Source: WASHINGTON STATE UNIVERSITY submitted to NRP
NUTRIENT EXCHANGE AND METABOLISM IN THE RHIZOBIUM-LEGUME SYMBIOSIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1000967
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Sep 1, 2013
Project End Date
Aug 31, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Inst of Biological Chemistry
Non Technical Summary
Forage legumes like alfalfa are important in state and national agriculture. These legumes acquire at least some of their value from a symbiotic interaction with bacteria, commonly called rhizobia. Because the rhizobia can reduce atmospheric nitrogen to ammonia and transfer this to the plant, legume crops can be grown without applying nitrogen-containing fertilizers, simultaneously avoiding an expensive input and problems associated with using nitrogen fertilizers, such as ground water contamination and soil acidification. Most ideas for sustainable agriculture include legumes as a major component since some of the nitrogen they produce is available to succeeding crops. Our focus is on the metabolism that underlies this symbiosis. Using various approaches grounded in bacterial genetics and physiology, we are attempting to understand what compounds the bacteria gets from its host plant and how use of these compounds is coupled to nitrogen fixation, a very energetically expensive process.
Animal Health Component
10%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031640104050%
2011640104010%
2031640110020%
2031640108010%
2031640102010%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1640 - Alfalfa;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology; 1080 - Genetics; 1020 - Physiology;
Goals / Objectives
The primary objectives are to investigate metabolism in the Medicago- Sinorhizobium (plant-bacterium) nitrogen fixing symbiosis and to identify factors that limit the productivity of the interaction. Sub objectives are to study the involvement of the bacterial nitrogen stress response in symbiotic metabolism, to analyze genetic and metabolic contributions to agronomic performance, to develop genetic materials for analyzing the bacteria, and to develop strategies for increasing the amount of nitrogen available for subsequent crops.
Project Methods
As implied by the descriptions of experiments planned and completed that are contained in the proposal, the laboratory's core expertise is in microbial genetics and physiology and in testing hypotheses about the symbiosis using bacterial mutants that we have constructed for specific purposes. This approach is integrated with a more extensive set of classical and molecular tools, including proteomics and transcriptomics, to try and generate a global description of the important components of the interaction between the symbionts. Examples of this approach, including the analysis of data from specific projects, can be seen in our published, peer-reviewed work. Several of the collaborations described in the proposal are a direct outgrowth of seminars given at meetings and, in addition, we share materials with scientists around the world. Members of the group give presentations at scientific meetings and the PL has also presented versions of the work at the annual meeting of Multistate Project W2147 "Managing Plant Microbe Interactions in Soil to Promote Sustainable Agriculture," for which he is Administrative Adviser, and of the US Dry Pea and Lentil Association. The primary evaluation criteria include publications of the work and funding of some aspects of the work by external funding agencies. In addition to peer-reviewed papers, the PL is coauthor of a chapter on the subject in the upcoming revision of a standard advanced text in plant biochemistry and molecular biology.

