Progress 03/01/11 to 02/28/15
Outputs
Target Audience:Academic colleagues and graduate students through departmental seminars and invited presentations. Peers at national and international scientific meetings. Invited lectures 2015 Society for Industrial Microbiology and Biotechnology, Philadelphia 2015 Battelle International Bioremediation Symposium, Miami 2015 Institute of Microbiology, Chinese Academy of Sciences, Beijing 2015 Peking University, College of Urban and Environmental Sciences 2015 Tsinghua University, College of Hydraulic and Hydroelectric Engineering 2015 University of Science and Technology, Beijing, Department of Environmental Engineering 2014 Nanjing Agricultural University, Department of Microbiology 2014 Nanjing University, School of the Environment 2014 Shanghai Jiao Tong University, School of Life Science and Biotechnology 2014 Xi' an Jiao Tong University, School of Energy and Power Engineering 2014 Division Lecture, American Society for Microbiology Annual Meeting, Boston 2013 University of Sydney, Department of Molecular Biology 2013 Rice University, Department of Civil and Environmental Engineering 2013 University of West Florida, Department of Biology Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities for graduate students and post doctoral researchers to attain higher levels of understanding and proficiency in biochemistry, microbial ecology, bioinformatics and molecular biology. Students wrote papers for publication, presented results at international meetings and collaborated with colleagues at a variety of institutions. How have the results been disseminated to communities of interest?Because the research is basic, dissemination has been primarily through publications and presentations in scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Allelopathy is the suppression of competing species through the release of chemical compounds. The persistence, availability and activity of allelochemicals is influenced by a range of biotic and abiotic factors, but the microbes responsible for destruction of the molecules and their ecological roles are largely unknown. If bacteria in the rhizosphere can biodegrade allelochemicals readily it would be unlikely that such compounds are important mediators of plant-plant competition or of resistance to fungal pathogens. We investigated three allelopathic compounds with disparate sources, structures, and activities: m-tyrosine in red fescue, resveratrol in peanuts, and 3-nitropropanol in crown vetch. The goal was to determine whether bacteria in the rhizosphere were capable of biodegradang the compounds and if so, to determine the degradation mechanisms and shed light on the ecological roles of bacteria in plant-microbe and plant-plant interactions. The work answered basic scientific questions as well as providing insight on practical problems of agricultural importance such as plant invasiveness, plant health, and nutritional status. Preliminary experiments revealed a large and diverse community of bacteria able to use the above allelopathic compounds as growth substrates. The results indicated that such bacteria were likely to be opportunistically degrading the chemicals rather than participating in highly specific interactions with the plants. We established the biodegradation mechanisms of the key chemicals, identified the genes from the genomes and provided information about the assembly and evolution of the pathways. We extended the approach to discover bacteria in progressively lower numbers growing on derivatives of resveratrol including pterostilbene and arachidin-3. Juglone is the major allelopathic chemical released by black walnut. Experiments with samples from the rhizosphere of black walnut revealed a large population of juglone degrading bacteria consistent with the above observations in peanuts. Bacteria able to degrade some, but not all of the juglone derivatives made by black walnut were also readily isolated and identified. The approach was extended to several other plants and allelopathic chemicals and the results consistently revealed robust populations of bacteria able to degrade the chemicals in the rhizosphere. A major conclusion was that the best known and simplest allelopathic chemicals are unlikely to be effective in the rhizosphere. Instead, a variety of derivatives of the basic compounds that seem more resistant to biodegradation are produced by the plants. Our working hypothesis is that the diversity of allelopathic chemicals and corresponding diversity of bacteria that degrade them is evidence of an ongoing evolutionary arms race. Simple allelopathic compoundscan lose their effectiveness due to development of resistance mechanisms in the target organisms. Our results indicate that evolution of biodegradation capabilities by soil bacteria would also serve as a strong selection for evolution of novel allelopathic chemicals. If the allelopathic chemicals play a major ecological role, the bacteria that degrade them must have a similarly important role. If bacteria can be encouraged to interfere with the allelopathic capabilities of invasive plants their invasiveness might be reduced. Similarly, if bacteria that degrade allelopathic chemicals of crop plants can be discouraged the plants might be less susceptible to competition from weeds and infection by pathogens. Specific accomplishments: Objective 1: Diverse bacteria were isolated and characterized from rhizospheres of red fescue, peanuts, black walnut and vetch plants with the ability to grow on m-tyrosine, resveratrol, arachidin-3, pterostilbene, juglone and 3-nitropropanol. Genome sequences of the key isolates were obtained and the details of the catabolic pathways and identities of the genes that encode the enzymes were established. 