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In Archaea and Bacteria, the arrays called CRISPRs for ‘clustered regularly interspaced short palindromic repeats’ and the CRISPR associated genes or cas provide adaptive immunity against viruses, plasmids and transposable elements. Short sequences called spacers, corresponding to fragments of invading DNA, are stored in-between repeated sequences. The CRISPR–Cas systems target sequences homologous to spacers leading to their degradation. To facilitate investigations of CRISPRs, we developed 12 years ago a website holding the CRISPRdb. We now propose CRISPRCasdb, a completely new version giving access to both CRISPRs and cas genes. We used CRISPRCasFinder, a program that identifies CRISPR arrays and cas genes and determine the system's type and subtype, to process public whole genome assemblies. Strains are displayed either in an alphabetic list or in taxonomic order. The database is part of the CRISPR-Cas++ website which also offers the possibility to analyse submitted sequences and to download programs. A BLAST search against lists of repeats and spacers extracted from the database is proposed. To date, 16 990 complete prokaryote genomes (16 650 bacteria from 2973 species and 340 archaea from 300 species) are included. CRISPR–Cas systems were found in 36% of Bacteria and 75% of Archaea strains. CRISPRCasdb is freely accessible at https://crisprcas.i2bc.paris-saclay.fr/.

Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2′ modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core.

A synthesis of phenotypic and quantitative genomic traits is provided for bacteria and archaea, in the form of a scripted, reproducible workflow that standardizes and merges 26 sources. The resulting unified dataset covers 14 phenotypic traits, 5 quantitative genomic traits, and 4 environmental characteristics for approximately 170,000 strain-level and 15,000 species-aggregated records. It spans all habitats including soils, marine and fresh waters and sediments, host-associated and thermal. Trait data can find use in clarifying major dimensions of ecological strategy variation across species. They can also be used in conjunction with species and abundance sampling to characterize trait mixtures in communities and responses of traits along environmental gradients., Measurement(s)Trait • phenotypic trait • quantitative genomic traitTechnology Type(s)digital curationFactor Type(s)habitat • speciesSample Characteristic - OrganismArchaea • BacteriaSample Characteristic - Locationglobal Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12221732

Summary: Euryarchaea from the genus Halorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: Halorhabdus utahensisAX-2T from the shallow Great Salt Lake of Utah, and Halorhabdus tiamateaSARL4BT from the Shaban deep-sea hypersaline anoxic lake (DHAL) in the Red Sea. We sequenced the H.tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H.tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant-derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low-oxygen conditions. Likewise, proteomics indicated quinone-mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. Halorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light-dependent enzymes (e.g. bacteriorhodopsin), suggesting that H.tiamatea evolution was mostly not governed by a cold, dark, anoxic deep-sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase-rich Halorhabdus members in the Mediterranean DHAL Medee, which supports that Halorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments., This study was supported by the EU FP7 project MAMBA (‘Marine Metagenomics for New Biotechnological Applications’, FP7‐KBBE‐2008–226977, the Spanish Ministry of Economy and Competitiveness (grant BIO2011–25012) and the Max Planck Society. H.T., O.V.G. and P.N.G. acknowledge the support of EU FP7 for the project MicroB3 (OCEAN‐2011‐287589).

The definitive version is available at ww3.interscience.wiley.com. En libre-accès sur Archimer : http://archimer.ifremer.fr/doc/2008/publication-6113.pdf; International audience; Spontaneous and induced abasic sites in hyperthermophiles DNA have long been suspected to occur at high frequency. Here, Pyrococcus abyssi was used as an attractive model to analyse the impact of such lesions onto the maintenance of genome integrity. We demonstrated that endogenous AP sites persist at a slightly higher level in P. abyssi genome compared with Escherichia coli. Then, the two replicative DNA polymerases, PabpolB and PabpolD, were characterized in presence of DNA containing abasic sites. Both Pabpols had abortive DNA synthesis upon encountering AP sites. Under running start conditions, PabpolB could incorporate in front of the damage and even replicate to the full-length oligonucleotides containing a specific AP site, but only when present at a molar excess. Conversely, bypassing activity of PabpolD was strictly inhibited. The tight regulation of nucleotide incorporation opposite the AP site was assigned to the efficiency of the proof-reading function, because exonuclease-deficient enzymes exhibited effective TLS. Steady-state kinetics reinforced that Pabpols are high-fidelity DNA polymerases onto undamaged DNA. Moreover, Pabpols preferentially inserted dAMP opposite an AP site, albeit inefficiently. While the template sequence of the oligonucleotides did not influence the nucleotide insertion, the DNA topology could impact on the progression of Pabpols. Our results are interpreted in terms of DNA damage tolerance.

