Browsing by Author "Amenabar, Maximiliano J."

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Acidianus Tailed Spindle Virus: a New Archaeal Large Tailed Spindle Virus Discovered by Culture-Independent Methods
(2016-04) Hochstein, Rebecca A.; Amenabar, Maximiliano J.; Munson-McGee, Jacob H.; Boyd, Eric S.; Young, Mark J.
The field of viral metagenomics has expanded our understanding of viral diversity from all three domains of life (Archaea, Bacteria, and Eukarya). Traditionally, viral metagenomic studies provide information about viral gene content but rarely provide knowledge about virion morphology and/or cellular host identity. Here we describe a new virus, Acidianus tailed spindle virus (ATSV), initially identified by bioinformatic analysis of viral metagenomic data sets from a high-temperature (80°C) acidic (pH 2) hot spring located in Yellowstone National Park, followed by more detailed characterization using only environmental samples without dependency on culturing. Characterization included the identification of the large tailed spindle virion morphology, determination of the complete 70.8-kb circular double-stranded DNA (dsDNA) viral genome content, and identification of its cellular host. Annotation of the ATSV genome revealed a potential three-domain gene product containing an N-terminal leucine-rich repeat domain, followed by a likely posttranslation regulatory region consisting of high serine and threonine content, and a C-terminal ESCRT-III domain, suggesting interplay with the host ESCRT system. The host of ATSV, which is most closely related to Acidianus hospitalis, was determined by a combination of analysis of cellular clustered regularly interspaced short palindromic repeat (CRISPR)/Cas loci and dual viral and cellular fluorescence in situ hybridization (viral FISH) analysis of environmental samples and confirmed by culture-based infection studies. This work provides an expanded pathway for the discovery, isolation, and characterization of new viruses using culture-independent approaches and provides a platform for predicting and confirming virus hosts. IMPORTANCE: Virus discovery and characterization have been traditionally accomplished by using culture-based methods. While a valuable approach, it is limited by the availability of culturable hosts. In this research, we report a virus-centered approach to virus discovery and characterization, linking viral metagenomic sequences to a virus particle, its sequenced genome, and its host directly in environmental samples, without using culture-dependent methods. This approach provides a pathway for the discovery, isolation, and characterization of new viruses. While this study used an acidic hot spring environment to characterize a new archaeal virus, Acidianus tailed spindle virus (ATSV), the approach can be generally applied to any environment to expand knowledge of virus diversity in all three domains of life.
Electron Acceptor Availability Alters Carbon and Energy Metabolism in a Thermoacidophile
(2018-05) Amenabar, Maximiliano J.; Colman, Daniel R.; Poudel, Saroj; Roden, Eric E.; Boyd, Eric S.
The thermoacidophilic Acidianus strain DS80 displays versatility in its energy metabolism and can grow autotrophically and heterotrophically with elemental sulfur (S°), ferric iron (Fe3+) or oxygen (O2) as electron acceptors. Here, we show that autotrophic and heterotrophic growth with S° as the electron acceptor is obligately dependent on hydrogen (H2) as electron donor; organic substrates such as acetate can only serve as a carbon source. In contrast, organic substrates such as acetate can serve as electron donor and carbon source for Fe3+ or O2 grown cells. During growth on S° or Fe3+ with H2 as an electron donor, the amount of CO2 assimilated into biomass decreased when cultures were provided with acetate. The addition of CO2 to cultures decreased the amount of acetate mineralized and assimilated and increased cell production in H2/Fe3+ grown cells but had no effect on H2/S° grown cells. In acetate/Fe3+ grown cells, the presence of H2 decreased the amount of acetate mineralized as CO2 in cultures compared to those without H2. These results indicate that electron acceptor availability constrains the variety of carbon sources used by this strain. Addition of H2 to cultures overcomes this limitation and alters heterotrophic metabolism.
Mechanisms of Mineral Substrate Acquisition in a Thermoacidophile
(2018-06) Amenabar, Maximiliano J.; Boyd, Eric S.
The thermoacidophile Acidianus is widely distributed in Yellowstone National Park hot springs that span large gradients in pH (1.60 to 4.84), temperature (42 to 90 degrees C), and mineralogical composition. To characterize the potential role of flexibility in mineral-dependent energy metabolism in contributing to the widespread ecological distribution of this organism, we characterized the spectrum of minerals capable of supporting metabolism and the mechanisms that it uses to access these minerals. The energy metabolism of Acidianus strain DS80 was supported by elemental sulfur (S-o), a variety of iron (hydr)oxides, and arsenic sulfide. Strain DS80 reduced, oxidized, and disproportionated S-o. Cells growing via S-o reduction and disproportionation did not require direct access to the mineral to reduce it, whereas cells growing via S-o oxidation did require direct access, observations that are attributable to the role of H2S produced by S-o reduction/disproportionation in solubilizing and increasing the bioavailability of S-o. Cells growing via iron (hydr) oxide reduction did not require access to the mineral, suggesting that the cells reduce Fe(III) that is being leached by the acidic growth medium. Cells growing via oxidation of arsenic sulfide with Fe(III) did not require access to the mineral to grow. The stoichiometry of reactants to products indicates that cells oxidize soluble As(III) released from oxidation of arsenic sulfide by aqueous Fe(III). Taken together, these observations underscore the importance of feedbacks between abiotic and biotic reactions in influencing the bioavailability of mineral substrates and defining ecological niches capable of supporting microbial metabolism. IMPORTANCE Mineral sources of electron donor and acceptor that support microbial metabolism are abundant in the natural environment. However, the spectrum of minerals capable of supporting a given microbial strain and the mechanisms that are used to access these minerals in support of microbial energy metabolism are often unknown, in particular among thermoacidophiles. Here, we show that the thermoacidophile Acidianus strain DS80 is adapted to use a variety of iron (hydro) oxide minerals, elemental sulfur, and arsenic sulfide to support growth. Cells rely on a complex interplay of abiologically and biologically catalyzed reactions that increase the solubility or bioavailability of minerals, thereby enabling their use in microbial metabolism.
