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. 2024 Jan 8;18(1):wrad012.
doi: 10.1093/ismejo/wrad012.

Dynamic nitrogen fixation in an aerobic endophyte of Populus

Andrew W Sher  1 , Jayde A Aufrecht  2 , Daisy Herrera  2 , Amy E Zimmerman  3 , Young-Mo Kim  3 , Nathalie Munoz  2 , Jesse B Trejo  3 , Vanessa L Paurus  3 , John B Cliff  2 , Dehong Hu  2 , William B Chrisler  2 , Robert J Tournay  1 , Emma Gomez-Rivas  1 , Galya Orr  2 , Amir H Ahkami  2 , Sharon L Doty  1
Affiliations

Dynamic nitrogen fixation in an aerobic endophyte of Populus

Andrew W Sher et al. ISME J. .

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 nitrogen-fixing 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.

Keywords: 15 N-tracking metabolomics; 15 N-tracking proteomics; Burkholderia; Populus; endophyte; microbial ecology; nanoSIMS; nitrogen fixation; nitrogenase expression; plant–microbe interactions.

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Conflict of interest statement

None declared.

Figures

Figure 1
Figure 1
WPB on NL-CCM plates displays heterogeneous growth patterns. (A) Top down view of plate shows large colonies intermixed with lawns of less vigorous growth. (B) Oblique view of plate shows raised, globular-like growth of the larger colonies.
Figure 2
Figure 2
The fluorescence images of WPB cells and direct quantification of nifH mRNA transcripts in single cells in a population. Closed dots indicate fluorescence blinking events. Open circles indicate actual transcript copies as determined based on blinking events per unit time. The scale bar is 1 μm. (A) Isolated cells; (B) aggregated cells.
Figure 3
Figure 3
Examples of spatial distribution of nitrogenase gene expression in WPB Pnif-GFP grown on nitrogen limited agar. Pools of cell suspensions across multiple experiments display heterogeneous expression patterns. (A) Pools of 7 μl from an OD600 0.4 cell suspension; (B) pools of 100 μl from an OD600 1.0 cell suspension.
Figure 4
Figure 4
(A) Representative hue saturation intensity (HSI) ion image of a15N-labeled WPB culture. Hues represent 15N atom% values of individual cells. Incorporation is heterogeneous in pure culture of WPB. (B) Histogram representing NanoSIMS analyses of three biological replicates of the in vitro15N2 fixation experiment. Bar height represents number of WPB cells in each enrichment class for each replicate. NanoSIMS was used to produce individual 15N atom% estimates from six separate images for each replicate. Cell numbers ranged from 440 to 682 individual cells and enrichments of 0.6–70.3 atom% between the three replicates.
Figure 5
Figure 5
Heatmap analysis of isotope enriched nitrogenous metabolites from B. vietnamiensis WPB. 15N-enriched metabolites were plotted based on the labeled fractions. Most of the nitrogenous metabolites were labeled under the 15N condition. Leucine was heavily co-eluted with other metabolite peaks so the shown background noise is high.
Figure 6
Figure 6
Atom% 15N enrichment of peptides from WPB cultures collected after 3 or 9 days of incubation with either 15N2 or 14N2, grouped by KEGG pathway. Datapoints represent individual peptides. Numbers at the bottom indicate the number of peptides detected in each treatment and time point. Overall, peptides associated with each pathway were significantly enriched in 15N2 vs. 14N2 treatments, as well as between 3 and 9 days of incubation (significance of pairwise comparisons shown on plots from Wilcoxon rank sum tests with Benjamini–Hochberg corrections for multiple testing), although individual peptide enrichment values varied. The y-axis is truncated to better show inter-treatment comparisons. Note that some pathway names have been abbreviated for visualization (“Nicotinate and nicotinamide metabolism” to “Nicotinate and nicotinamide”; “Tropane, piperidine and pyridine alkaloid biosynthesis” to “Tropane, piperidine, pyridine”).
Figure 7
Figure 7
Selected metabolites from the sectoring experiment with nifh gene expression. The abundance profiles of selected metabolites from the biomass with the expression of nifh gene (+ symbol) and without (− symbol). Polyamines (cadaverine and putrescine) were strongly correlated with the expression of the gene, and other metabolites were positively or negatively related with the expression. Peak area values of individual metabolites were normalized by corresponding dried biomass weight first, and then followed by z-score transformation. Abbreviation: 3HBA, 3-hydroxybutyric acid; EA, ethanol amine; OPC, o-phosphocolamine; GA, glutamic acid; PGA, pyroglutamic acid.
Figure 8
Figure 8
Confocal images of P. Trichocarpa roots growing inside a RhizoChip show (A) that WPB cells (fluorescent dots) express the nifH gene inside of root epidermal cells, and (B, C) fluorescence from nifH-expressing WPB cells is sometimes localized to spherical structures (referred to as nitrosomes in this work) roughly 3–5 μm in size inside of epidermal cells in the root elongation zone.
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