Clustered regularly interspaced short palindromic repeats (CRISPRs) constitute a particular family of tandem repeats found in a wide range of prokaryotic genomes (half of eubacteria and almost all archaea). They consist of a succession of highly conserved regions (DR) varying in size from 23 to 47 bp, separated by similarly sized unique sequences (spacer) of usually viral origin. A CRISPR cluster is flanked on one side by an AT-rich sequence called the leader and assumed to be a transcriptional promoter. Recent studies suggest that this structure represents a putative RNA-interference-based immune system. Here we describe CRISPRFinder, a web service offering tools to (i) detect CRISPRs including the shortest ones (one or two motifs); (ii) define DRs and extract spacers; (iii) get the flanking sequences to determine the leader; (iv) blast spacers against Genbank database and (v) check if the DR is found elsewhere in prokaryotic sequenced genomes. CRISPRFinder is freely accessible at http://crispr.u-psud.fr/Server/CRISPRfinder.php.

International audience; Unrecognized frameshifts, in-frame stop codons and sequencing errors lead to Interrupted CoDing Sequence (ICDS) that can seriously affect all subsequent steps of functional characterization, from in silico analysis to high-throughput proteomic projects. Here, we describe the Interrupted CoDing Sequence database containing ICDS detected by a similarity-based approach in 80 complete prokaryotic genomes. ICDS can be retrieved by species browsing or similarity searches via a web interface (http://www-bio3d-igbmc.u-strasbg.fr/ICDS/). The definition of each interrupted gene is provided as well as the ICDS genomic localization with the surrounding sequence. Furthermore, to facilitate the experimental characterization of ICDS, we propose optimized primers for re-sequencing purposes. The database will be regularly updated with additional data from ongoing sequenced genomes. Our strategy has been validated by three independent tests: (i) ICDS prediction on a benchmark of artificially created frameshifts, (ii) comparison of predicted ICDS and results obtained from the comparison of the two genomic sequences of Bacillus licheniformis strain ATCC 14580 and (iii) re-sequencing of 25 predicted ICDS of the recently sequenced genome of Mycobacterium smegmatis. This allows us to estimate the specificity and sensitivity (95 and 82%, respectively) of our program and the efficiency of primer determination.

International audience; This study describes reconstruction of two highly unusual archaeal genomes by de novo metagenomic assembly of multiple, deeply sequenced libraries from surface waters of Lake Tyrrell (LT), a hypersaline lake in NW Victoria, Australia. Lineage-specific probes were designed using the assembled genomes to visualize these novel archaea, which were highly abundant in the 0.1-0.8 μm size fraction of lake water samples. Gene content and inferred metabolic capabilities were highly dissimilar to all previously identified hypersaline microbial species. Distinctive characteristics included unique amino acid composition, absence of Gvp gas vesicle proteins, atypical archaeal metabolic pathways and unusually small cell size (approximately 0.6 μm diameter). Multi-locus phylogenetic analyses demonstrated that these organisms belong to a new major euryarchaeal lineage, distantly related to halophilic archaea of class Halobacteria. Consistent with these findings, we propose creation of a new archaeal class, provisionally named 'Nanohaloarchaea'. In addition to their high abundance in LT surface waters, we report the prevalence of Nanohaloarchaea in other hypersaline environments worldwide. The simultaneous discovery and genome sequencing of a novel yet ubiquitous lineage of uncultivated microorganisms demonstrates that even historically well-characterized environments can reveal unexpected diversity when analyzed by metagenomics, and advances our understanding of the ecology of hypersaline environments and the evolutionary history of the archaea.

International audience; A novel extrachromosomal element that we called pAMT11 was discovered in a deep-sea vent isolate belonging to the hyperthermophilic euryarchaeal order Thermococcales. It consists of a double-stranded DNA of 20,534bp which encodes 30 putative open reading frames (ORFs) of which six could be assigned to a putative function on the basis of sequence similarity to known genes or to protein domain families. Most of the ORFs of pAMT1 showed homology and synteny with a genomic island of Thermococcus kodakaraensis KOD1. This region, named TKV1, was previously described as a "virus-like integrated element" and assumed to integrate into the host chromosome by a site-specific recombination mechanism similar to that of Sulfolobus solfataricus virus 1. While most of the genes shared by pAMT11 and TKV1 encode putative membrane proteins presumably involved in virus particle formation, attempts to induce production of virus particles by mitomycin treatment of AMT11 cultures failed, suggesting that pAMT11 may represent the genome of a defective virus or a plasmid. Genomes of mobile elements usually contain two regions: a core of conserved genes mainly involved in replication, maintenance or spreading of the genetic element, and a variable set of accessory genes. Surprisingly, genes presumably implied in the replication process are quite divergent between TKV1 and pAMT11. Indeed, TKV1 possesses a MCM-like protein that may function as a replication initiator, while pAMT11 encodes a putative non-conventional protein distantly related to the Rep protein previously described in a small plasmid of Pyrococcus sp. strain JT1, assumed to replicate by a rolling-circle (RC) mechanism. However, in the case of pAMT11, this mode of plasmid replication could not be experimentally proven and is questionable given the lack of significant similarities with any other members of the RC-Rep superfamily and its unusual large size compared to other RC plasmids.

