring the optical density (A₅₉₅) in a spectrophotometer and establishing the ratio between A₅₉₅ and the cell concentration. One A₅₉₅ unit corresponded to 2.2 × 10⁷ colony-forming units/ml. Growth on urea as the nitrogen source was monitored on agar plates containing a modified EMM medium. The aforementioned supplements were added to final concentrations of 10 µg/ml, and urea (10 mM) was supplied in place of ammonium salt as the nitrogen source. Plasmids containing S. pombe DNA were propagated in Escherichia coli strains DH5F' (Life Technologies, Inc.) and XL1-Blue (Stratagene, Amsterdam) and the Dam strain GM2163 (New England Biolabs, Schwalbach, Germany). Recombinant E. coli strains were grown in LB medium supplemented with appropriate antibiotics.

Gene Interruption-- The nic1⁺ gene was cloned following amplification using total DNA of S. pombe wild-type strain 972 h as the template. Cells (10 ml) grown overnight in YES medium were harvested, washed in SCE buffer (900 mM sorbitol, 50 mM sodium citrate, 10 mM EDTA, pH 7.5), and resuspended in 350 µl SCE buffer containing 2-mercaptoethanol (0.8% w/v) and lyticase (Sigma) (1 mg/ml). After 30 min at 37 °C, the cells were pelleted and lysed by vigorous shaking after the addition of 250 µl of lysis buffer (10 mM Tris hydrochloride, pH 8.0, 100 mM NaCl, 2% w/v Triton X-100, 1% w/v SDS), 300 mg of acid-washed glass beads (Sigma), and 250 µl of phenol/chloroform/2-pentanol (25:24:1 v/v). After the addition of 200 µl of TE buffer (10 mM Tris hydrochloride, pH 8.0, 1 mM EDTA) and centrifugation, nucleic acids in the aqueous phase were precipitated with ethanol. The pellet was washed with 70% (w/v) ethanol, dried under vacuum, resuspended in 50 µl of distilled water containing 5 µg of RNase A, and stored at 4 °C. 1 µl of this sample was used for PCR (30 cycles of 20 s at 94 °C, 30 s at 50 °C, 2 min at 72 °C) in the presence of primer A (5'-GCGCCTCGAGATAAACACGTTTAAGCATCACC-3'), primer B (5'-CGAAAAAGAGCTCGGCATTAAACATACC-3') (Fig. 1), and 2.5 units of Taq DNA polymerase (Pan Systems, Aidenbach, Germany). The purified PCR product was incubated with restriction endonucleases XhoI and SacI and ligated into vector pBluescript II SK+ (Stratagene). For interruption of nic1⁺, this plasmid was isolated from E. coli GM2163. 246 base pair of nic1 were deleted by incubation with BclI and SpeI. The fragment was replaced by a 1.8-kilobase BamHI/XbaI fragment harboring the S. pombe ura4⁺ marker gene (a gift of Jürg Kohli, Universität Bern, Switzerland). The disrupted nic1 gene (nic1-1) was amplified by PCR (30 cycles of 20 s at 94 °C, 30 s at 45 °C, 2.5 min at 72 °C) using primer A, primer P4 (5'-CGGGAGCTCTCAAACCTTAGAATCCACTGTATCG-3') (Fig. 1) and Taq DNA polymerase. Approximately 20 µg of the purified PCR product was used to transform S. pombe FY254 by the method of Bähler et al. (11). A 20-ml culture of FY254 in YES was grown to a density of approximately 10⁷ cells/ml. The cells were washed twice with distilled water and once with LiAc/TE (100 mM lithium acetate, 10 mM Tris-hydrochloride, 1 mM EDTA, pH 7.5) and resuspended in 100 µl of LiAc/TE. 20 µg each of carrier DNA (sheared salmon sperm DNA, Stratagene), and the PCR product was added. After 10 min at room temperature, 260 µl of polyethylene glycol 4000 (40% w/v in LiAc/TE) was added, and the mixture was incubated at 30 °C for 1 h. Finally, 43 µl of dimethyl sulfoxide was added followed by heating to 42 °C for 5 min. The cells were washed and resuspended in 500 µl of distilled water. 250 µl was plated on uracil-free EMM agar. Colonies of ura4⁺-containing transformants appeared after 4 days and were purified by streaking on uracil-free agar plates. The disruption of nic1⁺ in recombinants was verified by PCR analysis (primer combinations P1 (5'-GGGCATATGTCTGAATATGTTAAACC-3')/P4 and P1/B) and Southern blotting (Fig. 1). For the latter purpose, a PCR product obtained with primers P1 and P4 and the nic1⁺ allele as the template was labeled with digoxigenin-11-dUTP (Roche Molecular Biochemicals) and used as the probe.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Strategy for cloning and interruption of nic1+. See "Materials and Methods" for details. Panel A shows the location of nic1+ on a 10-kilobase (kb) EcoRI fragment of chromosome III of S. pombe. The approximate location of primers used for mutant construction and verification is shown by arrows. Panels B and C, verification of the nic1-1 mutation by PCR (B) and Southern blotting probing EcoRI-digested total DNA with a labeled nic1+ fragment (C). S, DNA standard; 1, wild-type strain 972 h; 2, parental strain FY254; 3 and 4, two selected nic1-1 mutants. orf, open reading frame.

