Saroj Poudel

Defining Electron Bifurcation in the Electron Transferring Flavoprotein Family

Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.

Unification of [FeFe]-hydrogenases into three structural and functional groups

BACKGROUND [FeFe]-hydrogenases (Hyd) are structurally diverse enzymes that catalyze the reversible oxidation of hydrogen (H2). Recent biochemical data demonstrate new functional roles for these enzymes, including those that function in electron bifurcation where an exergonic reaction is coupled with an endergonic reaction to drive the reversible oxidation/production of H2. METHODS To identify the structural determinants that underpin differences in enzyme functionality, a total of 714 homologous sequences of the catalytic subunit, HydA, were compiled. Bioinformatics approaches informed by biochemical data were then used to characterize differences in inferred quaternary structure, HydA active site protein environment, accessory iron-sulfur clusters in HydA, and regulatory proteins encoded in HydA gene neighborhoods. RESULTS HydA homologs were clustered into one of three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while those in G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3) subunits. Variation in the HydA active site and accessory iron-sulfur clusters did not vary by group type. Group-specific regulatory genes were identified in the gene neighborhoods of both G2 and G3 Hyd. Analyses of purified G2 and G3 enzymes by mass spectrometry strongly suggest that they are post-translationally modified by phosphorylation. CONCLUSIONS These results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes, rather than by variation in HydA. GENERAL SIGNIFICANCE This classification scheme provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes.

Microbialite response to an anthropogenic salinity gradient in Great Salt Lake, Utah

A railroad causeway across Great Salt Lake, Utah (GSL), has restricted water flow since its construction in 1959, resulting in a more saline North Arm (NA; 24%-31% salinity) and a less saline South Arm (SA; 11%-14% salinity). Here, we characterized microbial carbonates collected from the SA and the NA to evaluate the effect of increased salinity on community composition and abundance and to determine whether the communities present in the NA are still actively precipitating carbonate or if they are remnant features from prior to causeway construction. SSU rRNA gene abundances associated with the NA microbialite were three orders of magnitude lower than those associated with the SA microbialite, indicating that the latter community is more productive. SSU rRNA gene sequencing and functional gene microarray analyses indicated that SA and NA microbialite communities are distinct. In particular, abundant sequences affiliated with photoautotrophic taxa including cyanobacteria and diatoms that may drive carbonate precipitation and thus still actively form microbialites were identified in the SA microbialite; sequences affiliated with photoautotrophic taxa were in low abundance in the NA microbialite. SA and NA microbialites comprise smooth prismatic aragonite crystals. However, the SA microbialite also contained micritic aragonite, which can be formed as a result of biological activity. Collectively, these observations suggest that NA microbialites are likely to be remnant features from prior to causeway construction and indicate a strong decrease in the ability of NA microbialite communities to actively precipitate carbonate minerals. Moreover, the results suggest a role for cyanobacteria and diatoms in carbonate precipitation and microbialite formation in the SA of GSL.

Identification of MicroRNAs and Transcript Targets in Camelina sativa by Deep Sequencing and Computational Methods

Camelina sativa is an annual oilseed crop that is under intensive development for renewable resources of biofuels and industrial oils. MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play key roles in diverse plant biological processes. Here, we conducted deep sequencing on small RNA libraries prepared from camelina leaves, flower buds and two stages of developing seeds corresponding to initial and peak storage products accumulation. Computational analyses identified 207 known miRNAs belonging to 63 families, as well as 5 novel miRNAs. These miRNAs, especially members of the miRNA families, varied greatly in different tissues and developmental stages. The predicted miRNA target genes are involved in a broad range of physiological functions including lipid metabolism. This report is the first step toward elucidating roles of miRNAs in C. sativa and will provide additional tools to improve this oilseed crop for biofuels and biomaterials.

Geobiological feedbacks and the evolution of thermoacidophiles

Oxygen-dependent microbial oxidation of sulfur compounds leads to the acidification of natural waters. How acidophiles and their acidic habitats evolved, however, is largely unknown. Using 16S rRNA gene abundance and composition data from 72 hot springs in Yellowstone National Park, Wyoming, we show that hyperacidic (pH<3.0) hydrothermal ecosystems are dominated by a limited number of archaeal lineages with an inferred ability to respire O2. Phylogenomic analyses of 584 existing archaeal genomes revealed that hyperacidophiles evolved independently multiple times within the Archaea, each coincident with the emergence of the ability to respire O2, and that these events likely occurred in the recent evolutionary past. Comparative genomic analyses indicated that archaeal thermoacidophiles from independent lineages are enriched in similar protein-coding genes, consistent with convergent evolution aided by horizontal gene transfer. Because the generation of acidic environments and their successful habitation characteristically require O2, these results suggest that thermoacidophilic Archaea and the acidity of their habitats co-evolved after the evolution of oxygenic photosynthesis. Moreover, it is likely that dissolved O2 concentrations in thermal waters likely did not reach levels capable of sustaining aerobic thermoacidophiles and their acidifying activity until ~0.8 Ga, when present day atmospheric levels were reached, a time period that is supported by our estimation of divergence times for archaeal thermoacidophilic clades.

