Uptake Hydrogenase (Hup) in Common Bean (Phaseolus vulgaris) Symbioses

Strains of Rhizobium forming nitrogen-fixing symbioses with common bean were systematically examined for the presence of the uptake hydrogenase (hup) structural genes and expression of uptake hydrogenase (Hup) activity. DNA with homology to the hup structural genes of Bradyrhizobium japonicum was present in 100 of 248 strains examined. EcoRI fragments with molecular sizes of approximately 20.0 and 2.2 kb hybridized with an internal SacI fragment, which contains part of both bradyrhizobial hup structural genes. The DNA with homology to the hup genes was located on pSym of one of the bean rhizobia. Hup activity was observed in bean symbioses with 13 of 30 strains containing DNA homologous with the hup structural genes. However, the Hup activity was not sufficient to eliminate hydrogen evolution from the nodules. Varying the host plant with two of the Hup⁺ strains indicated that expression of Hup activity was host regulated, as has been reported with soybean, pea, and cowpea strains.     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within Hup+Rhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H₂-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 H₂ oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H₂-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 H₂-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.     

The Relationship between H(2) Evolution and Acetylene Reduction in Pisum sativum-Rhizobium leguminosarum Symbioses Differing in Uptake Hydrogenase Activity

Peas (Pisum sativum L.) were inoculated with strains of Rhizobium leguminosarum having different levels of uptake hydrogenase (Hup) activity and were grown in sterile Leonard jars under controlled conditions. Rates of H₂ evolution and acetylene reduction were determined for intact nodulated roots at intervals after the onset of darkness or after removal of the shoots. Hup activity was estimated using treatment plants or equivalent plants from the growth chamber, by measuring the uptake of H₂ or ³H₂ in the presence of acetylene. In all cases, the rate of H₂ evolution was a continuous function of the rate of acetylene reduction. In symbioses with no demonstrable Hup activity, H₂ evolution increased in direct proportion to acetylene reduction and the slopes were similar with the Hup⁻ strains NA502 and 128C79. Hup activity was similar in strains 128C30 and 128C52 but significantly lower in strain 128C54. With these strains, the slopes of the H₂ evolution versus acetylene reduction curves initially increased with acetylene reduction, but became constant and similar to those for the Hup⁻ strains at high rates of acetylene reduction. On these parallel portions of the curves, the decreases in H₂ evolution by Hup⁺ strains were similar in magnitude to their H₂-saturated rates of Hup activity. The curvilinear relationship between H₂ evolution and acetylene reduction for a representative Hup⁺ strain (128C52) was the same, regardless of the experimental conditions used to vary the nitrogenase activity.     

Choice of hydrogen uptake (Hup) status in legume‐rhizobia symbioses

The H₂ is an obligate by-product of N-fixation. Recycling of H₂ through uptake hydrogenase (Hup) inside the root nodules of leguminous plants is often considered an advantage for plants. However, many of the rhizobium-legume symbioses found in nature, especially those used in agriculture are shown to be Hup⁻, with the plants releasing H₂ produced by nitrogenase activity from root nodules into the surrounding rhizosphere. Recent studies have suggested that, H₂ induces plant-growth-promoting rhizobacteria, which may explain the widespread of Hup⁻ symbioses in spite of the low energy efficiency of such associations. Wild legumes grown in Nova Scotia, Canada, were surveyed to determine if any plant-growth characteristics could give an indication of Hup choice in leguminous plants. Out of the plants sampled, two legumes, Securigera varia and Vicia cracca, showed Hup⁺ associations. Securigera varia exhibited robust root structure as compared with the other plants surveyed. Data from the literature and the results from this study suggested that plants with established root systems are more likely to form the energy-efficient Hup⁺ symbiotic relationships with rhizobia. Conversely, Hup⁻ associations could be beneficial to leguminous plants due to H₂-oxidizing plant-growth-promoting rhizobacteria that allow plants to compete successfully, early in the growing season. However, some nodules from V. cracca tested Hup⁺, while others were Hup⁻. This was similar to that observed in Glycine max and Pisum sativum, giving reason to believe that Hup choice might be affected by various internal and environmental factors.     

