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Two novel halophilic archaeal strains, CBA1133

In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Archaea, but its emergent function remains a mystery. To better understand the challenges associated with the establishment of pervasive negative supercoiling activity, we expressed the gyrase of the bacterium Thermotoga maritima in a naïve archaeon Thermococcus kodakarensis which naturally has positively supercoiled DNA. We found that the gyrase was catalytically active in T. kodakarensis leading to strong negative supercoiling of plasmid DNA which was stably maintained over at least eighty generations. An increased sensitivity of gyrase-expressing T. kodakarensis to ciprofloxacin suggested that gyrase also modulated chromosomal topology. Accordingly, global transcriptome analyses revealed large scale gene expression deregulation and identified a subset of genes responding to the negative supercoiling activity of gyrase. Surprisingly, the artificially introduced dominant negative supercoiling activity did not have a measurable effect on T. kodakarensis growth rate. Our data suggest that gyrase can become established in Thermococcales archaea without critically interfering with DNA transaction processes., Graphical Abstract Graphical AbstractIntroduction of DNA gyrase in Thermococcus kodakarensis flips its DNA supercoiling from positive to negative with only mild effects on its cell physiology.

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA–DNA and SegB–DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon–helix–helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA–SegB–DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.

Commonly used 16S rRNA gene primers do not detect the full range of archaeal diversity present in the vertebrate gut. As a result, several questions regarding the archaeal component of the gut microbiota remain, including which Archaea are host-associated, the specificities of such associations and the major factors influencing archaeal diversity. Using 16S rRNA gene amplicon sequencing with primers that specifically target Archaea, we obtained sufficient sequence data from 185 gastrointestinal samples collected from 110 vertebrate species that span five taxonomic classes (Mammalia, Aves, Reptilia, Amphibia and Actinopterygii), of which the majority were wild. We provide evidence for previously undescribed Archaea–host associations, including Bathyarchaeia and Methanothermobacter, the latter of which was prevalent among Aves and relatively abundant in species with higher body temperatures, although this association could not be decoupled from host phylogeny. Host phylogeny explained archaeal diversity more strongly than diet, while specific taxa were associated with both factors, and cophylogeny was significant and strongest for mammalian herbivores. Methanobacteria was the only class predicted to be present in the last common ancestors of mammals and all host species. Further analysis indicated that Archaea–Bacteria interactions have a limited effect on archaeal diversity. These findings expand our current understanding of Archaea–vertebrate associations., Analysis of the archaeal gut microbiota of 110 vertebrate species spanning five taxonomic classes revealed that host phylogeny could explain archaeal diversity.

Three novel halophilic archaea were isolated from seawater and sediment near Yeoungheungdo Island, Republic of Korea. The genome size and G + C content of the isolates MBLA0076T, MBLA0077T, and MBLA0078T were 3.56, 3.48, and 3.48 Mb and 61.7, 60.8, and 61.1 mol%, respectively. The three strains shared 98.5–99.5 % sequence similarity of the 16 S rRNA gene, whereas their sequence similarity to the 16 S rRNA gene of type strains was below 98.5 %. Phylogenetic analysis based on sequences of the 16 S rRNA and RNA polymerase subunit beta genes indicated that the isolates belonged to the genus Haloferax. The orthologous average nucleotide identity, average amino-acid identity, and in silico DNA-DNA hybridization values were below species delineation thresholds. Pan-genomic analysis indicated that the three novel strains and 11 reference strains had 8981 pan-orthologous groups in total. Fourteen Haloferax strains shared 1766 core pan-genome orthologous groups, which were mainly related to amino acid transport and metabolism. Cells of the three isolates were gram-negative, motile, red-pink pigmented, and pleomorphic. The strains grew optimally at 30 °C (MBLA0076T) and 40 °C (MBLA0077T, MBLA0078T) in the presence of 1.28 M (MBLA0077T) and 1.7 M (MBLA0076T, MBLA0078T) NaCl and 0.1 M (MBLA0077T), 0.2 M (MBLA0076T), and 0.3 M (MBLA0078T) MgCl2·6H2O at pH 7.0–8.0. Cells of all isolates lysed in distilled water; the minimum NaCl concentration necessary to prevent lysis was 0.43 M. The major polar lipids of the three strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and sulphated diglycosyl archaeol-1. Based on their phenotypic and genotypic properties, MBLA0076T, MBLA0077T, and MBLA0078T were described as novel species of Haloferax, for which we propose the names Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., respectively. The respective type strains of these species are MBLA0076T (= KCTC 4288T = JCM 34,169T), MBLA0077T (= KCTC 4289T = JCM 34,170T), and MBLA0078T (= KCTC 4290T = JCM 34,171T).

Fjords are semi-enclosed marine systems with unique physical conditions that influence microbial community composition and structure. Pronounced organic matter and physical condition gradients within fjords provide a natural laboratory for the study of changes in microbial community structure and metabolic potential in response to environmental conditions. Photosynthetic production in euphotic zones sustains deeper aphotic microbial activity via organic matter sinking, augmented by large terrestrial inputs. Previous studies do not consider both prokaryotic and eukaryotic communities when linking metabolic potential and activity, community composition, and environmental gradients. To address this gap we profiled microbial functional potential (Biolog Ecoplates), bacterial abundance, heterotrophic production (3H-Leucine incorporation), and prokaryotic/eukaryotic community composition (16S and 18S rRNA amplicon gene sequencing). Similar factors shaped metabolic potential, activity and community (prokaryotic and eukaryotic) composition across surface/near surface sites. However, increased metabolic diversity at near bottom (aphotic) sites reflected an organic matter influence from sediments. Photosynthetically produced particulate organic matter shaped the upper water column community composition and metabolic potential. In contrast, microbial activity at deeper aphotic waters were strongly influenced by other organic matter input than sinking marine snow (e.g. sediment resuspension of benthic organic matter, remineralisation of terrestrially derived organic matter, etc.), severing the link between community structure and metabolic potential. Taken together, different organic matter sources shape microbial activity, but not community composition, in New Zealand fjords.

