Amoebae interact with bacteria in diverse and multifaceted ways. Amoeba predation can serve as a selective pressure for the development of bacterial virulence traits. Bacteria may also adapt to life inside amoebae, resulting in symbiotic relationships (pathogenic or mutualistic). Indeed, amoebae are often infected with bacterial endosymbionts. Acanthamoeba and Hartmannella harbor symbionts from five distinct lineages within the Proteobacteria, Bacteroidetes, and Chlamydiae. Here, we PCR-screened an extensive collection of Dictyostelium discoideum wild isolates for the presence of bacterial symbionts. For the first time we found that obligate symbionts are surprisingly common in this highly-studied amoeba species, identified in 42% of screened isolates (N=730). Members of the environmental Chlamydiae are particularly prevalent. These strains are novel and phylogenetically distinct. We also found Amoebophilus symbionts in 8% of screened isolates (N=730). Under laboratory conditions, antibiotic-cured amoebae behave similarly to their endosymbiont-infected counterparts, suggesting that endosymbionts do not significantly impact host fitness, at least in the laboratory. We found several natural isolates were co-infected with different endosymbionts. The high prevalence and novelty of amoeba endosymbiont clades in the model organism D. discoideum opens the door to future research on the significance and mechanisms of amoeba-symbiont interactions.

Summary As predators of bacteria, amoebae select for traits that allow bacteria to become symbionts by surviving phagocytosis and exploiting the eukaryotic intracellular environment. Soil‐dwelling social amoebae can help us answer questions about the natural ecology of these amoeba‐bacteria symbioses along the pathogen‐mutualist spectrum. Our objective was to characterize the natural bacterial microbiome of phylogenetically and morphologically diverse social amoeba species using next‐generation sequencing of 16S rRNA amplicons directly from amoeba fruiting bodies. We found six phyla of amoeba‐associated bacteria: Proteobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, Firmicutes, and Acidobacteria. The most common associates of amoebae were classified to order Chlamydiales and genus Burkholderia‐Caballeronia‐Paraburkholderia. These bacteria were present in multiple amoeba species across multiple locations. While there was substantial intraspecific variation, there was some evidence for host specificity and differentially abundant taxa between different amoeba hosts. Amoebae microbiomes were distinct from the microbiomes of their soil habitat, and soil pH affected amoeba microbiome diversity. Alpha‐diversity was unsurprisingly lower in amoebae samples compared with soil, but beta‐diversity between amoebae samples was higher than between soil samples. Further exploration of social amoebae microbiomes may help us understand the roles of bacteria, host, and environment on symbiotic interactions and microbiome formation in basal eukaryotic organisms.

Amoebae interact with bacteria in multifaceted ways. Amoeba predation can serve as a selective pressure for the development of bacterial virulence traits. Bacteria may also adapt to life inside amoebae, resulting in symbiotic relationships. Indeed, particular lineages of obligate bacterial endosymbionts have been found in different amoebae. Here, we screened an extensive collection of Dictyostelium discoideum wild isolates for the presence of these bacterial symbionts using endosymbiont specific PCR primers. We find that these symbionts are surprisingly common, identified in 42% of screened isolates (N = 730). Members of the Chlamydiae phylum are particularly prevalent, occurring in 27% of the amoeba isolated. They are novel and phylogenetically distinct from other Chlamydiae. We also found Amoebophilus symbionts in 8% of screened isolates (N = 730). Antibiotic-cured amoebae behave similarly to their Chlamydiae or Amoebophilus-infected counterparts, suggesting that these endosymbionts do not significantly impact host fitness, at least in the laboratory. We found several natural isolates were co-infected with multiple endosymbionts, with no obvious fitness effect of coinfection under laboratory conditions. The high prevalence and novelty of amoeba endosymbiont clades in the model organism D. discoideum opens the door to future research on the significance and mechanisms of amoeba-symbiont interactions.