Structural and thermodynamic insights into the Cren7 mediated DNA organization in Crenarchaeota.

Archaea have histone homologues and chromatin proteins to organize their DNA into a compact form. This allows them to survive in extreme climates. Cren7 is one such chromatin protein conserved in Crenarchaeota. When Cren7 binds to model natural DNA, calf thymus DNA (CTD, 58% AT content) and polynucleotides under adverse solution conditions (high temperature, ionic strength), CD bands at 275-290 nm shift to higher wavelengths indicating structural changes in DNA. It formed a strong complex with CTD and poly(dA-dT)·poly(dA-dT), via a combination of electrostatic and non-electrostatic interactions. A low binding enthalpy indicated that the process was driven by entropy. The interaction was independent of the nature of the anions present in the solution. On studying the variation in protein affinity with salt concentration, it was estimated that the electrostatic interaction at the interface involves 3 pairs of ions at the protein-DNA interface. The affinity and binding site size decreased on changing the pH of the solution (between pH 6 and 8), but temperature did not result in such effects. Cren7 bound to 10 bp of DNA, increasing its flexibility and thermal stability by more than 30 °C. Increasing the amount of Cren7 produces cooperative structural transitions in DNAs without any similar transition in the protein. These crucial binding parameters, energetics, and structural changes decipher the mystery of Cren7 mediated DNA organization in Crenarchaeota.

Soil ammonia-oxidizing archaea in a paddy field with different irrigation and fertilization managements.

Because ammonia-oxidizing archaea (AOA) are ubiquitous and highly abundant in almost all terrestrial soils, they play an important role in soil nitrification. However, the changes in the structure and function of AOA communities and their edaphic drivers in paddy soils under different fertilization and irrigation regimes remain unclear. In this study, we investigated AOA abundance, diversity and activity in acid paddy soils by a field experiment. Results indicated that the highest potential ammonia oxidation (PAO) (0.011 µg NO 2 -  -N g-1 d.w.day-1) was found in T2 (optimal irrigation and fertilization)-treated soils, whereas the lowest PAO (0.004 µg NO 2 -  -N g-1 d.w.day-1) in T0 (traditional irrigation)- treated soils. Compared with the T0-treated soil, the T2 treatment significantly (P < 0.05) increased AOA abundances. Furthermore, the abundance of AOA was significantly (P < 0.01) positively correlated with pH, soil organic carbon (SOC), and PAO. Meanwhile, pH and SOC content were significantly (P < 0.05) higher in the T2-treated soil than those in the T1 (traditional irrigation and fertilization)- treated soil. In addition, these two edaphic factors further influenced the AOA community composition. The AOA phylum Crenarchaeota was mainly found in the T2-treated soils. Phylogenetic analysis revealed that most of the identified OTUs of AOA were mainly affiliated with Crenarchaeota. Furthermore, the T2 treatment had higher rice yield than the T0 and T1 treatments. Together, our findings confirm that T2 might ameliorate soil chemical properties, regulate the AOA community structure, increase the AOA abundance, enhance PAO and consequently maintain rice yields in the present study.

Convergent Evolution of a Promiscuous 3-Hydroxypropionyl-CoA Dehydratase/Crotonyl-CoA Hydratase in Crenarchaeota and Thaumarchaeota.

