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1.
Shoot/root grafting studies showed organ and host cultivar effects on net H 2 evolution from Pisum sativum L. root nodules. Net H 2 evolution from those nodules represents the sum of H 2 formed by Rhizobium nitrogenase and H 2 oxidized by any uptake hydrogenase present in the bacteria. Grafts between pea cultivars `JI1205' or `Alaska' and `Feltham First' in symbioses with R. leguminosarum 128C53 showed that shoots of both JI1205 and Alaska increased H 2 uptake significantly (P ≤ 0.05) in Feltham First root nodules. The same plants also had less net H 2 evolution at similar rates of C 2H 2 reduction than plants formed by grafting Feltham First shoots on Feltham First roots. Although JI1205 and Alaska shoots increased H 2-uptake activity of Feltham First root nodules 28 days after the graft was made, intermediate to high levels of H 2 uptake activity were still present in nodules on roots of both JI1205 and Alaska grafted to Feltham First shoots. These results indicate the presence of a transmissible shoot factor(s) which can increase uptake hydrogenase activity in a Rhizobium symbiont and show that root genotype also can influence that parameter. Parallel grafting experiments using the same pea cultivars in symbioses with R. leguminosarum strain 300, which lacks uptake hydrogenase activity, suggested that a transmissible shoot factor(s) altered H2 formation from nitrogenase by changing the electron allocation coefficient of that enzyme complex. The root and shoot factor(s) detected in this study had no permanent effect on strain 128C53. Bacterial cells isolated from Feltham First nodules with low H2 uptake activity formed root nodules on JI1205 and Alaska with high H2 uptake activity. Bacteroids isolated from nodules on intact JI1205, Alaska, or Feltham First plants with high, medium, or low H2 uptake activity, respectively, maintained those phenotypes during in vitro assays. 相似文献
2.
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). H 2 increased the rate of C 2H 2 reduction in the absence of added substrates. Malate also increased nitrogenase (C 2H 2) activity while decreasing the effect of H 2. At exogenous malate concentrations above 0.05 mM no effect of H 2 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 H 2 uptake was not coupled to ATP formation, H 2 increased the rate of C 2H 2 reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C 2H 2 reduction, but when H 2 was also included, the rate of C 2H 2 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 H 2 uptake is coupled to ATP formation, to the nitrogenase complex. 相似文献
3.
The effect of host plant cultivar on H 2 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 H 2 produced by the nitrogenase enzyme complex and H 2 oxidized by any uptake hydrogenase present in those bacterial cells. Relative efficiency (RE) calculated as RE = 1 − (H 2 evolved in air/C 2 H 2 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 H 2 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 C 2H 2 reduction on a whole-plant basis, nearly 24 times more H 2 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 H 2 uptake capacity of both strain 128C53 and the genetically related strain 3960. The 3H 2 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. 相似文献
4.
Hydrogen evolution from root nodules has been reported to make N 2 fixation by some legume- Rhizobium symbiotic systems inefficient. We have surveyed the extent of H 2 evolution and estimated relative efficiencies of nodules of Austrian winter peas formed by 15 strains of R. leguminosarum. Their rates of H 2 evolution in air were about 30% of the rates of H 2 evolution under an atmosphere in which N 2 was replaced by Ar. Relative efficiency values based on C 2H 2 reduction rates ranged from 0.55 to 0.80. With some of the strains, hydrogenase activities were demonstrated in intact nodules and in bacteroids, but the levels of activity were insufficient to recycle all the H 2 evolved by the nitrogenase system. In both intact nodules and bacteroids the hydrogenase is less sensitive to O 2 damage than the nitrogenase system, so H 2 uptake capacity was observed in intact nodules by suppressing the nitrogenase-dependent H 2 evolution with an atmosphere containing a high O 2 concentration, and in bacteroids by using aerobically prepared bacteroid suspensions. The hydrogenase activity of both was dependent on O 2 consumption. A K
mfor H 2 of near 4 M was determined in suspension of bacteroids from nodules formed by strains 128C53 and 128C56. 相似文献
5.
