Genome-based classification of the family Haloferacaceae and description of five novel species of Halobaculum.

The taxonomic status of some species of Halobellus, Haloferax, Halogranum, and Haloplanus within the family Haloferacaceae was elucidated by phylogenetic, phylogenomic, and comparative genomic analyses. The relative species of each genus should constitute a single species based on the overall genome-related indexes proposed for species demarcation. The cutoff values of AAI (72.1%), ANI (82.2%), and rpoB' gene similarity (90.7%) were proposed to differentiate genera within the family Haloferacaceae. According to these standards, a novel genus related to the genus Halobaculum was proposed to accommodate Halobaculum halophilum Gai3-2 T and Halobaculum salinum NJ-3-1 T. Five halophilic archaeal strains, DT31T, DT55T, DT92T, SYNS20T, and YSMS11T, isolated from a tidal flat and a marine solar saltern in China, were subjected to polyphasic classification. The phenotypic, phylogenetic, phylogenomic, and comparative genomic analyses revealed that strains DT31T (= CGMCC 1.18923 T = JCM 35417 T), DT55T (= CGMCC 1.19048 T = JCM 36147 T), DT92T (= CGMCC 1.19057 T = JCM 36148 T), SYNS20T (= CGMCC 1.62628 T = JCM 36154 T), and YSMS11T (= CGMCC 1.18927 T = JCM 34912 T) represent five novel species of the genus Halobaculum, for which the names, Halobaculum lipolyticum sp. nov., Halobaculum marinum sp. nov., Halobaculum litoreum sp. nov., Halobaculum halobium sp. nov., and Halobaculum limi sp. nov., are proposed.

Microscopic and metatranscriptomic analyses revealed unique cross-domain parasitism between phylum Candidatus Patescibacteria/candidate phyla radiation and methanogenic archaea in anaerobic ecosystems.

To verify whether members of the phylum Candidatus Patescibacteria parasitize archaea, we applied cultivation, microscopy, metatranscriptomic, and protein structure prediction analyses on the Patescibacteria-enriched cultures derived from a methanogenic bioreactor. Amendment of cultures with exogenous methanogenic archaea, acetate, amino acids, and nucleoside monophosphates increased the relative abundance of Ca. Patescibacteria. The predominant Ca. Patescibacteria were families Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, and the former showed positive linear relationships (r2 ≥ 0.70) Methanothrix in their relative abundances, suggesting related growth patterns. Methanothrix and Methanospirillum cells with attached Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae, respectively, had significantly lower cellular activity than those of the methanogens without Ca. Patescibacteria, as extrapolated from fluorescence in situ hybridization-based fluorescence. We also observed that parasitized methanogens often had cell surface deformations. Some Methanothrix-like filamentous cells were dented where the submicron cells were attached. Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae highly expressed extracellular enzymes, and based on structural predictions, some contained peptidoglycan-binding domains with potential involvement in host cell attachment. Collectively, we propose that the interactions of Ca. Yanofskyibacteriaceae and Ca. Minisyncoccaceae with methanogenic archaea are parasitisms.IMPORTANCECulture-independent DNA sequencing approaches have explored diverse yet-to-be-cultured microorganisms and have significantly expanded the tree of life in recent years. One major lineage of the domain Bacteria, Ca. Patescibacteria (also known as candidate phyla radiation), is widely distributed in natural and engineered ecosystems and has been thought to be dependent on host bacteria due to the lack of several biosynthetic pathways and small cell/genome size. Although bacteria-parasitizing or bacteria-preying Ca. Patescibacteria have been described, our recent studies revealed that some lineages can specifically interact with archaea. In this study, we provide strong evidence that the relationship is parasitic, shedding light on overlooked roles of Ca. Patescibacteria in anaerobic habitats.

Salinilacihabitans rarus gen. nov., sp. nov., Natrononativus amylolyticus gen. nov., sp. nov., Natronobeatus ordinarius gen. nov., sp. nov., and Halovivax gelatinilyticus sp. nov., halophilic archaea, isolated from a salt lake and soda lakes.

