Isolation and Genome Analysis of an Amoeba-Associated Bacterium Dyella terrae Strain Ely Copper Mine From Acid Rock Drainage in Vermont, United States

Abstract

Protozoa play important roles in microbial communities, regulating populations via predation and contributing to nutrient cycling. While amoebae have been identified in acid rock drainage (ARD) systems, our understanding of their symbioses in these extreme environments is limited. Here, we report the first isolation of the amoeba Stemonitis from an ARD environment as well as the genome sequence and annotation of an associated bacterium, Dyella terrae strain Ely Copper Mine, from Ely Brook at the Ely Copper Mine Superfund site in Vershire, Vermont, United States. Fluorescent in situ hybridization analysis showed this bacterium colonizing cells of Stemonitis sp. in addition to being outside of amoebal cells. This amoeba-resistant bacterium is Gram-negative with a genome size of 5.36 Mbp and GC content of 62.5%. The genome of the D. terrae strain Ely Copper Mine encodes de novo biosynthetic pathways for amino acids, carbohydrates, nucleic acids, and lipids. Genes involved in nitrate (1) and sulfate (7) reduction, metal (229) and antibiotic resistance (37), and secondary metabolite production (6) were identified. Notably, 26 hydrolases were identified by RAST as well as other biomass degradation genes, suggesting roles in carbon and energy cycling within the microbial community. The genome also contains type IV secretion system genes involved in amoebae resistance, revealing how this bacterium likely survives predation from Stemonitis sp. This genome analysis and the association of D. terrae strain Ely Copper Mine with Stemonitis sp. provide insight into the functional roles of amoebae and bacteria within ARD environments.

Keywords: Dyella terrae; Stemonitis sp.; acid rock drainage; amoeba-associated bacterium; amoebae.

Figure 1

Study site. Map and photograph of Ely Brook (EB-90M) at Ely Copper Mine Superfund site in Vershire, VT on 15 November 2018.

Figure 2

Differential interference contrast micrographs of isolated amoeba. Images show morphology features highlighting the diversity of shapes and sizes of the isolated amoeba. (A) Global view of the different shapes of amoebae. Panels (B) and (C) focus on rounded (arrows) and flagellated (arrows) amoebae, respectively. The bar length represents 10 µm for all panels.

Figure 3

Maximum likelihood tree based on near-complete 16S rRNA gene sequences showing the phylogeny of Dyella sp. Sequences of Frateuria were used as an outgroup. Bootstrap (left) and SH-like approximate likelihood ratio (right) support values are expressed in percentages at nodes. Bar represents 0.01 substitutions per nucleotide position. Circle and square symbols represent species isolated from water and soil, respectively. The colors of the symbols indicate whether the environment was acidic (red) or either neutral/no pH was reported (orange). Isolates without associated metadata have no symbol.

Figure 4

FISH showing Dyella in association with Stemonitis sp. FISH was performed using (A) a Stemonitis-specific Cy5 probe; (B) EUB3338 I-III FITC probes for labeling eubacteria; (C) DAPI; and (D) a Dyella-specific probe Cy3 probe. (E) The composite image of all labeling highlights the superimposition of eubacteria- and Dyella-specific FISH. The bar length represents 5 μm for all panels.

Figure 5

Three-dimensional FISH analysis of D. terrae strain Ely Copper Mine inside Stemonitis sp. FISH microscopy images were analyzed in-depth to determine the extracellular (panel A) and the intracellular (panel B) location of Dyella (in red) and other bacteria (in green, panel C). The margin images underneath and to the right of panels (A-C) represent projections through x-y and z planes, respectively. The colors white and blue correspond to the cy5 probe against Stemonitis and DAPI, respectively. The bar length represents 2 µm for all panels.