Proc. Natd. Acad. Sci. USA Vol. 91, pp. 5099-5103, May 1994 Microbiology Bacterial genes involved in incorporation of nickel into a (nikel-ontanig enme/nicke metabolism/nitrogen fixation) CHANGLIN Fu, SAM JAVEDAN, FARHAD MOSHIRI, AND ROBERT J. MAIER* Department of Biology, The Johns Hopkins University, Baltimore, MD 21218 Communicated by Christian B. Anfinsen, February 24, 1994

ABSTRACT Nickel is an essential component of all H2- important in regenerating energy for nitrogen fixation. Nickel uptake . A fgnt of DNA that complements a not only serves as an essential component of the B. japoni- H2-uptake-deflcient but nickeled mutant strain (JHK7) of cum hydrogenase enzyme (7) but also affects hydrogenase Bradyrhizobium japonicun was isolated and sequenced. This synthesis in wild-type strains of the bacterium (8). From use 4.5-kb DNA frgment contains four open reading frames of inhibitors of 63Ni uptake in B. japonicum JH, it has been designated as ORF1, hupN, hupO, and hupP, which encode suggested that the bulk of nickel is transported via a magne- polypeptides with predicted masses of 17, 40, 19, and 63.5 kDa, sium transport system (9). A nickel metabolism locus has respectively. The last three open reading frames (hupNOP) are been identified within the hydrogenase gene cluster of B. most likely organized as an operon with a putative o"-type japonicum (10). A mutation within this locus resulted in promoter. Based on its hydropathy profile, HupN is predicted strains that require higher levels of nickel than the wild type to be a transmembrane protein. It has 56% Identity to the for hydrogenase activity, and that produce an (apo)hydroge- previously described HoxN (high-amnity nickel transport pro- nase deficient in nickel content (10, 11). The mutant strain is tein) of Akaligenes euatophus. A subelone (pJF23) conain not deficient in nickel transport (10). In this study, we have the hupNOP genes excdudg ORF1 completely complemented isolated a fragment of DNA which complements the nickel- (in tram) strain JHK7 for hydrogenase activity In low nickel metabolism locus-deleted mutant strain. Here we report the conditions. pJFZ6 con ing only a functional hupN comple- cloning, sequence,t and characterization ofthe role of these mented the hydrogenase activity of mutant strain JHK7 to nickel-metabolism genes from B. japonicum. Complementa- 30-55% of the wild-type level. Mutant strain JHK70, with a tion and mutant analyses indicate that each ofthe three genes chromosomal deletion in hwpP but with an intact hupNO, (hupNOP) is needed for complete nickel incorporation into showed greater activities than pJF26-complemented JHK7 but the hydrogenase enzyme. hupO and hupP lack homology to still had lower activities than the wild type at all nickel levels known genes. Of particular comparison interest is the prod- tested. pJF25, containing the entire hupO and hupP, but uct of hupN, which has a predicted 56% identity to the without hupN (a portion of hupN was deleted), did not com- previously described HoxN (high-affinity nickel-specific plement hydrogenase activity of mutant strain JHK7. The transport protein) of A. eutrophus (3). results suggest that the products of the hupNOP operon are all involved in nickel incorporation/metabolism into the hydro- MATERIALS AND METHODS genase apoprotein. Based on (previous) nickel transport studies of strain JHK7, the hupNOP genes appear not to be involved Bacterial Strain and lamids. B. japonicum JH (12) was in nickel transport by whole cells. The deleterious effects on derived from strain USDA 110. Strain JHK7 (10) lacked a hydrogenase expression are most pronounced by lack of the large area of genetic information (hupNOP) (see text) for HupN . nickel metabolism. Strain JHK7) is a mutant lacking a 0.6-kb EcoRI fragment (within the hupP gene) and was created by kanamycin-cassette replacement (13). E. coli HB101, XL1- Nickel is an essential metal for proper functioning of several Blue (Stratagene), and S17-1 (14) were used -for genetic microbial , such as CO dehydrogenase, urease, manipulations. methylcoenzyme M reductase, and hydrogenase (1). Bacteria Cosmid pSH22 was isolated by Hom et al. (15) from a B. sequester nickel via a magnesium transport system and/or a japonicum gene bank that was constructed by Russell et al. high-affinity nickel-specific transport protein. Nickel- (16). pSH22 contains 23 kb of B. japonicum chromosomal specific transport operons have been reported in Alcaligenes DNA. It complements a variety of Hup- mutants and has eutrophus (2, 3) and Escherichia coli (4). Genes involved in been shown to contain the hydrogenase structural genes and intracellular nickel functions for E. coli hydrogenase (5) and other H2 uptake-related genes. pJF18-4, containing a 4.5-kb Klebsiella aerogenes urease (6) have recently been de- Bgl II fragment, was subcloned from a cosmid (map not scribed. GTP-binding domains in HypB of E. coli were shown) which was isolated from a B. japonicum strain 110 identified and are thought to be important in nickel metabo- gene bank (17). pJF23, containing the hupNOP putative lism (5). Specific functions of the gene products in intracel- operon, was subcloned from pJF18-4. DNA manipulation, lular nickel metabolism pathways are not known. Identifica- Southern blotting, and conjugation of plasmids from E. coli tion of additional genes or products would be helpful for strains into B. japonicum were previously described (10). understanding bacterial intracellular nickel metabolism. Growth Cots. B. japonicum JH, JHK7, and JHK70 Bradyrhizobium japonicum, a N2-fixing symbiont of soy- and mutant strains containing conjugant plasmids were bean, possesses a nickel-containing hydrogenase capable of grown in modified Bergersen's (MB) (18) medium at 300C recycling H2 evolved by the nitrogenase reaction. H2 is an with shaking at 150 rpm. Tetracycline (Tc) (75 was obligate product of nitrogenase , and its oxidation is pg/ml) Abbreviation: ORF, open reading frame. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L24432). 5099 Downloaded by guest on October 2, 2021 5100 Microbiology: Fu et A Proc. Nati. Acad. Sci. USA 91 (1994)

