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Abstract Background Collections of Clusters of Orthologous Genes (COGs) provide indispensable tools for comparative genomic analysis, evolutionary reconstruction and functional annotation of new genomes. Initially, COGs were made for all complete genomes of cellular life forms that were available at the time. However, with the accumulation of thousands of complete genomes, construction of a comprehensive COG set has become extremely computationally demanding and prone to error propagation, necessitating the switch to taxon-specific COG collections. Previously, we reported the collection of COGs for 41 genomes of Archaea (arCOGs). Here we present a major update of the arCOGs and describe evolutionary reconstructions to reveal general trends in the evolution of Archaea. Results The updated version of the arCOG database incorporates 91% of the pangenome of 120 archaea (251,032 protein-coding genes altogether) into 10,335 arCOGs. Using this new set of arCOGs, we performed maximum likelihood reconstruction of the genome content of archaeal ancestral forms and gene gain and loss events in archaeal evolution. This reconstruction shows that the last Common Ancestor of the extant Archaea was an organism of greater complexity than most of the extant archaea, probably with over 2,500 protein-coding genes. The subsequent evolution of almost all archaeal lineages was apparently dominated by gene loss resulting in genome streamlining. Overall, in the evolution of Archaea as well as a representative set of bacteria that was similarly analyzed for comparison, gene losses are estimated to outnumber gene gains at least 4 to 1. Analysis of specific patterns of gene gain in Archaea shows that, although some groups, in particular Halobacteria, acquire substantially more genes than others, on the whole, gene exchange between major groups of Archaea appears to be largely random, with no major ‘highways’ of horizontal gene transfer. Conclusions The updated collection of arCOGs is expected to become a key resource for comparative genomics, evolutionary reconstruction and functional annotation of new archaeal genomes. Given that, in spite of the major increase in the number of genomes, the conserved core of archaeal genes appears to be stabilizing, the major evolutionary trends revealed here have a chance to stand the test of time. Reviewers This article was reviewed by (for complete reviews see the Reviewers’ Reports section): Dr. PLG, Prof. PF, Dr. PL (nominated by Prof. JPG).

Abstract Background The virus-host arms race is a major theater for evolutionary innovation. Archaea and bacteria have evolved diverse, elaborate antivirus defense systems that function on two general principles: i) immune systems that discriminate self DNA from nonself DNA and specifically destroy the foreign, in particular viral, genomes, whereas the host genome is protected, or ii) programmed cell suicide or dormancy induced by infection. Presentation of the hypothesis Almost all genomic loci encoding immunity systems such as CRISPR-Cas, restriction-modification and DNA phosphorothioation also encompass suicide genes, in particular those encoding known and predicted toxin nucleases, which do not appear to be directly involved in immunity. In contrast, the immunity systems do not appear to encode antitoxins found in typical toxin-antitoxin systems. This raises the possibility that components of the immunity system themselves act as reversible inhibitors of the associated toxin proteins or domains as has been demonstrated for the Escherichia coli anticodon nuclease PrrC that interacts with the PrrI restriction-modification system. We hypothesize that coupling of diverse immunity and suicide/dormancy systems in prokaryotes evolved under selective pressure to provide robustness to the antivirus response. We further propose that the involvement of suicide/dormancy systems in the coupled antivirus response could take two distinct forms: 1) induction of a dormancy-like state in the infected cell to ‘buy time’ for activation of adaptive immunity; 2) suicide or dormancy as the final recourse to prevent viral spread triggered by the failure of immunity. Testing the hypothesis This hypothesis entails many experimentally testable predictions. Specifically, we predict that Cas2 protein present in all cas operons is a mRNA-cleaving nuclease (interferase) that might be activated at an early stage of virus infection to enable incorporation of virus-specific spacers into the CRISPR locus or to trigger cell suicide when the immune function of CRISPR-Cas systems fails. Similarly, toxin-like activity is predicted for components of numerous other defense loci. Implications of the hypothesis The hypothesis implies that antivirus response in prokaryotes involves key decision-making steps at which the cell chooses the path to follow by sensing the course of virus infection. Reviewers This article was reviewed by Arcady Mushegian, Etienne Joly and Nick Grishin. For complete reviews, go to the Reviewers’ reports section.