Nickel Accumulation in Growing Cells-- Nickel accumulation in S. pombe strains was analyzed by a modification of the method previously described for the analysis of recombinant E. coli (12, 13, 14). S. pombe cells were grown to an optical density (A₅₉₅) of approximately 1.5 in YES medium containing ⁶³NiCl₂ (24.4 TBq/mol) and the indicated supplements. Cells were harvested, washed twice with 50 mM Tris hydrochloride, pH 7.5, and concentrated 10-fold. The radioactivity of 50 µl of the cell suspension was quantitated by liquid scintillation counting in a Canberra-Packard 1600 TR counter using Zinsser Aquasafe 300 Plus as scintillation mixture. Nickel accumulation is expressed as pmol/10⁹ cells.

Nickel Transport-- S. pombe strains were grown in 10 ml of YES medium to an optical density (A₅₉₅) of approximately 5 absorbance units, harvested, washed twice, and resuspended in 10 ml of transport buffer (20 mM MES/NaOH, pH 6.2, 10 mM MgCl₂, 2% w/v D-glucose) and equilibrated at 30 °C in a water bath shaker for 5 min. ⁶³NiCl₂ (24.4 TBq/mol) was added to a final concentration of 10 nM. Samples (200 µl) were taken at the indicated time points, passed through Whatman glass microfiber filters (GF/C), and washed twice with 3.5 ml of glucose-free transport buffer. The radioactivity of filter-bound cells was analyzed by liquid scintillation counting. Nickel transport is expressed as pmol/10⁹ cells.

Urease Assay-- Cells were grown in 50 ml of YES medium overnight in the presence of the indicated supplements, harvested, washed, and resuspended in 2 ml potassium phosphate buffer (20 mM, pH 7.5). After two passages through a French pressure cell, the crude extracts were separated by ultracentrifugation (59,000 × g, 30 min, 4 °C). 20 µl of the supernatant (containing approximately 300 µg of protein) was added to 880 µl of potassium phosphate buffer (20 mM, pH 7.5), and the mixture was equilibrated at 37 °C. The reaction was started by the addition of 100 µl of a freshly prepared urea solution (100 mM). Urease activity was determined spectrophotometrically by quantitating the rate of ammonium ion released from urea. For this purpose, ammonium ion was converted into indophenol (15). Protein was estimated by a modification of the Lowry method (16). Urease activity is expressed in milliunits/mg of protein. One milliunit corresponds to 1 nmol of urea hydrolyzed/min.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Nic1p Is a Novel Member of a Family of Transition Metal Permeases-- The amino acid sequence alignment shown in Fig. 2 identified Nic1p of S. pombe as the first eukaryotic member of a family of transporters found in Gram-negative and Gram-positive bacteria (for a review, see Ref. 9). The bacterial counterparts consist of 337 to 381 amino acid residues and contain 8 transmembrane segments. Four characteristic amino acid signatures are conserved in these permeases, as follows. The motifs (R/K)HAXDADH(I/L) and FXXGHS(T/S)(V/I)V are located within transmembrane segments II and III, respectively, and have been shown to be critical for transport activity. Likewise, the motifs LGX(D/E)T(A/S)(T/S)E and GMXXXD(T/S)XD (located in transmembrane segments V and VI, respectively) are conserved and important for activity. A common feature of this family of membrane proteins is a highly charged hydrophilic loop connecting transmembrane segments IV and V. Deletions in this loop abolish activity (reviewed in Ref. 9). Hydropathy profile (9) and amino acid sequence alignments (Fig. 2) revealed that Nic1p is closely related to the bacterial counterparts. The aforementioned sequence motifs are fully conserved. The putative transmembrane helices IV and V of Nic1p are linked by a hydrophilic loop (residues 164 to 208) containing 12 potentially charged residues.

