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. 2020 Aug 25;11(4):e01917-20.
doi: 10.1128/mBio.01917-20.

Cooperation, Competition, and Specialized Metabolism in a Simplified Root Nodule Microbiome

Bridget L Hansen  1 , Rita de Cassia Pessotti  1 , Monika S Fischer  1 , Alyssa Collins  2 , Laila El-Hifnawi  2 , Mira D Liu  3 , Matthew F Traxler  4
Affiliations

Cooperation, Competition, and Specialized Metabolism in a Simplified Root Nodule Microbiome

Bridget L Hansen et al. mBio. .

Abstract

Microbiomes associated with various plant structures often contain members with the potential to make specialized metabolites, e.g., molecules with antibacterial, antifungal, or siderophore activities. However, when and where microbes associated with plants produce specialized metabolites, and the potential role of these molecules in mediating intramicrobiome interactions, is not well understood. Root nodules of legume plants are organs devoted to hosting symbiotic bacteria that fix atmospheric nitrogen and have recently been shown to harbor a relatively simple accessory microbiome containing members with the ability to produce specialized metabolites in vitro On the basis of these observations, we sought to develop a model nodule microbiome system for evaluating specialized microbial metabolism in planta Starting with an inoculum derived from field-grown Medicago sativa nodules, serial passaging through gnotobiotic nodules yielded a simplified accessory community composed of four members: Brevibacillus brevis, Paenibacillus sp., Pantoea agglomerans, and Pseudomonas sp. Some members of this community exhibited clear cooperation in planta, while others were antagonistic and capable of disrupting cooperation between other partners. Using matrix-assisted laser desorption ionization-imaging mass spectrometry, we found that metabolites associated with individual taxa had unique distributions, indicating that some members of the nodule community were spatially segregated. Finally, we identified two families of molecules produced by B. brevisin planta as the antibacterial tyrocidines and a novel set of gramicidin-type molecules, which we term the britacidins. Collectively, these results indicate that in addition to nitrogen fixation, legume root nodules are likely also sites of active antimicrobial production.

Keywords: antibiotic; microbial interactions; microbiome; root nodule; specialized metabolism.