Four halophilic archaea strains, AD-4T, CGA30T, CGA73T, and WLHSJ27T, were isolated from a salt lake and two soda lakes located in different regions of China. The 16S rRNA and rpoB' gene sequence similarities among strains AD-4T, CGA30T, CGA73T, WLHSJ27T, and the current species of the family Natrialbaceae were 90.9-97.5% and 83.1-91.8%, respectively. The phylogenetic and phylogenomic analyses revealed that these four strains separated from existing genera in the family Natrialbaceae and formed distant branches. The ANI, isDDH, and AAI values among these four strains and the current members of the family Natrialbaceae were 72-79%, 20-25%, and 63-73%, respectively, much lower than the threshold values for species demarcation. Strains AD-4T, CGA73T, and WLHSJ27T may represent three novel genera of the family Natrialbaceae according to the cutoff value of AAI (≤ 76%) proposed to differentiate genera within the family Natrialbaceae. These four strains could be distinguished from the related genera according to differential phenotypic characteristics. The major phospholipids of these four strains were identical while their glycolipid profiles were diverse. DGD-1 is a major glycolipid found in strain AD-4T, trace glycolipids, DGD-1, and S-DGD-1, and (or) S-TGD-1 was found in the other three strains. The major respiratory quinones detected in the four strains were menaquinone MK-8 and MK-8(H2). This polyphasic classification indicated that strains AD-4T, CGA73T, and WLHSJ27T represent three novel species of three new genera with the family Natrialbaceae, and strain CGA30T represents a novel species of Halovivax.

Proposed minimal standards for description of methanogenic archaea.

Methanogenic archaea are a diverse, polyphyletic group of strictly anaerobic prokaryotes capable of producing methane as their primary metabolic product. It has been over three decades since minimal standards for their taxonomic description have been proposed. In light of advancements in technology and amendments in systematic microbiology, revision of the older criteria for taxonomic description is essential. Most of the previously recommended minimum standards regarding phenotypic characterization of pure cultures are maintained. Electron microscopy and chemotaxonomic methods like whole-cell protein and lipid analysis are desirable but not required. Because of advancements in DNA sequencing technologies, obtaining a complete or draft whole genome sequence for type strains and its deposition in a public database are now mandatory. Genomic data should be used for rigorous comparison to close relatives using overall genome related indices such as average nucleotide identity and digital DNA-DNA hybridization. Phylogenetic analysis of the 16S rRNA gene is also required and can be supplemented by phylogenies of the mcrA gene and phylogenomic analysis using multiple conserved, single-copy marker genes. Additionally, it is now established that culture purity is not essential for studying prokaryotes, and description of Candidatus methanogenic taxa using single-cell or metagenomics along with other appropriate criteria is a viable alternative. The revisions to the minimal criteria proposed here by the members of the Subcommittee on the Taxonomy of Methanogenic Archaea of the International Committee on Systematics of Prokaryotes should allow for rigorous yet practical taxonomic description of these important and diverse microbes.

Cultivation and biogeochemical analyses reveal insights into methanogenesis in deep subseafloor sediment at a biogenic gas hydrate site.

Gas hydrates deposited in subseafloor sediments are considered to primarily consist of biogenic methane. However, little evidence for the occurrence of living methanogens in subseafloor sediments has been provided. This study investigated viable methanogen diversity, population, physiology and potential activity in hydrate-bearing sediments (1-307 m below the seafloor) from the eastern Nankai Trough. Radiotracer experiments, the quantification of coenzyme F430 and molecular sequencing analysis indicated the occurrence of potential methanogenic activity and living methanogens in the sediments and the predominance of hydrogenotrophic methanogens followed by methylotrophic methanogens. Ten isolates and nine representative culture clones of hydrogenotrophic, methylotrophic and acetoclastic methanogens were obtained from the batch incubation of sediments and accounted for 0.5-76% of the total methanogenic sequences directly recovered from each sediment. The hydrogenotrophic methanogen isolates of Methanocalculus and Methanoculleus that dominated the sediment methanogen communities produced methane at temperatures from 4 to 55 °C, with an abrupt decline in the methane production rate at temperatures above 40 °C, which is consistent with the depth profiles of potential methanogenic activity in the Nankai Trough sediments in this and previous studies. Our results reveal the previously overlooked phylogenetic and metabolic diversity of living methanogens, including methylotrophic methanogenesis.

