Eric S. Boyd

Engineering algae for biohydrogen and biofuel production.

There is currently substantial interest in utilizing eukaryotic algae for the renewable production of several bioenergy carriers, including starches for alcohols, lipids for diesel fuel surrogates, and H2 for fuel cells. Relative to terrestrial biofuel feedstocks, algae can convert solar energy into fuels at higher photosynthetic efficiencies, and can thrive in salt water systems. Recently, there has been considerable progress in identifying relevant bioenergy genes and pathways in microalgae, and powerful genetic techniques have been developed to engineer some strains via the targeted disruption of endogenous genes and/or transgene expression. Collectively, the progress that has been realized in these areas is rapidly advancing our ability to genetically optimize the production of targeted biofuels.

Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA(DeltaEFG).

Complex enzymes containing Fe–S clusters are ubiquitous in nature, where they are involved in a number of fundamental processes including carbon dioxide fixation, nitrogen fixation and hydrogen metabolism. Hydrogen metabolism is facilitated by the activity of three evolutionarily and structurally unrelated enzymes: the [NiFe]-hydrogenases, [FeFe]-hydrogenases and [Fe]-hydrogenases (Hmd). The catalytic core of the [FeFe]-hydrogenase (HydA), termed the H-cluster, exists as a [4Fe–4S] subcluster linked by a cysteine thiolate to a modified 2Fe subcluster with unique non-protein ligands. The 2Fe subcluster and non-protein ligands are synthesized by the hydrogenase maturation enzymes HydE, HydF and HydG; however, the mechanism, synthesis and means of insertion of H-cluster components remain unclear. Here we show the structure of HydAΔEFG (HydA expressed in a genetic background devoid of the active site H-cluster biosynthetic genes hydE, hydF and hydG) revealing the presence of a [4Fe–4S] cluster and an open pocket for the 2Fe subcluster. The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe–4S] subcluster by generalized host-cell machinery and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery. Insertion of the 2Fe subcluster presumably occurs through a cationically charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. The structure, together with phylogenetic analysis, indicates that HydA emerged within bacteria most likely from a Nar1-like ancestor lacking the 2Fe subcluster, and that this was followed by acquisition in several unicellular eukaryotes.

Metagenomes from high-temperature chemotrophic systems reveal geochemical controls on microbial community structure and function.

The 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.

(FeFe)- and (NiFe)-hydrogenase diversity, mechanism, and maturation

The [FeFe]- and [NiFe]-hydrogenases catalyze the formal interconversion between hydrogen and protons and electrons, possess characteristic non-protein ligands at their catalytic sites and thus share common mechanistic features. Despite the similarities between these two types of hydrogenases, they clearly have distinct evolutionary origins and likely emerged from different selective pressures. [FeFe]-hydrogenases are widely distributed in fermentative anaerobic microorganisms and likely evolved under selective pressure to couple hydrogen production to the recycling of electron carriers that accumulate during anaerobic metabolism. In contrast, many [NiFe]-hydrogenases catalyze hydrogen oxidation as part of energy metabolism and were likely key enzymes in early life and arguably represent the predecessors of modern respiratory metabolism. Although the reversible combination of protons and electrons to generate hydrogen gas is the simplest of chemical reactions, the [FeFe]- and [NiFe]-hydrogenases have distinct mechanisms and differ in the fundamental chemistry associated with proton transfer and control of electron flow that also help to define catalytic bias. A unifying feature of these enzymes is that hydrogen activation itself has been restricted to one solution involving diatomic ligands (carbon monoxide and cyanide) bound to an Fe ion. On the other hand, and quite remarkably, the biosynthetic mechanisms to produce these ligands are exclusive to each type of enzyme. Furthermore, these mechanisms represent two independent solutions to the formation of complex bioinorganic active sites for catalyzing the simplest of chemical reactions, reversible hydrogen oxidation. As such, the [FeFe]- and [NiFe]-hydrogenases are arguably the most profound case of convergent evolution. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

[FeFe]‐Hydrogenase Cyanide Ligands Derived From S‐Adenosylmethionine‐Dependent Cleavage of Tyrosine

Whats your poison? Hydrogenases catalyze the reversible formation of dihydrogen from two electrons and two protons. The maturation of the [FeFe]-hydrogenase active-site cofactor (H cluster) requires three gene products, HydE, HydF, and HydG. Cyanide has been characterized as one of the products of tyrosine cleavage by the S-adenosylmethionine-dependent enzyme HydG, clarifying its role in H-cluster biosynthesis. DOA=deoxyadenosine.

Isolation, Characterization, and Ecology of Sulfur-Respiring Crenarchaea Inhabiting Acid-Sulfate-Chloride-Containing Geothermal Springs in Yellowstone National Park

ABSTRACT Elemental sulfur (S0) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S0-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72°C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81°C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S0, and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S0 flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively.

A thermophilic bacterial origin and subsequent constraints by redox, light and salinity on the evolution of the microbial mercuric reductase.

