Both mutant strains nodulated soybean plants and fixed nitrogen at rates comparable to that of the wild-type strain. Carbon dioxide is an important metabolite in ...
Vol. 48, No. 2
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1984, p. 276-279
0099-2240/84/080276-04$02.00/0 Copyright © 1984, American Society for Microbiology
Nitrogen Fixation and Carbon Dioxide Assimilation in Rhizobium japonicum SUNDARAM S. MANIAN, ROBERT GUMBLETON, ANNE M. BUCKLEY, AND FERGAL O'GARA* Department of Microbiology, University College, Cork, Ireland Received 13 February 1984/Accepted 15 May 1984
In free-living Rhizobium japonicum cultures, the stimulatory effect of CO2 on nitrogenase (acetylene reduction) activity was mediated through ribulose bisphosphate carboxylase activity. Two mutant strains (CJ5 and CJ6) of R. japonicum defective in CO2 fixation were isolated by mitomycin C treatment. No ribulose bisphosphate carboxylase activity could be detected in strain CJ6, but a low level of enzyme activity was present in strain CJ5. Mutant strain CJ5 also exhibited pleiotropic effects on carbon metabolism. The mutant strains possessed reduced levels of hydrogen uptake, formate dehydrogenase, and phosphoribulokinase activities, which indicated a regulatory relationship between these enzymes. The C02-dependent stimulation of nitrogenase activity was not observed in the mutant strains. Both mutant strains nodulated soybean plants and fixed nitrogen at rates comparable to that of the wild-type strain. MATERIALS AND METHODS
Carbon dioxide is an important metabolite in many bacterial species, and it can be assimilated either autotrophically or heterotrophically. In the genus Rhizobium, CO2 is required to promote vegetative growth (8), and CO2 assimilation has been implicated in the regulation of important cellular processes such as hydrogen metabolism and nitrogen fixation (1, 11, 16). A stimulatory effect of CO2 on hydrogen uptake (Hup) activity has been reported and is thought to be mediated via a physiological effect on cellular metabolic processes rather than a specific effect on hydrogenase formation (11). The Hup system plays an important physiological role in nodule metabolism by recycling H2 and thereby recovering a part of the potential energy that is lost as a result of nitrogenase-catalyzed ATP-dependent H2 evolution (3). As the Hup system does not recycle the total amount of H2 evoJved in all Rhizobium-legume asssociations, an understanding of the factors that stimulate Hup activity is a prerequisite for enhancing symbiotic N2-fixation. Similarly, although it is known that CO2 is required for the expression or stimulation or both of nonsymbiotic nitrogenase activity under certain growth conditions (1, 16), the mechanism of this action is not clearly understood. The key autotrophic C02-fixing enzyme ribulose bisphosphate (RuBP) carboxylase does not appear to be present in all Rhizobium species. It has been detected in strains of Rhizobium japonicum (6, 14, 19) and Rhizobium meliloti (13). Although R. japonicum mutant strains defective in RuBP carboxylase activity have been reported (10), the physiological role and regulation of RuBP carboxylase in these agronomically important root nodule bacteria is not clear, and attempts to detect RuBP carboxylase in soybean bacteroids have been unsuccessful (18, 19). The present study was undertaken to learn the role of RuBP carboxylasedependent CO2 assimilation in nitrogen fixation in R. japonicum by using mutant strains unable to fix carbon dioxide (Cfx-). We found that in free-living R. japonicum cultures, the stimulation of nitrogenase activity by CO2 was mediated through RuBP carboxylase activity. The implications of this finding in attempting to increase the symbiotic performance of rhizobial inoculants are also discussed. *
Strains. R. japonicum CJ1 was isolated as described previously (14). R. japonicum CJ5 and CJ6 were mutants defective in RuBP carboxylase activity that were isolated in this study. Aerobic growth. The composition of the mineral salts medium used was described previously (15). All carbon sources were used at a final concentration of 0.1% (wt/vol), except formate, which was used at a final concentration of 0.15% (wt/vol). Cells from fully grown aerobic cultures in MSY medium (7) were centrifuged at room temperature, suspended in carbon- and nitrogen-free medium, and used to inoculate (2%) the growth medium. Cultures were incubated at 30°C with gentle rotation, and growth was followed by measuring the absorbance at 420 nm and also by determining the increase in cell protein. Microaerobic growth. The mineral salts medium used for microaerobic growth lacked ammonium sulfate. For malate growth conditions, the medium contained malate (0.4% [wt/ vol]) and glutamate (0.1% [wt/vol]). These two substrates were replaced by glutamate at 0.5% (wt/vol) for glutamate growth conditions. Cells from 40-h-old aerobic cultures in MSY medium were centrifuged at room temperature, suspended in carbon- and nitrogen-free medium, and used to inoculate (5%) 2 ml of medium in a 71-ml serum bottle essentially as described before (7). The serum bottles were then sealed, and the air within them was replaced by argon. The gas phase was then adjusted to 3% H2 or 1% CO2 or both as required. The gas phase also contained 1% acetylene for detection of nitrogenase activity. These cultures were incubated at 30°C with gentle rotation. After 48 h of incubation, 70 FLl of 02 was added to each bottle, at approximately 24-h intervals, to achieve a final PO2 of ca. 0.76 mmHg (101.31 Pa) as previously described (7). Samples of the gas phase (50 ,ul) were removed at different time periods for the determination of the rate of hydrogen consumption or ethylene formation. Data presented are the average of at least five replicate cultures. Mutagenesis and isolation of mutant strains. The mutagen used was mitomycin C (25 pLg/ml; obtained from Sigma London Chemical Company, Poole, Dorset, England), and the mutagenesis procedure used was described previously