Abstract Background In eukaryotes, the CMG (CDC45, MCM, GINS) complex containing the replicative helicase MCM is a key player in DNA replication. Archaeal homologs of the eukaryotic MCM and GINS proteins have been identified but until recently no homolog of the CDC45 protein was known. Two recent developments, namely the discovery of archaeal GINS-associated nuclease (GAN) that belongs to the RecJ family of the DHH hydrolase superfamily and the demonstration of homology between the DHH domains of CDC45 and RecJ, show that at least some Archaea possess a full complement of homologs of the CMG complex subunits. Here we present the results of in-depth phylogenomic analysis of RecJ homologs in archaea. Results We confirm and extend the recent hypothesis that CDC45 is the eukaryotic ortholog of the bacterial and archaeal RecJ family nucleases. At least one RecJ homolog was identified in all sequenced archaeal genomes, with the single exception of Caldivirga maquilingensis. These proteins include previously unnoticed remote RecJ homologs with inactivated DHH domain in Thermoproteales. Combined with phylogenetic tree reconstruction of diverse eukaryotic, archaeal and bacterial DHH subfamilies, this analysis yields a complex scenario of RecJ family evolution in Archaea which includes independent inactivation of the nuclease domain in Crenarchaeota and Halobacteria, and loss of this domain in Methanococcales. Conclusions The archaeal complex of a CDC45/RecJ homolog, MCM and GINS is homologous and most likely functionally analogous to the eukaryotic CMG complex, and appears to be a key component of the DNA replication machinery in all Archaea. It is inferred that the last common archaeo-eukaryotic ancestor encoded a CMG complex that contained an active nuclease of the RecJ family. The inactivated RecJ homologs in several archaeal lineages most likely are dedicated structural components of replication complexes. Reviewers This article was reviewed by Prof. Patrick Forterre, Dr. Stephen John Aves (nominated by Dr. Purificacion Lopez-Garcia) and Prof. Martijn Huynen. For the full reviews, see the Reviewers' Comments section.

Abstract Background The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated) proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Comparative analysis of the Cas protein sequences and structures led to the classification of the CRISPR-Cas systems into three Types (I, II and III). Results A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs) containing a distinct form of the RNA Recognition Motif (RRM) domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes. Conclusions Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure comparison continue to reveal unexpected homologous relationship among Cas proteins. Unification of Cas protein families previously considered unrelated provides for improvement in the classification of CRISPR-Cas systems and a reconstruction of their evolution. Open peer review This article was reviewed by Malcolm White (nominated by Purficacion Lopez-Garcia), Frank Eisenhaber and Igor Zhulin. For the full reviews, see the Reviewers' Comments section.

Abstract Several recent discoveries reveal unexpected versatility of the bacterial and archaeal cytoskeleton systems that are involved in cell division and other processes based on membrane remodeling. Here we apply methods for distant protein sequence similarity detection, phylogenetic approaches, and genome context analysis to described two previously unnoticed families of the FtsZ-tubulin superfamily. One of these families is limited in its spread to Proteobacteria whereas the other is represented in diverse bacteria and archaea, and might be the key component of a novel, multicomponent membrane remodeling system that also includes a Von Willebrand A domain-containing protein, a distinct GTPase and membrane transport proteins of the OmpA family. This article was reviewed by Purificación López-García and Gáspár Jékely; for complete reviews, see the Reviewers Reports section.