Anaerobic oxidation of petroleum hydrocarbons in enrichment cultures from sediments of the Gorevoy Utes natural oil seep under methanogenic and sulfate-reducing conditions.

This article presents the first experimental data on the ability of microbial communities from sediments of the Gorevoy Utes natural oil seep to degrade petroleum hydrocarbons under anaerobic conditions. Like in marine ecosystems associated with oil discharge, available electron acceptors, in particular sulfate ions, affect the composition of the microbial community and the degree of hydrocarbon conversion. The cultivation of the surface sediments under sulfate-reducing conditions led to the formation of a more diverse bacterial community and greater loss of n-alkanes (28%) in comparison to methanogenic conditions (6%). Microbial communities of both surface and deep sediments are more oriented to degrade polycyclic aromatic hydrocarbons (PAHs), to which the degree of the PAH conversion testifies (up to 46%) irrespective of the present electron acceptors. Microorganisms with the uncultured closest homologues from thermal habitats, sediments of mud volcanoes, and environments contaminated with hydrocarbons mainly represented microbial communities of enrichment cultures. The members of the phyla Firmicutes, Chloroflexi, and Caldiserica (OP5), as well as the class Deltaproteobacteria and Methanomicrobia, were mostly found in enrichment cultures. The influence of gas-saturated fluids may be responsible for the presence in the bacterial 16S rRNA gene libraries of the sequences of "rare taxa": Planctomycetes, Ca. Atribacteria (OP9), Ca. Armatimonadetes (OP10), Ca. Latescibacteria (WS3), Ca. division (AC1), Ca. division (OP11), and Ca. Parcubacteria (OD1), which can be involved in hydrocarbon oxidation.

Functional elucidation of TfuA in peptide backbone thioamidation.

YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser-Lys catalytic pair.

Engineered triply orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids.

Expanding and reprogramming the genetic code of cells for the incorporation of multiple distinct non-canonical amino acids (ncAAs), and the encoded biosynthesis of non-canonical biopolymers, requires the discovery of multiple orthogonal aminoacyl-transfer RNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl-tRNA synthetase (PylRS)/PyltRNA pairs are the most widely used system for genetic code expansion. Here, we reveal that the sequences of ΔNPylRS/ΔNPyltRNA pairs (which lack N-terminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and ΔNPyltRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with ΔNPyltRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/ΔNPyltRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/PyltRNA pairs. Finally, we diverge the ncAA specificity and decoding properties of each pair, within a triply orthogonal set, and direct the incorporation of three distinct non-canonical amino acids into a single polypeptide.

Supramolecular tholos-like architecture constituted by archaeal proteins without functional annotation.

Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.

Analysis of bacterial communities and characterization of antimicrobial strains from cave microbiota

Abstract In this study for the first-time microbial communities in the caves located in the mountain range of Hindu Kush were evaluated. The samples were analyzed using culture-independent (16S rRNA gene amplicon sequencing) and culture-dependent methods. The amplicon sequencing results revealed a broad taxonomic diversity, including 21 phyla and 20 candidate phyla. Proteobacteria were dominant in both caves, followed by Bacteroidetes, Actinobacteria, Firmicutes, Verrucomicrobia, Planctomycetes, and the archaeal phylum Euryarchaeota. Representative operational taxonomic units from Koat Maqbari Ghaar and Smasse-Rawo Ghaar were grouped into 235 and 445 different genera, respectively. Comparative analysis of the cultured bacterial isolates revealed distinct bacterial taxonomic profiles in the studied caves dominated by Proteobacteria in Koat Maqbari Ghaar and Firmicutes in Smasse-Rawo Ghaar. Majority of those isolates were associated with the genera Pseudomonas and Bacillus. Thirty strains among the identified isolates from both caves showed antimicrobial activity. Overall, the present study gave insight into the great bacterial taxonomic diversity and antimicrobial potential of the isolates from the previously uncharacterized caves located in the world's highest mountains range in the Indian sub-continent.