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Figures

FIG 1
FIG 1
16S community profiling of three different root nodule developmental phenotypes from agricultural M. sativa plants. (A) Images of young (Y, white), active (A, pink/red), and senescent (S, brown/green) nodules. (B) Relative abundance of each phylum based on 16S amplicon sequencing of young, active, and senescent root nodules from an agricultural field in Alturas, CA. (C) Relative abundance of each phylum, excluding Rhizobia.
FIG 2
FIG 2
Root nodule microbial community selection with M. sativa. (A) Schematic showing the workflow for creating the microbial community. (Step 1) Crushed nodules were inoculated onto gnotobiotic plant roots. Plants were incubated until nodules formed. (Step 2) Roots were then surface sterilized and nodules removed and sorted based on phenotype. (Step 3) Nodules were then crushed and resuspended in water. (Step 4) This homogenous mixture was used for three applications: for repeating the process by application to gnotobiotic roots (Step 4a), for environmental DNA extraction for 16S amplicon sequencing (Step 4b), and for isolation of bacteria (Step 4c). (B) Relative abundances of non-Ensifer operational taxonomic units (OTUs) at the genus level from each successive root nodule inoculum (“Round”) across the root nodule phenotypes. Black boxes indicate genera isolated after round 3 and used for subsequent experiments.
FIG 3
FIG 3
Interactions between members of the synthetic root nodule community. (A) Rate of reisolation of (or recovery) each bacterium from M. sativa roots that were inoculated with the bacterial isolates that resulted from the selection process detailed in Fig. 2. Experimental treatments were all possible combinations of these bacteria inoculated onto M. sativa roots with the essential nodulation strain, S. meliloti. These plants were grown until they developed root active nodules, and then bacteria were systematically reisolated from these root nodules. Recovery rates for each bacterium are relative to the recovery rate from plants that were inoculated with one bacterium plus S. meliloti. White boxes indicate that no bacterium was added to the inoculum. Gray, zero change; blue, negative relative recovery; yellow, positive relative recovery. (B) The number of bacterial colonies recovered from treatments 12, 9, 15, and 4 (see Fig. S3 for all other treatments). Numbers within gray circles labeled “None” represent the number of nodules where no bacterium was recovered, while numbers in colored circles represent the number of nodules where one or more bacteria were recovered. (C) Bioactivity agar plug diffusion assay of each member against a lawn of each member on root nodule agar medium. Red squares indicate that a zone of inhibition was observed, and tan squares indicate that no inhibition zone was observed. (D) Model summarizing the interactions observed for microbe recovery in planta (A and B) and in vitro (C).
FIG 4
FIG 4
Number of molecular features associated with the simplified nodule community in vitro and in planta. Molecular features identified via LC/MS from chemical extracts that were prepared from one of the three independent treatments of each microbe (present alone on agar plates [in vitro], inoculated onto M. sativa plants individually with S. meliloti, or inoculated onto M. sativa plants with all other members of the simplified nodule community) plus an additional control treatment of M. sativa plants inoculated solely with S. meliloti. (A) Analysis approach using nested Venn diagrams to identify features that are unique to each microbe followed by focusing on unique features associated with B. brevis compared to all other community members both in planta and in vitro. (B) Numbers of features that are unique to each microbe. (C) Numbers of features that are unique to B. brevis compared with the number of features identified in two different simplified communities lacking B. brevis in planta. The 13 features that are unique to B. brevis in vitro and in planta are highlighted as features of interest and detailed in Table S4.
FIG 5
FIG 5
MALDI images of the simplified community root nodule. A bright-field image is shown of a 20-μm-thick simplified community root nodule embedded in gelatin. The ion for heme B, m/z 616.2 (magenta), and an unidentified feature, m/z 536.4 (cyan), are unique to S. meliloti and colocalize. In contrast, the feature m/z 617.4 is associated with B. brevis, and an unidentified feature associated with the community, m/z 763.0, is spatially distinct (Overlay).
FIG 6
FIG 6
Tyrocidine A and britacidin A are detected in planta. (A) Tyrocidine A (amino acid position 1) and B (position 2) and britacidin A (position 3), B (position 4), and C (position 5) are produced by B. brevis. The asterisk (*) denotes that the indicated molecule was detected in planta (tyrocidine A and britacidin A). (B) Extracted ion chromatogram of the [M+H]+ tyrocidine A, m/z 1,270.66, from tyrocidine standard, B. brevis grown in vitro on root nodule medium, methanol extracts of community root nodules, and methanol extracts of S. meliloti-only root nodules. (C) Extracted ion chromatogram of the dominant isotope of the [M + 2H]2+ species of britacidin A, m/z 1,022.60, from B. brevis grown in vitro on root nodule medium, methanol extracts of community root nodules, and methanol extracts of S. meliloti-only root nodules.
FIG 7
FIG 7
Britacidin and gramicidin comparison at the chemical and genetic levels. (A) Chemical structure comparison of britacidin A to C and gramicidin A to D, with blue boxes highlighting the structural differences. (B) Comparison of gramicidin NRPS genes lgrA to lgrD (lgrA-D) from B. brevis ATCC 8185 and the britacidin NRPS genes briA-D from B. brevis Ag35. Amino acids incorporated by the AMP-binding domains (yellow) are listed within the gene described for LgrA-D. Stachelhaus alignments to genes LgrA-D were used for amino acid assignments for nonribosomal peptide-synthetase (NRPS)briA-D. (C) Table highlighting chemical positions 1, 7, 8, 11, and 13 (boxed in Fig. 7A) and their respective AMP-binding domain Stachelhaus codes, alignments, predictions, and observed amino acids.
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