Microbiology & Cell Biology
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Item Geobiological feedbacks, oxygen, and the evolution of nitrogenase(2019-02) Mus, Florence; Colman, Daniel R.; Peters, John W.; Boyd, Eric S.Biological nitrogen fixation via the activity of nitrogenase is one of the most important biological innovations, allowing for an increase in global productivity that eventually permitted the emergence of higher forms of life. The complex metalloenzyme termed nitrogenase contains complex iron-sulfur cofactors. Three versions of nitrogenase exist that differ mainly by the presence or absence of a heterometal at the active site metal cluster (either Mo or V). Mo-dependent nitrogenase is the most common while V-dependent or heterometal independent (Fe-only) versions are often termed alternative nitrogenases since they have apparent lower activities for N2 reduction and are expressed in the absence of Mo. Phylogenetic data indicates that biological nitrogen fixation emerged in an anaerobic, thermophilic ancestor of hydrogenotrophic methanogens and later diversified via lateral gene transfer into anaerobic bacteria, and eventually aerobic bacteria including Cyanobacteria. Isotopic evidence suggests that nitrogenase activity existed at 3.2 Ga, prior to the advent of oxygenic photosynthesis and rise of oxygen in the atmosphere, implying the presence of favorable environmental conditions for oxygen-sensitive nitrogenase to evolve. Following the proliferation of oxygenic phototrophs, diazotrophic organisms had to develop strategies to protect nitrogenase from oxygen inactivation and generate the right balance of low potential reducing equivalents and cellular energy for growth and nitrogen fixation activity. Here we review the fundamental advances in our understanding of biological nitrogen fixation in the context of the emergence, evolution, and taxonomic distribution of nitrogenase, with an emphasis placed on key events associated with its emergence and diversification from anoxic to oxic environments.Item Physiological adaptations to serpentinization in the Samail Ophiolite, Oman(2019-03) Fones, Elizabeth M.; Colman, Daniel R.; Kraus, Emily A.; Nothaft, Daniel B.; Poudel, Saroj; Rempfert, Kaitlin R.; Spear, John R.; Templeton, Alexis S.; Boyd, Eric S.Hydration of ultramafic rock during the geologic process of serpentinization can generate reduced substrates that microorganisms may use to fuel their carbon and energy metabolisms. However, serpentinizing environments also place multiple constraints on microbial life by generating highly reduced hyperalkaline waters that are limited in dissolved inorganic carbon. To better understand how microbial life persists under these conditions, we performed geochemical measurements on waters from a serpentinizing environment and subjected planktonic microbial cells to metagenomic and physiological analyses. Metabolic potential inferred from metagenomes correlated with fluid type, and genes involved in anaerobic metabolisms were enriched in hyperalkaline waters. The abundance of planktonic cells and their rates of utilization of select single-carbon compounds were lower in hyperalkaline waters than alkaline waters. However, the ratios of substrate assimilation to dissimilation were higher in hyperalkaline waters than alkaline waters, which may represent adaptation to minimize energetic and physiologic stress imposed by highly reducing, carbon-limited conditions. Consistent with this hypothesis, estimated genome sizes and average oxidation states of carbon in inferred proteomes were lower in hyperalkaline waters than in alkaline waters. These data suggest that microorganisms inhabiting serpentinized waters exhibit a unique suite of physiological adaptations that allow for their persistence under these polyextremophilic conditions.Item 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.Item Mixing of meteoric and geothermal fluids supports hyperdiverse chemosynthetic hydrothermal communities(2019-02) Colman, Daniel R.; Lindsay, Melody R.; Boyd, Eric S.Little is known of how mixing of meteoric and geothermal fluids supports biodiversity in non-photosynthetic ecosystems. Here, we use metagenomic sequencing to investigate a chemosynthetic microbial community in a hot spring (SJ3) of Yellowstone National Park that exhibits geochemistry consistent with mixing of a reduced volcanic gas-influenced end member with an oxidized near-surface meteoric end member. SJ3 hosts an exceptionally diverse community with representatives from similar to 50% of known higher-order archaeal and bacterial lineages, including several divergent deep-branching lineages. A comparison of functional potential with other available chemosynthetic community metagenomes reveals similarly high diversity and functional potentials (i.e., incorporation of electron donors supplied by volcanic gases) in springs sourced by mixed fluids. Further, numerous closely related SJ3 populations harbor differentiated metabolisms that may function to minimize niche overlap, further increasing endemic diversity. We suggest that dynamic mixing of waters generated by subsurface and near-surface geological processes may play a key role in the generation and maintenance of chemosynthetic biodiversity in hydrothermal and other similar environments.Item Metagenomes from high-temperature chemotrophic systems reveal geochemical controls on microbial community structure and