We found inconsistencies in the commonly used data for chlorophyll analysis in 80% acetone. Recently developed extinction coefficients for chlorophyll b in NN-dimethylformamide (DMF) based on values from 80% acetone are low as a result of these inconsistencies. We determined extinction coefficients of chlorophyll a (Chl a) and chlorophyll b (Chi b) in DMF for wavelengths of 618 to 665 nanometers. The (2) and soybeans (3).The extinction coefficients necessary for the quantification of Chl a, Chl b, and total Chl in DMF were determined by Moran (5). The extinction coefficient for Chl b was determined using the coefficient for Chl b in 80% actone determined by MacKinney (4). This coefficient is not consistent with the results of Vernon (10) which suggest that if the equations developed by Moran (5) are used, then the calculations for Chl b content may be off by more than 10%. An error also would be introduced into the calculation of total Chl. One objective of this study was to determine which extinction coefficients were most consistent for Chl a and b in 80% acetone and DMF.

Methanogenesis is an ancient metabolism of key ecological relevance, with direct impact on the evolution of Earth’s climate. Recent results suggest that the diversity of methane metabolisms and their derivations have probably been vastly underestimated. Here, by probing thousands of publicly available metagenomes for homologues of methyl-coenzyme M reductase complex (MCR), we have obtained ten metagenome-assembled genomes (MAGs) belonging to potential methanogenic, anaerobic methanotrophic and short-chain alkane oxidizing archaea. Five of these MAGs represent under-sampled (e.g., Verstraetearchaeota, Methanonatronarchaeia, ANME-1) or previously genomically undescribed (ANME-2c) archaeal lineages. The remaining five MAGs correspond to lineages that are only distantly related to previously known methanogens and span the entire archaeal phylogeny. Comprehensive comparative annotation significantly expands the metabolic diversity and energy conservation systems of MCR-bearing archaea. It also suggests the potential existence of a yet uncharacterized type of methanogenesis linked to short-chain alkane/fatty acid oxidation in a previously undescribed class of archaea (‘ Ca . Methanoliparia’). We redefine a common core of marker genes specific to methanogenic, anaerobic methanotrophic and short-chain alkane-oxidizing archaea, and propose a possible scenario for the evolutionary and functional transitions that led to the emergence of such metabolic diversity.

Tracing the origin of CRISPR-Cas CRISPR-Cas systems have transformed genome editing and other biotechnologies; however, the broader origins and diversity of RNA-guided nucleases have largely remained unexplored. Altae-Tran et al . show that three distinct transposon-encoded proteins, IscB, IsrB, and TnpB, are naturally occurring, reprogrammable RNA-guided DNA nucleases (see the Perspective by Rousset and Sorek). In addition to identifying diverse guide-encoding mechanisms, the authors elucidate the evolutionary relationship between IsrB, IscB, and CRISPR-Cas9. Overall, these newly characterized systems, called OMEGA (for obligate mobile element–guided activity) systems, are found in all domains of life and may be harnessed for biotechnology development. —DJ

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.

The Yellowstone geothermal complex contains over 10,000 diverse geothermal features that host numerous phylogenetically deeply rooted and poorly understood archaea, bacteria, and viruses. Microbial communities in high-temperature environments are generally less diverse than soil, marine, sediment, or lake habitats and therefore offer a tremendous opportunity for studying the structure and function of different model microbial communities using environmental metagenomics. One of the broader goals of this study was to establish linkages among microbial distribution, metabolic potential, and environmental variables. Twenty geochemically distinct geothermal ecosystems representing a broad spectrum of Yellowstone hot-spring environments were used for metagenomic and geochemical analysis and included approximately equal numbers of: (1) phototrophic mats, (2) “filamentous streamer” communities, and (3) archaeal-dominated sediments. The metagenomes were analyzed using a suite of complementary and integrative bioinformatic tools, including phylogenetic and functional analysis of both individual sequence reads and assemblies of predominant phylotypes. This volume identifies major environmental determinants of a large number of thermophilic microbial lineages, many of which have not been fully described in the literature nor previously cultivated to enable functional and genomic analyses. Moreover, protein family abundance comparisons and in-depth analyses of specific genes and metabolic pathways relevant to these hot-spring environments reveal hallmark signatures of metabolic capabilities that parallel the distribution of phylotypes across specific types of geochemical environments.