The autotrophic 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle functions in thermoacidophilic, (micro)aerobic, hydrogen-oxidizing Crenarchaeota of the order Sulfolobales as well as in mesophilic, aerobic, ammonia-oxidizing Thaumarchaeota. Notably, the HP/HB cycle evolved independently in these two archaeal lineages, and crenarchaeal and thaumarchaeal versions differ regarding their enzyme properties and phylogeny. These differences result in altered energetic efficiencies between the variants. Compared to the crenarchaeal HP/HB cycle, the thaumarchaeal variant saves two ATP equivalents per turn, rendering it the most energy-efficient aerobic pathway for carbon fixation. Characteristically, the HP/HB cycle includes two enoyl coenzyme A (CoA) hydratase reactions: the 3-hydroxypropionyl-CoA dehydratase reaction and the crotonyl-CoA hydratase reaction. In this study, we show that both reactions are catalyzed in the aforementioned archaeal groups by a promiscuous 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase (Msed_2001 in crenarchaeon Metallosphaera sedula and Nmar_1308 in thaumarchaeon Nitrosopumilus maritimus). Although these two enzymes are homologous, they are closely related to bacterial enoyl-CoA hydratases and were retrieved independently from the same enzyme pool by the ancestors of Crenarchaeota and Thaumarchaeota, despite the existence of multiple alternatives. This striking similarity in the emergence of enzymes involved in inorganic carbon fixation from two independently evolved pathways highlights that convergent evolution of autotrophy could be much more widespread than anticipated.IMPORTANCE Inorganic carbon fixation is the most important biosynthetic process on Earth and the oldest type of metabolism. The autotrophic HP/HB cycle functions in Crenarchaeota of the order Sulfolobales and in ammonia-oxidizing Archaea of the phylum Thaumarchaeota that are highly abundant in marine, terrestrial, and geothermal environments. Bioinformatic prediction of the autotrophic potential of microorganisms or microbial communities requires identification of enzymes involved in autotrophy. However, many microorganisms possess several isoenzymes that may potentially catalyze the reactions of the cycle. Here, we studied the enzymes catalyzing 3-hydroxypropionyl-CoA dehydration and crotonyl-CoA hydration in Nitrosopumilus maritimus (Thaumarchaeota) as well as in Metallosphaera sedula (Crenarchaeota). We showed that both reactions were catalyzed by homologous promiscuous enzymes, which evolved independently from each other from their bacterial homologs. Furthermore, the HP/HB cycle is of applied value, and knowledge of its enzymes is necessary to transfer them to a heterologous host for synthesis of various value-added products.

Metabolic versatility of freshwater sedimentary archaea feeding on different organic carbon sources.

Members of the phylum Bathyarchaeota and the class Thermoplasmata are widespread in marine and freshwater sediments where they have been recognized as key players in the carbon cycle. Here, we tested the responsiveness of archaeal communities on settled plant debris and sediment from a karstic lake to different organic carbon amendments (amino acids, plant-derived carbohydrates, and aromatics) using a lab-scale microcosm. Changes in the composition and abundance of sediment and biofilm archaeal communities in both DNA and RNA fractions were assessed by 16S rRNA gene amplicon sequencing and qPCR, respectively, after 7 and 30 days of incubation. Archaeal communities showed compositional changes in terms of alpha and beta diversity in relation to the type of carbon source (amino acids vs. plant-derived compounds), the nucleic acid fraction (DNA vs. RNA), and the incubation time (7 vs. 30 days). Distinct groups within the Bathyarchaeota (Bathy-15 and Bathy-6) and the Thermoplasmata (MBG-D) differently reacted to carbon supplements as deduced from the analysis of RNA libraries. Whereas Bathyarchaeota in biofilms showed a long-term positive response to humic acids, their counterparts in the sediment were mainly stimulated by the addition of tryptophan, suggesting the presence of different subpopulations in both habitats. Overall, our work presents an in vitro assessment of the versatility of archaea inhabiting freshwater sediments towards organic carbon and introduces settled leaf litter as a new habitat for the Bathyarchaeota and the Thermoplasmata.

Anaerobic digestion of spent mushroom substrate under thermophilic conditions: performance and microbial community analysis.

Spent mushroom substrate (SMS) is the residue of edible mushroom production occurring in huge amounts. The SMS residue can be digested for biogas production in the mesophilic anaerobic digestion. In the present study, performance of batch thermophilic anaerobic digestion (TAD) of SMS was investigated as well as the interconnected microbial population structure changes. The analyzed batch TAD process lasted for 12 days with the cumulative methane yields of 177.69 mL/g volatile solid (VS). Hydrolytic activities of soluble sugar, crude protein, and crude fat in SMS were conducted mainly in the initial phase, accompanied by the excessive accumulation of volatile fatty acids and low methane yield. Biogas production increased dramatically from days 4 to 6. The degradation rates of cellulose and hemicellulose were 47.53 and 55.08%, respectively. The high-throughput sequencing of 16S rRNA gene amplicons revealed that Proteobacteria (56.7%-62.8%) was the dominant phylum in different fermentative stages, which was highly specific compared with other anaerobic processes of lignocellulosic materials reported in the literature. Crenarchaeota was abundant in the archaea. The most dominant genera of archaea were retrieved as Methanothermobacter and Methanobacterium, but the latter decreased sharply with time. This study shows that TAD is a feasible method to handle the waste SMS.