Progress 09/01/13 to 08/31/18

Outputs
Target Audience:Stakeholders with interests in alfalfa, sustainable agriculture and plant/microbe interactions. Also nitrogen dynamics in agriculture. Fundamental scientists interested in energy transduction and the relationship of microbial physiology to establishment and maintainence of symbiotic and associative nitrogen-fixing relationships. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students obtained their Ph.D. degrees in 2018 and the laboratory hosted a CAHNRS intern and an NSF REU student. How have the results been disseminated to communities of interest?As publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Studies were carried out examining the effect of protease activity on the quality of nitrogen fixation. These showed that bacteria with mutations in various protease systems were more sensitive to stress than wild-type and that symbiotic performance was impaired by these mutations. Immunoprecipitation experiments indicated that the proteome associated with the HslUV and ClpXP proteases are enriched for proteins involved in pretein synthesis, suggesting that these systems are involved in eliminating poorly folded proteins or proteins that contain incorporation mistakes. In a separate project, the contribution of genes from Sinorhizobium medicae to the symbiosis between Sinorhizobium meliloti and Medicatgo truncatula was examined and it was established that three genes could increase symbiotic yield. The effect of two of these genes appear to be additive. A product of the nitrogen fixing but ineffective symbiosis formed by a mutant in GlnD can be catabolized by free-living bacteria, suggesting that this is normally eliminated in symbiosis.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Ogden AJ, McAleer JM, Kahn ML. 2019. Characterization of the Sinorhizobium meliloti HslUV and ClpXP protease systems in free-living and symbiotic states. J Bacteriol. Jan 22. pii: JB.00498-18. doi: 10.1128/JB.00498-18. [Epub ahead of print] PubMed PMID: 30670545.
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Aaron Joseph Ogden. 2018. Metabolic progression during development of symbiotic nitrogen fixation in the Medicago-Sinorhizobium symbiosis and the role of HslUV and ClpXP Protease machinery. Ph.D. thesis Molecular Plant Sciences. Washington State University.
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Katie Nicole Adolphsen. 2018. Rhizobial Genes that Contribute to Effective Symbiosis with Legumes. Ph.D. thesis Molecular Biosciences. Washington State University.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Stakeholders with interests in nitrogen dynamics in agriculture, especially related to sustainablility. This includes the development and improvement of currently available symbiotic and associative plant-microbe systems that fix nitrogen, especially with legumes. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several graduate and undergraduate students are being trained in biochemistry, microbiology and molecular biology through this and other projects associated with the laboratories involved. How have the results been disseminated to communities of interest?Presentations as talks and posters have been made at several national and international meetings. At the 20th International Congress on Nitrogen Fixation, both Dr. Kahn and Dr. Peters chaired sessions and presented research. Presentations were also made at the Annual meeting of the American Society for Plant Biology, the annual meeting of the American Horticultural Society and the American Chemical Society. What do you plan to do during the next reporting period to accomplish the goals?For the Kahn subgroup, the goal will be to publish information from several graduate student theses and for the work with the nitrogen fixing but ineffective symbiosis then text some of the implications of the work. For the Peters group, the goal will be to explore the implications of new mechanisms of electron transfer and to understand how to integrate some of this into a better understanding of how to make associative nitrogen fixing relationships more productive.