E fg *0 E E Bg s9 B B B so E E BEg E pSH22 L.a a s . m 0 2 a .. . 1

-- -- - HI ,4 - -0. H H H H

t9m E E fg pJF8-4 IaI'I I Al 1 kb II- Al EE ft pJF23 I I *I

FIG. 1. Restriction map ofplasmids pSH22, pJF18-4, and pJF23. hupS and hupL indicate the hydrogenase structural genes for the small and large subunits, respectively. The dashed-line arrows below pSH22 indicate the region that was found not to be genetically linked to the rest of the DNA on pSH22. Restriction sites: E, EcoRI; Bg, Bgi II; B, BamHI; H, HindIII; Al, AlwNI. added if the mutant contained plasmid. Nickel-free growth hr for derepression of hydrogenase activity. Hydrogenase medium and derepression medium (also nickel-free) were activity was measured amperometrically with 02 (air) as prepared as described (19). Nickel chloride was added to the (10). The cell number was determined after derepression medium at levels indicated in the figures. E. coli derepression from a standard curve of viable cell number cells containing cloned plasmid were grown in LB medium versus optical density at 540 nm. Data points are the average supplemented with antibiotics when necessary (10). of duplicates. DNA Sequence and Analysis. For DNA sequencing, the 4.5-kb Bgl II fragment ofpJF18-4 was cloned into the BamHI RESULTS site ofpBluescript II KS(+) (Stratagene) in two orientations, ofa Clone Tht Complements theNickel Metabolim forming pB2 and pB3. For convenient generation of nested Locus-Deeted Mutant JHK7. We previously identified a locus deletion mutants, subclones were made from pB2 at appro- (0.6-kb EcoRI fragment) within the hydrogenase gene cluster priate restriction sites. Overlapping nested deletions were (pSH22) ofB.japonicum that was involved in nickel incorpo- generated with the exonuclease III/mung bean deletion kit ration into the hydrogenase apoprotein but not involved in from Stratagene. Nested deletion clones that were to be used nickel transport by cells (10, 11). Since the cosmid (pSH22) did for sequencing were transformed into E. coli HB101 or not complement (in trans) mutant strain JHK7 (10), we probed XL1-Blue. Double-stranded template DNA was prepared by a different gene bank ofB.japonicum strain 110 (17) by using use of the Qiagen plasmid kit (Qiagen, Chatsworth, CA) or the 0.6-kb EcoRI fragment from pSH22 as a probe. A few Magic minipreps (Promega) system. In some cases, single- positive clones were identified; a 4.5-kb Bgl II fragment stranded template DNA was prepared from phagemids by encompassing the 0.6-kb EcoRI fragment from one positive infection ofE. coli XL1-Blue with helper phage M13 (with the clone was subcloned into the Bgl II site of pVK101 (20) (but protocol supplied by Stratagene). DNA templates were se- without the 1.7-kb cos-site Bgl II fragment), yielding pJF184 quenced by the Core Facility at the Johns Hopkins Univer- (Tcr) (Fig. 1). pJF18-4 was conjugated into the mutant JHK7. sity, using an automated DNA sequencer (Applied Biosys- Hydrogenase activity was determined in the presence of tems). Some sequencing was also done by use ofa Sequenase increasing amounts of nickel. Hydrogenase activity of the II kit (United States Biochemical) using [a-[35S]thio]dATP mutant (JHK7) was complemented to 94-100%o of wild type (New England Nuclear). In