1a) The catabolic pathways for resveratrol, pterostilbene, and arachidin-3. are all initiated by carotenoid cleaving monooxygenases (CCO) that seem to be widely distributed among the rhizosphere community. Isolates able to grow on arachidin-3 were able to assimilate only the non-prenylated ring. Similarly, pterostilbene degrading bacteria were not able to assimilate the methyl substituted ring. 1b) Genomes of resveratrol, pterostilbene, arachidin-3 and m-tyrosine degraders were sequenced and annotated. 1c) Candidate genes for key enzymes were expressed in E. coli to determine their roles. The CCO from resveratrol degraders has little activity with pterostilbene or arachidin-3 which suggests that prenylation of the molecule reduces the biodegradation of the allelopathic chemical. Objective 2: Preliminary experiments did not reveal effects of allelopathic compounds on bacteria other than induction of the catabolic pathways. The large and diverse array of bacteriathat degrade the allelopathic chemicals argues against highly specific symbiotic relationships so this area of inquiry was deemphasized. 2a). Transcriptomics assays revealed the upregulation of the key enzymes involved in m-tyrosine, resveratrol and pterostilbene biodegradation. The results provide unequivocal evidence for the participation of the genes. 2b). Juglans nigra (black walnut) was added to the list of plants to be examined. Extensive and diverse populations of juglone degrading bacteria were found in the rhizosphere of Juglans nigra. Homologs of juglone produced by the plants are substantially more resistant to biodegradation and likely are responsible for the allelopathic effects. Objective 3. The catabolic pathways are distributed widely among a surprising number of genera. The phylogenies indicate that the CCO genes are all related. 3a. Culture based comparison of the microbial communities in the rhizosphere of allelopathic plants revealed substantial enrichment of the bacteria able to degrade the allelochemicals in the rhizospheres of peanut plants compared to communities in nearby unplanted soil. Similar experiments with m-tyrosine and juglone in the rhizospheres of red fescue and black walnut indicated that the principles can be extrapolated to other systems. 3b. The phylogeny of the CCO enzymes indicate that the enzymes are more closely related to lignostilbene dioxygenases than to resveratrol cleavage enzymes from fungi where they probably serve as a resistance mechanism. The results provide the first rigorous evidence of the physiological role of the enzymes that cleave resveratrol in bacteria and indicate that the homologs that cleave pterostilbene and arachidin-3 are closely related. A key discovery is that the enzyme from resveratrol degrading isolates has much lower activity toward pterostilbene and arachidin 3. The findings suggest strongly that the addition of prenyl and methyl groups limits the biodegradation of the resveratrol homologs.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Vercammen, K., Q. Wei, D. Charlier, A. D�tsch, S. Haussler, S. Schulz, F. Salvi, G. Gadda, J.C Spain, M. L. Rybtke, T. Tolker-Nielsen, J. Dingemans, L. Ye, and P.Cornelis. 2015. Pseudomonas aeruginosa LysR PA4203 regulator NmoR acts as a repressor of the PA4202 nmoA gene encoding a nitronate monooxygenase. J. Bacteriol. 197:1026-1039.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Francis, K., S. Nishino, J.C. Spain and G. Gadda. 2012. A novel activity for fungal nitronate monooxygenase: detoxification of the metabolic inhibitor propionate-3-nitronate. Arch. Biochem. Biophys. 521:84-89.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Salvi, F., J. Agniswamy, H. Yuan, K. Vercammen, R. Pelicaen, P. Cornelius, J.C. Spain, I.T. Weber, and G. Gadda. 2014. The combined structural and kinetic characterization of a bacterial nitronate monooxygenase from Pseudomonas aeruginosa PAO1 establishes NMO class I and II. J. Biol. Chem. 289(34):23764-75.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Han, JI, JC Spain, JR Leadbetter, G. Ovchinnikova, LA Goodwin, CS Han, T Woyke, KW Davenport and P. Orwin. 2013. Genome of the root-associated plant growth-promoting bacterium Variovorax paradoxus strain EPS. Genome Announcements. 1:e00843-13.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Kurt, Z., M. Minoia, and J.C. Spain. 2015. Resveratrol as a growth substrate for bacteria from the rhizosphere. Environ. Microbiol. Submitted.
