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1.
The capacity of inducing a H 2-uptake hydrogenase in free-living cultures was examined in 21 strains of Rhizobium japonicum. Four strains were found to take up H 2 at rapid rates after 3 days of growth on agar slants inside sealed vials provided with an atmosphere of 5% H 2 in air. Soybean nodules from these strains lost little or no H 2 in air and their bacteroids oxidized H 2 at rates that were similar to those observed in free-living cultures. In contrast, three randomly chosen strains of R. japonicum that showed no H 2-uptake capacity in free-living state produced nodules which lost large amounts of H 2 and the corresponding bacteroids had no hydrogenase activity. A screening procedure is described for the selection of Rhizobium strains producing high energy-efficient nodules based on a test of their ability to induce a H 2-uptake hydrogenase in asymbiotic conditions. 相似文献
2.
A series of Rhizobium meliloti and Rhizobium trifolii strains were used as inocula for alfalfa and clover, respectively, grown under bacteriologically controlled conditions. Replicate samples of nodules formed by each strain were assayed for rates of H 2 evolution in air, rates of H 2 evolution under Ar and O 2, and rates of C 2H 2 reduction. Nodules formed by all strains of R. meliloti and R. trifolii on their respective hosts lost at least 17% of the electron flow through nitrogenase as evolved H 2. The mean loss from alfalfa nodules formed by 19 R. meliloti strains was 25%, and the mean loss from clover nodules formed by seven R. trifolii strains was 35%. R. meliloti and R. trifolii strains also were cultured under conditions that were previously established for derepression of hydrogenase synthesis. Only strains 102F65 and 102F51 of R. meliloti showed measurable activity under free-living conditions. Bacteroids from nodules formed by the two strains showing hydrogenase activity under free-living conditions also oxidized H 2 at low rates. The specific activity of hydrogenase in bacteroids formed by either strain 102F65 or strain 102F51 of R. meliloti was less than 0.1% of the specific activity of the hydrogenase system in bacteroids formed by H 2 uptake-positive Rhizobium japonicum USDA 110, which has been investigated previously. R. meliloti and R. trifolii strains tested possessed insufficient hydrogenase to recycle a substantial proportion of the H 2 evolved from the nitrogenase reaction in nodules of their hosts. Additional research is needed, therefore, to develop strains of R. meliloti and R. trifolii that possess an adequate H 2-recycling system. 相似文献
3.
H 2-uptake positive strains (122 DES and SR) and H 2-uptake negative strains SR2 and SR3 of Rhizobium japonicum were examined for ribulosebisphosphate (RuBP) carboxylase and H 2-uptake activities during growth conditions which induced formation of the hydrogenase system. The rate of 14CO 2 uptake by hydrogenase-derepressed cells was about 6-times greater in the presence than in the absence of H 2. RuBP carboxylase activity was observed in free-living R. japonicum strains 122 DES or SR only when the cells were derepressed for their hydrogenase system. Hydrogenase and RuBP carboxylase activities were coordinately induced by H 2 and both were repressed by added succinate. Hydrogenase-negative mutant strains SR2 and SR3 derived from R. japonicum SR showed no detecyable RuBP carboxylase activities under hydrogenase derepression conditions. No detectable RuBP carboxylase was observed in bacteroids formed by H 2-uptake positive strains R. japonicum 122 DES or SR. Propionyl CoA carboxylase activity was consistently observed in extracts of cells from free-living cultures of R. japonicum but activity was not appreciably influenced by the addition of H 2. Neither phosphoenolpyruvate carboxylase nor phosphoenolpyruvate carboxykinase activity was detected in extracts of R. japonicum.Abbreviations RuBP
Ribulose 1,5-bisphosphate
- (Na 2EDTA)
(Ethylenedinitrilo)-tetraacetic acid, disodium salt
- (propionyl CoA)
Propionyl coenzyme A
- (PEP)
Phosphoenolpyruvate
- (GSH)
Reduced glutathione
- (Tricine)
N-tris(hydroxymethyl)-methylglycine 相似文献
4.