some cases oligonucleotide prim- (JH) by pJF18-4 in the presence of0.2-5.0 pM nickel (Fig. 2), ers were synthesized and used for sequencingjunctions. Both indicating that pJF18-4 contains all the necessary nickel me- strands were sequenced. The nucleotide sequence data were tabolism genes required for hydrogenase activity. analyzed with the PC/GENE software package (IntelliGenet- JHK7 Lacks ABl Three Nickel Metabolism Genes (hupNOP). ics). The deduced sequences were compared with Because the Bgl II fragment of pJF18-4 (4.5 kb) was larger the Swiss-Prot protein sequence databank (release 26). than that covering the presumably same area ofpSH22 (Fig. Construction of Deletion Clones. In order to understand the 1), we isolated more clones and subsequently did Southern roles of each gene product, deletion plasmids were con- analysis (data not shown). It was found that a 3.8-kb EcoRI structed. pJF25 contained hupO and hupP but without the fragment on pSH22 (Fig. 1; the dashed-line arrows) was not entire hupN gene (a 742-bp Pvu I fragment in the hupN region was deleted; the deletion of the Pvu I fragment in the hupN 250 coding region yielded a premature stop codon, but the reading frames of the hupO and hupP were not affected). pJF26 contained a functional hupN only (hupOP genes were 200 disrupted after deletion of an Xho II-Aat II DNA fragment in the hupO region). pJF27 contained the entire hupP gene only (a 808-bp Sal I fragment in the hupN and hupO regions was 150 - deleted without changing the hupP reading frame). Derepression of Hydrogenase and Enzyme Assay. To dere- press hydrogenase activity ofB.japonicum JH or its derived P. 100tl mutants, cells were heterotrophically grown in the nickel-free MB medium until midlogarithmic phase, then harvested and x washed in carbon-free, nickel-free derepression medium (10). 50- The pelleted cells were finally resuspended in fresh carbon- free, nickel-free derepression medium to cell densities of 3-5 x 108 cells per ml, and 10-ml aliquots were dispensed into 0 150-ml serum-stoppered bottles. The appropriate amount of 0 1 2 3 4 5 nickel chloride (as indicated in the figures) was added. The bottles were then tightly closed with butyl rubber stoppers NiCl2, I&M and aluminum crimps. The gas phase was adjusted to 1% FiG. 2. Derepression of hydrogenase activity in strains JH (o), 02/5% C02/l0o H2/84% N2 as described (10). The bottles JHK7 (e), JHK7(pJF18-4) (A), and JHK7(pJF23) (v) as a function of were then incubated at 300C with shaking at 150 rpm for 20 nickel concentration. Downloaded by guest on October 2, 2021 Microbiology: Fu et A Proc. Natl. Acad. Sci. USA 91 (1994) 5101

Bj HupN MLPFSMTGLEKDHTRGVLILANAHRRSZRSRTASCAGPAVLPGGLITANIVAWAWAFALF 60 111 1 1 1:11 Ae HoxN MFQLLAGVRMNSTGRPRAKIILLYALLIAFNIG&WLCALAAF 42

Bj HupN ADRPVVNATALLAWVFGLRNAVDADRIA&IDNVVRSLMQTGGTPRSAGLYFALGNSSVVV 120 1:11:3: 11111: :111111111 1:111111:1:111:1 1 :111:1:11111111 Ae BoxN RDHPVLLGTALLAYGLGLRN&VDADULAAIDNVTRKLMQDGRRPITAGLWFSLGNSSVVV 102 Bj HupN VATJLLALGVVSLGGD-GLLKEIGSFIG&SVSALFLLVIAAINLAIFASLWRTFRKAREQ 179 : I :IsI: : :3: I :1I I I:: II III ::IIIII :1I: :jI :1II: A. HoxN LASVLIAVMATTLQZRLDRFHEVGSVIGTLASALFLFAIAAINLVILRSAYRAPRRVRRG 162 Bj HupN GIRDAAGLDALLAERGILVRLLGPMFRLVTKPWIHYPLGFLFGLGFDTATEIGLLSISAS 239