Expression of Chromosome-integrated Hydrogen-uptake Genes in Cicer-Rhizobium

Integration of H₂-uptake (hup) gene cosmid pHU52 into the chromosomal DNA, conferred H₂- uptake activity on the Hup^- Cicer-Rhizobium strain G36–84 in the free-living state and in nodules. In five transconjugants (G36–84:: Tn5:: pHU52) derepressed for hup gene expression, the specific Hup activity ranged from 158 to 256 nmole H₂ hr^-1 mg-¹ protein which was 42 to 64% lower than the activity obtained in transconjugant with pHU52 as an episome. Integration of the cosmid significantly improved the relative efficiency of symbiotic N₂-fixation by imparting H₂-recycling capability to Hup^- Cicer-Rhizobium. Demonstration of Hup activity in the nodules of field grown chickpea plants suggests that the integrated hup genes are stably maintained in natural environment     

Expression of hup Genes in Azorhizobium caulinodans and Their Coregulation With RuBP Carboxylase

In Azorhizobium caulinodans strain IRBG 46, H₂ evolved by nitrogenase Induced uptake hydrogenase ex-planta. The strain expressed an efficient H₂ recycling system under both symbiotic and ex-planta conditions. For the first time, a comparable relative efficiency of electron transfer to N₂ via nitrogenase under symbiotic as well as ex-planta conditions for both Hup? strain B11 as well as Hup⁺ strain IRBG 46, has been reported. The study also suggested a coordinate regulatory relationship between rubisco and hup genes.     

Sodium stimulation of uptake hydrogenase activity in symbiotic Rhizobium

Initial observations showed a 100% increase in H₂-uptake (Hup) activity of Rhizobium leguminosarum strain 3855 in pea root nodules (Pisum sativum L. cv Alaska) on plants growing in a baked clay substrate relative to those growing in vermiculite, and an investigation of nutrient factors responsible for the phenomenon was initiated. Significantly greater Hup activity was first measured in the clay-grown plants 24 days after germination, and higher activity was maintained relative to the vermiculite treatment until experiments were terminated at day 32. The increase in Hup activity was associated with a decrease in H₂ evolution for plants with comparable rates of acetylene reduction. Analyses of the clay showed that it contained more Na⁺ (29 versus 9 milligrams per kilogram) and less K⁺ (6 versus 74 milligrams per kilogram) than the vermiculite. Analyses of plants, however, showed a large increase in Na⁺ concentration of clay-grown plants with a much smaller reduction in K⁺ concentration. In tests with the same organisms in a hydroponic system with controlled pH, 40 millimolar NaCl increased Hup activity more than 100% over plants grown in solutions lacking NaCl. Plants with increased Hup activity, however, did not have greater net carbon or total nitrogen assimilation. KCl treatments from 5 to 80 millimolar produced slight increased in Hup activity at 10 millimolar KCl, and tests with other salts in the hydroponic system indicated that only Na⁺ strongly promoted Hup activity. Treating vermiculite with 50 millimolar NaCl increased Na⁺ concentration in pea plant tissue and greatly promoted Hup activity of root nodules in a manner analogous to the original observation with the clay rooting medium. A wider generality of the phenomenon was suggested by demonstrating that exogenous Na⁺ increased Hup activity of other R. leguminosarum strains and promoted Hup activity of R. meliloti strain B300 in alfalfa (Medicago sativa L.).     

Determination of the Hydrogenase Status of Individual Legume Nodules by a Methylene Blue Reduction Assay

We adapted a method for the rapid screening of colonies of free-living Rhizobium japonicum for hydrogenase activity to determine the hydrogenase status of individual soybean nodules. Crude bacteroid suspensions from nodules containing strains known to be hydrogen uptake positive (Hup⁺) caused a localized decolorization of filter paper disks, whereas suspensions from nodules arising from inoculation with hydrogen uptake-negative (Hup⁻) mutants or strains did not decolorize the disks. The reliability of the method was demonstrated by its successful application to 29 slow-growing rhizobia. The Hup phenotype on methylene blue filters agreed with that determined amperometrically with either methylene blue or oxygen as the electron acceptor.     