Although Rhizobium japonicum nodulates Vigna unguiculata and Macroptilium atropurpurem, little is known about the physiology of these symbioses. In this study, strains of R. japonicum of varying effectiveness on soybean were examined. The nonhomologous hosts were nodulated by all the strains tested, but effectiveness was not related to that of the homologous host. On siratro, compared to soybean, many strains reversed their relative effectiveness ranking. Both siratro and cowpea produced more dry matter with standard cowpea rhizobia CB756 and 176A22 than with the strains of R. japonicum. Strains USDA33 and USDA74 were more effective with siratro and cowpea than with soybean. The strain USDA122 expressed high rates of hydrogenase activity in symbiosis with the cowpea as well as the soybean host. The strains USDA61 and USDA74 expressed low levels of hydrogenase activity in symbiosis with cowpea, but no activity was found with soybean. Our results indicate host influence for the expression of hydrogenase activity, and suggest the possibility of host influence of nitrogenase for the allocation of electrons to N 2 and H +. 相似文献
5.
A sensitive tritium exchange assay was applied to the Rhizobium system for measuring the expression of uptake hydrogenase in free-living cultures of Rhizobium japonicum. Hydrogenase was detected about 45 hours after inoculation of cultures maintained under microaerophilic conditions (about 0.1% O 2). The tritium exchange assay was used to screen a variety of different strains of R. japonicum (including major production strains) with the findings that about 30% of the strains expressed hydrogenase activity with identical results being observed using an alternative assay based on uptake of H 2. The relative efficiency of intact soybean nodules inoculated with 10 different rhizobial strains gave results identical to those obtained using free-living cultures. The tritium exchange assay provides an easy, quick, and accurate assessment of H 2 uptake efficiency of intact nodules. 相似文献
6.
The interaction between the ATP-dependent evolution of H 2 catalyzed by nitrogenase and the oxidation of H 2 via a hydrogenase has been postulated to influence the efficiency of the N 2-fixing process in nodulated legumes. A comparative study using soybean ( Glycine max L. Merr.) cv. Anoka inoculated with either Rhizobium japonicum strain USDA 31 or USDA 110 and cowpea ( Vigna unguiculata L. Walp.) cv. Whippoorwill inoculated with Rhizobium strain 176A27 or 176A28 cultured on a N-free medium was conducted to address this question. Nodules from the Anoka cultivar inoculated with USDA 31 evolved H 2 in air and the H 2 produced accounted for about 30% of the energy transferred to the nitrogenase system during the period of active N 2 fixation. In contrast the same soybean cultivar inoculated with USDA 110 produced nodules with an active hydrogenase and consequently did not evolve H 2 in air. A comparison of Anoka soybeans inoculated with the two different strains of R. japonicum showed that mean rates of C 2H 2 reduction and O 2 consumption and mean mass of nodules taken at four times during vegetative growth were not significantly different. When compared to Anoka inoculated with USDA 31, the same cultivar inoculated with USDA 110 showed increases in total dry matter, per cent nitrogen, and total N2 fixed of 24, 7, and 31%, respectively. Cowpeas in symbiosis with the hydrogenase-producing strain 176A28 in comparison with the same cultivar inoculated with the H2-evolving strain 176A27 produced increases in plant dry weight and total N2 fixed of 11 and 15%, respectively. This apparent increase in the efficiency of N2 fixation for nodulated legumes capable of reutilizing the H2 evolved from nitrogenase is considered and it is concluded that provision of conclusive evidence of the role of the H2-recycling process in N2-fixing efficiency of legumes will require comparison of Rhizobium strains that are genetically identical with the exception of the presence of hydrogenase. 相似文献
7.