Ae HoxN GIYVEEDFDLLFGIRGFLARIFRPLFRFITRSWZMYPLGMLFALGFDTATEVALLGISTM 222 Bj HupN EAARGASLADVMVFPALFAAGMALVDTADSTLNVSAYRWAFVDPMRKLWYNLTITGASVA 299 11:11::: :311till1:11111:11 11:11 :11 11:::1:111:11:111 :1: Ae HoxN EASRGVPIWSILVFPALFTAGMALIDTIDSILMCGAYAWAYAKPVRKLYYNNTITFVSAI 282

Bj HupN VALFIGGIEALGLIGIRLDLSGGVWTLIDALNESLANVGLAVIALFAIAWLLSIVLYRRL 359

Ae BoxN VALIVGGIETLASWPTSSC 301

Bj BupN IAGSSGLADTEVLECADATEAV 381 FIG. 3. Alignment ofthe amino acid sequence ofB.japonicum HupN with A. eutrophus HoxN. Vertical lines indicate identical amino acids, and colons indicate conservative substitutions. Histidines conserved between HupN and HoxN are shown in bold type. genetically directly linked to the rest of the DNA on pSH22. membrane. Possible nickel-binding sites, Cys-Arg-Arg-Cys It is possible that this was due to DNA rearrangement events, at the N-terminus and His-Ala-His at the C terminus, were but this possibility was not investigated. After sequence present in the HupO sequence. HupP (63.5 kDa) did not have analysis, therefore, the previously created mutant strain significant homology to any of the proteins in the Swiss-Prot JHK7 (and similar mutants; see refs. 10 and 11) lacked the database. The motifs His-Gln-His, His-Cys, and Cys-Xaa9- entire nickel metabolism operon (hupNOP). Cys were also identified in this protein. DNA Sequence Analysi. To characterize the nickel metab- The IkupNOP Genes Are Required for Insertion ofNickd Into olism genes, we sequenced the entire 4.5-kb Bgl II fragment the Enzyme. Based on the nucleotide sequences, hupN, ofpJF184. Analysis ofthe nucleotide sequence revealed that hupO, and hupP (excluding ORF1) are most likely organized there were four open reading frames (ORFs), designated as as an operon and it was possible that only these three ORF1, hupN, hupO, and hupP, oriented inthe same direction products ofthe locus were involved in incorporation ofnickel as hupSL (hydrogenase structural genes). All ORFs started into the enzyme. To establish this, a 4-kb AlwNI-Bgl II with ATG and terminated with TGA except for hupP, which subclone (containing only hupNOP) was constructed in the terminated with TAG. Each of these ORFs was preceded by Bgl 11-blunted site ofpVK101, yielding pJF23 (Figs. 1 and 4). a putative ribosome . pJF23 was conjugated into the mutant JHK7. Hydrogenase ORF1 had 474 bp, encoding 158 aa with a predicted mass activity was determined after derepression in the presence of of 17 kDa. ORF1 was separated by 290 bp from the start various levels of nickel. pJF23, like pJF184, almost com- codon (ATG) ofhupN. A possible transcriptional termination loop was present between the end ofORF1 and the beginning of hupN. Apparently, ORF1 is located within a different ---r v F r (Veotorused) S Pv S Ap E E operon. A computer search (Swiss-Prot, release 26) of the ( pVK10Q ) pJFIl IlgSA ,IA - ORF1-predicted product did not reveal any highly homolo- S K Ev Pv Aa FvXAU ,,{,yX EV AOX gous proteins. The hupN, hupO, and hupP genes had 1143, 537, and 1752 pJF23 (Al) (B9D ( pVK101) bp, respectively, encoding proteins with predicted molecular masses of40, 19, and 63.5 kDa. hupNshared 20bp with hupO, Iso Pv PV (Bg whereas hupO and hupP were separated by 20 bp. Analysis of PJF25 ( pRK415) the 290-bp intergenic space between ORFi and hupN identi- (EV 9 (n)S (Aa) fied a sequence, 5'-CGQATCGCGCATITGCA-3', at positions pJF26 1I (pRK415) 794-809, that was simil to the common o54-binding consen- sus sequence 5'-T.GQYRYRN4ITIGCA-3' (21). K S S (Bd9 Most notably, HupN (40 kDa) had 56% identity with the pJF27 l~~I ( pRK415 ) previously described HoxN (high-affinity nickel transport I kb protein) in A. eutrophus (3). Analysis by the method ofKlein et al. (22) showed that the HupN would be classified as an FIG. 4. Restriction map of pJF18-4 and subclones (deletion integral membrane protein with six transmembrane seg- clones). Organization of the nickel metabolism genes (hupNOP) is ments. Seven