Origin and Evolution of Flavin-Based Electron Bifurcating Enzymes
(2018-08) Poudel, Saroj; Dunham, Eric C.; Lindsay, Melody R.; Amenabar, Maximiliano J.; Fones, Elizabeth M.; Colman, Daniel R.; Boyd, Eric S.
Twelve evolutionarily unrelated oxidoreductases form enzyme complexes that catalyze the simultaneous coupling of exergonic and endergonic oxidation–reduction reactions to circumvent thermodynamic barriers and minimize free energy loss in a process known as flavin-based electron bifurcation. Common to these 12 bifurcating (Bf) enzymes are protein-bound flavin, the proposed site of bifurcation, and the electron carrier ferredoxin. Despite the documented role of Bf enzymes in balancing the redox state of intracellular electron carriers and in improving the efficiency of cellular metabolism, a comprehensive description of the diversity and evolutionary history of Bf enzymes is lacking. Here, we report the taxonomic distribution, functional diversity, and evolutionary history of Bf enzyme homologs in 4,588 archaeal, bacterial, and eukaryal genomes and 3,136 community metagenomes. Bf homologs were primarily detected in the genomes of anaerobes, including those of sulfate-reducers, acetogens, fermenters, and methanogens. Phylogenetic analyses of Bf enzyme catalytic subunits (oxidoreductases) suggest they were not a property of the Last Universal Common Ancestor of Archaea and Bacteria, which is consistent with the limited and unique taxonomic distributions of enzyme homologs among genomes. Further, phylogenetic analyses of oxidoreductase subunits reveal that non-Bf homologs predate Bf homologs. These observations indicate that multiple independent recruitments of flavoproteins to existing oxidoreductases enabled coupling of numerous new electron Bf reactions. Consistent with the role of these enzymes in the energy metabolism of anaerobes, homologs of Bf enzymes were enriched in metagenomes from subsurface environments relative to those from surface environments. Phylogenetic analyses of homologs from metagenomes reveal that the earliest evolving homologs of most Bf enzymes are from subsurface environments, including fluids from subsurface rock fractures and hydrothermal systems. Collectively, these data suggest strong selective pressures drove the emergence of Bf enzyme complexes via recruitment of flavoproteins that allowed for an increase in the efficiency of cellular metabolism and improvement in energy capture in anaerobes inhabiting a variety of subsurface anoxic habitats where the energy yield of oxidation-reduction reactions is generally low.
Phylogenomic analysis of novel Diaforarchaea is consistent with sulfite but not sulfate reduction in volcanic environments on early Earth
(2020-02) Colman, Daniel R.; Lindsay, Melody R.; Amenabar, Maximiliano J.; Fernandes-Martins, Maria C.; Roden, Eric R.; Boyd, Eric S.
The origin(s) of dissimilatory sulfate and/or (bi)sulfite reducing organisms (SRO) remains enigmatic despite their importance in global carbon and sulfur cycling since at least 3.4 Ga. Here, we describe novel, deep-branching archaeal SRO populations distantly related to other Diaforarchaea from two moderately acidic thermal springs. Dissimilatory (bi)sulfite reductase homologs, DsrABC, encoded in metagenome assembled genomes (MAGs) from spring sediments comprise one of the earliest evolving Dsr lineages. DsrA homologs were expressed in situ under moderately acidic conditions. MAGs lacked genes encoding proteins that activate sulfate prior to (bi)sulfite reduction. This is consistent with sulfide production in enrichment cultures provided sulfite but not sulfate. We suggest input of volcanic sulfur dioxide to anoxic spring-water yields (bi)sulfite and moderately acidic conditions that favor its stability and bioavailability. The presence of similar volcanic springs at the time SRO are thought to have originated (>3.4 Ga) may have supplied (bi)sulfite that supported ancestral SRO. These observations coincide with the lack of inferred SO42− reduction capacity in nearly all organisms with early-branching DsrAB and which are near universally found in hydrothermal environments.
A review of the mechanisms of mineral-based metabolism in early Earth analog rock-hosted hydrothermal ecosystems
(2019-02) Amenabar, Maximiliano J.; Boyd, Eric S.
Prior to the advent of oxygenic photosynthesis similar to 2.8-3.2Ga, life was dependent on chemical energy captured from oxidation-reduction reactions involving minerals or substrates generated through interaction of water with minerals. Terrestrial hydrothermal environments host abundant and diverse non-photosynthetic communities and a variety of minerals that can sustain microbial metabolism. Minerals and substrates generated through interaction of minerals with water are differentially distributed in hot spring environments which, in turn, shapes the distribution of microbial life and the metabolic processes that support it. Emerging evidence suggests that terrestrial hydrothermal environments may have played a role in supporting the metabolism of the earliest forms of microbial life. It follows that these environments and their microbial inhabitants are increasingly being studied as analogs of early Earth ecosystems. Here we review current understanding of the processes that lead to variation in the availability of minerals or mineral-sourced substrates in terrestrial hydrothermal environments. In addition, we summarize proposed mechanisms of mineral substrate acquisition and metabolism in microbial cells inhabiting terrestrial hydrothermal environments, highlighting the importance of the dynamic interplay between biotic and abiotic reactions in influencing mineral substrate bioavailability. An emphasis is placed on mechanisms involved in the solubilization, acquisition, and metabolism of sulfur- and iron-bearing minerals, since these elements were likely integrated into the metabolism of the earliest anaerobic cells.