function(2010-03) Inskeep, William P.; Rusch, Douglas B.; Jay, Zackary J.; Herrgard, Markus J.; Kozubal, Mark A.; Richardson, Toby H.; Macur, Richard E.; Hamamura, Natsuko; Jennings, Ryan deM.; Fouke, Bruce W.; Reysenbach, Anna-Louise; Roberto, Frank; Young, Mark J.; Schwartz, Ariel; Boyd, Eric S.; Badger, Jonathan H.; Mathur, Eric J.; Ortmann, Alice C.; Bateson, Mary M.; Geesey, Gill G.; Frazier, MarvinThe Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14–15,000 Sanger reads per site) was obtained for five high-temperature (>65°C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O2 influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H2-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, Fe, O2) control microbial community structure and function in YNP geothermal springs.Item Effects of salinity on microbialite-associated production in Great Salt Lake, Utah(2019-03) Lindsay, Melody R.; Johnston, Rachel E.; Baxter, Bonnie K.; Boyd, Eric S.Microbialites, organosedimentary carbonate structures, cover approximately 20% of the basin floor in the south arm of Great Salt Lake, which ranges from ~12-15% salinity. Photosynthetic microbial mats associated with these benthic mounds contribute biomass that supports secondary production in the ecosystem, including that of the brine shrimp, Artemia franciscana. However, the effects of predicted increases in the salinity of the lake on the productivity and composition of these mats and on A. franciscana fecundity is not well documented. In the present study, we applied molecular and microcosm-based approaches to investigate the effects of changing salinity on i) the primary productivity, abundance, and composition of microbialite-associated mats of GSL, and ii) the fecundity and survivability of the secondary consumer, A. franciscana. When compared to microcosms incubated closest to the in situ measured salinity of 15.6%, the abundance of 16S rRNA gene templates increased in microcosms with lower salinities and decreased in those with higher salinities following seven weeks incubation. The abundance of 16S rRNA gene sequences affiliated with dominant primary producers, including the cyanobacterium Euhalothece and the diatom Navicula, increased in microcosms incubated at decreased salinity but decreased in microcosms incubated at increased salinity. Increased salinity also decreased the rate of primary production in microcosm assays containing mats incubated for 7 weeks and decreased the number of A. franciscana cysts that hatched and survived. These results indicate that an increase in the salinity of GSL is likely to negatively impact the productivity of microbialite communities and the fecundity and survivability of A. franciscana. These observations suggest that a sustained increase in the salinity of GSL and the effects this has on primary and secondary production could have an upward and negative cascading effect on higher trophic level ecological compartments that depend on A. franciscana as a food source, including a number of species of migratory birds.Item The path of electron transfer to nitrogenase in a phototrophic alpha-proteobacterium(2018-07) Fixen, Kathryn R.; Pal Chowdhury, Nilanjan; Martinez-Perez, Marta; Poudel, Saroj; Boyd, Eric S.; Harwood, Caroline S.The phototrophic alpha-proteobacterium, Rhodopseudomonas palustris, is a model for studies of regulatory and physiological parameters that control the activity of nitrogenase. This enzyme produces the energy-rich compound H2 , in addition to converting N2 gas to NH3 . Nitrogenase is an ATP-requiring enzyme that uses large amounts of reducing power, but the electron transfer pathway to nitrogenase in R. palustris was incompletely known. Here, we show that the ferredoxin, Fer1, is the primary but not sole electron carrier protein encoded by R. palustris that serves as an electron donor to nitrogenase. A flavodoxin, FldA, is also an important electron donor, especially under iron limitation. We present a model where the electron bifurcating complex, FixABCX, can reduce both ferredoxin and flavodoxin to transfer electrons to nitrogenase, and we present bioinformatic evidence that FixABCX and Fer1 form a conserved electron transfer pathway to nitrogenase in nitrogen-fixing proteobacteria. These results may be useful in the design of strategies to reroute electrons generated during metabolism of organic compounds to nitrogenase to achieve maximal activity.Item 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.Item Electron transfer to nitrogenase in different genomic and metabolic backgrounds(2018-02) Poudel, Saroj; Colman, Daniel R.; Fixen, Kathryn R.; Ledbetter, Rhesa N.; Zheng, Yanning; Pence, Natasha; Seefeldt, Lance C.; Peters, John W.; Hardwood, Caroline S.; Boyd, Eric S.Nitrogenase catalyzes the reduction of dinitrogen (N2) using low potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O2) sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd/Fld-reducing enzymes in 359 genomes of putative N2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified and their distributions largely corresponded to differences in the host cells' ability to integrate O2 or light into energy metabolism. Predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the level of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O2 or light into their energy metabolism. Acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex.Item 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.