Source: UNIV OF WISCONSIN submitted to NRP
GENETIC AND METABOLIC DETERMINANTS OF MICROBIAL INTERACTIONS IN THE RHIZOSPHERE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022881
Grant No.
2020-67012-31772
Cumulative Award Amt.
$164,786.00
Proposal No.
2019-07352
Multistate No.
(N/A)
Project Start Date
Jun 15, 2020
Project End Date
Aug 14, 2022
Grant Year
2020
Program Code
[A7201]- AFRI Post Doctoral Fellowships
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
WI INSTITUTE FOR DISCOVERY
Non Technical Summary
The plant root microbiome impacts plant health through growth promotion and suppression of infectious disease. Many microbiome interactions are mediated by bacterial secondary metabolites. This postdoctoral research project proposal aims to better understand the metabolites that mediate bacterial interactions in a rhizosphere model community. The central organism in this model system, Bacillus cereus UW85, has been used successfully as a biocontrol agent for alfalfa and soybean. However, variability in its performance and persistence remains a challenge. We hypothesize the variable effectiveness of B. cereus UW85 as a biocontrol agent is, in part, due to its interactions with other microbes in the rhizosphere community. Our model community, comprised of rhizosphere isolates, enables the coupled genetic and metabolomic investigation of secondary metabolic pathways within B. cereus UW85 that mediate multipartite community dynamics, both in vitro and on the plant host. Secondary metabolic pathways and their respective metabolites will be identified with modern genomic techniques and genetically verified. Transcriptomic studies will be performed to better understand the global changes in gene expression in response to B. cereus UW85 secondary metabolism for community members alone, in pairwise interactions, and in tripartite interactions. This proposed work expands the technical expertise of the Handelsman lab and the PD and is attainable within the training period. Further, it directly addresses the plant health and production priority of the AFRI Farm Bill. Investigations into the secondary metabolites and pathways involved in microbial interactions may inform novel soil management techniques or implementation of biocontrol agents to suppress plant disease.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240991100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The plant root microbiome impacts plant health through growth promotion and suppression of infectious disease. Many microbiome interactions are mediated by bacterial secondary metabolites. This postdoctoral research project proposal aims to better understand the metabolites that mediate bacterial interactions in a rhizosphere model community. The central organism in this model system, Bacillus cereus UW85, has been used successfully as a biocontrol agent for alfalfa and soybean. However, variability in its performance and persistence remains a challenge. We hypothesize the variable effectiveness of B. cereus UW85 as a biocontrol agent is, in part, due to its interactions with other microbes in the rhizosphere community. Our model community, comprised of rhizosphere isolates, enables the coupled genetic and metabolomic investigation of secondary metabolic pathways within B. cereus UW85 that mediate multipartite community dynamics, both in vitro and on the plant host. Secondary metabolic pathways and their respective metabolites will be identified with modern genomic techniques and genetically verified. Transcriptomic studies will be performed to better understand the global changes in gene expression in response to B. cereus UW85 secondary metabolism for community members alone, in pairwise interactions, and in tripartite interactions. This proposed work expands the technical expertise of the Handelsman lab and the PD and is attainable within the training period. Further, it directly addresses the plant health and production priority of the AFRI Farm Bill. Investigations into the secondary metabolites and pathways involved in microbial interactions may inform novel soil management techniques or implementation of biocontrol agents to suppress plant disease.
Project Methods
Objective 1: Determine the full metabolic potential of B. cereus UW85 with modern genomic techniques. When applied to seeds in the field, B. cereus UW85 abundance can decline to 0.1% of the initial inoculum in just the first few weeks of the growing season [5]. Due to nonuniformity in colonization and persistence in the rhizosphere microbial community, its use as a biocontrol agent to suppress plant disease has seen variable success. Through genetic manipulations of B. cereus UW85, I propose to investigate the role of secondary metabolism mediated interactions in a model rhizosphere community. The proposed experiments may provide insight into the B. cereus UW85 field colonization.1.1 Identify biosynthetic pathways and recent metabolic gene expansions in B. cereus UW85 via genome mining.1.2 Generate candidate mutants.Objective 2: Determine the effects of B. cereus UW85 secondary metabolism on interactions and emergent phenotypes within the model system. Preliminary data shown in Figure 2 indicate B. cereus can protect F. johnsoniae from the killing mediated by P. koreensis. I propose to conduct the first species-specific, coupled metabolomics and transcriptomics analysis of a defined microbial community to profile community-specific responses to specific secondary metabolites, both in the genes expressed and the molecules produced. I hypothesize that mutants for specific BGCs in Objective 1.2 will elicit differential responses in each species, both in pairwise studies with B. cereus and in the tripartite community. Experiments will be conducted with and without root exudate, such that plant-elicited responses will be captured. Mutants that affect the phenotypes of biofilm formation and/or colony spreading will be identified in Objective 2.1. This will generate a suite of candidate mutants to do LC-MS/MS characterization of the single-, double-, and triple-member metabolomes in Objective 2.2. In parallel, Objective 2.3 will identify the differential expression of genes that may be important for the interactions in the rhizosphere. Together, Objective 2 will prioritize mutants for analyses on the plant root in Objective 3.2.1 Determine the effects of metabolite-deficient B. cereus UW85 mutants on emergent community properties.2.2 Determine shifts in community metabolism via comparative LC-MS/MS of B. cereus UW85 mutants.2.3 Determine shifts in community transcription via comparative transcriptomics of B. cereus UW85 mutants.Objective 3: Determine the effects of B. cereus UW85 secondary metabolism on community root colonization. B. cereus mutants that have been prioritized by community phenotypes, metabolomes, and transcriptomes from Objective 2 will be inoculated onto soybean seeds to test for colonization success in the presence of the community. Between two and four mutants will be tested, including the mutant in which the zwittermicin pathway has been deleted. To assay colonization and translate findings from Objectives 1 and 2 directly to plant health, I will inoculate sterilized soybean seeds with equal populations of P. koreensis, F. johnsoniae, and B. cereus (either WT or mutant). Plants will be cultivated in growth chambers in sterilized soil. Roots will be collected daily for bacterial quantification on selective media as in Objective 2.3. Future directions will explore whether communities containing mutants protect the plant from infection by oomycete pathogens better than the WT, presumably due to B. cereus abundance and/or the absence of secondary metabolites (e.g. zwittermicin).

