Comparative proteomics of a versatile, marine, iron-oxidizing chemolithoautotroph.

This study conducted a comparative proteomic analysis to identify potential genetic markers for the biological function of chemolithoautotrophic iron oxidation in the marine bacterium Ghiorsea bivora. To date, this is the only characterized species in the class Zetaproteobacteria that is not an obligate iron-oxidizer, providing a unique opportunity to investigate differential protein expression to identify key genes involved in iron-oxidation at circumneutral pH. Over 1000 proteins were identified under both iron- and hydrogen-oxidizing conditions, with differentially expressed proteins found in both treatments. Notably, a gene cluster upregulated during iron oxidation was identified. This cluster contains genes encoding for cytochromes that share sequence similarity with the known iron-oxidase, Cyc2. Interestingly, these cytochromes, conserved in both Bacteria and Archaea, do not exhibit the typical ß-barrel structure of Cyc2. This cluster potentially encodes a biological nanowire-like transmembrane complex containing multiple redox proteins spanning the inner membrane, periplasm, outer membrane, and extracellular space. The upregulation of key genes associated with this complex during iron-oxidizing conditions was confirmed by quantitative reverse transcription-PCR. These findings were further supported by electromicrobiological methods, which demonstrated negative current production by G. bivora in a three-electrode system poised at a cathodic potential. This research provides significant insights into the biological function of chemolithoautotrophic iron oxidation.

Widespread energy limitation to life in global subseafloor sediments.

Microbial cells buried in subseafloor sediments comprise a substantial portion of Earth's biosphere and control global biogeochemical cycles; however, the rate at which they use energy (i.e., power) is virtually unknown. Here, we quantify organic matter degradation and calculate the power utilization of microbial cells throughout Earth's Quaternary-age subseafloor sediments. Aerobic respiration, sulfate reduction, and methanogenesis mediate 6.9, 64.5, and 28.6% of global subseafloor organic matter degradation, respectively. The total power utilization of the subseafloor sediment biosphere is 37.3 gigawatts, less than 0.1% of the power produced in the marine photic zone. Aerobic heterotrophs use the largest share of global power (54.5%) with a median power utilization of 2.23 × 10-18 watts per cell, while sulfate reducers and methanogens use 1.08 × 10-19 and 1.50 × 10-20 watts per cell, respectively. Most subseafloor cells subsist at energy fluxes lower than have previously been shown to support life, calling into question the power limit to life.

A Genus Definition for Bacteria and Archaea Based on a Standard Genome Relatedness Index.

Genus assignment is fundamental in the characterization of microbes, yet there is currently no unambiguous way to demarcate genera solely using standard genomic relatedness indices. Here, we propose an approach to demarcate genera that relies on the combined use of the average nucleotide identity, genome alignment fraction, and the distinction between type- and non-type species. More than 3,500 genomes representing type strains of species from >850 genera of either bacterial or archaeal lineages were tested. Over 140 genera were analyzed in detail within the taxonomic context of order/family. Significant genomic differences between members of a genus and type species of other genera in the same order/family were conserved in 94% of the cases. Nearly 90% (92% if polyphyletic genera are excluded) of the type strains were classified in agreement with current taxonomy. The 448 type strains that need reclassification directly impact 33% of the genera analyzed in detail. The results provide a first line of evidence that the combination of genomic indices provides added resolution to effectively demarcate genera within the taxonomic framework that is currently based on the 16S rRNA gene. We also identify the emergence of natural breakpoints at the genome level that can further help in the circumscription of taxa, increasing the proportion of directly impacted genera to at least 43% and pointing at inaccuracies on the use of the 16S rRNA gene as a taxonomic marker, despite its precision. Altogether, these results suggest that genomic coherence is an emergent property of genera in Bacteria and ArchaeaIMPORTANCE In recent decades, the taxonomy of Bacteria and Archaea, and therefore genus designation, has been largely based on the use of a single ribosomal gene, the 16S rRNA gene, as a taxonomic marker. We propose an approach to delineate genera that excludes the direct use of the 16S rRNA gene and focuses on a standard genome relatedness index, the average nucleotide identity. Our findings are of importance to the microbiology community because the emergent properties of Bacteria and Archaea that are identified in this study will help assign genera with higher taxonomic resolution.

Effects of trace element concentrations on culturing thermophiles.

The majority of microorganisms in natural environments resist laboratory cultivation. Sometimes referred to as 'unculturable', many phylogenetic groups are known only by fragments of recovered DNA. As a result, the ecological significance of whole branches of the 'tree of life' remains a mystery; this is particularly true when regarding genetic material retrieved from extreme environments. Geochemically relevant media have been used to improve the success of culturing Archaea and Bacteria, but these efforts have focused primarily on optimizing pH, alkalinity, major ions, carbon sources, and electron acceptor-donor pairs. Here, we cultured thermophilic microorganisms from 'Sylvan Spring' (Yellowstone National Park, USA) on media employing different trace element solutions, including one that mimicked the source fluid of the inocula. The growth medium that best simulated trace elements found in 'Sylvan Spring' produced a more diverse and faster growing mixed culture than media containing highly elevated trace element concentrations. The elevated trace element medium produced fewer phylotypes and inhibited growth. Trace element concentrations appear to influence growth conditions in extreme environments. Incorporating geochemical data into cultivation attempts may improve culturing success.

Energy sources for chemolithotrophs in an arsenic- and iron-rich shallow-sea hydrothermal system.