Abstract One of the hallmarks of eukaryotic information processing is the co-existence of 3 distinct, multi-subunit RNA polymerase complexes that are dedicated to the transcription of specific classes of coding or non-coding RNAs. Archaea encode only one RNA polymerase that resembles the eukaryotic RNA polymerase II with respect to the subunit composition. Here we identify archaeal orthologs of the eukaryotic RNA polymerase III subunit RPC34. Genome context analysis supports a function of this archaeal protein in the transcription of non-coding RNAs. These findings suggest that functional separation of RNA polymerases for protein-coding genes and non-coding RNAs might predate the origin of the Eukaryotes. Reviewers: This article was reviewed by Andrei Osterman and Patrick Forterre (nominated by Purificación López-García)

Abstract Background In eukaryotes, RNA interference (RNAi) is a major mechanism of defense against viruses and transposable elements as well of regulating translation of endogenous mRNAs. The RNAi systems recognize the target RNA molecules via small guide RNAs that are completely or partially complementary to a region of the target. Key components of the RNAi systems are proteins of the Argonaute-PIWI family some of which function as slicers, the nucleases that cleave the target RNA that is base-paired to a guide RNA. Numerous prokaryotes possess the CRISPR-associated system (CASS) of defense against phages and plasmids that is, in part, mechanistically analogous but not homologous to eukaryotic RNAi systems. Many prokaryotes also encode homologs of Argonaute-PIWI proteins but their functions remain unknown. Results We present a detailed analysis of Argonaute-PIWI protein sequences and the genomic neighborhoods of the respective genes in prokaryotes. Whereas eukaryotic Ago/PIWI proteins always contain PAZ (oligonucleotide binding) and PIWI (active or inactivated nuclease) domains, the prokaryotic Argonaute homologs (pAgos) fall into two major groups in which the PAZ domain is either present or absent. The monophyly of each group is supported by a phylogenetic analysis of the conserved PIWI-domains. Almost all pAgos that lack a PAZ domain appear to be inactivated, and the respective genes are associated with a variety of predicted nucleases in putative operons. An additional, uncharacterized domain that is fused to various nucleases appears to be a unique signature of operons encoding the short (lacking PAZ) pAgo form. By contrast, almost all PAZ-domain containing pAgos are predicted to be active nucleases. Some proteins of this group (e.g., that from Aquifex aeolicus) have been experimentally shown to possess nuclease activity, and are not typically associated with genes for other (putative) nucleases. Given these observations, the apparent extensive horizontal transfer of pAgo genes, and their common, statistically significant over-representation in genomic neighborhoods enriched in genes encoding proteins involved in the defense against phages and/or plasmids, we hypothesize that pAgos are key components of a novel class of defense systems. The PAZ-domain containing pAgos are predicted to directly destroy virus or plasmid nucleic acids via their nuclease activity, whereas the apparently inactivated, PAZ-lacking pAgos could be structural subunits of protein complexes that contain, as active moieties, the putative nucleases that we predict to be co-expressed with these pAgos. All these nucleases are predicted to be DNA endonucleases, so it seems most probable that the putative novel phage/plasmid-defense system targets phage DNA rather than mRNAs. Given that in eukaryotic RNAi systems, the PAZ domain binds a guide RNA and positions it on the complementary region of the target, we further speculate that pAgos function on a similar principle (the guide being either DNA or RNA), and that the uncharacterized domain found in putative operons with the short forms of pAgos is a functional substitute for the PAZ domain. Conclusion The hypothesis that pAgos are key components of a novel prokaryotic immune system that employs guide RNA or DNA molecules to degrade nucleic acids of invading mobile elements implies a functional analogy with the prokaryotic CASS and a direct evolutionary connection with eukaryotic RNAi. The predictions of the hypothesis including both the activities of pAgos and those of the associated endonucleases are readily amenable to experimental tests. Reviewers This article was reviewed by Daniel Haft, Martijn Huynen, and Chris Ponting.