Mercuric reductase (MerA) is central to the mercury (Hg) resistance (mer) system, catalyzing the reduction of ionic Hg to volatile Hg(0). A total of 213 merA homologues were identified in sequence databases, the majority of which belonged to microbial lineages that occupy oxic environments. merA was absent among phototrophs and in lineages that inhabit anoxic environments. Phylogenetic reconstructions of MerA indicate that (i) merA originated in a thermophilic bacterium following the divergence of the Archaea and Bacteria with a subsequent acquisition in Archaea via horizontal gene transfer (HGT), (ii) HGT of merA was rare across phylum boundaries and (iii) MerA from marine bacteria formed distinct and strongly supported lineages. Collectively, these observations suggest that a combination of redox, light and salinity conditions constrain MerA to microbial lineages that occupy environments where the most oxidized and toxic form of Hg, Hg(II), predominates. Further, the taxon-specific distribution of MerA with and without a 70 amino acid N-terminal extension may reflect intracellular levels of thiols. In conclusion, MerA likely evolved following the widespread oxygenation of the biosphere in a thermal environment and its subsequent evolution has been modulated by the interactions of Hg with the intra- and extracellular environment of the organism.

Molecular evidence for an active endogenous microbiome beneath glacial ice.

Geologic, chemical and isotopic evidence indicate that Earth has experienced numerous intervals of widespread glaciation throughout its history, with roughly 11% of present day Earth’s land surface covered in ice. Despite the pervasive nature of glacial ice both today and in Earth’s past and the potential contribution of these systems to global biogeochemical cycles, the composition and phylogenetic structure of an active microbial community in subglacial systems has yet to be described. Here, using RNA-based approaches, we demonstrate the presence of active and endogenous archaeal, bacterial and eukaryal assemblages in cold (0–1 °C) subglacial sediments sampled from Robertson Glacier, Alberta, Canada. Patterns in the phylogenetic structure and composition of subglacial sediment small subunit (SSU) ribosomal RNA (rRNA) assemblages indicate greater diversity and evenness than in glacial surface environments, possibly due to facilitative or competitive interactions among populations in the subglacial environment. The combination of phylogenetically more even and more diverse assemblages in the subglacial environment suggests minimal niche overlap and optimization to capture a wider spectrum of the limited nutrients and chemical energy made available from weathering of bedrock minerals. The prevalence of SSU rRNA affiliated with lithoautotrophic bacteria, autotrophic methane producing archaea and heterotrophic eukarya in the subglacial environment is consistent with this hypothesis and suggests an active contribution to the global carbon cycle. Collectively, our findings demonstrate that subglacial environments harbor endogenous active ecosystems that have the potential to impact global biogeochemical cycles over extended periods of time.

The modular respiratory complexes involved in hydrogen and sulfur metabolism by heterotrophic hyperthermophilic archaea and their evolutionary implications.

Hydrogen production is a vital metabolic process for many anaerobic organisms, and the enzyme responsible, hydrogenase, has been studied since the 1930s. A novel subfamily with unique properties was recently recognized, represented by the 14-subunit membrane-bound [NiFe] hydrogenase from the archaeon Pyrococcus furiosus. This so-called energy-converting hydrogenase links the thermodynamically favorable oxidation of ferredoxin with the formation of hydrogen and conserves energy in the form of an ion gradient. It is therefore a simple respiratory system within a single complex. This hydrogenase shows a modular composition represented by a Na(+)/H(+) antiporter domain (Mrp) and a [NiFe] hydrogenase domain (Mbh). An analysis of the large number of microbial genome sequences available shows that homologs of Mbh and Mrp tend to be clustered within the genomes of a limited number of archaeal and bacterial species. In several instances, additional genes are associated with the Mbh and Mrp gene clusters that encode proteins that catalyze the oxidation of formate, CO or NAD(P)H. The Mbh complex also shows extensive homology to a number of subunits within the NADH quinone oxidoreductase or complex I family. The respiratory-type membrane-bound hydrogenase complex appears to be closely related to the common ancestor of complex I and [NiFe] hydrogenases in general.

Diversity, Abundance, and Potential Activity of Nitrifying and Nitrate-Reducing Microbial Assemblages in a Subglacial Ecosystem

ABSTRACT Subglacial sediments sampled from beneath Robertson Glacier (RG), Alberta, Canada, were shown to harbor diverse assemblages of potential nitrifiers, nitrate reducers, and diazotrophs, as assessed by amoA, narG, and nifH gene biomarker diversity. Although archaeal amoA genes were detected, they were less abundant and less diverse than bacterial amoA, suggesting that bacteria are the predominant nitrifiers in RG sediments. Maximum nitrification and nitrate reduction rates in microcosms incubated at 4°C were 280 and 18.5 nmol of N per g of dry weight sediment per day, respectively, indicating the potential for these processes to occur in situ. Geochemical analyses of subglacial sediment pore waters and bulk subglacial meltwaters revealed low concentrations of inorganic and organic nitrogen compounds. These data, when coupled with a C/N atomic ratio of dissolved organic matter in subglacial pore waters of ∼210, indicate that the sediment communities are N limited. This may reflect the combined biological activities of organic N mineralization, nitrification, and nitrate reduction. Despite evidence of N limitation and the detection of nifH, we were unable to detect biological nitrogen fixation activity in subglacial sediments. Collectively, the results presented here suggest a role for nitrification and nitrate reduction in sustaining microbial life in subglacial environments. Considering that ice currently covers 11% of the terrestrial landmass and has covered significantly greater portions of Earth at times in the past, the demonstration of nitrification and nitrate reduction in subglacial environments furthers our understanding of the potential for these environments to contribute to global biogeochemical cycles on glacial-interglacial timescales.