Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H(2)-uptake hydrogenase positive (Hup) or negative (Hup) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H(2) oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H(2)-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H(2)-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup strains in this symbiotic species.     

Nif- Hup- mutants of Rhizobium japonicum.

Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity.     

Screening method for the detection of uptake hydrogenase activity of Rhizobium leguminosarum bacteroids

Abstract A method has been developed for screening Rhizobium leguminosarum wild-type strains and mutants for uptake hydrogenase (Hup) activity, using H₂-dependent methylene blue reduction. For this purpose, a simple device has been constructed which allows the simultaneous screening of 6 strains and 6 controls. Bacteroids of R. leguminosarum isolated from pea root nodules were suspended in buffer containing methylene blue and inhibitors of dehydrogenases. The suspensions were first sparged with argon (to remove oxygen) and then with hydrogen.     

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.     

Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum.

Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.     

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum.

A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHU1 containing hup genes of Bradyhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHU1 (5.9-kb HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. leguminosarum hup-specific region closely parallels that of B. japonicum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed Tn5 mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the Tn5 insertion strains in symbiosis with peas. Transposon Tn5 insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.     

Expression of uptake hydrogenase and hydrogen oxidation during heterotrophic growth of Bradyrhizobium japonicum.

Strains I-110 ARS, SR, USDA 136, USDA 137, and AK13 1c of Bradyrhizobium japonicum induced Hup activity when growing heterotrophically in medium with carbon substrate and NH4Cl in the presence of 2% H2 and 2% O2. Hup activity was induced during heterotrophic growth in the presence of carbon substrates, which were assimilated during the time of H2 oxidation. Strains I-110 ARS and SR grown heterotrophically or chemoautotrophically for 3 days had similar rates of H2 oxidation. Similar rates of Hup activity were also observed when cell suspensions were induced for 24 h in heterotrophic or chemoautotrophic growth medium with 1% O2, 10% H2, and 5% CO2 in N2. These results are contrary to the reported repression of Hup activity by carbon substrates in B. japonicum. Bradyrhizobial Hup activity during heterotrophic growth was limited by H2 and O2 and repressed by aerobic conditions, and CO2 addition had no effect. Nitrogenase and ribulosebisphosphate carboxylase activities were not detected in H2-oxidizing cultures of B. japonicum during heterotrophic growth. Immunoblot analysis of cell extracts with antibodies prepared against the 65-kilodalton subunit of uptake hydrogenase indicated that Hup protein synthesis was induced by H2 and repressed under aerobic conditions.     

Increased Nitrogenase-Dependent H(2) Photoproduction by hup Mutants of Rhodospirillum rubrum

Transposon Tn5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn5 insertions mapped at different positions within the same 13-kb EcoRI fragment (fragment E1). Hybridization experiments revealed homology to the structural hydrogenase genes hupSLM from Rhodobacter capsulatus and hupSL from Bradyrhizobium japonicum in a 3.8-kb EcoRI-ClaI subfragment of fragment E1. It is suggested that this region contains at least some of the structural genes encoding the nickel-dependent uptake hydrogenase of R. rubrum. At a distance of about 4.5 kb from the fragment homologous to hupSLM, a region with homology to a DNA fragment carrying hypDE and hoxXA from B. japonicum was identified. Stable insertion and deletion mutations were generated in vitro and introduced into R. rubrum by homogenotization. In comparison with the wild type, the resulting hup mutants showed increased nitrogenase-dependent H(2) photoproduction. However, a mutation in a structural hup gene did not result in maximum H(2) production rates, indicating that the capacity to recycle H(2) was not completely lost. Highest H(2) production rates were obtained with a mutant carrying an insertion in a nonstructural hup-specific sequence and with a deletion mutant affected in both structural and nonstructural hup genes. Thus, besides the known Hup activity, a second, previously unknown Hup activity seems to be involved in H(2) recycling. A single regulatory or accessory gene might be responsible for both enzymes. In contrast to the nickel-dependent uptake hydrogenase, the second Hup activity seems to be resistant to the metal chelator EDTA.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Further evidence that two unique subunits are essential for expression of hydrogenase activity in Rhizobium japonicum.