Progress 06/15/20 to 08/14/22

Outputs
Target Audience:The plant root microbiome impacts plant health through growth promotion and suppression of infectious disease. Many microbiome interactions are mediated by bacterial secondary metabolites. This postdoctoral research project proposal aims to better understand the metabolites that mediate bacterial interactions in a rhizosphere model community. The central organism in this model system, Bacillus cereus UW85, has been used successfully as a biocontrol agent for alfalfa and soybean. However, variability in its performance and persistence remains a challenge. We hypothesize the variable effectiveness of B. cereus UW85 as a biocontrol agent is, in part, due to its interactions with other microbes in the rhizosphere community. Our model community, comprised of rhizosphere isolates, enables the coupled genetic and metabolomic investigation of secondary metabolic pathways within B. cereus UW85 that mediate multipartite community dynamics, both in vitro and on the plant host. Secondary metabolic pathways and their respective metabolites will be identified with modern genomic techniques and genetically verified. Transcriptomic studies will be performed to better understand the global changes in gene expression in response to B. cereus UW85 secondary metabolism for community members alone, in pairwise interactions, and in tripartite interactions. This proposed work expands the technical expertise of the Handelsman lab and the PD and is attainable within the training period. Further, it directly addresses the plant health and production priority of the AFRI Farm Bill. Investigations into the secondary metabolites and pathways involved in microbial interactions may inform novel soil management techniques or implementation of biocontrol agents to suppress plant disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?For the PD, training at the interface of traditional microbiology, data analysis, metatranscriptomics, and metametabolomics. The PD has also mentored graduate and undergraduates on these projects. How have the results been disseminated to communities of interest?Open access publications and data sharing What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Yes. Development of these techniques has deepended our understanding of this model system, as evidenced but two major publications describing the transcriptomic and metabolomic changes unique to communities.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: MG Chevrette, CS Thomas, A Hurley, N Rosario-Mel�ndez, K Sankaran, Y Tu, A Hall, S Magesh, J Handelsman. "Microbiome composition modulates secondary metabolism in a multispecies bacterial community." 2022. Proceedings of the National Academy of Sciences. doi:10.1073/pnas.2212930119
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: A Hurley, MG Chevrette, N Rosario-Melendez, J Handelsman. "THOR's hammer: the antibiotic koreenceine drives gene expression in a model microbial community." 2022. mBio. doi:10.1128/mbio.02486-21