Abstract Background The prokaryotic toxin-antitoxin systems (TAS, also referred to as TA loci) are widespread, mobile two-gene modules that can be viewed as selfish genetic elements because they evolved mechanisms to become addictive for replicons and cells in which they reside, but also possess "normal" cellular functions in various forms of stress response and management of prokaryotic population. Several distinct TAS of type 1, where the toxin is a protein and the antitoxin is an antisense RNA, and numerous, unrelated TAS of type 2, in which both the toxin and the antitoxin are proteins, have been experimentally characterized, and it is suspected that many more remain to be identified. Results We report a comprehensive comparative-genomic analysis of Type 2 toxin-antitoxin systems in prokaryotes. Using sensitive methods for distant sequence similarity search, genome context analysis and a new approach for the identification of mobile two-component systems, we identified numerous, previously unnoticed protein families that are homologous to toxins and antitoxins of known type 2 TAS. In addition, we predict 12 new families of toxins and 13 families of antitoxins, and also, predict a TAS or TAS-like activity for several gene modules that were not previously suspected to function in that capacity. In particular, we present indications that the two-gene module that encodes a minimal nucleotidyl transferase and the accompanying HEPN protein, and is extremely abundant in many archaea and bacteria, especially, thermophiles might comprise a novel TAS. We present a survey of previously known and newly predicted TAS in 750 complete genomes of archaea and bacteria, quantitatively demonstrate the exceptional mobility of the TAS, and explore the network of toxin-antitoxin pairings that combines plasticity with selectivity. Conclusion The defining properties of the TAS, namely, the typically small size of the toxin and antitoxin genes, fast evolution, and extensive horizontal mobility, make the task of comprehensive identification of these systems particularly challenging. However, these same properties can be exploited to develop context-based computational approaches which, combined with exhaustive analysis of subtle sequence similarities were employed in this work to substantially expand the current collection of TAS by predicting both previously unnoticed, derived versions of known toxins and antitoxins, and putative novel TAS-like systems. In a broader context, the TAS belong to the resistome domain of the prokaryotic mobilome which includes partially selfish, addictive gene cassettes involved in various aspects of stress response and organized under the same general principles as the TAS. The "selfish altruism", or "responsible selfishness", of TAS-like systems appears to be a defining feature of the resistome and an important characteristic of the entire prokaryotic pan-genome given that in the prokaryotic world the mobilome and the "stable" chromosomes form a dynamic continuum. Reviewers This paper was reviewed by Kenn Gerdes (nominated by Arcady Mushegian), Daniel Haft, Arcady Mushegian, and Andrei Osterman. For full reviews, go to the Reviewers' Reports section.

Abstract Background Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved. Results We performed a comparative analysis of archaeal, eukaryotic, and bacterial B-family DNA polymerases, which are the main replicative polymerases in archaea and eukaryotes, combined with an analysis of domain architectures. Surprisingly, we found that eukaryotic Polymerase ε consists of two tandem exonuclease-polymerase modules, the active N-terminal module and a C-terminal module in which both enzymatic domains are inactivated. The two modules are only distantly related to each other, an observation that suggests the possibility that Pol ε evolved as a result of insertion and subsequent inactivation of a distinct polymerase, possibly, of bacterial descent, upstream of the C-terminal Zn-fingers, rather than by tandem duplication. The presence of an inactivated exonuclease-polymerase module in Pol ε parallels a similar inactivation of both enzymatic domains in a distinct family of archaeal B-family polymerases. The results of phylogenetic analysis indicate that eukaryotic B-family polymerases, most likely, originate from two distantly related archaeal B-family polymerases, one form giving rise to Pol ε, and the other one to the common ancestor of Pol α, Pol δ, and Pol ζ. The C-terminal Zn-fingers that are present in all eukaryotic B-family polymerases, unexpectedly, are homologous to the Zn-finger of archaeal D-family DNA polymerases that are otherwise unrelated to the B family. The Zn-finger of Polε shows a markedly greater similarity to the counterpart in archaeal PolD than the Zn-fingers of other eukaryotic B-family polymerases. Conclusion Evolution of eukaryotic DNA polymerases seems to have involved previously unnoticed complex events. We hypothesize that the archaeal ancestor of eukaryotes encoded three DNA polymerases, namely, two distinct B-family polymerases and a D-family polymerase all of which contributed to the evolution of the eukaryotic replication machinery. The Zn-finger might have been acquired from PolD by the B-family form that gave rise to Pol ε prior to or in the course of eukaryogenesis, and subsequently, was captured by the ancestor of the other B-family eukaryotic polymerases. The inactivated polymerase-exonuclease module of Pol ε might have evolved by fusion with a distinct polymerase, rather than by duplication of the active module of Pol ε, and is likely to play an important role in the assembly of eukaryotic replication and repair complexes. Reviewers This article was reviewed by Patrick Forterre, Arcady Mushegian, and Chris Ponting. For the full reviews, please go to the Reviewers' Reports section.