histidines, potential nickel , were pres- shown above pJF18-4. The hatched box below pJFl8-4 indicates a ent in the predicted HupN sequence. Four of those were region that was deleted in mutant strain JHK70. Dashed lines in conserved between HupN and the HoxN protein of A. pJF25, pJF26, and pJF27 indicate the areas deleted to form the eutrophus (Fig. 3). constructs. Restriction sites: E, EcoRI; Bg, Bgl Jl; S, Sal I; Ev, EcoRV; K, Kpn I; Bs, BssHII; Ap, Apa I; H, HindI; P, Pst I; Sm, HupO (19 kDa) did not show significant homology to any Sma I; Xb, Xba I; N, Not I; Sc, Sac I; Ss, Ssp I; Al, AlwNI; Mu, Mun ofthe gene products in the Swiss-Prot database. Based on the I; Pv, Pvu I; Aa, Aat II; X, Xho H. Restriction sites in parentheses Kyte-Doolittle scale (23), HupO contained an 11- to 15-aa indicate the sites which have been modified in the course of cloning stretch close to the N terminus exhibiting a hydrophobic and cannot be cut by these enzymes. Not all restriction sites are domain (data not shown). This hydrophobic domain may shown on subclones. pVK102, pVK101, and pRK415 have been serve as a membrane-spanning anchor for the protein to the described (20, 24). Downloaded by guest on October 2, 2021 5102 Microbiology: Fu et aL Proc. Natl. Acad. Sci. USA 91 (1994) surprising, since both proteins interact with nickel. There are seven histidines present in HupN. Four of those are con- served in B. japonicum HupN and A. eutrophus HoxN. The 200 conserved histidines may act as nickel ligands in both pro- teins. Conserved histidines in large subunits ofhydrogenases from diverse hydrogenase-containing bacteria and in ureases from jack bean and K. areogenes have been proposed to be 1507 nickel ligands (25, 26). Histidine-rich regions are also pro- posed to be involved as metal-binding sites for otherproteins, 2~~~~~~~~~~~~ such as UreE in K. aerogenes (27) and copper-containing 100 hemocyanin (28). Recently, Lee et al. (6) purified the UreE protein and demonstrated that it binds to and subsequently provides nickel for urease activity. Neither HupO nor HupP shows significant homology to any of the proteins in the Swiss-Prot database. However, the motifs Cys-Arg-Arg-Cys and His-Ala-His exist in HupO, and these regions could be directly involved in liganding nickel. 0 1 2 3 4 5 Analysis of mutants in these genes and use of various NiC12, plasmid constructs to complement mutants were useful to ItM conclude that HupN, HupO, and HupP all play roles in nickel FIG. 5. Derepression ofhydrogenase activity from strains JH (o), metabolism for hydrogenase. A deletion clone, pJF26, har- JHK7 (v), JHK70 (e), JHK7(pJF25) (A), JHK7(pJF26) (o), boring just the hupN gene complemented hydrogenase ac- JHK7(pJF27) (v), and JHK70(pJF23) (U). tivity of the mutant strain JHK7 to 30-55% of the wild-type level, whereas pJF25, containing intact hupOP but not hupN, pletely restored hydrogenase activity of the mutant strain was unable to restore appreciable activity to the hupNOP JHK7 to the wild-type level (Fig. 2); this result indicates that deletion strain (JHK7). Similarly, the plasmid pJF27, carry- the products of the hupNOP genes are sufficient to permit ing a deletion in hupNO, could not complement strain JHK7. incorporation of nickel into the enzyme. Although the specific deletion of hupP showed less effect on To investigate the roles of each gene product, deletion hydrogenase activity in response to nickel levels than the clones were constructed and conjugated into the mutant deletion of hupN and hupNO, a role for the HupP protein in strain JHK7 (in which the hupNOP operon was deleted) to maximal expression of hydrogenase activity cannot be elim- address trans complementation. pJF27, containing only hupP inated, particularly at very low nickel levels (e.g., 0.1-0.5 (Fig. 4), did not complement hydrogenase activity of the ,gM; see Figs. 2 and 5). mutant (Fig. 5). Similarly, pJF25, containing hupO and hupP Recently the product of the