Progress 06/15/20 to 06/14/21

Outputs
Target Audience:Target audiences include the Tiny Earth network (tinyearth.wisc.edu), which reaches over 10000 undergraduates per year. Efforts in developing teaching materials and workshops for Tiny Earth classes have been developed and disseminated to the network of instructors. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided opportunities to learn new microbiological, computational, and pedegogical techniques. How have the results been disseminated to communities of interest?Publications are in prep. What do you plan to do during the next reporting period to accomplish the goals?Publish results and follow up on new hypotheses.

Impacts
What was accomplished under these goals? New methods for genome mining of biosynthetic gene clusters have been designed and deployed to describe the full biosynthetic potential of all organisms in the model. Transcriptomics and metabolomics as proposed has been completed and analyzed. Two publications are in prep describing general and biosynthetic transcriptomic shifts in the different community condtions, respectively.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Marc G Chevrette, Athina Gavrilidou, Shrikant Mantri, Nelly Selem-Mojica, Nadine Ziemert, Francisco Barona-G�mez. The confluence of big data and evolutionary genome mining for the discovery of natural products. Natural Product Reports. DOI:10.1039/D1NP00013F
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Marc Chevrette, Bradley Himes, Camila Carlos-Shanley. Nutrient availability shifts the biosynthetic potential of soil-derived microbial communities. bioRxiv. DOI:10.1101/2021.09.02.458721
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Humberto E Ortega, Vitor B Lourenzon, Marc G Chevrette, Leonardo LG Ferreira, Ren� F Ramos Alvarenga, Weilan GP Melo, Tiago Ven�ncio, Cameron R Currie, Adriano D Andricopulo, Tim S Bugni, M�nica T Pupo. Antileishmanial macrolides from ant-associated Streptomyces sp. ISID311. Bioorganic & Medicinal Chemistry. DOI:10.1016/j.bmc.2021.116016
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Amanda Hurley, Marc G Chevrette, Deepa D Acharya, Gabriel L Lozano, Manuel Garavito, Jen Heinritz, Luis Balderrama, Mara Beebe, Martel L DenHartog, Kamiyah Corinaldi, Renee Engels, Alyssa Gutierrez, Orli Jona, Josephine HI Putnam, Brody Rhodes, Tiffany Tsang, Simon Hernandez, Carol Bascom-Slack, Jessamina E Blum, Paul A Price, Debra Davis, Joanna Klein, Joshua Pultorak, Nora L Sullivan, Nigel J Mouncey, Pieter C Dorrestein, Sarah Miller, Nichole A Broderick, Jo Handelsman. Tiny earth: a big idea for STEM education and antibiotic discovery. mBio. DOI:10.1128/mBio.03432-20
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Eric JN Helfrich, Reiko Ueoka, Marc G Chevrette, Franziska Hemmerling, Xiaowen Lu, Stefan Leopold-Messer, Hannah A Minas, Adrien Y Burch, Steven E Lindow, J�rn Piel, Marnix H Medema. Evolution of combinatorial diversity in trans-acyltransferase polyketide synthase assembly lines across bacteria. Nature Communications. DOI:10.1038/s41467-021-21163-x
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Benjamin D Lee, Anthony Gitter, Casey S Greene, Sebastian Raschka, Finlay Maguire, Alexander J Titus, Michael D Kessler, Alexandra J Lee, Marc G Chevrette, Paul Allen Stewart, Thiago Britto-Borges, Evan M Cofer, Kun-Hsing Yu, Juan Jose Carmona, Elana J Fertig, Alexandr A Kalinin, Beth Signal, Benjamin J Lengerich, Timothy J Triche Jr, Simina M Boca. Ten Quick Tips for Deep Learning in Biology. arXiv. https://arxiv.org/abs/2105.14372