Impacts
What was accomplished under these goals? We have further characterized the unusual metabolite that accumulates in an ineffective nitrogen-fixing alfalfa symbiosis and are exploring its metabolism in both plant and bacteria. A model for this unusual interaction has been developed and is being tested. Proteomics work has shown that several proteins are expressed at higher levels in nodules than in free-living growth. Among these are protease complexes and the significance to this for nodule development is being explored. Work with the Sinorhizobium cold shock RNA binding proteins has shown that these proteins are expressed in a developmentally significant way during nodule development. Immunoprecipitation experiments using CspA-GFP fusion proteins has shown that several sRNA molecules are bound to the CspA proteins and in in vitro tests we have shown that binding to these RNA molecules involves high-affinity interactions including cooperativity and that the protein-RNA interaction can allow folded RNA to escape metastable complexes. We have identified a gene in Sinorhizobium medicae that increases nodulation in S. meliloti and have shown that this gene has significant consequences on gene expression in the latter. The gene is needed for normal nodulation by S. medicae and can stimulate nodulation by Rhizobium leguminosarum on pea and lentil, suggesting that it acts through some general mechanism. In work related to the original objectives of the project, Dr. Peters' group has shown that electron bifurcation using NADH as substrate is a plausible mechanism for generating low potential electrons that can participate in nitrogen fixation. The description of flavoprotein mechanisms that can carry out the needed reactions is a fundamental step toward understanding how general metabolism can contribute to nitrogen fixation. In addition, his group has been exploring the consequences of ammonium export from nitrogen fixing bacteria, with the goal of trying to develop associative nitrogen fixing relationships between diazotrophs and crop plants that will improve nitrogen availability.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Ogden AJ, Gargouri M, Park J, Gang DR, Kahn ML. Integrated analysis of zone-specific protein and metabolite profiles within nitrogen-fixing Medicago truncatula-Sinorhizobium medicae nodules. PLoS One. 2017 Jul 10;12(7):e0180894. doi: 10.1371/journal.pone.0180894.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hagberg KL, Yurgel SN, Mulder M, Kahn ML. Interaction between Nitrogen and Phosphate Stress Responses in Sinorhizobium meliloti. Front Microbiol. 2016 Nov 30;7:1928.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: A. Garcia-Costas, S. Poudel, A-F. Miller, G.J. Schut, R.N. Ledbetter, K. Fixen, L.C. Seefeldt, M.W.W. Adams, C.S. Harwood, E.S. Boyd, J.W. Peters. Defining electron bifurcation in the electron transferring flavoprotein Family J. Bacteriol. doi: 10.1128/JB.00440-17.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: J.H. Artz, O.A. Zadvornyy, D.W. Mulder, P.W. King, J.W. Peters. Structural characterization of poised states in the oxygen sensitive hydrogenases and nitrogenases Meth. in Enzymol. 595:213-259 (2017)
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: B.M. Barney, M.H. Plunkett, V. Natarajan, F. Mus, J.W. Peters. Transcriptional analysis of an ammonium-excreting strain of Azotobacter vinelandii deregulated for nitrogen fixation App. Environ. Microbiol. doi: 10.1128/AEM.01534-17. [Epub ahead of print] (2017)
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: R.N. Ledbetter, A.M. Garcia Costas, C.E. Lubner, D.W. Mulder, M. Tokmina-Lukaszewska, J.H. Artz, A. Patterson, T.S. Magnuson, Z.J.H Jay, D. Duan, J. Miller, M.H. Plunkett, J.P. Hoben, B.M. Barney, R.P. Carlson, A-F. Miller, B. Bothner, P.W. King, J.W. Peters, Peters, L.C. Seefeldt. The electron bifurcating FixABCX protein complex from Azotobacter vinelandii: Generation of low-potential reducing equivalents for nitrogenase catalysis Biochemistry 56:4177-4190 (2017)
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: F. Mus, A. Tseng, R. Dixon, J.W. Peters. Diazotrophic growth allows Azotobacter vinelandii to overcome the deleterious effects of a glnE deletion App. Environ. Micro. 83:13 (2017)


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Stakeholders with interests in alfalfa, sustainable agriculture and plant/microbe interactions. Also nitrogen dynamics in agriculture. Changes/Problems:Under project modifications. At the end of 2016, Dr. John Peters was added to the project to increase its focus on the mechanism of nitrogenase in nitrogen fixation of free-living bacteria and in symbiosis. Dr. Svetlana Yurgel continues to consult with the project from her position at Dalhousie University, including in manuscript preparation and student training. What opportunities for training and professional development has the project provided?The project involves the participation of four graduate students who are investigating various aspects of the symbiosis. One of these should finish her Ph.D. in early 2017; the others plan to finish later in the year. One undergraduate was trained in the RNA binding protein project. How have the results been disseminated to communities of interest?Results have been published in open literature and presented at scientific meetings, including the ""RNA structure meets function" EMBO meeting, June 12-15, 2016, Stockholm, Sweden What do you plan to do during the next reporting period to accomplish the goals?Continue research and prepare considerable unpublished research for publication.