Roles of Leu28 side chain intercalation in the interaction between Cren7 and DNA.

Crenarchaeal chromatin protein Cren7 binds double-stranded DNA in the minor groove, introducing a sharp single-step DNA kink. The side chain of Leu28, a residue conserved among all Cren7 homologs, intercalates into the kinked DNA step. In the present study, we replaced Leu28 with a residue containing a hydrophobic side chain of different sizes (i.e. L28A, L28V, L28I, L28M and L28F). Both the stability of the Cren7-DNA complex and the ability of Cren7 to constrain DNA supercoils correlated well with the size of the intercalated side chain. Structural analysis shows that L28A induces a kink (∼43°), nearly as sharp as that produced by wild-type Cren7 (∼48°), in the bound DNA fragment despite the lack of side chain intercalation. In another duplex DNA fragment, L28F inserts a large hydrophobic side chain deep into the DNA step, but introduces a smaller kink (∼39°) than that formed by the wild-type protein (∼50°). Mutation of Leu28 into methionine yields two protein conformers differing in loop ß3-ß4 orientation, DNA-binding surface and DNA geometry in the protein-DNA structure. Our results indicate that side chain intercalation is not directly responsible for DNA kinking or bending by Cren7, but plays a critical role in the stabilization of the Cren7-DNA complex. In addition, the flexibility of loop ß3-ß4 in Cren7, as revealed in the crystal structure of L28M-DNA, may serve a role in the modulation of chromosomal organization and function in the cell.

Comparative analysis of codon usage bias in Crenarchaea and Euryarchaea genome reveals differential preference of synonymous codons to encode highly expressed ribosomal and RNA polymerase proteins.

The present study was undertaken to investigate the pattern of optimal codon usage in Archaea. Comparative analysis was executed to understand the pattern of codon usage bias between the high expression genes (HEG) and the whole genomes in two Archaeal phyla, Crenarchaea and Euryarchaea. The G+C% of the HEG was found to be less in comparison to the genome G+C% in Crenarchaea, whereas reverse was the case in Euryarchaea. The preponderance of U/A ending codons that code for HEG in Crenarchaea was in sharp contrast to the C/G ended ones in Euryarchaea. The analysis revealed prevalence of Uending codons even within theWWY(nucleotide ambiguity code) families in Crenarchaea vis-à-vis Euryarchaea, bacteria and Eukarya. No plausible interpretation of the observed disparity could be made either in the context of tRNA gene composition or genome G+C%. The results in this study attested that the preferential biasness for codons in HEG of Crenarchaea might be different from Euryarchaea. The main highlights are (i) varied CUB in the HEG and in the whole genomes in Euryarchaea and Crenarchaea. (ii) Crenarchaea was found to have some unusual optimal codons (OCs) compared to other organisms. (iii) G+C% (and GC3) of the HEG were different from the genome G+C% in the two phyla. (iv) Genome G+C% and tRNA gene number failed to explain CUB in Crenarchaea. (v) Translational selection is possibly responsible for A+T rich OCs in Crenarchaea.

Protein phosphorylation and its role in archaeal signal transduction.

Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies.

Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment.

Biological features can be inferred, based on genomic data, for many microbial lineages that remain uncultured. However, cultivation is important for characterizing an organism's physiology and testing its genome-encoded potential. Here we use single-cell genomics to infer cultivation conditions for the isolation of an ectosymbiotic Nanoarchaeota ('Nanopusillus acidilobi') and its host (Acidilobus, a crenarchaeote) from a terrestrial geothermal environment. The cells of 'Nanopusillus' are among the smallest known cellular organisms (100-300 nm). They appear to have a complete genetic information processing machinery, but lack almost all primary biosynthetic functions as well as respiration and ATP synthesis. Genomic and proteomic comparison with its distant relative, the marine Nanoarchaeum equitans illustrate an ancient, common evolutionary history of adaptation of the Nanoarchaeota to ectosymbiosis, so far unique among the Archaea.

Sulfate Reduction and Inorganic Carbon Assimilation in Acidic Thermal Springs of the Kamchatka Peninsula.

Thermoacidophilic sulfate reduction remains a poorly studied process, which was investigated in the present work. Radioisotope analysis with 35S-Iabeled sulfate was used to determine the rates of dissimilatory sulfate reduction in acidic thermal springs of Kamchatka, Russia. Sulfate reduction rates were found to vary from 0.054 to 12.9 nmol S04/(cm3 day). The Neftyanaya ploshchadka spring (Uzon caldera, 60'C, pH 4.2) and Oreshek spring (Mutnovskii volcano, 91'C, pH 3.5) exhibited the highest activity of sulfate-reducing prokaryotes. Stable enrich- ment'cultures reducing sulfate at pH and temperature values close to'the environmental ones were obtained from these springs. Analysis of the 16S rRNA gene sequences revealed that'a chemolithoautotrophic bacterium Ther- modesufobium sp. 3127-1 was responsible for sulfate reduction in the enrichment from the Oil Site spring. A chemoorganoheterotrophic archaeon Vulcanisaeta sp. 3102-1 (phylum Crenarchaeota) was identified in the en- richment from Oreshek spring. Thus, dissimilatory sulfate reduction under thermoacidophilic conditions was demonstrated and the agents responsible for this process were revealed.

Auxotrophy and intrapopulation complementary in the 'interactome' of a cultivated freshwater model community.

Microorganisms are usually studied either in highly complex natural communities or in isolation as monoclonal model populations that we manage to grow in the laboratory. Here, we uncover the biology of some of the most common and yet-uncultured bacteria in freshwater environments using a mixed culture from Lake Grosse Fuchskuhle. From a single shotgun metagenome of a freshwater mixed culture of low complexity, we recovered four high-quality metagenome-assembled genomes (MAGs) for metabolic reconstruction. This analysis revealed the metabolic interconnectedness and niche partitioning of these naturally dominant bacteria. In particular, vitamin- and amino acid biosynthetic pathways were distributed unequally with a member of Crenarchaeota most likely being the sole producer of vitamin B12 in the mixed culture. Using coverage-based partitioning of the genes recovered from a single MAG intrapopulation metabolic complementarity was revealed pointing to 'social' interactions for the common good of populations dominating freshwater plankton. As such, our MAGs highlight the power of mixed cultures to extract naturally occurring 'interactomes' and to overcome our inability to isolate and grow the microbes dominating in nature.

Uncultured Desulfobacteraceae and Crenarchaeotal group C3 incorporate 13C-acetate in coastal marine sediment.

Stable isotope probing (SIP) of deoxyribonucleic acid (DNA) was used to identify microbes incorporating (13) C-labeled acetate in sulfate-reducing sediment from Aarhus Bay, Denmark. Sediment was incubated in medium containing 10 mM sulfate and different (13) C-acetate (10, 1, 0.1 mM) concentrations. The resultant changes in microbial community composition were monitored in total and SIP-fractionated DNA during long-term incubations. Chemical analyses demonstrated metabolic activity in all sediment slurries, with sulfate-reducing activity largely determined by initial acetate concentrations. Sequencing of 16S rRNA gene PCR amplicons showed that the incubations shifted the bacterial but not the archaeal community composition. After 3 months of incubation, only sediment slurries incubated with 10 mM (13) C-acetate showed detectable (13) C-DNA labeling. Based on 16S rRNA and dsrB gene PCR amplicon sequencing, the (13) C-labeled DNA pool was dominated by a single type of sulfate reducer representing a novel genus in the family Desulfobacteraceae. In addition, members of the uncultivated Crenarchaeotal group C3 were enriched in the (13) C-labeled DNA. Our results were reproducible across biological replicate experiments and provide new information about the identities of uncultured acetate-consuming bacteria and archaea in marine sediments.

Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota.

UNLABELLED: Autotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylum Crenarchaeota. Aerobic members of the order Sulfolobales utilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobic Thermoproteales use the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways in Archaea is limited. We applied a comparative genomics approach to predict novel autotrophic regulons in the Crenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in the Sulfolobales (HHC box) and Thermoproteales (DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in all Sulfolobales genomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed by in vitro binding assays with the recombinant HhcR protein from Metallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the order Thermoproteales. DhcR in Thermoproteus neutrophilus (Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data in Metallosphaera and Thermoproteus spp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in the Crenarchaeota. IMPORTANCE: Little is known about transcriptional regulation of carbon dioxide fixation pathways in Archaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages of Archaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages of Crenarchaeota and to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays in Metallosphaera spp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways in Archaea.

