Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea

Abstract

Background: Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order.

Results: We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis.

Conclusion: The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that subtle, but important genomic differences, coupled with unknown differences in gene expression, distinguish these organisms enough to allow for co-existence. Overall this study reveals shared features of organisms from the Thermoplasmatales lineage and provides new insights into the functioning of AMD communities.

Figure 1

16S rRNA tree indicating the possibility of a candidate class that includes Iplasma.Ferroplasma acidarmanus is Fer1 and Fer2. Bootstrap values are shown at branch splits. Gene start and stop positions and Genbank accession numbers are listed after organism names.

Figure 2

Cluster of unique genes in Gplasma. Arrows are proportional to the length of each gene and indicate its direction of transcription. The gene numbers are shown inside the arrows. All genes are from contig number 13327. Motif and domain-based annotations are shown above the arrows. Genes with no annotations are hypothetical proteins. Rhod indicates a rhodanese-like domain.

Figure 3

Cryo-EM of surface-layer on an AMD plasma cell from the Richmond Mine. Insets show a higher magnification. Arrows point to putative surface-layer proteins. Panel A and panel B show evidence of proteinaceous surface layers in two different cells collected from the Richmond Mine AMD.

Figure 4

Cryo-electron microscopy of AMD plasma cells. Panel A and panel B show evidence of flagella on two different cells collected from the Richmond Mine AMD. Arrows point to flagella. The box surrounds a potential motor protein complex.

Figure 5

Cryo-electron microscopy of AMD plasma cells with putative pili. Panel A and panel B show evidence of pili on two different cells collected from the Richmond Mine AMD. Arrows point to pili. Vesicle-like structures are delineated by a single membrane layer around an ovoid shape in each cell’s cytoplasm.