The hydrothermally influenced sediments of Tutum Bay, Ambitle Island, Papua New Guinea, are ideal for investigating the chemolithotrophic activities of micro-organisms involved in arsenic cycling because hydrothermal vents there expel fluids with arsenite (As(III)) concentrations as high as 950 µg L(-1) . These hot (99 °C), slightly acidic (pH ~6), chemically reduced, shallow-sea vent fluids mix with colder, oxidized seawater to create steep gradients in temperature, pH, and concentrations of As, N, Fe, and S redox species. Near the vents, iron oxyhydroxides precipitate with up to 6.2 wt% arsenate (As(V)). Here, chemical analyses of sediment porewaters from 10 sites along a 300-m transect were combined with standard Gibbs energies to evaluate the energy yields (-ΔG(r)) from 19 potential chemolithotrophic metabolisms, including As(V) reduction, As(III) oxidation, Fe(III) reduction, and Fe(II) oxidation reactions. The 19 reactions yielded 2-94 kJ mol(-1) e(-) , with aerobic oxidation of sulphide and arsenite the two most exergonic reactions. Although anaerobic As(V) reduction and Fe(III) reduction were among the least exergonic reactions investigated, they are still potential net metabolisms. Gibbs energies of the arsenic redox reactions generally correlate linearly with pH, increasing with increasing pH for As(III) oxidation and decreasing with increasing pH for As(V) reduction. The calculated exergonic energy yields suggest that micro-organisms could exploit diverse energy sources in Tutum Bay, and examples of micro-organisms known to use these chemolithotrophic metabolic strategies are discussed. Energy modeling of redox reactions can help target sampling sites for future microbial collection and cultivation studies.

Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and bacteria.

Thermophilic and hyperthermophilic Archaea and Bacteria have been isolated from marine hydrothermal systems, heated sediments, continental solfataras, hot springs, water heaters, and industrial waste. They catalyze a tremendous array of widely varying metabolic processes. As determined in the laboratory, electron donors in thermophilic and hyperthermophilic microbial redox reactions include H2, Fe(2+), H2S, S, S2O3(2-), S4O6(2-), sulfide minerals, CH4, various mono-, di-, and hydroxy-carboxylic acids, alcohols, amino acids, and complex organic substrates; electron acceptors include O2, Fe(3+), CO2, CO, NO3(-), NO2(-), NO, N2O, SO4(2-), SO3(2-), S2O3(2-), and S. Although many assimilatory and dissimilatory metabolic reactions have been identified for these groups of microorganisms, little attention has been paid to the energetics of these reactions. In this review, standard molal Gibbs free energies (DeltaGr(0)) as a function of temperature to 200 degrees C are tabulated for 370 organic and inorganic redox, disproportionation, dissociation, hydrolysis, and solubility reactions directly or indirectly involved in microbial metabolism. To calculate values of DeltaGr(0) for these and countless other reactions, the apparent standard molal Gibbs free energies of formation (DeltaG(0)) at temperatures to 200 degrees C are given for 307 solids, liquids, gases, and aqueous solutes. It is shown that values of DeltaGr(0) for many microbially mediated reactions are highly temperature dependent, and that adopting values determined at 25 degrees C for systems at elevated temperatures introduces significant and unnecessary errors. The metabolic processes considered here involve compounds that belong to the following chemical systems: H-O, H-O-N, H-O-S, H-O-N-S, H-O-C(inorganic), H-O-C, H-O-N-C, H-O-S-C, H-O-N-S-C(amino acids), H-O-S-C-metals/minerals, and H-O-P. For four metabolic reactions of particular interest in thermophily and hyperthermophily (knallgas reaction, anaerobic sulfur and nitrate reduction, and autotrophic methanogenesis), values of the overall Gibbs free energy (DeltaGr) as a function of temperature are calculated for a wide range of chemical compositions likely to be present in near-surface and deep hydrothermal and geothermal systems.

Calculation of the standard molal thermodynamic properties of aqueous biomolecules at elevated temperatures and pressures II. Unfolded proteins.

Equations of state for completely unfolded proteins have been generated from group additivity algorithms and the revised Helgeson-Kirkham-Flowers (HKF) equations of state to compute the standard molal thermodynamic properties of these molecules at elevated temperatures and pressures. The requisite equations of state parameters were computed from those of groups retrieved by regression of experimental calorimetric and densimetric data reported in the literature. This approach permits calculation of the standard molal thermodynamic properties as a function of temperature and pressure for any completely unfolded protein for which the amino acid sequence is known. Calculations of this kind have been carried out for 11 thermophilic proteins. The thermodynamic properties reported below can be combined with those for protein unfolding to compute the corresponding properties of completely folded (i.e. native) proteins.

Energetics of amino acid synthesis in hydrothermal ecosystems.

Thermodynamic calculations showed that the autotrophic synthesis of all 20 protein-forming amino acids was energetically favored in hot (100 degrees C), moderately reduced, submarine hydrothermal solutions relative to the synthesis in cold (18 degrees C), oxidized, surface seawater. The net synthesis reactions of 11 amino acids were exergonic in the hydrothermal solution, but all were endergonic in surface seawater. The synthesis of the requisite amino acids of nine thermophilic and hyperthermophilic proteins in a 100 degreesC hydrothermal solution yielded between 600 and 8000 kilojoules per mole of protein, which is energy that is available to drive the intracellular synthesis of enzymes and other biopolymers in hyperthermophiles thriving in these ecosystems.