Differential Expression of Uptake Hydrogenase Activity in Free Living Cells and Nodule Bacteroids of Azorhizobium caulinodans

An oxygen sensitive mutant of Azorhizobium caulinodans strain IRBG 46 in which N₂ fixation ability was affected, was previously isolated by NTG mutagenesis. Now, the mutation has been shown to affect H₂- uptake hydrogenase (Hup) activity under symbiotic conditions. However, free living Hup activity remained unaffected. Thus the mutant is Hup^- under symbiotic conditions and Hup⁺ under free living condtions. A possible regulatory link between N₂ fixation and H₂ uptake system has been discussed.     

Hydrogen Recycling by Rhizobium leguminosarum Isolates and Growth and Nitrogen Contents of Pea Plants (Pisum sativum L.)

The ability to recycle H₂ evolved by nitrogenase is thought to be of importance in increasing the efficiency of N₂ fixation and to be a factor in increasing plant yield in symbiotic systems. To determine whether this ability is a significant factor in the Rhizobium leguminosarum-Pisum sativum L. system, plants were inoculated with R. leguminosarum isolates which differed in their ability to oxidize H₂ and in their relative efficiency of N₂ fixation. These plants were grown at three levels of irradiance and harvested after 3, 4, and 5 weeks of growth for determination of C₂H₂ reduction, H₂ evolution and uptake, plant dry weight, and N content. Plants inoculated with uptake hydrogenase-positive (Hup⁺) isolates did not exhibit higher dry weight or N content than those inoculated with Hup⁻ isolates under any of the growth conditions studied. The efficiency of the nitrogenase system of Hup⁻ isolates increased at a low irradiance, a factor which may allow them to compete successfully with Hup⁺ isolates. In some Hup⁺R. leguminosarum isolates, H₂ oxidation is coupled to ATP formation, whereas in others, it is not. There were no differences in plant dry weight and N content in plants inoculated with the two types and grown for 5 weeks at three irradiance levels. The addition of H₂ to Hup⁺ nodules whose supply of photosynthate had been removed by stem excision did not increase C₂H₂ reduction in either coupled or uncoupled types.     

Transposon Tn5-Generated Bradyrhizobium japonicum Mutants Unable To Grow Chemoautotrophically with H2

Twelve Tn5-induced mutants of Bradyrhizobium japonicum unable to grow chemoautotrophically with CO₂ and H₂ (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% O₂. 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 CO₂ 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.     

Rhizobium japonicum Serogroup and Hydrogenase Phenotype Distribution in 12 States

A survey was conducted in 1980 on 972 isolates of Rhizobium japonicum obtained from 65 soybean field locations in 12 states. Isolates were examined for the hydrogenase (Hup) phenotype and somatic serogroup identity. Only 20% of the isolates were Hup⁺, with a majority of Hup⁻ isolates occurring in 10 of the 12 states. The most predominant serogroup was 31 (21.5%), followed by 123 (13.6%). Although most serogroups contained a majority of Hup⁻ isolates, marked differences occurred. None of the isolates in serogroup 135 were Hup⁺, but 93% of the isolates in serogroup 122 were Hup⁺. The serogroups with relatively high frequencies of Hup⁺ isolates (122 and 110) constitute only a small part (<5% each) of the R. japonicum field population in the 12 states.     