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(12). Clones that could not grow autotrophically on the mineral medium and were white on formate-tetrazolium plates (12) were selected. These clones were unable to grow in formate minimal medium. Two clones with reversion frequencies of approximately 10-9 (CJ5 and CJ6) were selected and used in this study. Whole cell assays. Hydrogen uptake activity in whole cells was determined by following the uptake of hydrogen gas, using gas chromatography (Perkin-Elmer model Sigma 3 equipped with a thermal conductivity detector and a molecular sieve column [no. 5A] with N2 as the carrier gas) as described previously (15). Maximum hydrogenase activity was detected 80 to 100 h after inoculation. Nitrogenase activity was assayed by the acetylene reduction procedure, using gas chromatography (Perkin-Elmer model Sigma 3 equipped with a flame ionization detector and Porapak N [100-200 mesh] column) as described before (12). Maximum nitrogenase activity was detected 130 to 150 h after inoculation. Whole cell CO- uptake was determined by adding 25 ,ul of NaH14CO3 (87.5 nmol; specific activity, 57 mCi/mmol; obtained from Amersham International PLC, Amersham, Buckinghamshire, England) to the growth medium (2 ml) just before inoculation of the medium and then assaying portions (50 ,ul) of the medium removed at regular intervals for incorporation of radioactivity into acid-stable product. Maximum CO2 uptake was detected 60 to 80 h after inoculation. Enzyme assays in cell-free extracts. Cell-free extracts were prepared by sonication as described previously (14). Formate dehydrogenase was assayed spectrophotometrically by following the formate-dependent reduction of NAD+ at 340 nm as described by Johnson et al. (4). RuBP carboxylase was assayed radiometrically by following the RuBP-dependent incorporation of 14Co0 (as NaH14CO3) into acid-stable product as described by Wishnick and Lane (20). Phosphoribulokinase was assayed radiometrically in a coupled reaction with excess RuBP carboxylase. The ribulose-5-phosphate-dependent incorporation of 14C02 (as NaHt4CO3) into acid-stable product was followed as described by Leadbeater et al. (5). RuBP carboxylase from spinach was obtained from Sigma (London). At least three replicate cultures were set up for all enzyme assays. Soluble protein was estimated by the procedure of Lowry et al. (9), and whole cell protein was estimated by the method of Drews (2). Immunodiffusion experiments. The presence of RuBP carboxylase antigen in cell extracts was determined by doublediffusion experiments as described by Ouchterlony (17). R. japonicum RuBP carboxylase antibody (18) was a gift from H. J. Evans, Laboratory for Nitrogen Fixation Research, Oregon State University, Corvallis. Plant nodulation tests. R. japonicum CJ1 and CJ6 were tested for their symbiotic properties essentially as described
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previously (12). Nodule nitrogenase assays was performed 6-week-old plants, and plant dry weight and total plant nitrogen tests were performed on 10-week-old plants as described previously (12). At least eight replicate cultures of each strain were used for plant tests. Bacteria were reisolated from nodules essentially as described previously (16) and tested for their appropriate genetic markers.
on
RESULTS RuBP carboxylase-mediated stimulation of nitrogenase activity. Cultures of R. japonicum CJ1 grown microaerobically on glutamate did not induce any nitrogenase (acetylene reduction) activity (Table 1). Significant levels of nitrogenase activity were induced when H2 alone or both H2 and CO, were added to the gas phase. The C02-dependent stimulation of nitrogenase activity occurred only in the presence of H2 and also required the induction of RuBP carboxylase activity. The levels of pyruvate carboxylase, an anapleurotic CO2-fixing enzyme, were not significantly altered by the presence or absence of H2 and CO2 in the gas phase (data not shown). The level of RuBP carboxylase activity decreased from 10.5 to 1.5 nmol/min per mg of protein when malate replaced glutamate as the carbon source. This indicated that RuBP carboxylase activity was under catabolite repression control during nonsymbiotic N2-fixing conditions. C02, either alone or in combination with H2, had no stimulatory effect on the nitrogenase activity (ca. 20 nmol/h per mg of protein) of
malate-grown cultures. This lack of stimulation of nitrogenase activity by CO2 was probably due to the low level of RuBP carboxylase activity observed under these conditions. These results clearly show that though H2-dependent RuBP carboxylase-mediated CO2 uptake has the potential to support nitrogenase activity, the RuBP carboxylase-mediated stimulation of nitrogenase activity is dependent on the nature of the carbon source(s) present in the medium. Isolation and characterization of Cfx mutants of R. japonicum. The nature of the C02-dependent stimulation of nitrogenase activity was further studied by isolating and characterizing two R. japonicum mutant strains defective in RuBP carboxylase-dependent CO2 fixation. These strains (CJ5 and CJ6) were isolated as mutants unable to grow autotrophically on minimal medium plates or in formate minimal medium. The two mutant strains differed in their ability to utilize other carbon sources. The specific growth rate of strain CJ6 was not significantly different from that of the wild-type strain CJ1 in minimal media containing a variety of different carbon sources (Table 2), indicating that this mutant strain was not defective in ammonia assimilation enzymes or other central pathways of carbon metabolism. The specific growth rate of strain CJ5 on the different carbon sources was significantly reduced, indicating that the mutation(s) in the strain exerted pleiotropic effects. Biochemical characterization of the mutant strains showed that strain CJ6 did not possess any
TABLE 1. Relationship between RuBP carboxylase, Hup, and nitrogenase activities in free-living R. japonicurn cultures" Addition(s) to phase
None CO, H2 H, + CO2
gas
Nitrogenase activity (nmol/h per mg of
protein) < 1.0
