Progress 09/15/01 to 09/30/05
Outputs
The alanine excretion model implies the generation of pyruvate in the plant portion of the nodule after transamination to form glutamate for ureide biosynthesis. Pyruvate metabolism in the plant cytoplasm was examined and there was no evidence for a metabolic pathway that would recycle pyruvate to malate. However, it found that bacteroids could take up pyruvate and support alanine excretion. In the presence of malate plus pyruvate, alanine excretion doubled relative to malate alone. This suggested a revision of the original alanine excretion model with which the pyruvate produced in the plant after transamination could be directly returned to the bacteroid for further metabolism. Acetaldehyde and ethanol are two possible metabolic products of pyruvate metabolism. Exogenous acetaldehyde did not support alanine excretion but exogenous ethanol permitted alaine excretion to about 40% of that of malate alone. These results imply that bacteroids may receive multiple carbon
substrates during symbiosis and that alalnine remains a primary nitrogen excretion product.
Impacts
The reduction of atmospheric dinitrogen into ammonium is an energy intensive process. Given the energy intensive nature of the catalytic process, it would seem intuitively obvious that the transport of the reduced nitrogen would be facile and efficient. Alanine provides a convenient vehicle by which to transport nitrogen from the bacteria to the plant. Determining the metabolic events of alanine excretion is necessary if the nitrogen fixation process is ever to be expanded to crop plants not able to do so at present.
Publications
- N.W. Oehrle, R. Shah, B. Gentry, and D. W. Emerich 2005.Rapid, multiphasic attachment of Bradyrhizobium japonicum soybean roots. Symbiosis 39: 21-26
|
Progress 01/01/04 to 12/31/04
Outputs
Alanine was found to be excreted by isolated soybean nodule bacteroids under microaerophilic conditions in preference to ammonia. To determine the metabolic significance of alanine excretion, an alanine dehydrogenase was isolated and purified from soybean nodule bacteroids. Kinetic analysis demonstrated it to strongly favor alanine synthesis as opposed to alanine breakdown. To determine whether alanine excretion performed a metabolic role in symbiosis, mutants in alanine dehydrogenase were created. A gene located in the symbiotic island of the B. japonicum chromosome believed to be alanine dehydrogenase was isolated and interrupted with a kanamycin cassette. This mutant had no effect on symbiosis; the bacteroids still reduced atmospheric nitrogen, excreted alanine and had normal levels of alanine dehydrogenase. A second putative alanine dehydrogenase (ALADH-2) was found and this gene was interrupted. This mutant also had no effect on symbiosis; the bacteroids still
reduced atmospheric nitrogen, excreted alanine and had near normal levels of alanine dehydrogenase. Attempts to identify additional alanine dehydrogenases by genome searching and genetic screens of random mutant populations are ongoing. The effect of exogenous carbon substrates was determined. Pyruvate alone was able to support alanine excreted but only to about 30% of that of succinate or malate. Pyruvate plus succinate or malate increased alanine excretion by 50% over that of succinate or malate alone. To determine the metabolic origin of alanine, 13C-labeled metabolites were supplied to bacteroids under conditions in which they excrete alanine. The metabolites were: 13C-succinate alone, 13C-pyurvate alone, 13C-succinate plus 12C-pyruvate and 13C-pyurvate plus 12C-succinate. Exudates and cellular metabolites were collected from bacteroids incubated in each set of metabolites. Samples were analyzed both by 13C-Nulcear Magnetic Resonance and HPLC. Both the intracellular and
extracellular samples indicated that the citric acid cycle was not operating in bacteroids under conditions in which alanine was being excreted. Additional analysis of the metabolites is ongoing.
Impacts
Nitrogen is the common limiting nutrient in agriculture with the occasional exception of water. The transport of fixed nitrogen from the bacteroid to the plant is not known. If it is transported by transport proteins, these proteins can be upregulated to enhance nitrogen transport to the plant seed.
