Evaluation of effectiveness in mutant strains of rhizobium by acetylene reduction relative to other criteria of N2 fixation

Schwinghamer, E. A.; Evans, Harold J.; Dawson, M. D.

doi:10.1007/bf01378210

Cited by 72 publications

(32 citation statements)

References 20 publications

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“…Nodulation assays were performed as described previously by Balatti et al (2). Acetylene reduction rates were determined by the method of Schwinghamer et al (35).…”

Section: Methodsmentioning

confidence: 99%

A Functional myo -Inositol Dehydrogenase Gene Is Required for Efficient Nitrogen Fixation and Competitiveness of Sinorhizobium fredii USDA191 To Nodulate Soybean ( Glycine max [L.] Merr.)

Jiang¹,

Krishnan²,

Kim

et al. 2001

J Bacteriol

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Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.

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“…Nodulation assays were performed as described previously by Balatti et al (2). Acetylene reduction rates were determined by the method of Schwinghamer et al (35).…”

Section: Methodsmentioning

confidence: 99%

A Functional myo -Inositol Dehydrogenase Gene Is Required for Efficient Nitrogen Fixation and Competitiveness of Sinorhizobium fredii USDA191 To Nodulate Soybean ( Glycine max [L.] Merr.)

Jiang¹,

Krishnan²,

Kim

et al. 2001

J Bacteriol

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“…To analyze the symbiotic proficiency of the S. meliloti mutants, plant tests were carried out as previously described (29). Dry weight measurements of shoots of plants were performed after 4 weeks of plant cultivation (38).…”

Section: Methodsmentioning

confidence: 99%

Stationary-phase mutants of Sinorhizobium meliloti are impaired in stationary-phase survival or in recovery to logarithmic growth

et al. 1997

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A screening method was used to identify Sinorhizobium meliloti mutants which are affected in stationaryphase survival. Of 20,000 individual colonies mutagenized with transposon Tn5-B20, 10 mutant strains which showed poor or no survival in the stationary phase were identified. Analyses of expression patterns of the promoterless lacZ genes in the mutant strains revealed individual induction patterns. Most strains were induced in stationary phase as well as under carbon limitation and in pure H 2 O, but none of the mutants was induced under heat, alkali stress conditions, or low oxygen tension. Plant inoculation tests revealed that the symbiotic proficiency of the mutants was not affected. Two mutants, however, showed gene induction not only in the stationary phase under free-living conditions but also in the bacteroid state. A long-term starvation test was carried out to examine the ability of the 10 mutants to survive prolonged stationary-phase conditions. All mutants showed a clear decrease in the colony-forming ability under the chosen experimental conditions. Staining with green and red fluorescent nucleic acid stain showed that the mutants fell into two different classes. Seven mutants died during stationary phase; the three other mutants remained viable but did not resume growth after prolonged starvation. Five of the ten Tn5-B20 insertions were cloned from the genomes of the mutant strains. Nucleotide sequence analyses established that the transposon had inserted in five distinctive genes. Database searches revealed that four of the tagged loci corresponded to already characterized genes whose gene products are involved in important cellular processes such as amino acid metabolism or aerobic respiration.

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“…Acetylene (C2H2) reduction assays of excised nodules were performed as described previously (23 (24) was alleviated by continuously maintaining a potential of +0.6 V across the electrode. Hydrogen evolution and acetylene reduction assays were conducted on soybeans in situ in Saran bags (25).…”

Section: Introductionmentioning

confidence: 99%

Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts

Schubert

Evans

1976

Proc. Natl. Acad. Sci. U.S.A.

446

276

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Nitrogenase-dependent hydrogen evolution from detached legume nodules and from reaction mixtures containing cell-free nitrogenase has been well established, but the overall effect of hydrogen evolution on the efficiency of nitrogen fixation in vivo has not been critically assessed. This paper describes a survey which revealed that hydrogen evolution is a general phenomenon associated with nitrogen fixation by many nodulated nitrogen-fixing symbionts. An evaluation of the magnitude of energy loss in terms of the efficiency of electron transfer to nitrogen, via nitirogenase, in excised nodules suggested that hydrogen production may severely reduce nitrogen fixation in many legumes where photosynthate supply is a factor limiting fixation. With most symbionts. including soybeans, only 40-60% of the electron fow to nitrogenase was transferred to nitrogen. The remainder was lost through hydrogen evolution. In situ measurements of hydrogen evolution and acetylene reduction by nodulated soybeans confirmed the results obtained with excised nodules. In an atmosphere of air, a major portion of the total electron flux available for the reduction of atmospheric nitrogen by either excised nodules or intact nodulated plants was utilized in the production of hydrogen gas. Some nonleguminous symbionts, such as Alnus rubra, and a few legumes (i.e., Vigns sinensis) apparently have evolved mechanisms of minimizing net hydrogen production, thus increasin their efficiency of electron. transfer to nitrogen. Our res ts indicate that the extent of hydrogen evolution during nitrogen reduction is a major factor affecting the efficiency of nitrogen fixation by many agronomically important legumes. The increasing world population and depletion of fossil fuel supplies have stimulated renewed interest in methods of increasing agricultural productivity while minimizing the consumption of fossil fuels. A major factor limiting agricultural production is nitrogen fertilizer, the synthesis of which consumes major quantities of energy. Approximately 3% (600 X 109 cubic feet; 16.8 X 109 meter3) of the natural gas consumed in the United States in 1973 was used for the synthesis of 17 X 106 U.S. tons (153 X 108 kg) of anhydrous ammonia (1, 2). About 10 X 106 U.S. tons of synthetic ammonia were used for nitrogen fertilizer to supply a portion of an annual agricultural nitrogen demand of about 18 X 106 U.S. tons (1, 2). A large part of the remaining need for nitrogen in agriculture was supplied by nitrogen-fixing organisms, such as legumes, which utilize photosynthetically stored solar energy to reduce atmospheric nitrogen to ammonia. Because the biological nitrogen-fixing process is not dependent upon nonrenewable energy resources, its use in agriculture should be maximized. Factors limiting biological nitrogen fixation therefore deserve thorough investigation (3,4).One characteristic of all cell-free nitrogenase preparations that might limit nitrogen fixation is the release of hydrogen gas concomitant with nitrogen reduction (5-11). During thi...

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Evaluation of effectiveness in mutant strains of rhizobium by acetylene reduction relative to other criteria of N2 fixation

Cited by 72 publications

References 20 publications

A Functional myo -Inositol Dehydrogenase Gene Is Required for Efficient Nitrogen Fixation and Competitiveness of Sinorhizobium fredii USDA191 To Nodulate Soybean ( Glycine max [L.] Merr.)

A Functional myo -Inositol Dehydrogenase Gene Is Required for Efficient Nitrogen Fixation and Competitiveness of Sinorhizobium fredii USDA191 To Nodulate Soybean ( Glycine max [L.] Merr.)

Stationary-phase mutants of Sinorhizobium meliloti are impaired in stationary-phase survival or in recovery to logarithmic growth

Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts

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