Eight strains of Rhizobium lacking hydrogenase uptake (Hup) activity and 17 transconjugant strains carrying the hup cosmids pHU1, pHU52, or pHU53 (G. R. Lambert, M. A. Cantrell, F. J. Hanus, S. A. Russell, K. R. Haddad, and H. J. Evans, Proc. Natl. Acad. Sci. USA, 82:3232-3236, 1985) were screened for Hup activity and the presence of immunologically detectable hydrogenase polypeptides. Crude extracts of these strains were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with affinity-purified antibodies against the two subunits of purified hydrogenase (Mr 60,000 and 30,000). Derepressed transconjugants carrying the cosmid pHU52 were Hup+ and contained detectable levels of both hydrogenase subunit polypeptides. Non-derepressed strains, Hup- parent strains, and strains carrying cosmids other than pHU52 did not express Hup activity and contained no immunologically detectable protein. These data provide further evidence for the essential involvement of the smaller (Mr 30,000) subunit in the expression of hydrogenase activity in Rhizobium japonicum and suggest that the determinants for hydrogenase subunit synthesis are present on pHU52.     

Evaluation and improvement of the energy efficiency of nitrogen fixation in Lotus corniculatus nodules induced by Rhizobium loti strains indigenous to Uruguay

In order to evaluate energy efficiency of nitrogen fixation by the Lotus corniculatus/Rhizobium loti symbiosis, Uruguayan R. loti strains were tested for hydrogen-uptake (Hup) status. Nodules induced in L. corniculatus by all eight R. loti strains tested evolved high amounts of hydrogen (2.0–8.7 mol H2/h.g nodule fresh weight). This production of hydrogen corresponds to 38–69% of total nitrogenase activity estimated as acetylene reduction, suggesting that hydrogen is not recycled within these nodules. This was confirmed by the lack of hydrogenase activity in bacteroid suspensions. Additionally, no hybridization signals were observed in total DNA restriction digests from these strains when a DNA fragment containing part of hydrogenase structural genes from Rhizobium leguminosarum bv. viciae was used as probe. Cosmid pHU52, containing the complete gene cluster required for hydrogen oxidation in Bradyrhizobium japonicum, was introduced into two R. loti strains. Transconjugants from only one of the strains were able to express hydrogenase activity in vegetative cells incubated under the derepression conditions described for B. japonicum. Bacteroids induced by both transconjugant strains in L. corniculatus and Lotus tenuis expressed hydrogenase activity in nodules. The level of hydrogenase activity induced in L. tenuis nodules was two-fold higher than those induced in L. corniculatus. This implies the existence of a strong host effect on hydrogenase expression in this symbiotic system.     

Transposon Tn5-Generated Bradyrhizobium japonicum Mutants Unable To Grow Chemoautotrophically with H(2)

Twelve Tn5-induced mutants of Bradyrhizobium japonicum unable to grow chemoautotrophically with CO(2) and H(2) (Aut) were isolated. Five Aut mutants lacked hydrogen uptake activity (Hup). The other seven Aut mutants possessed wild-type levels of hydrogen uptake activity (Hup), both in free-living culture and symbiotically. Three of the Hup mutants lacked hydrogenase activity both in free-living culture and as nodule bacteroids. The other two mutants were Hup only in free-living culture. The latter two mutants appeared to be hypersensitive to repression by oxygen, since Hup activity could be derepressed under 0.4% O(2). All five Hup mutants expressed both ex planta and symbiotic nitrogenase activities. Two of the seven Aut Hup mutants expressed no free-living nitrogenase activity, but they did express it symbiotically. These two strains, plus one other Aut Hup mutant, had CO(2) fixation activities 20 to 32% of the wild-type level. The cosmid pSH22, which was shown previously to contain hydrogenase-related genes of B. japonicum, was conjugated into each Aut mutant. The Aut Hup mutants that were Hup both in free-living culture and symbiotically were complemented by the cosmid. None of the other mutants was complemented by pSH22. Individual subcloned fragments of pSH22 were used to complement two of the Hup mutants.     

Regulation of two nickel-requiring (inducible and constitutive) hydrogenases and their coupling to nitrogenase in Methylosinus trichosporium OB3b.

Two uptake hydrogenases were found in the obligate methanotroph Methylosinus trichosporium OB3b; one was constitutive, and a second was induced by H2. Both hydrogenases could be assayed by measuring methylene blue reduction anaerobically or by coupling their activity to nitrogenase acetylene reduction activity in vivo in an O2-dependent reaction. The H2 concentration for half-maximal activity of the inducible and constitutive hydrogenases in both assays was 0.01 and 0.5 bar (1 and 50 kPa), respectively, making it easy to distinguish these enzymes from one another both in vivo and in vitro. Hydrogen uptake was shown to be coupled to ATP synthesis in methane-starved cells. Methane, methanol, formate, succinate, and glucose all repressed the H2-mediated synthesis of the inducible hydrogenase. Furthermore, this enzyme was only expressed in N-starved cultures and was repressed by NH4+ and NO3-; synthesis of the constitutive hydrogenase was not affected by excess N in the growth medium. In nickel-free, EDTA-containing medium, the activities of these two enzymes were negligible; however, both enzyme activities appeared rapidly following the addition of nickel to the culture. Chloramphenicol, when added along with nickel, had no effect on the rapid appearance of either the constitutive or inducible activity, indicating that nickel is not required for synthesis of the hydrogenase apoproteins. These observations all suggest that these hydrogenases are nickel-containing enzymes. Finally, both hydrogenases were soluble and could be fractionated by 20% ammonium sulfate; the constitutive enzyme remained in the supernatant solution, while the inducible enzyme was precipitated under these conditions.     