Publications
- N.W. Oehrle, L.S. Green, D.B. Karr and D.W. Emerich. 2004. The HFC/HCFC breakdown product of trifluoroacetic acid (TFA) and its effects on the symbiosis between Bradyrhizobium japonicum and soybean (Glycine max). Soil Biol. Biochem. 36:333-342.
|
Progress 01/01/03 to 12/31/03
Outputs
Alanine was found to be excreted by isolated soybean nodule bacteroids under microaerophilic conditions in preference to ammonia. To determine the metabolic significance of alanine excretion, an alanine dehydrogenase was isolated and purified from soybean nodule bacteroids. Kinetic analysis demonstrated it to strongly favor alanine synthesis as opposed to alanine breakdown. To determine whether alanine excretion performed a metabolic role in symbiosis, mutants in alanine dehydrogenase were created. A gene located in the symbiotic island of the B. japonicum chromosome believed to be alanine dehydrogenase was isolated and interrupted with a kanamycin cassette. This mutant had no effect on symbiosis, the bacteroids still reduced atmospheric nitrogen, excreted alanine and had normal levels of alanine dehydrogenase. A second putative alanine dehydrogenase (ALADH-2) was found and this gene was interrupted. Many attempts, utilizing various positional interruptions, failed to
isolate a mutant in ALADH-2. Several isolates were recently obtained in which the entire open reading frame and promoter region was removed and replaced with a streptomycin cassette. These isolates display a severely retarded growth rate on all media that have been tried. Sufficient genomic DNA has been isolated and analysis is currently being conducted to verify the mutation.
Impacts
Nitrogen is the common limiting nutrient in agriculture with the occasional exception of water. The transport of fixed nitrogen from the bacteroid to the plant is not known. If it is transported by transport proteins, these proteins can be upregulated to enhance nitrogen transport to the plant seed.
Publications
- L.S. Green, J.K. Waters, S. Ko, and D.W. Emerich. 2003. Comparative analysis of the Bradyrhizobium japonicum sucA region. Can. J. Microbiol. 49:237-243.
- D.B. Karr, N.W. Oehrle and D.W. Emerich. 2003. Recovery of nitrogenase from aerobically isolated soybean nodule bacteroids. Plant and Soil. 257:27-33.
- N.W. Oehrle, L.S. Green, D.B. Karr and D.W. Emerich. 2004. The HFC/HCFC breakdown product of trifluoroacetic acid (TFA) and its effects on the symbiosis between Bradyrhizobium japonicum and soybean (Glycine max). Soil Biol. Biochem. 36:333-342.
|
Progress 01/01/02 to 12/31/02
Outputs
The underlying hypothesis of this project is that soybean nodule bacteroids excrete alanine as their primary nitrogen product of symbiotic nitrogen fixation. To examine this hypothesis, two primary avenues of investigation are being pursued. The first is the creation of an alanine dehydrogenase-null mutant of B. japonicum. Since the publication of the B. japonicum genome we have been able to obtain the gene via PCR and are constructing the null-mutant. The second is the characterization of pyruvate, orthophosphate dikinase (PPDK) found in the uninfected cells of soybean nodules. which may participate in an alanine-malate cycle similar to that of C-4 photosynthesis. Soybean nodule PPDK has been partially purified and through the use of anti-PPDK antibody from maize, the enzyme has been cytolocalized to the uninfected cells of the nodule.
Impacts
The nitrogen exchange process in soybean nodules, which provides the plant with nitrogen is a key step that reduces the plant's requirement for fertilizer nitrogen. Determining the steps of this pathway will help clarify future research efforts to increase the ability of the soybean to provide its own nitrogen requirement.
Publications
- No publications reported this period
|
Progress 01/01/01 to 12/31/01
Outputs
The overall objective of this proposal is to determine the role of alanine in the soybean-B. japonicum symbiosis. The initial experimental procedure was to generate a mutant of B. japonicum deficient in alanine dehydrogenase and to determine its symbiotic phenotype. A second alanine dehydrogenase was found in the genome of B. japonicum. The two enzymes are highly similar in their N-terminal ends but are quite different in the remainder of their sequence. Each enzyme may have a unique physiological role in this dimorphic organism. The second alanine dehydrogenase is being isolated via the polymerase chain reaction and will be genetically inactivated by insertion of a kanamycin resistance cassette.
Impacts
The presence of two alanine dehydrogenases suggests that each of the two enzymes performs a different metabolic role. Genetic inactivation will determine the role of each. If alanine is a primary transport compound from the bacteroid to the plant, then the process can be genetically modified to improve the nitrogen fixation capacity of the symbiosis and enhance plant productivity.
Publications
- No publications reported this period
|
|