A pathway for biological methane production using bacterial iron-only nitrogenase

Methane (CH4) is a potent greenhouse gas that is released from fossil fuels and is also produced by microbial activity, with at least one billion tonnes of CH4 being formed and consumed by microorganisms in a single year1. Complex methanogenesis pathways used by archaea are the main route for bioconversion of carbon dioxide (CO2) to CH4 in nature2–4. Here, we report that wild-type iron-iron (Fe-only) nitrogenase from the bacterium Rhodopseudomonas palustris reduces CO2 simultaneously with nitrogen gas (N2) and protons to yield CH4, ammonia (NH3) and hydrogen gas (H2) in a single enzymatic step. The amount of CH4 produced by purified Fe-only nitrogenase was low compared to its other products, but CH4 production by this enzyme in R. palustris was sufficient to support the growth of an obligate CH4-utilizing Methylomonas strain when the two microorganisms were grown in co-culture, with oxygen (O2) added at intervals. Other nitrogen-fixing bacteria that we tested also formed CH4 when expressing Fe-only nitrogenase, suggesting that this is a general property of this enzyme. The genomes of 9% of diverse nitrogen-fixing microorganisms from a range of environments encode Fe-only nitrogenase. Our data suggest that active Fe-only nitrogenase, present in diverse microorganisms, contributes CH4 that could shape microbial community interactions.Here the authors show that a bacterial iron-only nitrogenase can generate CH4 in a single step, which can consequently be used for growth by a CH4-utilizing Methylomonas strain.

The deep, hot biosphere: Twenty-five years of retrospection

Twenty-five years ago this month, Thomas Gold published a seminal manuscript suggesting the presence of a “deep, hot biosphere” in the Earth’s crust. Since this publication, a considerable amount of attention has been given to the study of deep biospheres, their role in geochemical cycles, and their potential to inform on the origin of life and its potential outside of Earth. Overwhelming evidence now supports the presence of a deep biosphere ubiquitously distributed on Earth in both terrestrial and marine settings. Furthermore, it has become apparent that much of this life is dependent on lithogenically sourced high-energy compounds to sustain productivity. A vast diversity of uncultivated microorganisms has been detected in subsurface environments, and we show that H2, CH4, and CO feature prominently in many of their predicted metabolisms. Despite 25 years of intense study, key questions remain on life in the deep subsurface, including whether it is endemic and the extent of its involvement in the anaerobic formation and degradation of hydrocarbons. Emergent data from cultivation and next-generation sequencing approaches continue to provide promising new hints to answer these questions. As Gold suggested, and as has become increasingly evident, to better understand the subsurface is critical to further understanding the Earth, life, the evolution of life, and the potential for life elsewhere. To this end, we suggest the need to develop a robust network of interdisciplinary scientists and accessible field sites for long-term monitoring of the Earth’s subsurface in the form of a deep subsurface microbiome initiative.

The DUF59 Containing Protein SufT Is Involved in the Maturation of Iron-Sulfur (FeS) Proteins during Conditions of High FeS Cofactor Demand in Staphylococcus aureus

Proteins containing DUF59 domains have roles in iron-sulfur (FeS) cluster assembly and are widespread throughout Eukarya, Bacteria, and Archaea. However, the function(s) of this domain is unknown. Staphylococcus aureus SufT is composed solely of a DUF59 domain. We noted that sufT is often co-localized with sufBC, which encode for the Suf FeS cluster biosynthetic machinery. Phylogenetic analyses indicated that sufT was recruited to the suf operon, suggesting a role for SufT in FeS cluster assembly. A S. aureus ΔsufT mutant was defective in the assembly of FeS proteins. The DUF59 protein Rv1466 from Mycobacterium tuberculosis partially corrected the phenotypes of a ΔsufT mutant, consistent with a widespread role for DUF59 in FeS protein maturation. SufT was dispensable for FeS protein maturation during conditions that imposed a low cellular demand for FeS cluster assembly. In contrast, the role of SufT was maximal during conditions imposing a high demand for FeS cluster assembly. SufT was not involved in the repair of FeS clusters damaged by reactive oxygen species or in the physical protection of FeS clusters from oxidants. Nfu is a FeS cluster carrier and nfu displayed synergy with sufT. Furthermore, introduction of nfu upon a multicopy plasmid partially corrected the phenotypes of the ΔsufT mutant. Biofilm formation and exoprotein production are critical for S. aureus pathogenesis and vancomycin is a drug of last-resort to treat staphylococcal infections. Defective FeS protein maturation resulted in increased biofilm formation, decreased production of exoproteins, increased resistance to vancomycin, and the appearance of phenotypes consistent with vancomycin-intermediate resistant S. aureus. We propose that SufT, and by extension the DUF59 domain, is an accessory factor that functions in the maturation of FeS proteins. In S. aureus, the involvement of SufT is maximal during conditions of high demand for FeS proteins.

H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle

Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP+ oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron‑sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.

Two functionally distinct NADP(+)-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus

Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP+ oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed, depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes, and NfnII does not catalyze the NfnI bifurcating reaction. Instead, it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and also to catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown.