Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria

Abstract

Symbiotic associations can allow an organism to acquire novel traits by accessing the genetic repertoire of its partner. In the Dictyostelium discoideum farming symbiosis, certain amoebas (termed "farmers") stably associate with bacterial partners. Farmers can suffer a reproductive cost but also gain beneficial capabilities, such as carriage of bacterial food (proto-farming) and defense against competitors. Farming status previously has been attributed to amoeba genotype, but the role of bacterial partners in its induction has not been examined. Here, we explore the role of bacterial associates in the initiation, maintenance, and phenotypic effects of the farming symbiosis. We demonstrate that two clades of farmer-associated Burkholderia isolates colonize D. discoideum nonfarmers and infectiously endow them with farmer-like characteristics, indicating that Burkholderia symbionts are a major driver of the farming phenomenon. Under food-rich conditions, Burkholderia-colonized amoebas produce fewer spores than uncolonized counterparts, with the severity of this reduction being dependent on the Burkholderia colonizer. However, the induction of food carriage by Burkholderia colonization may be considered a conditionally adaptive trait because it can confer an advantage to the amoeba host when grown in food-limiting conditions. We observed Burkholderia inside and outside colonized D. discoideum spores after fruiting body formation; this observation, together with the ability of Burkholderia to colonize new amoebas, suggests a mixed mode of symbiont transmission. These results change our understanding of the D. discoideum farming symbiosis by establishing that the bacterial partner, Burkholderia, is an important causative agent of the farming phenomenon.

Keywords: Burkholderia; Dictyostelium; mutualism; social amoeba; symbiosis.

Fig. 1.

Burkholderia phylogeny. Phylogenetic tree based on 400-nt 16S rRNA gene sequences showing relatedness of D. discoideum-associated Burkholderia isolates with environmental Burkholderia species using the pathogenic Burkholderia pseudomallei as an outgroup. The current collection of D. discoideum-associated Burkholderia isolates falls within two distinct clades, referred to here as clades B1 and B2.

Fig. 2.

Exposure of D. discoideum to Burkholderia results in stable bacterial carriage. (A) The percent of bacteria-positive sori from 10 farmers and 10 nonfarmers after exposure to K. pneumoniae, E. coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and the indicated Burkholderia isolates was detected by spotting individual sorus contents on nutrient medium and observing bacterial growth. All the tested Burkholderia isolates and the P. aeruginosa strain do not support D. discoideum growth and development without the addition of a bacterial food source. Therefore, to ensure D. discoideum growth during exposure to these strains, they were mixed at 10% by volume with a culture of K. pneumoniae after both cultures were preset to an OD₆₀₀ of 2 in KK2 buffer (denoted by 10%). The other four bacteria were edible and did not need to be mixed with K. pneumonia for D. discoideum to proliferate. Error bars represent SEM. (B) Images of bacterial growth after the contents of one sorus from the indicated D. discoideum clone were plated on nutrient medium. (C) Percent of bacteria-positive sori from six farmer and six nonfarmer D. discoideum clones after exposure to the indicated bacterial isolates (transfer 1) and after subsequent social cycles on K. pneumoniae alone (transfers 2–5). Error bars represent SEM. (D) Representative agarose gel images of PCR amplification of genes specific to eukaryotes, eubacteria, Burkholderia, and Klebsiella from DNA isolated from the sorus contents of farmer (QS70 and QS11) and nonfarmer (QS9) D. discoideum clones (the superscript “col” indicates precolonization with the indicated Burkholderia isolate).

Fig. S1.

Carriage of non-Burkholderia isolates associated with farmers. (A) Percent of bacteria-positive sori from 10 farmers and 10 nonfarmers detected by spotting individual sorus contents on nutrient medium and observing bacterial growth after exposure to K. pneumoniae and to farmer-associated P. fluorescens 2, P. fluorescens 3, Flavobacterium, or Stenotrophomonas isolates. All strains were tested as a mixture at 10% by volume with a culture of K. pneumoniae after both cultures were preset to an OD₆₀₀ of 2 in KK2 buffer (denoted by 10%). Strains that were edible and could support D. discoideum growth without the addition of K. pneumoniae also were tested as pure cultures. Error bars represent SEM. (B) Percent of bacteria-positive sori from six farmer and six nonfarmer D. discoideum clones after exposure to the indicated bacterial isolates (transfer 1) and after subsequent social cycles on K. pneumoniae alone (transfers 2 and 3). Error bars represent SEM.

Fig. S2.

Secondary carriage of S. marcescens by colonized D. discoideum clones. Bacterial growth after plating the contents of one sorus from the indicated D. discoideum clone on nutrient medium after growth on a red strain of S. marcescens. The observation of red-pigmented bacterial growth from spotted sorus contents indicates that S. marcescens is carried by Burkholderia-colonized clones.

Fig. 3.

Colonization of D. discoideum with Burkholderia confers differential costs and benefits depending on dispersal conditions. (A) Total D. discoideum spore counts after growth on live K. pneumoniae (K.pneu abundant) for three replicates each of four farmers, six uncolonized nonfarmers, and six nonfarmers colonized via pregrowth with 10% of the indicated Burkholderia isolate (^col). Error bars represent SEM. (B) Total spore counts after growth on dead K. pneumoniae (K.pneu scarce) for three replicates each of six uncolonized nonfarmers (uncolonized) and six nonfarmers colonized with Burkholderia via pregrowth with 10% B1qs70, B1nc21, B2qs11, or B2nc28 (Burk^col). Error bars represent SEM.

Fig. S3.

The cost of colonization by Burkholderia isolates from clade B2 depends on dosage and host clone genotype. Total spore counts after growth on live K. pneumoniae (K.pneu abundant) for three replicates each of the original host clone (QS11 for B2qs11 and NC28 for B2nc28) and six nonfarmer clones precolonized by 10% or 0.1% by B2qs11 or B2nc28. Error bars represent SEM.

Fig. 4.

Burkholderia and its associated effects can be eliminated from original hosts by antibiotic treatment. (A) Percent of bacteria-positive sori for 10 farmers with or without prior tetracycline treatment. Error bars represent SEM. (B) Percent of bacteria-positive sori over the course of five social cycles with K. pneumoniae for 10 farmers pretreated with tetracycline. Error bars represent SEM. (C) PCR amplification of eukaryote-, eubacteria-, Burkholderia-, and Klebsiella-specific genes from DNA isolated from the sorus contents of representative tetracycline-treated farmers. (D) Total spore counts after growth on live (food-rich) or dead (food-scarce) K. pneumoniae for three replicates of four farmers with or without prior tetracycline treatment. Error bars represent SEM.

Fig. 5.

Burkholderia and K. pneumoniae can be visualized inside spores after fruiting. (A and B) Burkholderia-negative spores from a tetracycline-treated farmer clone QS70 (A) and a nonfarmer QS9 (B) after growth on K. pneumoniae-GFP and stained with calcofluor-white (pseudocolored yellow) show no evidence of bacterial carriage. (C and D) Spores from a tetracycline-treated farmer clone QS70 (C) and a nonfarmer QS9 (D) after growth with 10% B1qs70-RFP (pseudocolored magenta) and 90% K. pneumoniae-GFP (green) stained with calcofluor-white (pseudocolored yellow) show B1qs70-RFP and K. pneumoniae-GFP inside a portion of the spores.