Fifty-four strains of Bradyrhizobium sp. ( Lupinus) from worldwide collections were screened by a colony hybridization method for the presence of DNA sequences homologous to the structural genes of the Bradyrhizobium japonicum hydrogenase. Twelve strains exhibited strong colony hybridization signals, and subsequent Southern blot hybridization experiments showed that they fell into two different groups on the basis of the pattern of EcoRI fragments containing the homology to the hup probe. All strains in the first group (UPM860, UPM861, and 750) expressed uptake hydrogenase activity in symbiosis with Lupinus albus, Lupinus angustifolius, Lupinus luteus, and Ornithopus compressus, but both the rate of H 2 uptake by bacteroids and the relative efficiency of N 2 fixation (RE = 1 - [H 2 evolved in air/acetylene reduced]) by nodules were markedly affected by the legume host. L. angustifolius was the less permissive host for hydrogenase expression in symbiosis with the three strains (average RE = 0.76), and O. compressus was the more permissive (average RE = 1.0). None of the strains in the second group expressed hydrogenase activity in lupine nodules, and only one exhibited low H 2-uptake activity in symbiosis with O. compressus. The inability of these putative Hup + strains to induce hydrogenase activity in lupine nodules is discussed on the basis of the legume host effect. Among the 42 strains showing no homology to the B. japonicum hup-specific probe in the colony hybridization assay, 10 were examined in symbiosis with L. angustifolius. The average RE for these strains was 0.51. However, one strain, IM43B, exhibited high RE values (higher than 0.80) and high levels of hydrogenase activity in symbiosis with L. angustifolius, L. albus, and L. luteus. In Southern blot hybridization experiments, no homology was detected between the B. japonicum hup-specific DNA probe and total DNA from vegetative cells or bacteroids from strain IM43B even under low stringency hybridization conditions. We conclude from these results that strain IM43B contains hup DNA sequences different from those in B. japonicum and in other lupine rhizobia strains. 相似文献
9.
Utilisation (uptake) of hydrogen gas by whole cells of Rhizobium japonicum was found to be influenced by the carbon source(s) present in the growth medium, with activity being highest in a medium containing sugars. Tricarboxylic acid cycle intermediates, such as malate, significantly reduced H 2 utilisation. No reduction in the hydrogenase activity is observed when the enzyme is assayed directly by the tritium exchange method, indicating that the decrease in hydrogen uptake activity is not due to repression of hydrogenase biosynthesis. Cyclic AMP was found to alleviate the inhibition of H 2 uptake by malate, and this requires new protein synthesis. Addition of chloramphenicol or rifampicin simultaneously with cyclic AMP eliminated the stimulation of H 2 uptake in the malate medium. These results show that in R. japonicum cyclic AMP plays a major role in the regulation of H 2 metabolism. 相似文献
10.
Rates of respiratory CO 2 loss and nitrogenase activities of H 2 uptake-negative mutant strains and H 2 uptake-positive revertant strains of Rhizobium japonicum have been investigated. Two-dimensional gel protein patterns of bacteroids formed by inoculation of soybeans ( Glycine max L.) with these two strains show that they are closely related and revealed only one obvious difference between them. On the basis of molecular weight standards, it was concluded that the missing protein spot in the H 2 uptake-negative mutant strain could be caused by a failure of the mutant to synthesize hydrogenase. Nodules formed by the H 2 uptake-negative mutant strain evolved respiratory CO 2 at a rate of about 10% higher than that of nodules formed by the H 2 uptake-positive revertant strain. During short-term experiments employed, rates of both C 2H 2 reduction and 15N 2 fixation varied considerably among replicate samples and no statistically significant differences between mutant and revertant strains were observed. It was observed that increasing the partial pressure of O 2 over nodules significantly decreased the proportion of nitrogenase electrons allocated to H +. 相似文献
12.
Some strains of rhizobia possess a hydrogenase system which catalyzes the oxidation of the H 2 that is evolved from nitrogenase during N 2 fixation. Oxidation of H 2 by a hydrogen uptake positive strain of provides energy for support of the N 2 fixation reactions and protects nitrogenase from O 2 damage 相似文献
13.