Role of uptake hydrogenase in providing reductant for nitrogenase in Rhizobium leguminosarum bacteroids

The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H₂ increased the rate of C₂H₂ reduction in the absence of added substrates. Malate also increased nitrogenase (C₂H₂) activity while decreasing the effect of H₂. At exogenous malate concentrations above 0.05 mM no effect of H₂ was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H₂ uptake was not coupled to ATP formation, H₂ increased the rate of C₂H₂ reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C₂H₂ reduction, but when H₂ was also included, the rate of C₂H₂ reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H₂ uptake is coupled to ATP formation, to the nitrogenase complex.     

Comparative characterization of two distinct hydrogenases from Anabaena sp. strain 7120

Two distinct hydrogenases, hereafter referred to as "uptake" and "reversible" hydrogenase, were extracted from Anabaena sp. strain 7120 and partially purified. The properties of the two enzymes were compared in cell-free extracts. Uptake hydrogenase was largely particulate, and although membrane bound, it could catalyze an oxyhydrogen reaction. Particulate and solubilized uptake hydrogenase could catalyze H2 uptake with a variety of artificial electron acceptors which had midpoint potentials above 0 mV. Reversible hydrogenase was soluble, could donate electrons rapidly to electron acceptors of both positive and negative midpoint potential, and could evolve H2 rapidly when provided with reduced methyl viologen. Uptake hydrogenase was irreversibly inactivated by O2, whereas reversible hydrogenase was reversibly inactivated and could be reactivated by exposure to dithionite or H2. Reversible hydrogenase was stable to heating at 70 degrees C, but uptake hydrogenase was inactivated with a half-life of 12 min at this temperature. Uptake hydrogenase was eluted from Sephadex G-200 in a single peak of molecular weight 56,000, whereas reversible hydrogenase was eluted in two peaks with molecular weights of 165,000 and 113,000. CO was competitive with H2 for each enzyme; the Ki's for CO were 0.0095 atm for reversible hydrogenase and 0.039 atm for uptake hydrogenase. The pH optima for H2 evolution and H2 uptake by reversible hydrogenase were 6 and 9, respectively. Uptake hydrogenase existed in two forms with pH optima of 6 and 8.5. Both enzymes had very low Km's for H2, and neither was inhibited by C2H2.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H(2) consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H(2) evolution and the light-dependent H(2) production in the presence of thiosulfate. Both Hox(+) and Hup(+) mutants demonstrated light-dependent H(2) uptake stimulated by CO(2) but only the Hup(+) mutant was able to mediate O(2)-dependent H(2) consumption in the dark. The ability of the Hox(+) mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO(2), H(2), light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Hydrogen metabolism of Azospirillum brasilense in nitrogen-free medium

Production of H2 by Azospirillum brasilense under N2-fixing conditions was studied in continuous and batch cultures. Net H2 production was consistently observed only when the gas phase contained CO. Nitrogenase activity (C2H2 reduction) and H2 evolution (in the presence of 5% CO) showed a similar response to O2 and were highest at 0.75% dissolved O2. Uptake hydrogenase activity, ranging from 0.3 to 2.5 mumol H2/mg protein per hour was observed in batch cultures under N2. Such rates were more than sufficient to recycle nitrogenase-produced H2. Tritium-exchange assay showed that H2 uptake was higher under Ar than under N2. Uptake hydrogenase was strongly inhibited by CO and C2H2. Cyclic GMP inhibited both nitrogenase and uptake hydrogenase activities.     

Effect of Plasmid pIJ1008 from Rhizobium leguminosarum on Symbiotic Function of Rhizobium meliloti

Plasmid pIJ1008, which carries determinants for uptake hydrogenase (Hup) activity, was transferred from Rhizobium leguminosarum to Rhizobium meliloti without impairing the capacity of the latter species to form root nodules on alfalfa. The plasmid was still present in rhizobia reisolated from the root nodules of 12 different alfalfa cultivars, but only low levels of Hup activity were detected in alfalfa.