Genome-Resolved Proteomic Stable Isotope Probing of Soil Microbial Communities Using 13CO2 and 13C-Methanol

Zhou Li, Qiuming Yao, Xuan Guo, Alexander Crits-Christoph, Melanie A. Mayes, William Judson Hervey IV, Sarah L. Lebeis, Jillian F. Banfield, Gregory B. Hurst, Robert L. Hettich, Chongle Pan

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26 Scopus citations

Abstract

Stable isotope probing (SIP) enables tracking the nutrient flows from isotopically labeled substrates to specific microorganisms in microbial communities. In proteomic SIP, labeled proteins synthesized by the microbial consumers of labeled substrates are identified with a shotgun proteomics approach. Here, proteomic SIP was combined with targeted metagenomic binning to reconstruct metagenome-assembled genomes (MAGs) of the microorganisms producing labeled proteins. This approach was used to track carbon flows from 13CO2 to the rhizosphere communities of Zea mays, Triticum aestivum, and Arabidopsis thaliana. Rhizosphere microorganisms that assimilated plant-derived 13C were capable of metabolic and signaling interactions with their plant hosts, as shown by their MAGs containing genes for phytohormone modulation, quorum sensing, and transport and metabolism of nutrients typical of those found in root exudates. XoxF-type methanol dehydrogenases were among the most abundant proteins identified in the rhizosphere metaproteomes. 13C-methanol proteomic SIP was used to test the hypothesis that XoxF was used to metabolize and assimilate methanol in the rhizosphere. We detected 7 13C-labeled XoxF proteins and identified methylotrophic pathways in the MAGs of 8 13C-labeled microorganisms, which supported the hypothesis. These two studies demonstrated the capability of proteomic SIP for functional characterization of active microorganisms in complex microbial communities.

Original languageEnglish
Article number2706
JournalFrontiers in Microbiology
Volume10
DOIs
StatePublished - Dec 6 2019

Funding

This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research, the ORNL Plant-Microbe Interfaces Scientific Focus Area project, and the DOE Grants DOE-SC10010566 and DE-SC0020356. WH acknowledges support We thank Ton Gorissen at the Isolife for assistance with the plant growth and 13CO2 labeling. This research used resources of the Oak Ridge Leadership Computing Facility and Department of Defense?s High Performance Computing Modernization Program Application Software Initiative (HASI) under the U.S. Army Corps of Engineers, Engineer Research and Development Center (ERDC) by the Department of the Army. Funding. This work was supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research, the ORNL Plant-Microbe Interfaces Scientific Focus Area project, and the DOE Grants DOE-SC10010566 and DE-SC0020356. WH acknowledges support from the HASI program and Naval Research Laboratory core funding. MM acknowledges funding support from U.S. Department of Energy (DOE), Office of Science Early Career Award. ORNL is managed by UT-Battelle, LLC, under contract DE-AC05-00OR22725 for the U.S. Department of Energy. The opinions and assertions contained herein are those of the authors and are not to be construed as those of the U.S. Navy, Military Service at large, and/or the U.S. Government. Vandenkoornhuyse, P., Mahé, S., Ineson, P., Staddon, P., Ostle, N., Cliquet, J.-B., et al. (2007). Active root-inhabiting microbes identified by rapid incorporation of plant-derived carbon into RNA. Proc. Natl. Acad. Sci. U.S.A. 104, 16970–16975. doi: 10.1073/pnas.070590 2104 Washburn, M. P., Wolters, D., and Yates, J. R. I. I. I. (2001). Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19:242. doi: 10.1038/85686 Wi´sniewski, J. R., Zougman, A., Nagaraj, N., and Mann, M. (2009). Universal sample preparation method for proteome analysis. Nat. Methods 6:359. doi: 10.1038/nmeth.1322 Wu, M., and Eisen, J. A. (2008). A simple, fast, and accurate method of phylogenomic inference. Genome Biol. 9:R151. doi: 10.1186/gb-2008-9-10-r151 Yao, Q., Li, Z., Song, Y., Wright, S. J., Guo, X., Tringe, S. G., et al. (2018). Community proteogenomics reveals the systemic impact of phosphorus availability on microbial functions in tropical soil. Nat. Ecol. Evol. 2:499. doi: 10.1038/s41559-017-0463-5 Zhalnina, K., Louie, K. B., Hao, Z., Mansoori, N., da Rocha, U. N., Shi, S., et al. (2018). Dynamic root exudate chemistry and microbial substrate preferences drive patterns in rhizosphere microbial community assembly. Nat. Microbiol. 3:470. doi: 10.1038/s41564-018-0129-3 Disclaimer: This manuscript has been authored in part by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the U.S. Department of Energy (DOE). The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for U.S. Government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan). The opinions and assertions contained herein are those of the authors and are not to be construed as those of the U.S. Government.

Keywords

  • metagenomic analyses
  • metaproteomic analysis
  • microbial ecology
  • rhizosphere
  • stable isotope probing

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