The Relative Abundance and Transcriptional Activity of Marine Sponge-Associated Microorganisms Emphasizing Groups Involved in Sulfur Cycle.

During the last decades, our knowledge about the activity of sponge-associated microorganisms and their contribution to biogeochemical cycling has gradually increased. Functional groups involved in carbon and nitrogen metabolism are well documented, whereas knowledge about microorganisms involved in the sulfur cycle is still limited. Both sulfate reduction and sulfide oxidation has been detected in the cold water sponge Geodia barretti from Korsfjord in Norway, and with specimens from this site, the present study aims to identify extant versus active sponge-associated microbiota with focus on sulfur metabolism. Comparative analysis of small subunit ribosomal RNA (16S rRNA) gene (DNA) and transcript (complementary DNA (cDNA)) libraries revealed profound differences. The transcript library was predominated by Chloroflexi despite their low abundance in the gene library. An opposite result was found for Acidobacteria. Proteobacteria were detected in both libraries with representatives of the Alpha- and Gammaproteobacteria related to clades with presumably thiotrophic bacteria from sponges and other marine invertebrates. Sequences that clustered with sponge-associated Deltaproteobacteria were remotely related to cultivated sulfate-reducing bacteria. The microbes involved in sulfur cycling were identified by the functional gene aprA (adenosine-5'-phosphosulfate reductase) and its transcript. Of the aprA sequences (DNA and cDNA), 87 % affiliated with sulfur-oxidizing bacteria. They clustered with Alphaproteobacteria and with clades of deep-branching Gammaproteobacteria. The remaining sequences clustered with sulfate-reducing Archaea of the phylum Euryarchaeota. These results indicate an active role of yet uncharacterized Bacteria and Archaea in the sponge's sulfur cycle.

Impact of cystic fibrosis disease on archaea and bacteria composition of gut microbiota.

Cystic fibrosis is often associated with intestinal inflammation due to several factors, including altered gut microbiota composition. In this study, we analyzed the fecal microbiota among patients with cystic fibrosis of 10-22 years of age, and compared the findings with age-matched healthy subjects. The participating patients included 14 homozygotes and 14 heterozygotes with the delF508 mutation, and 2 heterozygotes presenting non-delF508 mutations. We used PCR-DGGE and qPCR to analyze the presence of bacteria, archaea and sulfate-reducing bacteria. Overall, our findings confirmed disruption of the cystic fibrosis gut microbiota. Principal component analysis of the qPCR data revealed no differences between homozygotes and heterozygotes, while both groups were distinct from healthy subjects who showed higher biodiversity. Archaea were under the detection limit in all homozygotes subjects, whereas methanogens were detected in 62% of both cystic fibrosis heterozygotes and healthy subjects. Our qPCR results revealed a low frequency of sulfate-reducing bacteria in the homozygote (13%) and heterozygote (13%) patients with cystic fibrosis compared with healthy subjects (87.5%). This is a pioneer study showing that patients with cystic fibrosis exhibit significant reduction of H2-consuming microorganisms, which could increase hydrogen accumulation in the colon and the expulsion of this gas through non-microbial routes.

Activation of CO2-reducing methanogens in oil reservoir after addition of nutrient.

Nutrient addition as part of microbial enhanced oil recovery (MEOR) operations have important implications for more energy recovery from oil reservoirs, but very little is known about the in situ response of microorganisms after intervention. An analysis of two genes as biomarkers, mcrA encoding the key enzyme in methanogenesis and fthfs encoding the key enzyme in acetogenesis, was conducted during nutrient addition in oil reservoir. Clone library data showed that dominant mcrA sequences changed from acetoclastic (Methanosaetaceae) to CO2-reducing methanogens (Methanomicrobiales and Methanobacteriales), and the authentic acetogens affiliated to Firmicutes decreased after the intervention. Principal coordinates analysis (PCoA) and Jackknife environment clusters revealed evidence on the shift of the microbial community structure among the samples. Quantitative analysis of methanogens via qPCR showed that Methanobacteriales and Methanomicrobiales increased after nutrient addition, while acetoclastic methanogens (Methanosaetaceae) changed slightly. Nutrient treatment activated native CO2-reducing methanogens in oil reservoir. The high frequency of Methanobacteriales and Methanomicrobiales (CO2-reducers) after nutrient addition in this petroleum system suggested that CO2-reducing methanogenesis was involved in methane production. The nutrient addition could promote the methane production. The results will likely improve strategies of utilizing microorganisms in subsurface environments.