Abstract A widespread and highly conserved family of apparently inactivated derivatives of archaeal B-family DNA polymerases is described. Phylogenetic analysis shows that the inactivated forms comprise a distinct clade among archaeal B-family polymerases and that, within this clade, Euryarchaea and Crenarchaea are clearly separated from each other and from a small group of bacterial homologs. These findings are compatible with an ancient duplication of the DNA polymerase gene followed by inactivation and parallel loss in some of the lineages although contribution of horizontal gene transfer cannot be ruled out. The inactivated derivative of the archaeal DNA polymerase could form a complex with the active paralog and play a structural role in DNA replication. Reviewers This article was reviewed by Purificacion Lopez-Garcia and Chris Ponting. For the full reviews, please go to the Reviewers' Reports section.

Abstract Background The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia. Results We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C1-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a major upward shift in the isoelectric points of proteins. Conclusion The results of genome analysis of M. infernorum support the monophyly of the PVC superphylum. M. infernorum possesses a streamlined genome but seems to have acquired numerous genes including those for enzymes of methylotrophic pathways via horizontal gene transfer, in particular, from Proteobacteria. Reviewers This article was reviewed by John A. Fuerst, Ludmila Chistoserdova, and Radhey S. Gupta.

Abstract Background The F- and V-type ATPases are rotary molecular machines that couple translocation of protons or sodium ions across the membrane to the synthesis or hydrolysis of ATP. Both the F-type (found in most bacteria and eukaryotic mitochondria and chloroplasts) and V-type (found in archaea, some bacteria, and eukaryotic vacuoles) ATPases can translocate either protons or sodium ions. The prevalent proton-dependent ATPases are generally viewed as the primary form of the enzyme whereas the sodium-translocating ATPases of some prokaryotes are usually construed as an exotic adaptation to survival in extreme environments. Results We combine structural and phylogenetic analyses to clarify the evolutionary relation between the proton- and sodium-translocating ATPases. A comparison of the structures of the membrane-embedded oligomeric proteolipid rings of sodium-dependent F- and V-ATPases reveals nearly identical sets of amino acids involved in sodium binding. We show that the sodium-dependent ATPases are scattered among proton-dependent ATPases in both the F- and the V-branches of the phylogenetic tree. Conclusion Barring convergent emergence of the same set of ligands in several lineages, these findings indicate that the use of sodium gradient for ATP synthesis is the ancestral modality of membrane bioenergetics. Thus, a primitive, sodium-impermeable but proton-permeable cell membrane that harboured a set of sodium-transporting enzymes appears to have been the evolutionary predecessor of the more structurally demanding proton-tight membranes. The use of proton as the coupling ion appears to be a later innovation that emerged on several independent occasions. Reviewers This article was reviewed by J. Peter Gogarten, Martijn A. Huynen, and Igor B. Zhulin. For the full reviews, please go to the Reviewers' comments section.

