Abstract
Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogenfixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ∼11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant endophyte interactions.
Original language | English |
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Article number | wrad012 |
Journal | ISME Journal |
Volume | 18 |
Issue number | 1 |
DOIs | |
State | Published - Jan 1 2024 |
Externally published | Yes |
Funding
This study was supported by the EMSL user grant #50774 and the United States Department of Energy Office of Science, Office of Biological and Environmental Research (BER), grant no. DESC0021137.
Funders | Funder number |
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United States Department of Energy Office of Science | |
Biological and Environmental Research | DESC0021137 |
Biological and Environmental Research |
Keywords
- 15 N-tracking metabolomics
- 15 N-tracking proteomics
- Burkholderia
- Populus
- endophyte
- microbial ecology
- nanoSIMS
- nitrogen fixation
- nitrogenase expression
- plant-microbe interactions