Transposon Tn 5 mutagenesis was used to isolate mutants of Rhodospirillum rubrum which lack uptake hydrogenase (Hup) activity. Three Tn 5 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. 相似文献
14.
The hydrogenase system which catalyzes the oxyhydrogen reaction in soybean nodules produced by strains of is located in the bacteroids. The hydrogenase complex in intact bacteroids has an apparent K m for H 2 of 2.8 μM and an apparent K m for O 2 of 1.3 μM. The addition of hydrogen to bacteroids increases oxygen uptake but decreases respiratory CO 2 production, indicating a conservation of endogenous substrates. After correction for the effect of hydrogen on endogenous respiration a ratio of 1.9 ± 0.1 for H 2 to O 2 uptake was determined. Bacteroids from greenhouse or field-grown soybeans that evolved hydrogen showed no measurable oxyhydrogen reaction activity whereas consistent activity was demonstrated by bacteroids from soybean nodules that evolved little or no H 2. 相似文献
15.
Four strains of Rhizobium (R. trifolii RCL10, R. japonicum S19 and SB16, and Rhizobium sp. NEA4) were demonstrated to grow lithoautotrophically with molecular hydrogen as sole electron donor and with ammonium or with N 2 as N source. All of them showed ribulose-1,5-bisphosphate carboxylase activity and hydrogenase (H 2-uptake) activity with methylene blue and oxygen as electron acceptors. For R. japonicum SB 16, a doubling time under autotrophic conditions of 30 h and a specific hydrogenase activity (methylene blue reduction) in crude extracts of 1.4 U/mg protein were calculated. Rhizobium hydrogenase is a membrane-bound enzyme. It is mainly detectable in particulate cell fractions, it cross-reacts with the antibodies of the membrane-bound hydrogenase of Alcaligenes eutrophus, and is unable to reduce NAD. The isolated hydrogenase is a relatively oxygen-sensitive enzyme with a half-life of three days when stored at 4°C under air. 相似文献
16.
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. 相似文献
17.
Occurrence and localization of an uptake hydrogenase were examined in three strains of the blue-green alga, Anabaena. In vivo H 2 uptake was detected (0.60-1.44 μmoles/[mg of chlorophyll a per hour]) in all three strains when grown with N 2 as the sole source of nitrogen. H 2 uptake ( in vivo and in vitro) was severely suppressed in cultures grown on NH 4+ and lacking heterocysts. H 2 uptake in cell-free extracts could be readily measured with a methyl viologen-ferricyanide electron acceptor system. Solubilization kinetics during cavitation of aerobically grown Anabaena 7120 indicates that the uptake hydrogenase is localized solely in the heterocyst. When the same organism is grown on N 2/CO 2, vegetative cells may account for up to 21% of the total hydrogenase activity in the filaments. The results are discussed in terms of a proposed functional relationship between nitrogenase and hydrogenase. 相似文献
18.