Impacts
What was accomplished under these goals? We have further characterized the unusual metabolite that accumulates in an ineffective nitrogen-fixing alfalfa symbiosis and are exploring its metabolism in both plant and bacteria. Proteomics of the nodules indicates that few bacterial proteins are changed in the interaction, the most notable of which are early steps in the synthesis of a few amino acids. We are not sure how to interpret these results. Work with the cold shock RNA binding proteins has identified several mRNA molecules that are bound to the proteins in vivo, including some that suggest that the proteins are involved in a cascade regulation via control of RNA structure. We have developed a novel assay for RNA-protein interaction and have shown that these proteins can convert RNA structures folded into metastable states into more stable and assay-active structures. We have also identified a gene in Sinorhizobium medicae that increases nodulation in S. meliloti and have shown that this gene has significant consequences on gene expression in the latter. The gene appears to interact with plant mediated nodulation inhibition. RNA-seq data has been gathered to investigate changes that occur as the result of phosphate and nitrogen stress.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hagberg KL, Yurgel SN, Mulder M, Kahn ML. 2016. Interaction between Nitrogen and Phosphate Stress Responses in Sinorhizobium meliloti. Frontiers in Microbiology: Microbial Physiology and Metabolism. doi: 10.3389/fmicb.2016.01928


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Stakeholders with interests in alfalfa, sustainable agriculture and plant/microbe interactions. Also nitrogen dynamics in agriculture. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project involves the participation of four graduate students who are investigating various aspects of the symbiosis. At the beginning of 2015 a senior investigator in the laboratory, who began here as a postdoctoral scientist, left for an Associate Professor position at Dalhousie University in Canada. How have the results been disseminated to communities of interest?The project involves the participation of four graduate students who are investigating various aspects of the symbiosis. At the beginning of 2015 a senior investigator in the laboratory, who began here as a postdoctoral scientist, left for an Associate Professor position at Dalhousie University in Canada. What do you plan to do during the next reporting period to accomplish the goals?Continue research and prepare considerable unpublished research for publication.

Impacts
What was accomplished under these goals? We have further characterized the unusual metabolite that accumulates in an ineffective nitrogen-fixing alfalfa symbiosis and are exploring its metabolism in both plant and bacteria. Work with the cold shock RNA binding proteins has identified several mRNA molecules that are bound to the proteins in vivo, including some that suggest that the proteins are involved in a cascade regulation via control of RNA structure. We have also identified a gene in Sinorhizobium medicae that increases nodulation in S. meliloti and have shown that this gene has significant consequences on gene expression in the latter. A published manuscript from the laboratory contributed to the idea that riboflavin synthesis occurs in multi protein complexes and that one rationale for this may be to channel metabolites and lower the impact of potentially toxic molecules formed during riboflavin biosynthesis.

Publications

  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Humann JL, Kahn ML. 2015. Genes involved in desiccation resistance of rhizobia and other bacteria. Chapter 39 in Biological Nitrogen Fixation F. deBruijn, ed. John Wiley and Sons. pp 397-403
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Long SR, Kahn ML, Seefeldt, L, Tsay, Y-F, Kopriva S 2015 Nitrogen and Sulfur Chapter 16, Biochemistry and Molecular Biology of Plants. Ed: BB Buchanan, W Gruissem, RL Jones. Am Soc Plant Biol., Rockville MD pp. 711-768
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Frelin O, Huang L, Hasnain G, Jeffryes JG, Ziemak MJ, Rocca JR, Wang B, Rice J, Roje S, Yurgel SN, Gregory JF 3rd, Edison AS, Henry CS, de Cr�cy-Lagard V, Hanson AD. A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis. Biochem J. 2015 Feb 15;466(1):137-45. doi: 10.1042/BJ20141237.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Stakeholders with interests in alfalfa, sustainable agriculture and plant/microbe interactions. Also nitrogen dynamics in agriculture. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Four graduate students are being trained in the projects and several undergraduates have participated in the work. How have the results been disseminated to communities of interest? Via presentations at meetings and publication of refereed journal articles. What do you plan to do during the next reporting period to accomplish the goals? We plan to further characterize each of the aspects described above. We hope to isolate mutants defective in the production of the unique metabolite, to refine the localization and pathway organization of metabolism in root nodules using new instrumentation on campus, and to follow the RNA binding protein related processes in nodules. Since the primary investigator on the riboflavin project has taken a job elsewhere, we are uncertain about the future of this subproject.