- Type:
Journal Articles
Status:
Other
Year Published:
2015
Citation:
Yu, R-Q., Z. Kurt, F. Hei and J.C. Spain. 2015. Pterostilbene degradation pathways in Sphingobium isolated from the rhizosphere. Appl. Environ. Microbiol. In preparation.
- Type:
Journal Articles
Status:
Other
Year Published:
2015
Citation:
Kurt, Z., C.A. Gulvik, E. Lang, S. Craven, M. Minoa, S.F. Nishino, and J.C. Spain. 2015. Biochemical and genetic determination of the meta-tyrosine biodegradation pathway in bacteria from the rhizosphere. Appl. Environ. Microbiol. In preparation.
- Type:
Journal Articles
Status:
Other
Year Published:
2015
Citation:
Fida, T.T., F. Medina-Bolivar, T. Yang and J.C. Spain. 2015. Biodegradation of arachidin-3 by peanut rhizosphere isolate Massilia sp. JS1662. Appl. Environ. Microbiol. In preparation.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Kurt, Z., M. Minoa, and J.C. Spain. 2013. Resveratrol biodegradation by Acinetobacter. 113th General Meeting of the American Society for Microbiology
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Smitherman, C., K. Francis, S. Nishino, JC Spain, and G. Gadda. 2011. Detoxification of a lethal toxin by nitronate monooxygenase. International Symposium on Flavins and Flavoproteins, Berkeley, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Gadda, G., K. Francis, S. Nishino, and JC Spain. 2011. Propionate-3-nitronate oxidation by fungal nitronate monooxygenase: a 60- year mystery solved. Gordon Research Conference on Enzymes, Coenzymes and Metabolic Pathways.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Spain, J.C. 2015. Evolution of metabolic diversity. Society for Industrial Microbiology and Biotechnology Annual Meeting. Philadelphia
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Francis, K., C. Smitherman, S. Nishino, J.C. Spain and G. Gadda. 2013. The biochemistry of the metabolic poison propionate 3-nitronate and its conjugate acid, 3-nitropropionate. IUBMB Life. 65:759-768.
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Progress 03/01/13 to 02/28/14
Outputs
Target Audience: Academic colleagues through: departmental seminar at Rice University; Undergraduate and graduate students through special projects in the lab; Presentation and extended discussions at USDA Peanut Lab in Dawson GA; presentations and extended discussions at CSIRO Ecosystem Sciences/Australia National University, Canberra, Australia Changes/Problems: Post doc got a good offer and left unexpectedly. Delays in recruiting a new post doc led to delays in expending funds during CY 2013. We request a no cost extension until 31 Dec. 2014. What opportunities for training and professional development has the project provided? One undergraduate student conducted laboratory studies on the elimination of allelopathic effects by specific bacteria isolated for the ability to degrade the chemicals. The student gained considerable insight about the scientific method and laboratory research. His results have provided the basis for current studies by one of the senior investigators on the elimination of m-Tyrosine toxicity by bacteria. Two post docs have participated and gained mastery of techniques for isolation of key bacteria from the rhizosphere and for determining catabolic pathways. How have the results been disseminated to communities of interest? Spain spent three months at the Ecosystem Sciences Division of the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia's national science agency where he established a collaboration with scientists on the impact of allelopathy in the success of invasive species. The ongoing project leverages the work at Georgia Tech on the three allelopathic chemicals above and expands the approach to several additional systems including invasives. Seminars and extended discussions at CSIRO served to engage the interest of a wider audience including plant taxonomists, biochemists, bioinformaticists, and weed specialists. What do you plan to do during the next reporting period to accomplish the goals? Additional genes described above will be expressed in E. coli and the remaining catabolic pathways will be established rigorously. Biodegradation of pterostilbene and other arachidin-3 will be explored more extensively because they seem to be more important in the allelopathic responses of peanuts and grapes. The collaborations with Fabricio Medina Bolivar at the University of Arkansas; Victor Sobolev at USDA, Dawson; and Gunjan Pandey and John Oakeshott at CSIRO will continue during the coming year as we expand to consider additional allelopathic plants and chemicals to evaluate the applicability of our findings. Results continue to support the emerging realization that the bacteria that degrade allelopathic chemicals seem to be closely associated with the plants that produce them. New isolates will be characterized and their catabolic pathways determined. The mystery of how the allelopathic chemicals can be effective will be explored. Attempts will be made to evaluate the flux of model allelopathic chemicals in simple greenhouse systems.