View larger version (105K): [in this window] [in a new window]   Fig. 2.   Alignment of nickel permeases. Comparison of the amino acid sequences of Nic1p and nine related proteins from Ralstonia eutropha (ReHoxN), Helicobacter pylori (HpNixA), Mycobacterium avium (M.avi), Mycobacterium tuberculosis (MtNicT), Bradyrhizobium japonicum (BjHupN), Rhodococcus rhodochrous (RrNhlF), Salmonella enterica serovar typhimurium (Sa.typ), Staphylococcus aureus (St.aur), and Yersinia pestis (Y.pes). Identical residues are marked by an asterisk, and similar residues are marked by a colon.

Interruption of the nic1⁺ Gene-- nic1⁺is located on chromosome III of S. pombe between the long terminal repeat of a Tf2-type retrotransposon and an open reading frame of unknown function. The strategy for gene interruption is illustrated in Fig. 1. 246 base pairs of nic1⁺ were deleted and replaced by an approximately 1.8-kilobase ura4⁺ marker gene (17). Using 20 µg of the amplified construct for transformation into S. pombe FY254, approximately 1,000 transformants were obtained on uracil-free EMM agar plates. Two transformants were chosen and shown by PCR (Fig. 1B) and Southern blotting (Fig. 1C) to contain the disrupted nic1 allele. Both mutants grew normally in mineral medium in the presence of ammonium salt as the nitrogen source as well as in complex medium, indicating that nic1⁺ is dispensable under both conditions.

Nickel Uptake-- Based on the similarity to the bacterial nickel permeases, we suspected that Nic1p plays a role in nickel transport into S. pombe cells. To investigate this hypothesis, nickel accumulation of growing cells of the S. pombe nic1-1 mutant was compared with metal uptake by the parental strain under various conditions (Table I). In the presence of 5 µM ⁶³NiCl₂ in complex medium, both strains accumulated high amounts of Ni²⁺ ion. The addition of magnesium salt to the medium resulted in a 20-fold decrease in nickel accumulation. At a low Ni²⁺ concentration (100 nM), the nic1-1 mutation caused a moderately reduced Ni²⁺ accumulation when the medium was not supplemented with Mg²⁺ ion. In the presence of 10 mM Mg²⁺, however, a strong effect was observed. Although 10⁹ cells of the parental strain accumulated 48 pmol of nickel, metal accumulation of the nic1-1 mutant decreased to 4 pmol of nickel/10⁹ cells (Table I). A series of nickel accumulation experiments performed at substrate concentrations between 25 and 150 nM in the presence of 10 mM MgCl₂ (Fig. 3) confirmed the assumption that Nic1p acts as a high affinity nickel transporter in S. pombe. This conclusion was further substantiated by uptake assays with resting cells in buffer. While the nic1-1 mutant was unable to transport ⁶³Ni²⁺ over the 50-min test period, significant transport was observed for the parental strain (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   The nic1-1 mutation blocks nickel accumulation in S. pombe. Cells were grown in the presence of 63NiCl2 in YES medium containing 10 mM MgCl2, harvested, washed, and concentrated. 63Ni accumulation was determined by liquid scintillation counting. The values are the means of duplicates. Circles, strain FY254; squares, mutant strain.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Nickel transport of resting cells is abolished in the nic1-1 mutant. Washed cells were resuspended in 20 mM MES buffer, pH 6.2, containing 2% D-glucose, and 10 mM MgCl2. After the addition of 10 nM 63NiCl2, timed aliquots were passed through glass microfiber filters. The radioactivity of washed filters was quantitated by liquid scintillation counting. Circles, strain FY254; squares, mutant strain.