Water hypoxia (DO 2 mg/L) is a growing global environmental concern that has the potential to significantly influence not only the aquatic ecosystem but also the benthic sedimentary ecosystem. The Changjiang River Estuary hypoxia, classified as one of the world's largest seasonal hypoxic water basins, has been reported to be expanding rapidly in recent decades. However, the microbial community dynamics and responses to this water hypoxia are still unclear. In this study, we examined the abundance, community composition, and distribution of sedimentary archaea, one important component of microbial communities in the Changjiang River Estuary and the East China Sea (ECS). Our results indicated that

In the present study, we report the complete genome sequencing of Haloterrigena daqingensis species. The genome of H. daqingensis JX313T consisted of a circular chromosome with three plasmids. The genome size and G+C content were estimated to be 3835796 bp and 61.7%, respectively. A total of 4158 genes were predicted with six rRNAs and 45 tRNAs. Metabolic pathway analysis suggests that H. daqingensis JX313T codes for all the necessary genes responsible to sustain its life at saline environment. The pan-genome analysis suggests that the number of singleton-gene between H. daqingensis and other Haloterrigena species varied. The study not only helps us understand H. daqingensis strategy for dealing with high stress, but it also provides an overview of its genomic makeup.

Transcription initiates at promoters, DNA regions recognized by a DNA-dependent RNA polymerase. We previously identified horizontally acquired Escherichia coli promoters from which the direction of transcription was unclear. In the present study, we show that more than half of these promoters are bidirectional and drive divergent transcription. Using genome-scale approaches, we demonstrate that 19% of all transcription start sites detected in E. coli are associated with a bidirectional promoter. Bidirectional promoters are similarly common in diverse bacteria and archaea, and have inherent symmetry: specific bases required for transcription initiation are reciprocally co-located on opposite DNA strands. Bidirectional promoters enable co-regulation of divergent genes and are enriched in both intergenic and horizontally acquired regions. Divergent transcription is conserved among bacteria, archaea and eukaryotes, but the underlying mechanisms for bidirectionality are different.

A haloalkaliphilic strain JWXQ-INN-674T was isolated from the water sample of a soda lake in Inner Mongolia Autonomous Region, China. Cells of the strain were coccoid, motile, and strictly aerobic. The strain was able to grow in presence of 2.6–5.4 M NaCl (optimum concentration is 3.4 M) at 30–50 °C (optimum temperature is 42 °C) and pH 7–9.5 (optimum pH is 9.0). The 16S rRNA gene sequence of strain JWXQ-INN-674T showed 95.3–96.6% similarity to members of the genus Natronorubrum of the family Natrialbaceae. The whole genome sequencing of strain JWXQ-INN-674T revealed a genome size of 4.56 M bp and a DNA G + C content of 62.5 mol%. Genome relatedness of strain JWXQ-INN-674T and other species in the genus Natronorubrum was analyzed by average nucleotide identity and digital DNA–DNA hybridization with the values of 76.8–90.6 and 23.1–39.3%, respectively. The strain possessed the polar lipids phosphatidylglycerol and methylated phosphatidylglycerol phosphate lipid. No glycolipids were detected. Based on phylogenetic analysis, phenotypic characteristics, chemotaxonomic properties and genome relatedness, the isolate was proposed as the type strain of a novel species of genus Natronorubrum, Natronorubrum halalkaliphilum sp. nov. (type strain JWXQ-INN-674T = CGMCC 1.17283T = JCM 34245T).

Six novel halophilic archaeal strains of XZYJT10

A novel methanogenic strain, CaP3V-MF-L2AT, was isolated from an exploratory oil well from Cahuita National Park, Costa Rica. The cells were irregular cocci, 0.8–1.8 μm in diameter, stained Gram-negative and were motile. The strain utilized H2/CO2, formate and the primary and secondary alcohols 1-propanol and 2-propanol for methanogenesis, but not acetate, methanol, ethanol, 1-butanol or 2-butanol. Acetate was required as carbon source. The novel isolate grew at 25–40 °C, pH 6.0–7.5 and 0–2.5% (w/v) NaCl. 16S rRNA gene sequence analysis revealed that the strain is affiliated to the genus Methanofollis. It shows 98.8% sequence similarity to its closest relative Methanofollis ethanolicus. The G + C content is 60.1 mol%. Based on the data presented here type strain CaP3V-MF-L2AT (= DSM 113321T = JCM 39176T) represents a novel species, Methanofollis propanolicus sp. nov.

Halophilic archaea are the dominant type of microorganisms in hypersaline environments. The diversity of halophilic archaea in Zehrez-Chergui (Saharian chott) was analyzed and compared by both analysis of a library of PCR amplified 16S rRNA genes and by cultivation approach. This work, represents the first of its type in Algeria. A total cell count was estimated at 3.8 × 10

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, phosphodiester linkages that create the repeating sugarphosphate backbone of DNA. Can this linkage selectivity observed in nature be overcome by design to produce non-natural nucleic acids? Here, we report that structure-guided redesign of an archaeal DNA polymerase (9°N) enables a new polymerase activity that is undetectable in the wild type enzyme: catalyzing the formation of N3’→P5’ phosphoramidate linkages in the presence of 3’-amino-2’,3’-dideoxynucleoside 5’-triphosphate (3’-NH2-ddNTP) building blocks. Replacing a highly conserved metal-binding aspartate in the 9°N active site (Asp-404) with asparagine was key to the emergence of this unnatural enzyme activity. Molecular dynamics simulations provided insights into how a single substitution could enhance the productive positioning of the 3’-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface with substitutions in the finger subdomain led to a quadruple-mutant variant (9°N-NRQS) that incorporated 3’-NH2-ddNTPs into a 3’-amino-primer on various DNA templates. This work presents the first example of an active-site substitution of a metal-binding residue that leads to a novel activity in a DNA polymerase, and sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.