Co-occurence of Crenarchaeota, Thermoplasmata and methanogens in anaerobic sludge digesters.

16S rRNA Crenarchaeota and Thermoplasmata sequences retrieved from 22 anaerobic digesters were analysed. 4.8 and 0.53 % of archaeal sequences were simultaneously affiliated to these lineages. A core of 2 operational taxonomic units (OTUs) representing 0.6 to -33.6 % of all archaeal sequences were defined for the Crenarchaeotes and identified to already known but not yet cultivable organisms in almost half of the digesters sampled. For the Thermoplasmata, apparently less abundant with 0.7 to -4.7 % of the archaeal sequences, 3 OTUs were identified. We showed here that Crenarchaeotes coexist with methanogens and are particularly abundant when Arch I lineage (also called WSA2 by Hugenholtz) is dominant in digesters. Moreover, Thermoplasmata were detected when Crenarchaeota were present. Interactions between methanogens, Crenarchaeotea and Thermoplamata were thus discussed.

Interrogation of Chesapeake Bay sediment microbial communities for intrinsic alkane-utilizing potential under anaerobic conditions.

Based on the transient exposure of Chesapeake Bay sediments to hydrocarbons and the metabolic versatility of known anaerobic alkane-degrading microorganisms, it was hypothesized that distinct Bay sediment communities, governed by geochemical gradients, would have intrinsic alkane-utilizing potential under sulfate-reducing and/or methanogenic conditions. Sediment cores were collected along a transect of the Bay. Community DNA was interrogated via pyrosequencing of 16S rRNA genes, PCR of anaerobic hydrocarbon activation genes, and qPCR of 16S rRNA genes and genes involved in sulfate reduction/methanogenesis. Site sediments were used to establish microcosms amended with n-hexadecane under sulfate-reducing and methanogenic conditions. Sequencing of 16S rRNA genes indicated that sediments associated with hypoxic water columns contained significantly greater proportions of Bacteria and Archaea consistent with syntrophic degradation of organic matter and methanogenesis compared to less reduced sediments. Microbial taxa frequently associated with hydrocarbon-degrading communities were found throughout the Bay, and the genetic potential for hydrocarbon metabolism was demonstrated via the detection of benzyl-(bssA) and alkylsuccinate synthase (assA) genes. Although microcosm studies did not indicate sulfidogenic alkane degradation, the data suggested that methanogenic conversion of alkanes was occurring. These findings highlight the potential role that anaerobic microorganisms could play in the bioremediation of hydrocarbons in the Bay.

The archaeal lipidome in estuarine sediment dominated by members of the Miscellaneous Crenarchaeotal Group.

The anoxic sediments of the White Oak River estuary comprise a distinctive sulfate-methane transition zone (SMTZ) and natural enrichment of the archaea affiliated with the Miscellaneous Crenarchaeotal Group (MCG). Archaeal biphytanes were generally depleted in (13) C, with δ(13) C values being less than -35‰, indicative of production by active sedimentary archaeal populations. Multivariate analysis of the downcore distributions of 63 lipid biomarkers identified three major groups of lipids that were enriched in the surface, SMTZ or subsurface depths. Intact polar lipids with phosphatidylglycerol headgroups and glycerol dibiphytanyl glycerol tetraethers containing one, two or three cyclopentane rings were enriched at the base of the SMTZ and likely represent the accumulated product of a small but active ANME-1 community. The recently identified butanetriol dibiphytanyl glycerol tetraethers (BDGT), which increased relatively to other lipids with depth, were correlated with the relative abundance of MCG in archaeal 16S rRNA clone libraries, and were (13) C depleted throughout the depth profile, suggesting BDGT lipids as putative biomarkers of an MCG community that may either be autotrophic or feeding on (13) C-depleted organic substrates transported by porewater.

Urea uptake and carbon fixation by marine pelagic bacteria and archaea during the Arctic summer and winter seasons.