Our data are compatible with the view that S. pombe is able to transport Ni²⁺ ion by nonspecific Mg²⁺ uptake systems. A similar situation has been reported for S. cerevisiae (18). Lesions in the two S. cerevisiae genes ALR1 and ALR2 were found to produce a magnesium-deficient phenotype while conferring increased resistance to certain metal ions including the divalent ions of the transition metals copper, manganese, nickel, and zinc. Alr1p and Alr2p belong to the CorA family of membrane transporters, the most widespread type of nonspecific Mg²⁺ uptake system in bacteria and archaea (19). Alr1p- and Alr2p-like proteins are also encoded in the genome of S. pombe. The respective open reading frames (O13779 and O13657) together with Alr1p and Alr2p contain large N-terminal extensions compared with their prokaryotic counterparts and represent a CorA subfamily (19).

At very low Ni²⁺ concentrations and high molar ratios of Mg²⁺ to Ni²⁺, Ni²⁺ uptake of S. pombe was dependent on Nic1p. This result indicated that nonspecific systems contribute little to Ni²⁺ uptake under conditions that reflect the situation in the natural environment.

Selectivity of Nic1p-- To test the specificity of Nic1p, the effect of cadmium, cobalt, copper, manganese, and zinc ions on nickel accumulation of S. pombe FY254 was investigated. For this purpose, the cells were grown in YES medium containing 100 nM ⁶³NiCl₂ and 10 mM MgCl₂. Under these conditions, high level nickel accumulation is dependent on Nic1p. The competing metal ions were added to final concentrations of 1 µM. With the exception of Co²⁺, none of the metal ions caused significant inhibition of nickel accumulation (data not shown). The effect of Co²⁺ was analyzed in more detail. Fig. 5 illustrates that Co²⁺ was an inhibitor. At a 50-fold excess, Co²⁺ ion abolished ⁶³Ni²⁺ accumulation in S. pombe. We then addressed the question of whether Nic1p is capable of transporting Co²⁺ ion. The nic1-1 mutant and its parental strain FY254 were grown in YES medium supplemented with ⁵⁷CoCl₂ at concentrations between 100 and 500 nM in the presence of 10 mM MgCl₂. Both strains were able to accumulate cobalt, and no obvious difference was found under any conditions tested (data not shown). This result suggested that Nic1p is not the main mediator of Co²⁺ uptake. The observed Co²⁺ accumulation could be due to the activity of a nonspecific transition metal transporter of the Nramp (natural resistance-associated macrophage protein) family. Three Nramp-like proteins (Smf1p, Smf2p, and Smf3p) have been identified in S. cerevisiae, and a homologous open reading frame (Q10177) is also encoded in the genome of S. pombe (see Refs. 20 and 21 for recent reviews).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent inhibition of nickel accumulation by Co2+ ion. S. pombe FY254 was grown in YES medium in the presence of 100 nM 63NiCl2, 10 mM MgCl2, and CoCl2 at the indicated concentrations. The values represent the means of duplicates.

Physiological Role of Nic1p-- To elucidate the physiological consequences of the nic1-1 mutation, we first monitored growth on mineral agar plates containing 10 mM urea as the nitrogen source (not shown). Although S. pombe FY254 formed colonies similar to those observed on ammonium-containing plates after 3 to 4 days, the nic1-1 mutant failed to grow on urea under standard conditions. Growth of the mutant was partially restored by adding nickel salt at µM concentrations to the medium. At Ni²⁺ concentrations above 500 µM, growth of both strains as well as the wild-type strain 972 h was completely inhibited. S. pombe harbors a putative metallothionein and has recently been shown to produce a phytochelatin synthase (22). Metallothioneins and phytochelatins mainly mediate resistance toward cadmium, copper, and zinc and apparently do not allow S. pombe to escape nickel toxicity under the conditions tested.