BackgroundIn symbiotic legume nodules, endosymbiotic rhizobia (bacteroids) fix atmospheric N 2, an ATP-dependent catalytic process yielding stoichiometric ammonium and hydrogen gas (H 2). While in most legume nodules this H 2 is quantitatively evolved, which loss drains metabolic energy, certain bacteroid strains employ uptake hydrogenase activity and thus evolve little or no H 2. Rather, endogenous H 2 is efficiently respired at the expense of O 2, driving oxidative phosphorylation, recouping ATP used for H 2 production, and increasing the efficiency of symbiotic nodule N 2 fixation. In many ensuing investigations since its discovery as a physiological process, bacteroid uptake hydrogenase activity has been presumed a single entity. Methodology/Principal Findings
Azorhizobium caulinodans, the nodule endosymbiont of Sesbania rostrata stems and roots, possesses both orthodox respiratory ( exo-)hydrogenase and novel ( endo-)hydrogenase activities. These two respiratory hydrogenases are structurally quite distinct and encoded by disparate, unlinked gene-sets. As shown here, in S. rostrata symbiotic nodules, haploid A. caulinodans bacteroids carrying single knockout alleles in either exo- or- endo-hydrogenase structural genes, like the wild-type parent, evolve no detectable H 2 and thus are fully competent for endogenous H 2 recycling. Whereas, nodules formed with A. caulinodans exo-, endo-hydrogenase double-mutants evolve endogenous H 2 quantitatively and thus suffer complete loss of H 2 recycling capability. More generally, from bioinformatic analyses, diazotrophic microaerophiles, including rhizobia, which respire H 2 may carry both exo- and endo-hydrogenase gene-sets. Conclusions/SignificanceIn symbiotic S. rostrata nodules, A. caulinodans bacteroids can use either respiratory hydrogenase to recycle endogenous H 2 produced by N 2 fixation. Thus, H 2 recycling by symbiotic legume nodules may involve multiple respiratory hydrogenases. 相似文献
19.
The prospects of developing strains of legume nodule bacteria that provide higher productivity of leguminous plants are described. The generic, biochemical, physiological, regulatory, and economic constraints that govern the ability of private and public efforts to construct better inoculants for legume nodulation are discussed. Success in constructing better inoculants requires a two-pronged approach. First, strains need to be improved in order to compete successfully with indigenous strains for root nodulation of legumes. Several loci have been identified to date that affect competitiveness for strain nodule occupancy. Usually mutations in these loci affect the ability of a strain to form nodules rapidly and efficiently. Other loci, such as those that confer antibiotic production, can be added to strains to enhance nodulation competitiveness when co-inoculated with antibiotic-sensitive strains. Second, the inoculum strains must be improved with respect to symbiotic nitrogen fixation. Efforts to enhance the symbiotic productivity of legume nodule bacteria either by mutation or genetic engineering are also described. The best characterized example of these is the hydrogenase system. Due to nitrogenase-dependent catalysis of proton reduction, diazotrophs evolve large amounts of H 2. An approach to maximize the efficiency of symbiotic N 2 fixation, and therefore of legume productivity, is to construct strains of Rhizobium with the ability to oxidize this otherwise wasted H 2. The electrons produced by H 2 oxidation are funneled through energy-conserving electron transport chains. Our knowledge of the genetics and biochemistry of H 2 oxidation in Bradyrhizobium japonicum and Rhizobium leguminosarum has developed rapidly in recent years. At least 20 genes are needed for these bacteria to manufacture and efficiently express a nickel-containing H 2-uptake hydrogenase. These genes include those encoding regulatory elements, posttranslational processing enzymes, nickel-sensing and nickel-metabolism proteins, and electron transport components for integrating the electrons from H 2 oxidation into the respiratory chain. Some of the components for oxidizing H 2 in the symbiotic N 2 fixing bacteria are distinct from the analogous components in (nonsymbiotic) H 2 oxidizing bacteria. 相似文献
20.
A method has been developed for the rapid screening of Rhizobium japonicum colonies for hydrogenase activity based on their ability to reduce methylene blue in the presence of respiratory inhibitors and hydrogen. Hydrogen uptake-positive (Hup +) colonies derepressed for hydrogenase activity were visualized by their localized decolorization of filter paper disks impregnated with the dye. Appropriate responses were seen with a number of Hup + and Hup − wild-type strains of R. japonicum as well as Hup − mutants. Its specificity was further confirmed in selected strains on the basis of comparisons with chemolithotrophic growth and the presence of other genetic markers. Utilization of the method in identifying Hup + colonies among 16,000 merodiploid derivatives of the Hup − mutant strain PJ17nal containing cloned DNA fragments of the Hup + strain 122 DES has demonstrated its applicability as a screening procedure in the genetic analysis of the R. japonicum hydrogen uptake system. 相似文献
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