Plant roots and soil prokaryotes primarily interact with each other in the rhizosphere. Changes in the rhizosphere prokaryotic structure are influenced by several factors. In this study, the community structure of the Potentilla anserina L. rhizosphere prokaryotes was identified and evaluated by high-throughput sequencing technology in different continuous cropping fields and developmental stages of the plant. In total, 2 archaeal (Euryarchaeota and Thaumarchaeota) and 26 bacterial phyla were identified in the P. anserina rhizosphere. The bacterial community was mainly composed of Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Planctomycetes, Proteobacteria, and Verrucomicrobia. Moreover, the prokaryotic community structure of the rhizosphere varied significantly during plant development. Our results provide new insights into the dynamics of the P. anserina rhizosphere prokaryotic community and may provide useful information for enhancing the growth and development of P. anserina through artificial control of the soil prokaryotes.

Acid mine drainage (AMD) harbors all three life forms in spite of its toxic and hazardous nature. In comparison to bacterial diversity, an in-depth understanding of the archaeal diversity in AMD and their ecological significance remain less explored. Archaeal populations are known to play significant roles in various biogeochemical cycles within the AMD ecosystem, and it is imperative to have a deeper understanding of archaeal diversity and their functional potential in AMD system. The present study is aimed to understand the archaeal diversity of an AMD sediment of Malanjkhand Copper Project, India through archaea specific V6 region of 16S rRNA gene amplicon sequencing. Geochemical data confirmed the acidic, toxic, heavy metal-rich nature of the sample. Archaea specific V6-16S rRNA gene amplicon data showed a predominance of Thermoplasmata (BSLdp215, uncultured Thermoplasmata, and Thermoplasmataceae) and Nitrososphaeria (Nitrosotaleaceae) members constituting ~ 95% of the archaeal community. Uncultured members of Bathyarchaeia, Group 1.1c, Hydrothermarchaeota, and Methanomassiliicoccales along with Methanobacteriaceae, Methanocellaceae, Haloferaceae, Methanosaetaceae, and Methanoregulaceae constituted the part of rare taxa. Analysis of sequence reads indicated that apart from their close ecological relevance, members of the Thermoplasmata present in Malanjkhand AMD were mostly involved in chemoheterotrophy, Fe/S redox cycling, and with heavy metal resistance, while the Nitrososphaeria members were responsible for ammonia oxidation and fixation of HCO3− through 3-hydroxypropionate/4-hydroxybutyrate cycle at low pH and oligotrophic environment which subsequently played an important role in nitrification process in AMD sediment. Overall, the present study elucidated the biogeochemical significance of archaeal populations inhabiting the toxic AMD environment.

The vertical distribution of subseafloor archaeal communities is thought to be primarily controlled by in situ conditions in sediments such as the availability of electron acceptors and donors, although sharp community shifts have also been observed at lithological boundaries suggesting that at least a subset of vertically stratified Archaea form a long-term genetic record of coinciding environmental conditions that occurred at the time of sediment deposition. To substantiate this possibility, we performed a highly resolved 16S rRNA gene survey of vertically stratified archaeal communities paired with paleo-oceanographic proxies in a sedimentary record from the northern Red Sea spanning the last glacial-interglacial cycle (i.e., marine isotope stages 1-6; MIS1-6). Our results show a strong significant correlation between subseafloor archaeal communities and drastic paleodepositional changes associated with glacial low vs. interglacial high stands (ANOSIM; R = .73; p = .001) and only a moderately strong correlation with lithological changes. Bathyarchaeota, Lokiarchaeota, MBGA, and DHVEG-1 were the most abundant identified archaeal groups. Whether they represented ancient cell lines from the time of deposition or migrated to the specific sedimentary horizons after deposition remains speculative. However, we show that the majority of sedimentary archaeal tetraether membrane lipids were of allochthonous origin and not produced in situ. Slow post-burial growth under energy-limited conditions would explain why the downcore distribution of these dominant archaeal groups still indirectly reflect changes in the paleodepositional environment that prevailed during the analyzed marine isotope stages. In addition, archaea seeded from the overlying water column such as Thaumarchaeota and group II and III Euryarchaeota, which were likely not have been able to subsist after burial, were identified from a lower abundance of preserved sedimentary DNA signatures, and represented direct markers of paleoenvironmental changes in the Red Sea spanning the last six marine isotope stages.

Ammonia-oxidising archaea (AOA) and ammonia-oxidising bacteria (AOB) are ammonium oxidising prokaryotes that can drive soil nitrification in wetlands. During the restoration of wetlands, different types of land use soils (agricultural soil [AS], restored wetland soil [RS], and natural wetland soil [NWS]) are present. However, studies on the effects of changes in the types of land use in wetlands during restoration on nitrification and the community composition of AOA and AOB are still not well understood. In this study, the differences in the potential nitrification rate (PNR) and community composition of AOA and AOB in AS, RS, and NWS were compared and discussed. The results indicated that the PNRs in the AS, RS, and NWS were on the same order of magnitude. Nitrification was mainly driven by AOB. High-throughput sequencing results showed that the genus