We next investigated the role of Nic1p in urea metabolism by quantitative urease assays (Table II). Upon growth of the cells in standard YES medium, soluble extracts of the nic1-1 mutant and its parental strain contained urease-specific activities of approximately 200 to 250 milliunits/mg of protein. These values were in good agreement with those published previously (6). The addition of NiCl₂ (5 µM) to the growth medium resulted in a slightly increased urease activity of strain FY254. Surprisingly, high concentrations of MgCl₂ (20 mM) had almost no effect on urease activity of both strains. Under comparable conditions, efficient Ni²⁺ uptake was dependent on Nic1p (Table I, Figs. 3 and 4). We hypothesized that low level Ni²⁺ uptake, which could be due to a nonspecific Nramp-like transporter, is sufficient for maximal urease activity under these conditions. Therefore, a nickel-complexing agent (NTA), which had proved to efficiently inhibit nonspecific nickel uptake in bacteria (13), was added to the growth medium. 50 µM NTA led to a small but significant decrease of urease activity in the absence of Nic1p. The addition of a combination of NTA and Mg²⁺, however, gave a strong response. Although the parental strain was almost unaffected, urease activity in the mutant was below the threshold of the assay. Since the natural habitats of S. pombe contain nutrients with strong metal-complexing capacity and since the molar ratio of Mg²⁺ to Ni²⁺ is generally very high, the latter growth conditions reflect the situation in the environment.

Our results identified Nic1p as an important auxiliary factor for urease activity in S. pombe, a finding that may be of general significance for the analysis of urease biosynthesis in eukaryotes. Additional urease accessory genes in fission yeast have been tentatively mapped (23). The genome sequencing project revealed homologs of the bacterial urease operon proteins UreD and UreF, which are essential for urease metallocenter assembly (4). An isolog with high similarity to the bacterial UreG, a GTPase that is important for nickel incorporation into urease apoprotein (24), is not obvious from the S. pombe genome sequence.

Although at present we have only indirect data on the localization of Nic1p, our results strongly suggest that it represents a plasma-membrane transporter. Protein-sorting signals are not obvious from the primary structure, and recent work on the Nramp homolog Smf1p of S. cerevisiae has demonstrated that sorting is a mechanism of post-translational activity control of plasma-membrane transporters in yeast (25, 26).

Compared with its bacterial relatives, Nic1p has a unique specificity. Ni²⁺ transport by HoxN of Ralstonia eutropha, for instance, is not inhibited by Co²⁺, and HoxN does not transport Co²⁺ ion (14). On the other hand, Ni²⁺ uptake by NhlF of Rhodococcus rhodochrous is specifically inhibited by Co²⁺, and this permease is able to transport Co²⁺ ion (14). Nic1p seems to be a third type of nickel permease, since Co²⁺ was an inhibitor but, if at all, only a weak substrate for transport. Understanding the molecular basis of the differences in ion selectivity is a challenging problem.

	ACKNOWLEDGEMENTS

We are indebted to B. Friedrich and E. Schwartz (Humboldt-Universität zu Berlin) for continous support and critical comments on the manuscript, respectively. We thank S. Forsburg (The Salk Institute, La Jolla, CA), N. Käufer (Technische Universität Braunschweig, Germany), and J. Kohli (Universität Bern, Switzerland) for S. pombe strains and cloned marker genes and U. Eckhardt (Humboldt-Universität zu Berlin) for initial advice on handling S. pombe. The sequence data were produced by the S. pombe sequencing group at the Sanger Center (Hinxton, Cambridge, UK) and can be obtained from their website. We thank the Sanger Center for rapid delivery of cosmid clones.

	FOOTNOTES

^* This work was funded by grant Ei 374/1-2 from the Deutsche Forschungsgemeinschaft (to T. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Humboldt-Universität zu Berlin, Institut für Biologie/Mikrobiologie, Chausseestrae 117, 10115 Berlin, Germany. Tel.: 49-30-2093-8103; Fax: 49-30-2093-8102; E-mail: thomas.eitinger@rz.hu-berlin.de.

Published, JBC Papers in Press, March 31, 2000, DOI 10.1074/jbc.M001978200

	ABBREVIATIONS

The abbreviations used are: EMM, Edinburgh minimal medium; PCR, polymerase chain reaction; MES, 4-morpholineethanesulfonic acid; Nramp, natural resistance-associated macrophage protein; NTA, nitrilotriacetate.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

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