Nitrogenase (EC 1.7.99.2) activity in pea ( Pisum savitum) nodules formed after infection with Rhizobium leguminosarum (lacking uptake hydrogenase) was measured as acetylene reduction, H 2 evolution in air and H 2 evolution in Ar:O 2. With detached roots the relative efficiency, calculated from acetylene reduction, showed a decrease (from 55 to below 0%) with increasing temperature. With excised nodules and isolated bacteroids similar results were obtained. However, the relative efficiency calculated from H 2 evolution in Ar:O 2 was unaffected by temperature. Measurements on both excised nodules and isolated bacteroids showed a marked difference between acetylene reduction and H 2 evolution in Ar:O 2 with increased temperature, indicating that either acetylene reduction or H 2 evolution in Ar:O 2 are inadequate measures of nitrogenase activity at higher temperature. 相似文献
6.
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
7.
The H 2 uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H 2 evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N 2-fixing conditions have the highest H 2 uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction ( Mr less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H 2 oxidation and H 2 evolution at rates of 3000 and 5.9 μmol H 2 consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum. 相似文献
8.
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
9.
Summary Hydrogen uptake is thought to increase the efficiency of nitrogen fixation by recycling H 2 produced by nitrogenase that would otherwise be lost by diffusion. Here we demonstrate the capacity of eight Rhizobium strains to take up molecular hydrogen. Uptake by nodule homogenates from Robinia pseudoacacia was measured amperometrically under nitrogenase repression. Markedly lower activities were found than in soybean nodules. In addition hydrogenase activity was detected by the ability of bacteroids to reduce methylene blue in the presence of hydrogen. It was demonstrated that hydrogenase structural genes are present in the black locust symbiont, Rhizobium sp. strain R1, using hybridization with a plasmid, which contained hydrogenase genes from R. leguminosarum bv. viceae. 相似文献
10.
A heterotrophic semisolid medium was used with two sensitive assay methods, C 2H 2 reduction and O 2-dependent tritium uptake, to determine nitrogenase and hydrogenase activities, respectively. Organisms known to be positive for both activities showed hydrogenase activity in both the presence and absence of 1% C 2H 2, and thus, it was possible to test a single culture for both activities. Hydrogen uptake activity was detected for the first time in N 2-fixing strains of Pseudomonas stutzeri. The method was then applied to the most-probable-number method of counting N 2-fixing and H 2-oxidizing bacteria in some natural systems. The numbers of H 2-oxidizing diazotrophs were considerably higher in soil surrounding nodules of white beans than they were in the other systems tested. This observation is consistent with reports that the rhizosphere may be an important ecological niche for H 2 transformation. 相似文献
11.
Acetylene reduction, deuterium uptake and hydrogen evolution were followed in in-vivo cultures of Azospirillum brasilense, strain Sp 7, by a direct mass-spectrometric kinetic method. Although oxygen was needed for nitrogenase functioning, the enzyme was inactivated by a fairly low oxygen concentration in the culture and an equilibrium had to be found between the rate of oxygen diffusion and bacterial respiration. A nitrogenase-mediated hydrogen evolution was observed only in the presence of carbon monoxide inhibiting the uptake hydrogenase activity which normally recycles all the hydrogen produced. However, under anaerobic conditions and in the presence of deuterium, a bidirectional hydrogenase activity was observed, consisting in D 2 uptake and in H 2 and HD evolution. In contrast to the nitrogenase-mediated H 2 production, this anaerobic H 2 and HD evolution was insensitive to the presence of acetylene and was partly inhibited by carbon monoxide. It was moreover relatively unaffected by the deuterium partial pressure. These results suggest that the anaerobic H 2 and HD evolution can be ascribed to a reverse hydrogenase activity under conditions where D 2 is saturating the uptake process and scavenging the electron acceptors. Although the activities of both nitrogenase and hydrogenase were thus clearly differentiated, a close relationship was found between their respective functioning conditions. 相似文献
12.
Acetylene reduction assays were shown to inactivate uptake hydrogenase activity to different extents in one Casuarina and two Alnus symbioses. Inactivation was found to be caused by C 2H 2 and not by C 2H 4. Acetylene completely inactivated the hydrogenase activity of intact root systems of Alnus incana inoculated with Frankia strain Avcl1 in 90 minutes, as shown by a drop in the relative efficiency of nitrogenase from 1.0 to 0.73. The hydrogenase of Frankia preparations (containing vesicles) and of cell-free extracts (not containing vesicles) from the same symbiosis was much more susceptible to acetylene inactivation. Cell-free extracts lost all hydrogenase activity after 5 minutes of exposure to acetylene. The hydrogenase activity of intact root systems of Casuarina obesa was less sensitive to acetylene than that of root systems of A. incana, since the relative efficiency of nitrogenase changed only from 1.0 to 0.95 over 90 minutes. Frankia preparations and cell-free extracts of C. obesa still retained hydrogenase activity after a 10 minute-exposure to acetylene. 相似文献
13.