Impacts
What was accomplished under these goals?
Objective 1: During the previous reporting periods diverse bacteria were isolated from rhizosphere of red fescue, peanuts and vetch plants with the ability to grow on m-tyrosine, resveratrol, and 3-nitropropanol/3-nitropropionic acid. During this reporting period A range of stilbenes are produced by peanut plants and biodegraded by associated bacteria. In addition to resveratrol and pterostilbene we isolated bacteria able to degrade arachidin-3 which suggests that all of the allelopathic chemicals are biodegradable. The isolates were able to assimilate only the non-prenylated ring of arachidin-3 and the behavior and effects of the other portion of the molecule are under investigation. 1a) The catabolic pathways for resveratrol, pterostilbene, and arachidin-3 are all initiated by carotenoid cleaving monooxygenases (CCO)that seems to be widely distributed among the rhizosphere community. 1b) Genomes have been annotated for the initial nitropropanol and resveratrol degraders. Additional genomes are being sequenced for pterostilbene degraders and m-tyrosine degraders. 1c) Candidate genes are being expressed to determine their roles in the pathways. The CCO has been expressed in E. coli where it catalyzes cleavage of resveratrol. Current experiments will determine the substrate ranges of the CCO enzymes from the various pathways and isolates. Objective 2: Preliminary experiments have not revealed effects of allelopathic compounds on bacteria other than induction of the catabolic pathways . The large and diverse array of bacteria able to degrade the allelopathic chemicals argues against highly specific symbiotic relationships so this area of inquiry has been deemphasized. 2a). Transcriptomics assays have not started yet. 2b). The emphasis this year has been on m-tyrosine and there are extensive and diverse populations of m-tyrosine degrading bacteria not only in the rhizosphere of Festuca rubra, but also in soil not associated with m-tyrosine producing plants. Preliminary experiments indicate that addition of m-tyrosine degrading bacteria to sterile soil can dramatically reduce the allelopathic effects of m-tyrosine, but the ubiquitous presence of m-tyrosine degraders raises questions about the effectiveness of m-tyrosine as an allelopathic chemical. Objective 3. The catabolic pathways are distributed widely among a surprising number of genera. The phylogeny of the genes and enzymes will be established when more genomes have been sequenced to allow comparison. Additional genomes were sequenced in this reporting period for isolates that degrade m-tyrosine, pterostilbene and nitropropanol. Bioinformatic analyses are ongoing.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Francis, K., C. Smitherman, S. Nishino, J.C. Spain and G. Gadda. 2013. The biochemistry of the metabolic poison propionate 3-nitronate and its conjugate acid, 3-nitropropionate. IUBMB Life. 65:759-768.
Francis, K., S. Nishino, J.C. Spain and G. Gadda. 2012. A novel activity for fungal nitronate monooxygenase: detoxification of the metabolic inhibitor propionate-3-nitronate. Arch. Biochem. Biophys. 521:84-89.