Impacts
What was accomplished under these goals? A metabolite that is found in much higher levels in symbiotic relationships between alfalfa and a bacterial GlnD mutant was purified earlier and its identity was confirmed by NMR and de novo synthesis. Quantification of the metabolite is in progress as are experiments to determine the origin of its biosynthetic precursors. Experiments to localize metabolites and proteins within developing alfalfa and M. truncatula root nodules are continuing. Principal component analysis of the data shows that the regions under analysis are distinct and the determinants are being used to generate a pattern of how metabolism shifts during nodule development. Sinorhizobial RNA binding proteins have been isolated and their binding to bacterial RNA molecules have been examined. These are highly expressed in nodules and may be involved in regulating the differentiation of bacteria into bacteroids as the capacity for nitrogen fixation develops. Riboflavin biosynthesis by rhizobia has been studied and the unusual aspects of the initial reactions in S. meliloti are being examined. The work has led to new insights about the control of riboflavin and the operation of the riboflavin biosynthetic complex.

Publications

  • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: Humann JL, Kahn ML. 2014. Genes involved in desiccation resistance of rhizobia and other bacteria. in Biological Nitrogen Fixation F. deBruijn, ed. John Wiley and Sons. (invited review, in press)
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2014 Citation: Long SR, Kahn ML, Seefeldt, L, Tsay, Y-F, Kopriva S 2014 Nitrogen and Sulfur Chapter 16, Biochemistry and Molecular Biology of Plants. Ed: BB Buchanan, W Gruissem, RL Jones. Am Soc Plant Biol., Rockville MD (in press)
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Frelin O, Huang L, Hasnain G, Jeffryes JG, Ziemak MJ, Rocca JR, Wang B, RiceJ, Roje S, Yurgel SN, Gregory JF, Edison AS, Henry CS, de Cr�cy-Lagard V, Hanson AD. A directed-overflow and damage-control N-glycosidase in riboflavin biosynthesis. Biochem J. 2015 Feb 15;466(1):137-45.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Yurgel SN, Rice J, Domreis E, Lynch J, Sa N, Qamar Z, Rajamani S, Gao M, Roje S, Bauer WD. Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of the bifunctional RibBA protein. Mol Plant Microbe Interact. 2014 May;27(5):437-45.


Progress 09/01/13 to 09/30/13

Outputs
Target Audience: Sustainable agriculture, scientists interested in plant-microbe interactions, microbial geneticists, and physiologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Four graduate students and a postdoctoral scholar are currently being trained in the laboratory, working on projects related to this one. In addition, the laboratory is working with several undergraduates who are helping with the work. How have the results been disseminated to communities of interest? At this time, the conference papers listed are the results disseminated. What do you plan to do during the next reporting period to accomplish the goals? We will be working theObjectives and subobjectivesas described: 1.Investigate metabolism in the Medicago- Sinorhizobium (plant-bacterium) nitrogen fixing symbiosis and to identify factors that limit the productivity of the interaction. a. study the involvement of the bacterial nitrogen stress response in symbiotic metabolism b.analyze genetic and metabolic contributions to agronomic performance c. develop genetic materials for analyzing the bacteria, and to d.develop strategies for increasing the amount of nitrogen available for subsequent crops.

Impacts
What was accomplished under these goals? The project has been active for one month. No progress to report at this time.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Yurgel SN, Qu Y, Rice JT, Lipton M, and Kahn ML 2013 Global proteome analysis of Sinorhizobium medicaeMedicago truncatula Jemalong A17 symbiosis: new approach and new insights. Abstract 22nd North American Symbiotic Nitrogen Fixation meeting, Minneapolis, July, 2013
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Hagberg K, Yurgel SN, Rice JT, Mulder M, and Kahn ML. 2013. The analysis of cross-talk between nitrogen and phosphate stress responses in Sinorhizobium meliloti. Abstract 22nd North American Symbiotic Nitrogen Fixation meeting, Minneapolis, July, 2013