International audience; Archaeal viruses, or archaeoviruses, display a wide range of virion morphotypes. Whereas the majority of those morphotypes are unique to archaeal viruses, some are more widely distributed across different cellular domains. Tailed double-stranded DNA archaeoviruses are remarkably similar to viruses of the same morphology (order Caudovirales) that infect many bacterial hosts. They have, so far, only been found in one phylum of the archaea, the Euryarchaeota, which has led to controversial hypotheses about their origin. In the present paper, we describe the identification and analysis of a putative provirus present in the genome of a mesophilic thaumarchaeon. We show that the provirus is related to tailed bacterial and euryarchaeal viruses and encodes a full complement of proteins that are required to build a tailed virion. The recently discovered wide distribution of tailed viruses in Euryarchaeota and the identification of a related provirus in Thaumarchaeota, an archaeal phylum which might have branched off before the separation of Crenarchaeota and Euryarchaeota, suggest that an association of these viruses with Archaea might be more ancient than previously anticipated.

Background In Archeae and Bacteria, the repeated elements called CRISPRs for "clustered regularly interspaced short palindromic repeats" are believed to participate in the defence against viruses. Short sequences called spacers are stored in-between repeated elements. In the current model, motifs comprising spacers and repeats may target an invading DNA and lead to its degradation through a proposed mechanism similar to RNA interference. Analysis of intra-species polymorphism shows that new motifs (one spacer and one repeated element) are added in a polarised fashion. Although their principal characteristics have been described, a lot remains to be discovered on the way CRISPRs are created and evolve. As new genome sequences become available it appears necessary to develop automated scanning tools to make available CRISPRs related information and to facilitate additional investigations. Description We have produced a program, CRISPRFinder, which identifies CRISPRs and extracts the repeated and unique sequences. Using this software, a database is constructed which is automatically updated monthly from newly released genome sequences. Additional tools were created to allow the alignment of flanking sequences in search for similarities between different loci and to build dictionaries of unique sequences. To date, almost six hundred CRISPRs have been identified in 475 published genomes. Two Archeae out of thirty-seven and about half of Bacteria do not possess a CRISPR. Fine analysis of repeated sequences strongly supports the current view that new motifs are added at one end of the CRISPR adjacent to the putative promoter. Conclusion It is hoped that availability of a public database, regularly updated and which can be queried on the web will help in further dissecting and understanding CRISPR structure and flanking sequences evolution. Subsequent analyses of the intra-species CRISPR polymorphism will be facilitated by CRISPRFinder and the dictionary creator. CRISPRdb is accessible at http://crispr.u-psud.fr/crispr

The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor.

Background As bacteria, motile archaeal species swim by means of rotating flagellum structures driven by a proton gradient force. Interestingly, experimental data have shown that the archaeal flagellum is non-homologous to the bacterial flagellum either in terms of overall structure, components and assembly. The growing number of complete archaeal genomes now permits to investigate the evolution of this unique motility system. Results We report here an exhaustive phylogenomic analysis of the components of the archaeal flagellum. In all complete archaeal genomes, the genes coding for flagellum components are co-localized in one or two well-conserved genomic clusters showing two different types of organizations. Despite their small size, these genes harbor a good phylogenetic signal that allows reconstruction of their evolutionary histories. These support a history of mainly vertical inheritance for the components of this unique motility system, and an interesting possible ancient horizontal gene transfer event (HGT) of a whole flagellum-coding gene cluster between Euryarchaeota and Crenarchaeota. Conclusion Our study is one of the few exhaustive phylogenomics analyses of a non-informational cell machinery from the third domain of life. We propose an evolutionary scenario for the evolution of the components of the archaeal flagellum. Moreover, we show that the components of the archaeal flagellar system have not been frequently transferred among archaeal species, indicating that gene fixation following HGT can also be rare for genes encoding components of large macromolecular complexes with a structural role.

Analysis of the complete genome indicates that insertion sequences (ISs) are abundant in the radio-resistant bacterium Deinococcus radiodurans. By developing a forward mutagenesis assay to detect any inactivation events in D. radiodurans, we found that in the presence of an active mismatch repair system 75% of the mutations to trimethoprim-resistance (Tmp(R)) resulted from an IS insertion into the thyA coding region. Analysis of their distribution among the spontaneous Tmp(R) mutants indicated that five different ISs were transpositionally active. A type II Miniature Inverted-repeat Transposable Element (MITE), related to one of the deinococcal ISs, was also discovered as an insertion into thyA. Seven additional genomic copies of this MITE element were identified by BLASTN. Gamma-ray irradiation of D. radiodurans led to an increase of up to 10-fold in the frequency of Tmp(R) mutants. Analysis of the induced mutations in cells exposed to 10 kGy indicated that gamma-irradiation induced transposition of ISDra2 approximately 100-fold. A 50-fold induction of ISDra2 transposition was also observed in cells exposed to 600 J m(-2) UV-irradiation. Point mutations to rifampicin resistance (Rif(R)) were also induced by gamma-irradiation to reach a plateau at 2 kGy. The plateau value represented a 16-fold increase in the mutant frequency over the background. Although error-free repair strategies predominate in D. radiodurans, an upregulation of transposition, as well as induction of point mutations in cells recovering from DNA damage, provide a genetic variability that may have long-term evolutionary consequences on the fitness of this organism in its habitat.