Conservation in Soil of H2 Liberated from N2 Fixation by Hup- Nodules

Pigeon peas (Cajanus cajan) were grown in large soil columns (90-cm length by 30-cm diameter) and inoculated with four different strains of cowpea rhizobia, which varied with respect to hydrogen uptake activity (Hup). Despite the profuse liberation of H₂ from Hup^- nodules in vitro, H₂ gas was not detected in any of the soil columns. When H₂ was injected into the columns, the rates of consumption were highest in the treatments (including control) containing Hup^- nodules (218 and 177 nmol · h⁻¹ · cm⁻²) and lowest in the Hup⁺ treatments (158, 92, and 64 nmoles · h⁻¹ · cm⁻²). In situ H₂ uptake rates in small soil cores at fixed distances from the nodules decreased exponentially with distance from the nodule (R² = 0.99). This decrease in H₂ consumption was associated with a similar decrease in numbers of H₂-oxidizing chemolithotrophic bacteria as determined by the most-probable-number method. On the basis of two equations derived separately upon diffusive theory (Fix's Law) and kinetic theory (Michaelis-Menten), the empirically derived rate constants and coefficients indicated that all of the H₂ emitted from Hup^- nodules would be consumed by H₂-oxidizing bacteria within a 3- to 4.5-cm radius of the nodule surface. It is concluded that H₂ is not lost from the soil-plant ecosystem during N₂ fixation in C. cajan but is conserved by H₂-oxidizing bacteria.     

Competitive nodulation blocking of Afghanistan pea is determined by nodDABC and nodFE alleles in Rhizobium leguminosarum

Summary One well-defined competitive interaction amongst rhizobia is that between compatible and non-compatible strains of Rhizobium leguminosarum with respect to the nodulation of some primitive pea genotypes. The Middle Eastern pea cv Afghanistan is nodulated effectively can R. leguminosarum TOM, but its capacity to nodulate can be blocked if a mixed inoculation is made with R. leguminosarum PF₂. This PF₂ phenotype (Cnb) is encoded by its symbiotic plasmid and cosmid clones thereof. We found that Cnb is also encoded by the well-characterized Sym plasmid pRL1JI of R. leguminosarum strain 248. We have isolated and characterized a 6.9 kb HindIII fragment of pSymPF₂ which confers the Cnb⁺ phentoype on other (Cnb^–) rhizobia. A Tn5 site-directed Cnb^– mutant was constructed by homogenotization and was also found to be Nod^– on the European pea cv Rondo. DNA hybridization and complementation analysis indicated that the 6.9 kb Cnb⁺ fragment contained the nodD, nodABC and nodFE operons. Analysis of the Cnb phenotype of nod::Tn5 alleles of pRL1JI showed that mutations of nodC, nodD or nodE all abolished Cnb activity whereas mutants in nodI and nodJ reduced activity to 50% of the wild-type level.     

Influence of the Bradyrhizobium japonicum Hydrogenase on the Growth of Glycine and Vigna Species

The effect of the Bradyrhizobium japonicum hydrogenase on nitrogen fixation was evaluated by comparing the growth of Vigna and Glycine species inoculated with a Hup mutant and its Hup⁺ revertant. In all experiments, the growth of plants inoculated with the strain without hydrogenase was at least equal to the growth of the strain with hydrogenase. For Glycine usuriensis and Glycine max cv. Hodgson in liquid culture, the growth was higher with the Hup⁻ strain. It is possible that reduced rates of nitrogen fixation in the presence of hydrogenase are due to O₂ depletion caused by the hydrogen oxidizing, since the oxygen pressure in the air appears to be a limiting factor of symbiotic nitrogen fixation in the soybean.     

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Summary An hydrogenase-deficient (Hup^–) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup^– mutant, strain JP91, selected by its inability to grow autotrophically (Aut^– phenotype) together with other Hup^– mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup^– mutants, span 15–20 kb of DNA.     