CRISPR-Cas systems provide the small RNA-based adaptive immunity to defend against invasive genetic elements in archaea and bacteria. Organisms of Sulfolobales, an order of thermophilic acidophiles belonging to the Crenarchaeotal Phylum, usually contain both type I and type III CRISPR-Cas systems. Two species, Saccharolobus solfataricus and Sulfolobus islandicus, have been important models for CRISPR study in archaea, and knowledge obtained from these studies has greatly expanded our understanding of molecular mechanisms of antiviral defense in all three steps: adaptation, expression and crRNA processing, and interference. Four subtypes of CRISPR-Cas systems are common in these organisms, including I-A, I-D, III-B, and III-D. These cas genes form functional modules, e.g., all genes required for adaptation and for interference in the I-A immune system are clustered together to form aCas and iCas modules. Genetic assays have been developed to study mechanisms of adaptation and interference by different CRISPR-Cas systems in these model archaea, and these methodologies are useful in demonstration of the protospacer-adjacent motif (PAM)-dependent DNA interference by I-A interference modules and multiple interference activities by III-B Cmr systems. Ribonucleoprotein effector complexes have been isolated for Sulfolobales III-B and III-D systems, and their biochemical characterization has greatly enriched the knowledge of molecular mechanisms of these novel antiviral immune responses.

BackgroundDNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3′-5′ editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex fromThermococcus kodakarensisusing single-particle cryo-electron microscopy (EM).ResultsTwo distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 fromPyrococcus abyssionto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a β-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro.ConclusionsBased on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life.

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the ‘parental’ DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication.

Haloferax volcanii is, to our knowledge, the only prokaryote known to tolerate CRISPR-Cas-mediated damage to its genome in the WT background; the resulting cleavage of the genome is repaired by homologous recombination restoring the WT version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalyzed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centered on the targeting site. Furthermore, we observed naive spacer acquisition at a low level in WT Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2, and Cas4. Taken together, these findings indicate that naive adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.

Significance The Mre11/Rad50 (M/R) complex forms the core of an essential DNA-repair complex, conserved in all divisions of life. Here we investigate this complex from the thermophilic archaeon Sulfolobus acidocaldarius using real-time atomic force microscopy. We demonstrate that the coiled-coil regions of Rad50 facilitate M/R interaction with DNA and permit substrate translocation until a free end is encountered. We also observe that the M/R complex drives unprecedented unwinding of the DNA duplexes. Taking these findings together, we provide a model for how the M/R complex can identify DNA double-strand breaks and orchestrate repair events., Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1–2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.

DNA can be preserved in marine and freshwater sediments both in bulk sediment and in intact, viable resting stages. Here, we assess the potential for combined use of ancient, environmental, DNA and timeseries of resurrected long-term dormant organisms, to reconstruct trophic interactions and evolutionary adaptation to changing environments. These new methods, coupled with independent evidence of biotic and abiotic forcing factors, can provide a holistic view of past ecosystems beyond that offered by standard palaeoecology, help us assess implications of ecological and molecular change for contemporary ecosystem functioning and services, and improve our ability to predict adaptation to environmental stress., Ellegaard et al. discuss the potential for using ancient environmental DNA (eDNA), combined with resurrection ecology, to analyse trophic interactions and evolutionary adaptation to changing environments. Their Review suggests that these techniques will improve our ability to predict genetic and phenotypic adaptation to environmental stress.

RNA polymerases (RNAPs) carry out transcription in the three domains of life, Bacteria, Archaea, and Eukarya. Transcription initiation is highly regulated by a variety of transcription factors, whose number and subunit complexity increase during evolution. This process is regulated in Bacteria by the σ factor, while the three eukaryotic RNAPs require a complex set of transcription factors (TFs) and a TATA-binding protein (TBP). The archaeal transcription system appears to be an ancestral version of the eukaryotic RNAPII, requiring transcription factor B (TFB), TBP, and transcription factor E (TFE). The function of the bacterial sigma (σ) factor has been correlated to the roles played by the eukaryotic RNAP II and the archaeal RNAP. In addition, σ factors, TFB, and TFIIB all contain multiple DNA binding helix-turn-helix (HTH) structural motifs; although TFIIB and TFB display two HTH domains, while the bacterial σ factor spans 4 HTH motifs. The sequence similarities and structure alignments of the bacterial σ factor, eukaryotic TFIIB, and archaeal TFB evidence that these three proteins are homologs.Key Points• Transcription initiation is highly regulated by TFs.• Transcription is finely regulated in all domains of life by different sets of TFs.• Specific TFs in Bacteria, Eukarya and Archaea are homologs.

Three halophilic archaeal strains, NEN8

Archaea participate in biogeochemical cycles in aquatic ecosystems, and deciphering their community dynamics and assembly mechanisms is key to understanding their ecological functions. Here, sediments from 12 selected reservoirs from the Wujiang and Pearl River basins in southwest China were investigated using 16S rRNA Illumina sequencing and quantitative PCR for archaeal abundance and richness in all seasons. Generally, archaeal abundance and α-diversity were significantly correlated with temperature; however, β-diversity analysis showed that community structures varied greatly among locations rather than seasons, indicating a distance-decay pattern with geographical variation. The null model revealed the major contribution of stochasticity to archaeal community assembly, which was further confirmed by the neutral community model that could explain 71.7% and 90.2% of the variance in archaeal assembly in the Wujiang and Pearl River basins, respectively. Moreover, sediment total nitrogen and organic carbon levels were significantly correlated with archaeal abundance and α-diversity. Interestingly, these nutrient levels were positively and negatively correlated, respectively, with the abundance of methanogenic and ammonia-oxidized archaea: the dominant sedimentary archaea in these reservoirs. Taken together, this work systematically characterized archaeal community profiles in reservoir sediments and demonstrated the combined action of stochastic processes and nutrient load in shaping archaeal communities in reservoir ecosystems.

