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Hydrogenase Enzyme (Nikel-Ontanig Enme/Nicke Metabolism/Nitrogen Fixation) CHANGLIN Fu, SAM JAVEDAN, FARHAD MOSHIRI, and ROBERT J

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Hydrogenase Enzyme (Nikel-Ontanig Enme/Nicke Metabolism/Nitrogen Fixation) CHANGLIN Fu, SAM JAVEDAN, FARHAD MOSHIRI, and ROBERT J Proc. Natd. Acad. Sci. USA Vol. 91, pp. 5099-5103, May 1994 Microbiology Bacterial genes involved in incorporation of nickel into a hydrogenase enzyme (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 hydrogenases. 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 product. 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 enzymes, 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 catalysis, 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 electron acceptor (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.
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