Iron Supplementation Eliminates Antagonistic Interactions Between Root-Associated Bacteria

Abstract

The rhizosphere microbiome (rhizobiome) plays a critical role in plant health and development. However, the processes by which the constituent microbes interact to form and maintain a community are not well understood. To investigate these molecular processes, we examined pairwise interactions between 11 different microbial isolates under select nutrient-rich and nutrient-limited conditions. We observed that when grown with media supplemented with 56 mM glucose, two microbial isolates were able to inhibit the growth of six other microbes. The interaction between microbes persisted even after the antagonistic microbe was removed, upon exposure to spent media. To probe the genetic basis for these antagonistic interactions, we used a barcoded transposon library in a proxy bacterium, Pseudomonas putida, to identify genes which showed enhanced sensitivity to the antagonistic factor(s) secreted by Acinetobacter sp. 02. Iron metabolism-related gene clusters in P. putida were implicated by this systems-level analysis. The supplementation of iron prevented the antagonistic interaction in the original microbial pair, supporting the hypothesis that iron limitation drives antagonistic microbial interactions between rhizobionts. We conclude that rhizobiome community composition is influenced by competition for limiting nutrients, with implications for growth and development of the plant.

Keywords: Acinetobacter; Pseudomonas putida; RB-TnSeq; composition and function; iron depletion; microbe-macroorganism interaction; rhizobiota.

FIGURE 1

Workflow for bacterial chemical genomic screen. Rhizobacteria mediate complex interactions in the rhizosphere (green-shaded box). After isolation, rhizobacteria are screened for potential interactions against one another and with a random barcode transposon sequencing (RB-TnSeq) library of a model microorganism (orange-shaded box). Finally, RB-TnSeq data are analyzed and used to characterize and validate microbe-microbe interactions (blue-shaded box).

FIGURE 2

Methodology for microbial interaction screen. (A) Multiple rhizobacteria are used to inoculate LB plates, either concurrently or staggered such that the vertical, “primary” species was grown for 2 days before the introduction of a secondary species. For staggered inoculation assays, half of the primary inoculum was grown on top of a nitrocellulose membrane that was removed prior to secondary inoculation. (B) Representative images of staggered inoculation assays with Acinetobacter sp. 02 grown in co-culture with six rhizobacterial species. Dashed lines indicate the location of a 0.44-μm nitrocellulose membrane prior to removal. Brightness and contrast have been uniformly edited to increase visibility.

FIGURE 3

Results of concurrent inoculation interaction screen. Interactions observed on (A) LB or (B) 0.1× LB supplemented with 55 mM D-glucose. Rows represent primary, vertically streaked species and columns represent secondary, horizontally streaked species. The same color legend is used for both (A) and (B).

FIGURE 4

Results of staggered inoculation interaction screen. Interactions observed on (A) LB or (B) LB supplemented with 55 mM D-glucose. Rows represent primary, vertically streaked species and columns represent secondary, horizontally streaked species. n ≥ 3. Where interactions were variable by colony, the most frequently observed interaction is displayed.

FIGURE 5

Pseudomonas putida KT2440 RB-TnSeq results. (A) Scatterplot of gene fitness values from P. putida KT2440 grown in Acinetobacter sp. 02 supernatant vs. control media. (B) The top 10 depleted genes in library populations grown in supernatant and relevant log₂-fold decreases relative to the population at Time 0.

FIGURE 6

Iron supplementation in Acinetobacter sp. 02 concurrent interaction assay. (A) Acinetobacter colonies were streaked vertically on LB with or without 100 μM supplemental FeCl₃ followed by the perpendicular inoculation of the secondary species indicated. Dashed boxes indicate interactions that change depending on iron supplementation. n ≥ 3. (B) A magnified view of the region indicated with dashed white boxes from (A).