A novel haloarchaeal species designated as MBLA0099

Two extremely halophilic strains, designated SYSU A558-1T and SYSU A121-1, were isolated from a saline sediment sample collected from Aiding salt lake, China. Cells of strains SYSU A558-1T and SYSU A121-1 were Gram-stain-negative, coccoid, and non-motile. The isolates were aerobic and grew at NaCl concentration of 10-30% (optimum, 20-22%), at 20-55ºC (optimum, 37-42ºC) and at pH 6.5-8.5 (optimum, 7.0-8.0). Cells lysed in distilled water. Major polar lipids were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, disulphated diglycosyl diether-1 and one unidentified glycolipid. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the two strains SYSU A558-1T and SYSU A121-1 were closely related to the membranes of the genus Haloterrigena. Phylogenetic trees based on strains SYSU A558-1T and SYSU A121-1 16S rRNA gene sequence, rpoB' gene sequence and concatenation of 87 protein markers demonstrated a robust clade with Haloterrigena turkmenica, Haloterrigena salifodinae and Haloterrigena salina. The genomic DNA G+C contents of strains SYSU A558-1T and SYSU A121-1 were 65.8 and 65.0%, respectively. Phenotypic, chemotaxonomic characteristics and phylogenetic properties suggested that the two strains SYSU A558-1T and SYSU A121-1 represent a novel species of the genus Haloterrigena, for which the name Haloterrigena gelatinilytica sp. nov. is proposed. The type strain is SYSU A558-1T (= KCTC 4259T = CGMCC 1.15953T).

The origin of the eukaryotic cell is a major open question in biology. Asgard archaea are the closest known prokaryotic relatives of eukaryotes, and their genomes encode various eukaryotic signature proteins, indicating some elements of cellular complexity prior to the emergence of the first eukaryotic cell. Yet, microscopic evidence to demonstrate the cellular structure of uncultivated Asgard archaea in the environment is thus far lacking. We used primer-free sequencing to retrieve 715 almost full-length Loki- and Heimdallarchaeota 16S rRNA sequences and designed novel oligonucleotide probes to visualize their cells in marine sediments (Aarhus Bay, Denmark) using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Super-resolution microscopy revealed 1–2 µm large, coccoid cells, sometimes occurring as aggregates. Remarkably, the DNA staining was spatially separated from ribosome-originated FISH signals by 50–280 nm. This suggests that the genomic material is condensed and spatially distinct in a particular location and could indicate compartmentalization or membrane invagination in Asgard archaeal cells.

It has been predicted that 30-80% of archaeal genomes remain annotated as hypothetical proteins with no assigned gene function. Further, many archaeal organisms are difficult to grow or are unculturable. To overcome these technical and experimental hurdles, we have developed a high-throughput functional genomics screen that utilizes capillary electrophoresis (CE) to identify nucleic acid modifying enzymes based on activity rather than sequence homology. Here, we describe a functional genomics screening workflow to find DNA modifying enzyme activities encoded by the hyperthermophile Thermococcus kodakarensis (T. kodakarensis). Large DNA insert fosmid libraries representing a ∼5-fold average coverage of the T. kodakarensis genome were prepared in E. coli. RNA-seq showed a high fraction (84%) of T. kodakarensis genes were transcribed in E. coli despite differences in promoter structure and translational machinery. Our high-throughput screening workflow used fluorescently labeled DNA substrates directly in heat-treated lysates of fosmid clones with capillary electrophoresis detection of reaction products. Using this method, we identified both a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and a novel AP lyase DNA repair enzyme family (termed 'TK0353') found only in a small subset of Thermococcales. The screening methodology described provides a fast and efficient way to explore the T. kodakarensis genome for a variety of nucleic acid modifying activities and may have implications for similar exploration of enzymes and pathways that underlie core cellular processes in other Archaea. IMPORTANCE This study provides a rapid, simple, high-throughput method to discover novel archaeal nucleic acid modifying enzymes by utilizing a fosmid genomic library, next-generation sequencing and capillary electrophoresis. The method described here provides details necessary to create 384-well fosmid library plates from Thermococcus kodakarensis genomic DNA, sequence 384-well fosmids plates using Illumina next generation sequencing and perform high-throughput functional read-out assays using capillary electrophoresis to identify a variety of nucleic acid modifying activities including DNA cleavage and ligation. We used this approach to identify a new DNA endonuclease activity for a previously described RNA endonuclease (Nob1) and identify a novel AP lyase enzyme (TK0353) that lacks sequence homology to known nucleic acid modifying enzymes.

Replicative DNA polymerases cannot initiate DNA synthesis de novo and rely on dedicated RNA polymerases, primases, to generate a short primer. This primer is then extended by the DNA polymerase. In diverse archaeal species, the primase has long been known to have the ability to synthesize both RNA and DNA. However, the relevance of these dual nucleic acid synthetic modes for productive primer synthesis has remained enigmatic. In the current work, we reveal that the ability of primase to polymerize DNA serves dual roles in promoting the hand-off of the primer to the replicative DNA polymerase holoenzyme. First, it creates a 5′-RNA-DNA-3′ hybrid primer which serves as an optimal substrate for elongation by the replicative DNA polymerase. Second, it promotes primer release by primase. Furthermore, modeling and experimental data indicate that primase incorporates a deoxyribonucleotide stochastically during elongation and that this switches the primase into a dedicated DNA synthetic mode polymerase., DNA primases initiate a short primer before handing off to DNA polymerases to continue replication. Here the authors reveal a unique ability of archaeal primases to first synthesize RNA before stochastically incorporating a deoxyribonucleotide and further extending the primer as DNA.

Three halophilic archaeal strains, Gai1-5

All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases.