How Arctic climate change might translate into alterations of biogeochemical cycles of carbon (C) and nitrogen (N) with respect to inorganic and organic N utilization is not well understood. This study combined 15N uptake rate measurements for ammonium, nitrate, and urea with 15N- and 13C-based DNA stable-isotope probing (SIP). The objective was to identify active bacterial and archeal plankton and their role in N and C uptake during the Arctic summer and winter seasons. We hypothesized that bacteria and archaea would successfully compete for nitrate and urea during the Arctic winter but not during the summer, when phytoplankton dominate the uptake of these nitrogen sources. Samples were collected at a coastal station near Barrow, AK, during August and January. During both seasons, ammonium uptake rates were greater than those for nitrate or urea, and nitrate uptake rates remained lower than those for ammonium or urea. SIP experiments indicated a strong seasonal shift of bacterial and archaeal N utilization from ammonium during the summer to urea during the winter but did not support a similar seasonal pattern of nitrate utilization. Analysis of 16S rRNA gene sequences obtained from each SIP fraction implicated marine group I Crenarchaeota (MGIC) as well as Betaproteobacteria, Firmicutes, SAR11, and SAR324 in N uptake from urea during the winter. Similarly, 13C SIP data suggested dark carbon fixation for MGIC, as well as for several proteobacterial lineages and the Firmicutes. These data are consistent with urea-fueled nitrification by polar archaea and bacteria, which may be advantageous under dark conditions.

Crenarchaeal heterotrophy in salt marsh sediments.

Mesophilic Crenarchaeota (also known as Thaumarchaeota) are ubiquitous and abundant in marine habitats. However, very little is known about their metabolic function in situ. In this study, salt marsh sediments from New Jersey were screened via stable isotope probing (SIP) for heterotrophy by amending with a single (13)C-labeled compound (acetate, glycine or urea) or a complex (13)C-biopolymer (lipids, proteins or growth medium (ISOGRO)). SIP incubations were done at two substrate concentrations (30-150 µM; 2-10 mg ml(-1)), and (13)C-labeled DNA was analyzed by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes. To test for autotrophy, an amendment with (13)C-bicarbonate was also performed. Our SIP analyses indicate salt marsh crenarchaea are heterotrophic, double within 2-3 days and often compete with heterotrophic bacteria for the same organic substrates. A clone library of (13)C-amplicons was screened to find matches to the (13)C-TRFLP peaks, with seven members of the Miscellaneous Crenarchaeal Group and seven members from the Marine Group 1.a Crenarchaeota being discerned. Some of these crenarchaea displayed a preference for particular carbon sources, whereas others incorporated nearly every (13)C-substrate provided. The data suggest salt marshes may be an excellent model system for studying crenarchaeal metabolic capabilities and can provide information on the competition between crenarchaea and other microbial groups to improve our understanding of microbial ecology.

Predominant Acidilobus-like populations from geothermal environments in yellowstone national park exhibit similar metabolic potential in different hypoxic microbial communities.

High-temperature (>70°C) ecosystems in Yellowstone National Park (YNP) provide an unparalleled opportunity to study chemotrophic archaea and their role in microbial community structure and function under highly constrained geochemical conditions. Acidilobus spp. (order Desulfurococcales) comprise one of the dominant phylotypes in hypoxic geothermal sulfur sediment and Fe(III)-oxide environments along with members of the Thermoproteales and Sulfolobales. Consequently, the primary goals of the current study were to analyze and compare replicate de novo sequence assemblies of Acidilobus-like populations from four different mildly acidic (pH 3.3 to 6.1) high-temperature (72°C to 82°C) environments and to identify metabolic pathways and/or protein-encoding genes that provide a detailed foundation of the potential functional role of these populations in situ. De novo assemblies of the highly similar Acidilobus-like populations (>99% 16S rRNA gene identity) represent near-complete consensus genomes based on an inventory of single-copy genes, deduced metabolic potential, and assembly statistics generated across sites. Functional analysis of coding sequences and confirmation of gene transcription by Acidilobus-like populations provide evidence that they are primarily chemoorganoheterotrophs, generating acetyl coenzyme A (acetyl-CoA) via the degradation of carbohydrates, lipids, and proteins, and auxotrophic with respect to several external vitamins, cofactors, and metabolites. No obvious pathways or protein-encoding genes responsible for the dissimilatory reduction of sulfur were identified. The presence of a formate dehydrogenase (Fdh) and other protein-encoding genes involved in mixed-acid fermentation supports the hypothesis that Acidilobus spp. function as degraders of complex organic constituents in high-temperature, mildly acidic, hypoxic geothermal systems.