hypB gene from E. coli was but without hupN (Fig. 4), did not complement the activity of reported to be required for insertion of nickel into E. coli the mutant (Fig. 5). Activity of mutant strain JHK7 was hydrogenase (5). The results suggested that HypB catalyzes restored to 30-55% ofthe wild-type level by pJF26, which has donation of nickel to the hydrogenase apoprotein. GTP only a functional hupN gene (Figs. 4 and 5). Hydrogenase hydrolysis is proposed to be utilized to reverse the interaction activity of a different mutant strain, JHK70, with a chromo- between either HypB or another nickel-binding protein and somal deletion in hupP but with the entire hupN and hupO hydrogenase after the nickel has been incorporated (5). Very genes was higher than that of the pJF26-complemented JHK7 recently, we have identified a hypB gene homologous to the but still lower than the wild type; this was especially evident E. coli hypB located =5.5 kb downstream from the end ofthe at low concentrations of nickel (<0.5 ,uM). Strain JHK70, hydrogenase structural genes ofB.japonicum (29). B.japoni- lacking hupP, was fully complemented to wild-type levels of cum HypB has an extremely histidine-rich region (24 his- activity by pJF23 (Fig. 5). The mutant and complementation tidines in a 39-aa stretch) and GTP-binding domains (29). results indicate that all three genes (hupNOP) are important Recently, we have expressed and purified B. japonicum for nickel-dependent activity but that hupN plays the most HypB protein by chromatography on a nickel-affinity column significant role. (data not shown). We propose that HypB binds to and accumulates nickel in a GTP-requiring reaction for its sub- sequent mobilization into the enzyme. It is possible that DISCUSSION HypB and products of hupNOP may work together or We have subcloned a 4.5-kb fragment of B.japonicum DNA individually in sequential steps of mobilization of nickel into which complemented the previously created nickel metabo- the hydrogenase enzyme. lism locus-deleted mutant JHK7. Nucleotide sequence anal- We thank Roxann Ingersoll, Tim Raeke, and Elizabeth Nantha- ysis of the 4.5-kb Bgl II fragment revealed four ORFs (ORF1 kumar (at the Core Facility of the Johns Hopkins University) for and hupNOP). The hupN, hupO, and hupP genes are most assistance with DNA sequence determination. This work was sup- likely organized as an operon preceded by a putative o54-type ported by Department of Energy Grant DE-FG02-89ER14011. promoter. pJF23, containing hupNOP (excluding ORF1), 1. Hausinger, R. F. (1987) Microbiol. Rev. 51, 22-42. fully complemented the hydrogenase activity of the mutant 2. Eberz, G., Eitinger, T. & Friedrich, B. (1989) J. Bacteriol. 171, strain JHK7, indicating that the products of hupNOP play a 1340-1345. role in nickel metabolism. Further complementation studies 3. Eitinger, T. & Friedrich, B. (1991) J. Biol. Chem. 266, 3222- indicated that the products of hupN and hupO (especially 3227. HupN) probably play the more crucial roles in this nickel 4. Navarro, C., Wu, L.-F. & Mandrand-Berthelot, M.-A. (1993) metabolism. Mol. Microbiol. 9, 1181-1191. 5. Maier, T., Jacobi, A., Sauter, M. & Bock, A. (1993) J. HupN was predicted to be a transmembrane protein with Bacteriol. 175, 630-635. six membrane-spanning segments. Most notably, HupN 6. Lee, M. H., Pankratz, H. S., Wang, S., Scott, R. A., Finne- showed considerable identity (56%6) to the previously de- gan, M. G., Johnson, M. K., Ippolito, J. A., Christianson, scribed HoxN (high-affinity nickel-specific transport protein) D. W. & Hausinger, R. P. (1993) Protein Sci. 2, 1042-1052. ofA. eutrophus, indicating that some common domains exist 7. Stults, L., O'Hara, E. & Maier, R. J. (1984) J. Bacteriol. 159, in B. japonicum HupN and A. eutrophus HoxN. This is not 153-158. Downloaded by guest on October 2, 2021 Microbiology: Fu et al. Proc. Nat!. Acad. Sci. USA 91 (1994) 5103

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