Historical DNA analysis of archival samples has added new dimensions to population genetic studies, enabling spatiotemporal approaches for reconstructing population history and informing conservation management. Here we tested the efficacy of Genotyping-in-Thousands by sequencing (GT-seq) for collecting targeted single nucleotide polymorphism genotypic data from archival scale samples, and applied this approach to a study of kokanee salmon (Oncorhynchus nerka) in Kluane National Park and Reserve (KNPR; Yukon, Canada) that underwent a severe 12-year population decline followed by a rapid rebound. We genotyped archival scales sampled pre-crash and contemporary fin clips collected post-crash, revealing high coverage (> 90% average genotyping across all individuals) and low genotyping error (

With increasing temperature, nucleobases in DNA become increasingly damaged by hydrolysis of exocyclic amines. The most prominent damage includes the conversion of cytosine to uracil and adenine to hypoxanthine. These damages are mutagenic and put the integrity of the genome at risk if not repaired appropriately. Several archaea live at elevated temperatures and thus, are exposed to a higher risk of deamination. Earlier studies have shown that DNA polymerases of archaea have the property of sensing deaminated nucleobases in the DNA template and thereby stalling the DNA synthesis during DNA replication providing another layer of DNA damage recognition and repair. However, the structural basis of uracil and hypoxanthine sensing by archaeal B‐family DNA polymerases is sparse. Here we report on three new crystal structures of the archaeal B‐family DNA polymerase from Thermococcus kodakarensis (KOD) DNA polymerase in complex with primer and template strands that have extended single stranded DNA template 5’‐overhangs. These overhangs contain either the canonical nucleobases as well as uracil or hypoxanthine, respectively, and provide unprecedented structural insights into their recognition by archaeal B‐family DNA polymerases., Due to their life in hot environments, archaea are increasingly affected by deamination of the bases cytosine and adenine. To prevent mutations, archaeal DNA polymerases seem to recognize these base changes by a so‐called “read‐ahead” mechanism and react accordingly. Here we show by means of crystal structures how Thermococcus kodakarensis (KOD) DNA polymerase binds uracil and hypoxanthine in a special binding pocket and thus can recognize these bases on the single stranded template overhang already six nucleotides before the active site.

The Dallol protovolcanic area on the Danakil Depression (Afar region, Ethiopia) exhibits unique hydrothermal manifestations in hypersaline context, yielding varied polyextreme physicochemical conditions. Previous studies identified a wide archaeal diversity in less extreme brines but failed to identify microorganisms thriving in either high-chaotropicity, low-water-activity brines or hyperacidic-hypersaline Na-Fe-rich brines. Recently, we accessed several small lakes under intense degassing activity adjacent to the Round Mountain, west to the Dallol dome (Western Canyon Lakes; WCL1-5). They exhibited intermediate parameter combinations (pH~5, 34-41% NaCl-dominated salts with relatively high levels of chaotropic Mg-Ca salts) that should allow to better constrain life limits. These lakes were overwhelmingly dominated by Archaea, encompassing up to 99% of prokaryotic 16S rRNA gene amplicon sequences in metabarcoding studies. The majority belonged to Halobacteriota and Nanohaloarchaeota, the latter representing up to half of prokaryotic sequences. Optical and epifluorescence microscopy showed active cells in natural samples and diverse morphotypes in enrichment cultures. Scanning electron microscopy coupled with energy-dispersive x-ray spectroscopy revealed tiny cells (200-300 nm diameter) epibiotically associated with somewhat larger cells (0.6-1 μm) but also the presence of silica-dominated precipitates of similar size and shape, highlighting the difficulty of distinguishing microbes from mineral biomorphs in this kind of low-biomass systems. This article is protected by copyright. All rights reserved.

Archaeal sequences have been detected in human colostrum and milk, but no studies have determined whether living archaea are present in either of these fluids. Methanogenic archaea are neglected since they are not detected by usual molecular and culture methods. By using improved DNA detection protocols and microbial culture techniques associated with antioxidants previously developed in our center, we investigated the presence of methanogenic archaea using culture and specific Methanobrevibacter smithii and Methanobrevibacter oralis real-time PCR in human colostrum and milk. M. smithii was isolated from 3 colostrum and 5 milk (day 10) samples. M. oralis was isolated from 1 milk sample. For 2 strains, the genome was sequenced, and the rhizome was similar to that of strains previously isolated from the human mouth and gut. M. smithii was detected in the colostrum or milk of 5/13 (38%) and 37/127 (29%) mothers by culture and qPCR, respectively. The different distribution of maternal body mass index according to the detection of M. smithii suggested an association with maternal metabolic phenotype. M. oralis was not detected by molecular methods. Our results suggest that breastfeeding may contribute to the vertical transmission of these microorganisms and may be essential to seed the infant’s microbiota with these neglected critical commensals from the first hour of life.

Summary Faecal contamination is one of the major factors affecting biological water quality. In this study, we investigated microbial taxonomic diversity of faecally polluted lotic ecosystems in Norway. These ecosystems comprise tributaries of drinking water reservoirs with moderate and high faecal contamination levels, an urban creek exposed to extremely high faecal pollution and a rural creek that was the least faecally polluted. The faecal water contamination had both anthropogenic and zoogenic origins identified through quantitative microbial source tracking applying host‐specific Bacteroidales 16S rRNA genetic markers. The microbial community composition revealed that Proteobacteria and Bacteroidetes (70–90% relative abundance) were the most dominant bacterial phyla, followed by Firmicutes, especially in waters exposed to anthropogenic faecal contamination. The core archaeal community consisted of Parvarchaeota (mainly in the tributaries of drinking water reservoirs) and Crenarchaeota (in the rural creek). The aquatic microbial diversity was substantially reduced in water with severe faecal contamination. In addition, the community compositions diverge between waters with dominant anthropogenic or zoogenic pollution origins. These findings present novel interpretations of the effect of anthropo‐zoogenic faecal water contamination on microbial diversity in lotic ecosystems.