Host Plant Cultivar Effects on Hydrogen Evolution by Rhizobium leguminosarum

The effect of host plant cultivar on H₂ evolution by root nodules was examined in symbioses between Pisum sativum L. and selected strains of Rhizobium leguminosarum. Hydrogen evolution from root nodules containing Rhizobium represents the sum of H₂ produced by the nitrogenase enzyme complex and H₂ oxidized by any uptake hydrogenase present in those bacterial cells. Relative efficiency (RE) calculated as RE = 1 − (H₂ evolved in air/C₂ H₂ reduced) did not vary significantly among `Feltham First,' `Alaska,' and `JI1205' peas inoculated with R. leguminosarum strain 300, which lacks uptake hydrogenase activity (Hup⁻). That observation suggests that the three host cultivars had no effect on H₂ production by nitrogenase. However, RE of strain 128C53 was significantly (P ≤ 0.05) greater in symbiosis with cultivar JI1205 than in root nodules of Feltham First. At a similar rate of C₂H₂ reduction on a whole-plant basis, nearly 24 times more H₂ was evolved from the Feltham First/128C53 symbiosis than from the JI1205/128C53 association. Root nodules from the Alaska/128C53 symbiosis had an intermediate RE over the entire study period, which extended from 21 to 36 days after planting. Direct assays of uptake hydrogenase by two methods showed significant (P ≤ 0.05) host cultivar effects on H₂ uptake capacity of both strain 128C53 and the genetically related strain 3960. The ³H₂ incorporation assay showed that strains 128C53 and 3960 in symbiosis with Feltham First had about 10% of the uptake hydrogenase activity measured in root nodules of Alaska or JI1205. These data are the first demonstration of significant host plant effects on rhizobial uptake hydrogenase in a single plant species.     

Construction of a Gene Bank of Azorhizobium IRBG-46: Isolation of hup Genes

Genomic DNA from an efficient Hup⁺ Sesbania-Azorhizobium strain IRBG-46 was isolated, partially digested with EcoRI and fractionated on a 10–40% sucrose density gradient to obtain DNA fragments in the size range of 15–23 kb. In order to isolate hup genes from this strain, a gene bank was constructed in Escherichia coil HB101 using a mobilizable plasmid vector pRK290 having a EcoRI cloning site. Approximately 2x10⁴ Tc-resistant transformants were pooled to constitute the gene bank. Using 12.9 kb EcoRI fragment of cosmid pHU52 as a heterologous hup probe, a total of 2,000 clones were screened by colony hybridization. Five positive clones confirmed by secondary screening and ex planta uptake hydrogenase activity were identified. An insert size in the range of 15–22 kb was revealed by restriction analysis with EcoRI. These five recombinant plasmids containing Hup-determlnants of Azorhizobium IRBG-46 have been designated as pSRH1, pSRH2, pSRH3, pSRH4 and pSRH5. These plasm ids were transferred into Hup^- Cicer-Rhizobium strain Rcd 301 to check the expression of hup genes in the new genetic background. In the transconjugants so obtained, the hup genes were found to express under ex planta conditions, and uptake hydrogenase activity ranged from 134 to 392 nmol H₂ taken up per h per mg protein.     

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 H₂ 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 H₂ evolution and the light-dependent H₂ production in the presence of thiosulfate. Both Hox⁺ and Hup⁺ mutants demonstrated light-dependent H₂ uptake stimulated by CO₂ but only the Hup⁺ mutant was able to mediate O₂-dependent H₂ 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, CO₂, H₂, 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.     

Attachment of Rhizobium to Legume Roots as the Basis for Specific Interactions

Experiments were conducted to elucidate the basis of the observation that different strains of Rhizobium infect particular legumes. Rhizobia specific for a variety of legumes were grown with ¹³PO^2?₄ and exposed to pea roots (Pisum sativum L.), R. leguminosarum 128C53, which nodulates pea, did not attach to the roots in greater numbers than those strains of rhizobia incapable of infecting pea roots. A complex of R. leguminosarum 128C53 conjugated to a fluorochrome-labeled antibody exhibited a striking attachment to the tips of pea root hairs, where infection normally occurs, but this fluorescent complex also bound to the root hairs of Canavalia en siformis DC., Lupinus polyphyllus Lindl., Trifolium pratense L., and Medicago sativa L., which are not infected by this bacterium. A reproducible, quantitative technique developed for studying interactions between fluorochrome-labeled lectins and rhizobia revealed no relationship between lectin-Rhizobium interactions and the capacity to infect a plant. The data are interpreted as suggesting that simple attachment of Rhizobium to a legume root is not the basis of host-symbiont specificity in this system.