Geothermal springs within Yellowstone National Park (YNP) often contain arsenic (As) at concentrations of 10-40 microM, levels that are considered toxic to many organisms. Arsenite (As(III)) is often the predominant valence state at the point of discharge but is rapidly oxidized to arsenate (As(V)) during transport in shallow surface water. The current study was designed to establish rates and possible mechanisms of As(III) oxidation and to characterize the geochemical environment associated with predominant microbial mats in a representative acid-sulfate-chloride (pH 3.1) thermal (58-62 degrees C) spring in Norris Basin, YNP. At the spring origin, total soluble As was predominantly As(III) at concentrations of 33 microM. No oxidation of As(III) was detected over the first 2.7 m downstream from the spring source, corresponding to an area dominated by a yellow filamentous S0-rich microbial mat However, rapid oxidation of As(III) to As(V) was observed between 2.7 and 5.6 m, corresponding to termination of the S0-rich mats, decreases in dissolved sulfide, and commencement of a brown Fe/As-rich mat. Rates of As(II) oxidation were estimated, yielding an apparent first-order rate constant of 1.2 min(-1) (half-life = 0.58 min). The oxidation of As(III) was shown to require live organisms present just prior to and within the Fe/As-rich mat. Complementary analytical tools used to characterize the brown mat revealed an As:Fe molar ratio of 0.7 and suggested that this filamentous microbial mat contains iron(III) oxyhydroxide coprecipitated with As(V). Results from the current work are the first to provide a comprehensive characterization of microbially mediated As(III) oxidation and the geochemical environments associated with microbial mats in acid-sulfate-chloride springs of YNP.

Microbial populations responsible for the oxidation and reduction of As were examined in unsaturated (aerobic) soil columns treated with 75 microM arsenite [As(III)] or 250 microM arsenate [As(V)]. Arsenite [As(III)] was rapidly oxidized to As(V) via microbial activity, whereas no apparent reduction of As(V) was observed in the column experiments. Eight aerobic heterotrophic bacteria with varying As redox phenotypes were isolated from the same columns. Three isolates, identified as Agrobacterium tumefaciens-, Pseudomonas fluorescens-, and Variovorax paradoxus-like organisms (based on 16S sequence), were As(III) oxidizers, and all were detected in community DNA fingerprints generated by PCR coupled with denaturing gradient gel electrophoresis. The five other isolates were identified (16S gene sequence) as A. tumefaciens, Flavobacterium sp., Microbacterium sp., and two Arthrobacter sp. -like organisms and were shown to rapidly reduce As(V) under aerobic conditions. Although the two A. tumefaciens-like isolates exhibited opposite As redox activity,their 16S rDNA sequences (approximately 1400 bp) were 100% identical, and both were shown to contain putative arsC genes. Our results support the hypothesis that bacteria capable of either oxidizing As(III) or reducing As(V) coexist and are ubiquitous in soil environments, suggesting that the relative abundance and metabolic activity of specific microbial populations plays an important role in the speciation of inorganic As in soil pore waters.

Chemolithotrophic micro‐organisms are important primary producers in high‐temperature geothermal environments and may catalyse a number of different energetically favourable redox reactions as a primary energy source. Analysis of geochemical constituents followed by chemical speciation and subsequent calculation of reaction free energies (ΔGrxn) is a useful tool for evaluating the thermodynamic favourability and potential energy available for microbial metabolism. The primary goal of this study was to examine relationships among geochemical gradients and microbial population distribution, and to evaluate the utility of energetic approaches for predicting microbial metabolism from free‐energy calculations, utilizing as examples, several geothermal habitats in Yellowstone National Park where thorough geochemical and phylogenetic analyses have been performed. Acidic (pH ∼ 3) and near‐neutral (pH ∼ 6–7) geothermal springs were chosen for their range in geochemical properties. Aqueous and solid phase samples obtained from the source pools and the outflow channels of each spring were characterized for all major chemical constituents using laboratory and field methods to accurately measure the concentrations of predominant oxidized and reduced species. Reaction free energies (ΔGrxn) for 33 oxidation–reduction reactions potentially important to chemolithotrophic micro‐organisms were calculated at relevant spring temperatures after calculating ion activities using an aqueous equilibrium model. Free‐energy values exhibit significant variation among sites for reactions with pH dependence. For example, free‐energy values for reactions involving Fe3+ are especially variable across sites due in large part to the pH dependence of Fe3+ activity, and exhibit changes of up to 40 kJ mol−1 electron from acidic to near neutral geothermal springs. Many of the detected 16S rRNA gene sequences represent organisms whose metabolisms are consistent with exergonic processes. However, sensitivity analyses demonstrated that reaction free energies do not generally represent the steep gradients in local geochemical conditions resulting from air–water gas exchange and solid phase deposition that are important in defining microbial habitats and 16S rRNA gene sequence distribution within geothermal outflow channels.