The diversity and community composition of archaea in soil samples from three wetlands (SP1, SP2, and SP3) of Ebinur Lake were studied by constructing 16S rDNA cloning library. The correlation between the diversity of archaea and soil environmental factors was analyzed by CANOCO software. The aim of this study was to reveal the differences of community structures of archaea in different sample sites, to provide a theoretical basis for further study on degradation and restoration of Ebinur Lake wetland. The results showed that Euryarchaeota accounted for 57.1% was the most dominant phylum observed, followed by Thaumarchaeota and Crenarchaeota for the three wetland soil analyzed. Compared with SP3 site, the proportions of Euryarchaeota were decreased by 16.70% and 31.78%, while Thaumarchaeota increased by 7.26% and 17.64% in the SP1 and SP2, respectively. Crenarchaeota was found only in SP3. Shannon–wiener diversity indices in SP1, SP2, and SP3 sites were 3.44, 3.87, and 3.94, respectively, indicating that the diversity of archaea in three plots was: SP3 > SP2 > SP1. Redundancy analysis (RDA) showed that electrical conductivity (EC), soil moisture (SM), hydrogen potential (pH), and soil organic matter content (SOM) may affect archaeal communities. Compared to EC and pH, SM and SOM may have a greater impact on the community composition of archaea.

An extremely halophilic archaeon, strain ZY21T, was isolated from a subterranean rock salt sample in Yunnan, China. Colonies of strain ZY21T on nutrient-rich agar plates are orange, wet and transparent. Cells are pleomorphic, motile, Gram-stain negative and lyse in distilled water. Cells can grow at 20–55 °C (optimum 42 °C), in the presence of 15–30% (w/v) NaCl (optimum 18–20%) and at pH 6.0–9.5 (optimum 7.5). Mg2+ is required for growth (optimum 0.3 M). The major polar lipids of strain ZY21T are phosphatidylglycerol, phosphatidylglycerol sulfate and phosphatidylglycerol phosphate methyl ester, sulfated mannosyl-glucosyl-glycerol diether-1 and seven unidentified glycolipids. Sequence similarity searches with the 16S rRNA gene and rpoB′ gene showed that strain ZY21T is closely related to Halobellus rufus CBA1103T (sequence similarities: 97.5% for 16S rRNA gene and 93.3% for rpoB′ gene). The DNA G+C content of strain ZY21T was determined to be 63.0 mol% based on the draft genome sequence. Genome-based sequence similarity analysis showed that the values of the ANI, AAI, and DDH were far below the boundary for delineation of new species. Phenotypic, chemotaxonomic characteristics and phylogenetic properties suggest that strain ZY21T represents a novel species in the genus Halobellus, for which the name Halobellus captivus sp. nov. is proposed. The type strain is ZY21T (= CGMCC 1.16343T = NBRC 113439T).

Summary Waste rice straw (RS) is generated in massive quantities around the world and is often burned, creating greenhouse gas and air quality problems. Anaerobic digestion (AD) may be a better option for RS management, but RS is presumed to be comparatively refractory under anaerobic conditions without pre‐treatment or co‐substrates. However, this presumption assumes frequent reactor feeding regimes but less frequent feeding may be better for RS due to slow hydrolysis rates. Here, we assess how feeding frequency (FF) and organic loading rate (OLR) impacts microbial communities and biogas production in RS AD reactors. Using 16S rDNA amplicon sequencing and bioinformatics, microbial communities from five bench‐scale bioreactors were characterized. At low OLR (1.0 g VS l−1 day−1), infrequently fed units (once every 21 days) had higher specific biogas yields than more frequent feeding (five in 7 days), although microbial community diversities were statistically similar (P > 0.05; ANOVA with Tukey comparison). In contrast, an increase in OLR to 2.0 g VS l−1 day−1 significantly changed Archaeal and fermenting Eubacterial sub‐communities and the least frequency fed reactors failed. ‘Stable’ reactors were dominated by Methanobacterium, Methanosarcina and diverse Bacteroidetes, whereas ‘failed’ reactors saw shifts towards Clostridia and Christensenellaceae among fermenters and reduced methanogen abundances. Overall, OLR impacted RS AD microbial communities more than FF. However, combining infrequent feeding and lower OLRs may be better for RS AD because of higher specific yields.

Genomic organization impacts accessibility and movement of information processing systems along DNA. DNA-bound proteins dynamically dictate gene expression and provide regulatory potential to tune transcription rates to match ever-changing environmental conditions. Archaeal genomes are typically small, circular, gene dense, and organized either by histone proteins that are homologous to their eukaryotic counterparts, or small basic proteins that function analogously to bacterial nucleoid proteins. We review here how archaeal genomes are organized and how such organization impacts archaeal gene expression, focusing on conserved DNA-binding proteins within the clade and the factors that are known to impact transcription initiation and elongation within protein-bound genomes.

The discovery of the archaeal domain of life is tightly connected to an in-depth analysis of the prokaryotic RNA world. In addition to Carl Woese's approach to use the sequence of the 16S rRNA gene as phylogenetic marker, the finding of Karl Stetter and Wolfram Zillig that archaeal RNA polymerases (RNAPs) were nothing like the bacterial RNAP but are more complex enzymes that resemble the eukaryotic RNAPII was one of the key findings supporting the idea that archaea constitute the third major branch on the tree of life. This breakthrough in transcriptional research 40years ago paved the way for in-depth studies of the transcription machinery in archaea. However, although the archaeal RNAP and the basal transcription factors that fine-tune the activity of the RNAP during the transcription cycle are long known, we still lack information concerning the architecture and dynamics of archaeal transcription complexes. In this context, single-molecule measurements were instrumental as they provided crucial insights into the process of transcription initiation, the architecture of the initiation complex and the dynamics of mobile elements of the RNAP. In this review, we discuss single-molecule approaches suitable to examine molecular mechanisms of transcription and highlight findings that shaped our understanding of the archaeal transcription apparatus. We furthermore explore the possibilities and challenges of next-generation single-molecule techniques, for example, super-resolution microscopy and single-molecule tracking, and ask whether these approaches will ultimately allow us to investigate archaeal transcription in vivo.