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Progress 03/01/12 to 02/28/13
Outputs
Target Audience: Academic colleagues through departmental seminar at Rice University Undergraduate and graduate students through special projects in the lab Presentation and extended discussions at USDA Peanut Lab in Dawson GA Products Type Status Year Published NIFA Support Acknowledged Journal Articles Published 2012 YES Citation Francis, K., S. Nishino, J.C. Spain and G. Gadda. 2012. A novel activity for fungal nitronate monooxygenase: detoxification of the metabolic inhibitor propionate-3-nitronate. Arch. Biochem. Biophys. 521:84-89. Conference Proceedings Nishino, S.F., S. Craven, E. Lang, and J.C. Spain. 2011. Biodegradation of m-tyrosine by rhizosphere bacteria. 111th General Meeting of the American Society for Microbiology Changes/Problems: Post doc got a good offer and left unexpectedly. Delays in recruiting a new post doc led to delays in expending funds. We will request a no cost extension later in the year. This report was previously submitted in error in mid 2013 as the report for the period ending Feb 2012. A duplicate is submitted here to allow the system to open access to the 2014 report link as suggested by Adam Preuter. What opportunities for training and professional development has the project provided? One undergraduate students conducted laboratory studies on the elimination of allelopathic effects by specific bacteria isolated for the ability to degrade the chemicals. The student gained considerable insight about the scientific method and laboratory research. His results will provide the basis for future studies by one of the senior investigators on the elimination of mTyrosine toxicity by bacteria. Post doctoral research associate has developed skills in pathway determination and in communicating results. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? The candidate genes described above will be expressed in E. coli and the catabolic pathways will be established rigorously. Biodegradation of pterostilbene and other resveratrol homologs will be explored because it turns out that resveratrol may be a minor player in allelopathic response of peanuts and grapes. A collaboration has been established with Fabricio Medina Bolivar at the University of Arkansas. Prof Bolivar has developed an in vitro system of peanut hairy root cultures that produce large amounts of several stilbenes including resveratrol, pterostilbene and arachidins. We will establish the capabilities of bacteria from peanut rhizosphere to degrade all of them simultaneously. Nitropropanol/nitropropionic acid degrading bacteria have been isolated from leaves and roots of crown vetch. The mystery of how the allelopathic chemicals can coexist with the bacteria that degrade them will be explored. Attempts will be made to determine the flux of the compounds. mTyrosine degrading bacteria from the rhizosphere of red fescue will be tested for the ability to ameliorate the allelopathic effects of red fescue on other plant species. Transcriptomic analyses will allow evaluation of the effects of the allelopathic chemicals on the bacteria that degrade them in order to reveal any evidence of symbiosis.
Impacts
What was accomplished under these goals?
Objective 1: During the previous reporting period bacteria were isolated from rhizosphere of red fescue, peanuts and vetch plants with the ability to grow on m-tyrosine, resveratrol, and 3-nitropropanol/3-nitropropionic acid. The results established a proof of concept and allowed preliminary determination of the catabolic pathways. During this reporting period more extensive investigation revealed the diversity of each microbial community associated with the plants and indicated that biodegradation of allelopathic compounds is supporting a substantial guild of microbes around each of the plants. Investigation of peanut and grape plants revealed that a range of stilbenes are produced by the plants and biodegraded by associated bacteria. In particular, pterostilbene (a methylated derivative of resveratrol) seems to be produced by the plants and biodegraded by soil bacteria and even by endophytes. 1a) The catabolic pathway for resveratrol clearly is initiated by a carotenoid cleaving monooxygenase (CCO)that seems to be widely distributed among the rhizosphere community. Currently we are developing probes for the genes to allow quantification of the bacteria containing the genes. 1b) Genomes have been annotated for the initial nitropropanol and resveratrol degraders. Additional genomes are being sequenced. 1c) Candidate genes are being expressed to determine their roles in the pathways. The CCO has been expressed in E. coli where it catalyzes cleavage of resveratrol. Objective 2: Preliminary experiments have not revealed effects of allelopathic compounds on bacteria other than induction of the catabolic pathways, but such experiments are in the very early stages. The large and diverse array of bacteria able to degrade the allelopathic chemicals argues against highly specific symbiotic relationships. 2a). Transcriptomics assays have not started yet. 2b). The emphasis this year has been on resveratrol and there are extensive and diverse populations of resveratrol and pterostilbene degraders in the rhizosphere of peanuts and grapes. Surprisingly, preliminary experiments with surface sterilized plants indicate that some of the degrading bacteria are endophytes. Objective 3. The catabolic pathways are distributed widely among a surprising number of genera. The phylogeny of the genes and enzymes will be established when more genomes have been sequenced to allow comparison.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Francis, K., S. Nishino, J.C. Spain and G. Gadda. 2012. A novel activity for fungal nitronate monooxygenase: detoxification of the metabolic inhibitor propionate-3-nitronate. Arch. Biochem. Biophys. 521:84-89.
Conference Proceedings Nishino, S.F., S. Craven, E. Lang, and J.C. Spain. 2011. Biodegradation of m-tyrosine by rhizosphere bacteria. 111th General Meeting of the American Society for Microbiology
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