Changes in microbial communities associated with gas hydrates in subseafloor sediments from the Nankai Trough.

Little is known about the microbial distribution patterns in subseafloor sediments. This study examines microbial diversity and activities in sediments of the Nankai Trough, where biogenic gas hydrates are deposited. Illumina sequencing of 16S rRNA genes revealed that the prokaryotic community structure is correlated with hydrate occurrence and depth but not with the sedimentary facies. The bacterial phyla 'Atribacteria' lineage JS1 and Chloroflexi dominated in all samples, whereas lower taxonomic units of Chloroflexi accounted for community variation related to hydrate saturation. In archaeal communities, 'Bathyarchaeota' was significantly abundant in the hydrate-containing samples, whereas Marine Benthic Group-B dominated in the upper sediments without hydrates. mcrA gene sequences assigned to deeply branching groups and ANME-1 were detected only in hydrate-containing samples. A predominance of hydrogenotrophic methanogens, Methanomicrobiales and Methanobacteriales, over acetoclastic methanogens was found throughout the depth. Incubation tests on hydrate-containing samples with a stable isotope tracer showed anaerobic methane oxidation activities under both low- and seawater-like salinity conditions. These results indicate that the distribution patterns of microorganisms involved in carbon cycling changed with gas hydrate occurrence, possibly because of the previous hydrate dissociation followed by pore water salinity decrease in situ, as previously proposed by a geochemical study at the study site.

Signal transduction in light-oxygen-voltage receptors lacking the adduct-forming cysteine residue.

Light-oxygen-voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications.

Anaerobic alkane biodegradation by cultures enriched from oil sands tailings ponds involves multiple species capable of fumarate addition.

A methanogenic short-chain alkane-degrading culture (SCADC) was enriched from oil sands tailings and transferred several times with a mixture of C6, C7, C8 and C10 n-alkanes as the predominant organic carbon source, plus 2-methylpentane, 3-methylpentane and methylcyclopentane as minor components. Cultures produced ∼40% of the maximum theoretical methane during 18 months incubation while depleting the n-alkanes, 2-methylpentane and methylcyclopentane. Substrate depletion correlated with detection of metabolites characteristic of fumarate activation of 2-methylpentane and methylcyclopentane, but not n-alkane metabolites. During active methanogenesis with the mixed alkanes, reverse-transcription PCR confirmed the expression of functional genes (assA and bssA) associated with hydrocarbon addition to fumarate. Pyrosequencing of 16S rRNA genes amplified during active alkane degradation revealed enrichment of Clostridia (particularly Peptococcaceae) and methanogenic Archaea (Methanosaetaceae and Methanomicrobiaceae). Methanogenic cultures transferred into medium containing sulphate produced sulphide, depleted n-alkanes and produced the corresponding succinylated alkane metabolites, but were slow to degrade 2-methylpentane and methylcyclopentane; these cultures were enriched in Deltaproteobacteria rather than Clostridia. 3-Methylpentane was not degraded by any cultures. Thus, nominally methanogenic oil sands tailings harbour dynamic and versatile hydrocarbon-degrading fermentative syntrophs and sulphate reducers capable of degrading n-, iso- and cyclo-alkanes by addition to fumarate.

DNA stable-isotope probing of oil sands tailings pond enrichment cultures reveals different key players for toluene degradation under methanogenic and sulfidogenic conditions.