Although arctic and subarctic lakes are important sources of methane, the emission of which will increase due to the melting of permafrost, the processes related to the methane cycle in such environments are far from being comprehensively understood. Here we studied the microbial communities in the near-bottom water layer and sediments of the meromictic subarctic Lake Svetloe using high-throughput sequencing of the 16S rRNA and methyl coenzyme M reductase subunit A genes. Hydrogenotrophic methanogens of the order Methanomicrobiales were abundant, both in the water column and in sediments, while the share of acetoclastic Methanosaetaceae decreased with the depth of sediments. Members of the Methanomassiliicoccales order were absent in the water but abundant in the deep sediments. Archaea known to perform anaerobic oxidation of methane were not found. The bacterial component of the microbial community in the bottom water layer included oxygenic (Cyanobacteria) and anoxygenic (Chlorobi) phototrophs, aerobic Type I methanotrophs, methylotrophs, syntrophs, and various organotrophs. In deeper sediments the diversity of the microbial community decreased, and it became dominated by methanogenic archaea and the members of the Bathyarchaeota, Chloroflexi and Deltaproteobacteria. This study shows that the sediments of a subarctic meromictic lake contain a taxonomically and metabolically diverse community potentially capable of complete mineralization of organic matter.

Candidatus Hydrothermarchaeota, formally called Marine Benthic Group E, has often been detected in iron- and sulfur-rich marine environments, such as hydrothermal vents and cold seeps. However, their ecology and physiology remain unclear. Cultivated representatives of this group are still lacking and only several metagenome-assembled genomes (MAGs) and single-amplified genomes (SAGs) are available from two deep-sea hydrothermal areas, the Juan de Fuca Ridge (JdFR) and Guaymas Basin (GB), in the north-east Pacific. We herein report four MAGs of Ca. Hydrothermarchaeota recovered from hydrothermally-inactive metal sulfide deposits at the Southern Mariana Trough (SMT) in the north-west Pacific. A phylogenetic analysis indicated that the MAGs of the SMT were distinct from those of the JdFR and GB at the genus or potentially family level. Ca. Hydrothermarchaeota MAGs from the SMT commonly possessed putative genes for carboxydotrophic and hydrogenotrophic respiration using oxidized chemical species of sulfur as electron acceptors and also for carbon fixation, as reported previously in MAGs/SAGs from the JdFR and GB. This result strongly supports Ca. Hydrothermarchaeota containing anaerobic chemolithoautotrophs using carbon monoxide and/or hydrogen as electron donors. A comparative genome analysis highlighted differences in the capability of nitrogen fixation between MAGs from the SMT and the other fields, which are consistent with environmental differences in the availability of nitrogen sources for assimilation between the fields. Based on the wide distribution in various areas, abundance, and metabolic potential of Ca. Hydrothermarchaeota, they may play a role in the biogeochemical cycling of carbon, nitrogen, sulfur, and iron in marine environments, particularly in deep-sea hydrothermal fields.

The DNA translocation activity of the minichromosome maintenance (MCM) complex powers DNA strand separation of the replication forks of eukaryotes and archaea. Here we illustrate an atomic level mechanism for this activity with a crystal structure of an archaeal MCM hexamer bound to single-stranded DNA and nucleotide cofactors. Sequence conservation indicates this rotary mechanism is fully possible for all eukaryotes and archaea. The structure definitively demonstrates the ring orients during translocation with the N-terminal domain leading, indicating that the translocation activity could also provide the physical basis of replication initiation where a double-hexamer idly encircling double-stranded DNA transforms to single-hexamers that encircle only one strand. In this mechanism, each strand binds to the N-terminal tier of one hexamer and the AAA+ tier of the other hexamer such that one ring pulls on the other, aligning equivalent interfaces to enable each hexamer to pull its translocation strand outside of the opposing hexamer., Eukaryotes and archaea use a heximeric ring-shaped MCM helicase to unwind the DNA template during replication. Here the authors present a crystal structure of the MCM complex from archaeon S. solfataricus bound to single-stranded DNA, and to a combination of ADP, and ATP-mimic, ADP-BeF3.

Casposons are a group of bacterial and archaeal DNA transposons encoding a specific integrase, termed casposase, which is homologous to the Cas1 enzyme responsible for the integration of new spacers into CRISPR loci. Here, we characterized the sequence motifs recognized by the casposase from a thermophilic archaeon Aciduliprofundum boonei. We identified a stretch of residues, located in the leader region upstream of the actual integration site, whose deletion or mutagenesis impaired the concerted integration reaction. However, deletions of two-thirds of the target site were fully functional. Various single-stranded 6-FAM-labelled oligonucleotides derived from casposon terminal inverted repeats were as efficiently incorporated as duplexes into the target site. This result suggests that, as in the case of spacer insertion by the CRISPR Cas1–Cas2 integrase, casposon integration involves splaying of the casposon termini, with single-stranded ends being the actual substrates. The sequence critical for incorporation was limited to the five terminal residues derived from the 3′ end of the casposon. Furthermore, we characterize the casposase from Nitrosopumilus koreensis, a marine member of the phylum Thaumarchaeota, and show that it shares similar properties with the A. boonei enzyme, despite belonging to a different family. These findings further reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the CRISPR–Cas systems.