Several strains of Rhizobium resistant to spectinomycin also had nitrogenase activity (C 2H 2 reduction and H 2 production) in static culture under 95% Ar/1%O 2/4%C 2H 2. This relationship between nitrogenase activity and spectinomycin resistance was observed in both fast-growing ( R. trifolii and R. leguminosarum) and slow-growing ( R. japonicum) rhizobia. The effect of different media and various carbon sources on nitrogenase activity was investigated in more detail in R. trifolii strain TlSp. This communication demonstrates that fast-growing rhizobia can have nitrogenase activity in the absence of any plant component. 相似文献
14.
Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H 2 evolution. The uptake hydrogenase was identified in all N 2-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N 2-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N 2-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H 2 production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl
chlorophyll
- MV
methyl viologen 相似文献
15.
Summary Hydrogenase and nitrogenase activities of sulfate-reducing bacteria allow their adaptation to different nutritional habits even under adverse conditions. These exceptional capabilities of adaptation are important factors in the understanding of their predominant role in problems related to anaerobic metal corrosion. Although the D 2–H + exchange reaction indicated that Desulfovibrio desulfuricans strain Berre-Sol and Desulfovibrio gigas hydrogenases were reversible, the predominant activity in vivo was hydrogen uptake. Hydrogen production was restricted to some particular conditions such as sulfate or nitrogen starvation. Under diazotrophic conditions, a transient hydrogen evolution was followed by uptake when dinitrogen was effectively fixed. In contrast, hydrogen evolution proceeded when acetylene was substituted as the nitrogenase substrate. Hydrogen can thus serve as an electron donor in sulfate reduction and nitrogen metabolism. 相似文献
17.
Nitrogenase activity in the Gunnera Nostoc symbiosis is shown to respond dramatically to the addition of glucose. H 2 can replace glucose in stimulating nitrogenase activity, but there is no H 2 stimulation in the presence of excess glucose. Net hydrogen evolution is strongly stimulated by addition of glucose. We postulate that carbohydrate supply and uptake hydrogenase can moderate the apparent activity of nitrogenase by supplying reductant and/or ATP. The recycling of a large proportion of the electron flux in nitrogenase through uptake hydrogenase maintains a high level of potential nitrogenase ready to take advantage of an influx of carbohydrate. 相似文献
18.
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 H 2 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 H 2 or 3H 2 in the presence of acetylene. In all cases, the rate of H 2 evolution was a continuous function of the rate of acetylene reduction. In symbioses with no demonstrable Hup activity, H 2 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 H 2 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 H 2 evolution by Hup + strains were similar in magnitude to their H 2-saturated rates of Hup activity. The curvilinear relationship between H 2 evolution and acetylene reduction for a representative Hup + strain (128C52) was the same, regardless of the experimental conditions used to vary the nitrogenase activity. 相似文献
19.
The ability to recycle H 2 evolved by nitrogenase is thought to be of importance in increasing the efficiency of N 2 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 H 2 and in their relative efficiency of N 2 fixation. These plants were grown at three levels of irradiance and harvested after 3, 4, and 5 weeks of growth for determination of C 2H 2 reduction, H 2 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, H 2 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 H 2 to Hup + nodules whose supply of photosynthate had been removed by stem excision did not increase C 2H 2 reduction in either coupled or uncoupled types. 相似文献
20.
Root respiration associated with symbiotic fixation in soybean ( Glycine max [L.] Merr.) was estimated by four methods. Averaged over the life of the plant, the root respires 5.8 milligrams C per milligram N accumulated from fixation. When nitrogenase (C2H2) activity and root respiration were decreased by treating roots briefly with 1.0 atmosphere O2, the respiration associated with nitrogenase was estimated as 2.10 micromoles CO2 per micromole C2H4. When nitrogenase activity and respiration were decreased by addition of nitrate, the respiration associated with fixation was calculated as 2.90 micromoles CO2 per micromole C2H4. Removing nodules from roots decreased fixation and root respiration, and the ratio was 4.08 micromoles CO2 per micromole C2H4. When soybean plants were kept in prolonged darkness, then returned to light, the associated drop and recovery of respiration and nitrogenase activity had a ratio of 4.36 micromoles CO2 per micromole C2H2. 相似文献
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