Oil sands tailings ponds are anaerobic repositories of fluid wastes produced by extraction of bitumen from oil sands ores. Diverse indigenous microbiota biodegrade hydrocarbons (including toluene) in situ, producing methane, carbon dioxide and/or hydrogen sulfide, depending on electron acceptor availability. Stable-isotope probing of cultures enriched from tailings associated specific taxa and functional genes to (13)C6- and (12)C7-toluene degradation under methanogenic and sulfate-reducing conditions. Total DNA was subjected to isopycnic ultracentrifugation followed by gradient fraction analysis using terminal restriction fragment length polymorphism (T-RFLP) and construction of 16S rRNA, benzylsuccinate synthase (bssA) and dissimilatory sulfite reductase (dsrB) gene clone libraries. T-RFLP analysis plus sequencing and in silico digestion of cloned taxonomic and functional genes revealed that Clostridiales, particularly Desulfosporosinus (136 bp T-RF) contained bssA genes and were key toluene degraders during methanogenesis dominated by Methanosaeta. Deltaproteobacterial Desulfobulbaceae (157 bp T-RF) became dominant under sulfidogenic conditions, likely because the Desulfosporosinus T-RF 136 apparently lacks dsrB and therefore, unlike its close relatives, is presumed incapable of dissimilatory sulfate reduction. We infer incomplete oxidation of toluene by Desulfosporosinus in syntrophic association with Methanosaeta under methanogenic conditions, and complete toluene oxidation by Desulfobulbaceae during sulfate reduction.

Low-temperature anaerobic digestion is associated with differential methanogenic protein expression.

Anaerobic digestion (AD) is an attractive wastewater treatment technology, leading to the generation of recoverable biofuel (methane). Most industrial AD applications, carry excessive heating costs, however, as AD reactors are commonly operated at mesophilic temperatures while handling waste streams discharged at ambient or cold temperatures. Consequently, low-temperature AD represents a cost-effective strategy for wastewater treatment. The comparative investigation of key microbial groups underpinning laboratory-scale AD bioreactors operated at 37, 15 and 7°C was carried out. Community structure was monitored using 16S rRNA clone libraries, while abundance of the most prominent methanogens was investigated using qPCR. In addition, metaproteomics was employed to access the microbial functions carried out in situ. While δ-Proteobacteria were prevalent at 37°C, their abundance decreased dramatically at lower temperatures with inverse trends observed for Bacteroidetes and Firmicutes. Methanobacteriales and Methanosaeta were predominant at all temperatures investigated while Methanomicrobiales abundance increased at 15°C compared to 37 and 7°C. Changes in operating temperature resulted in the differential expression of proteins involved in methanogenesis, which was found to occur in all bioreactors, as corroborated by bioreactors' performance. This study demonstrated the value of employing a polyphasic approach to address microbial community dynamics and highlighted the functional redundancy of AD microbiomes.

The dynamic nature of genomes across the tree of life.

Genomes are dynamic in lineages across the tree of life. Among bacteria and archaea, for example, DNA content varies throughout life cycles, and nonbinary cell division in diverse lineages indicates the need for coordination of the inheritance of genomes. These observations contrast with the textbook view that bacterial and archaeal genomes are monoploid (i.e., single copied) and fixed both within species and throughout an individual's lifetime. Here, we synthesize information on three aspects of dynamic genomes from exemplars representing a diverse array of bacterial and archaeal lineages: 1) ploidy level variation, 2) epigenetic mechanisms, and 3) life cycle variation. For example, the Euryarchaeota analyzed to date are all polyploid, as is the bacterium Epulopiscium that contains up to tens of thousands of copies of its genome and reproduces by viviparity. The bacterium Deinococcus radiodurans and the archaeon Halobacterium sp. NRC-1 can repair a highly fragmented genome within a few hours. Moreover, bacterial genera such as Dermocarpella and Planctomyces reproduce by fission (i.e., generating many cells from one cell) and budding, respectively, highlighting the need for regulation of genome inheritance in these lineages. Combining these data with our previous work on widespread genome dynamics among eukaryotes, we hypothesize that dynamic genomes are a rule rather than the exception across the tree of life. Further, we speculate that all domains may have the ability to distinguish germline from somatic DNA and that this ability may have been present the last universal common ancestor.