Abstract Intracellular vesicle traffic that enables delivery of proteins between the endoplasmic reticulum, Golgi and various endosomal subcompartments is one of the hallmarks of the eukaryotic cell. Its evolutionary history is not well understood but the process itself and the core vesicle traffic machinery are believed to be ancient. We show here that the 4-vinyl reductase (V4R) protein domain present in bacteria and archaea is homologous to the Bet3 subunit of the TRAPP1 vesicle-tethering complex that is conserved in all eukaryotes. This suggests, for the first time, a prokaryotic origin for one of the key eukaryotic trafficking proteins. Reviewers This article was reviewed by Gaspar Jekely and Mark A. Ragan

Abstract Although most of the key components of the transcription apparatus, and in particular, RNA polymerase (RNAP) subunits, are conserved between archaea and eukaryotes, no archaeal homologs of the small RPB8 subunit of eukaryotic RNAP have been detected. We report that orthologs of RPB8 are encoded in all sequenced genomes of hyperthermophilic Crenarchaeota and a recently sequenced "korarchaeal" genome, but not in Euryarchaeota or the mesophilic crenarchaeon Cenarchaeum symbiosum. These findings suggest that all 12 core subunits of eukaryotic RNAPs were already present in the last common ancestor of the extant archaea. Open peer review This article was reviewed by Purificacion Lopez-Garcia and Chris Ponting.

Abstract Background An evolutionary classification of genes from sequenced genomes that distinguishes between orthologs and paralogs is indispensable for genome annotation and evolutionary reconstruction. Shortly after multiple genome sequences of bacteria, archaea, and unicellular eukaryotes became available, an attempt on such a classification was implemented in Clusters of Orthologous Groups of proteins (COGs). Rapid accumulation of genome sequences creates opportunities for refining COGs but also represents a challenge because of error amplification. One of the practical strategies involves construction of refined COGs for phylogenetically compact subsets of genomes. Results New Archaeal Clusters of Orthologous Genes (arCOGs) were constructed for 41 archaeal genomes (13 Crenarchaeota, 27 Euryarchaeota and one Nanoarchaeon) using an improved procedure that employs a similarity tree between smaller, group-specific clusters, semi-automatically partitions orthology domains in multidomain proteins, and uses profile searches for identification of remote orthologs. The annotation of arCOGs is a consensus between three assignments based on the COGs, the CDD database, and the annotations of homologs in the NR database. The 7538 arCOGs, on average, cover ~88% of the genes in a genome compared to a ~76% coverage in COGs. The finer granularity of ortholog identification in the arCOGs is apparent from the fact that 4538 arCOGs correspond to 2362 COGs; ~40% of the arCOGs are new. The archaeal gene core (protein-coding genes found in all 41 genome) consists of 166 arCOGs. The arCOGs were used to reconstruct gene loss and gene gain events during archaeal evolution and gene sets of ancestral forms. The Last Archaeal Common Ancestor (LACA) is conservatively estimated to possess 996 genes compared to 1245 and 1335 genes for the last common ancestors of Crenarchaeota and Euryarchaeota, respectively. It is inferred that LACA was a chemoautotrophic hyperthermophile that, in addition to the core archaeal functions, encoded more idiosyncratic systems, e.g., the CASS systems of antivirus defense and some toxin-antitoxin systems. Conclusion The arCOGs provide a convenient, flexible framework for functional annotation of archaeal genomes, comparative genomics and evolutionary reconstructions. Genomic reconstructions suggest that the last common ancestor of archaea might have been (nearly) as advanced as the modern archaeal hyperthermophiles. ArCOGs and related information are available at: ftp://ftp.ncbi.nih.gov/pub/koonin/arCOGs/. Reviewers This article was reviewed by Peer Bork, Patrick Forterre, and Purificacion Lopez-Garcia.

Abstract Background All archaeal and many bacterial genomes contain Clustered Regularly Interspaced Short Palindrome Repeats (CRISPR) and variable arrays of the CRISPR-associated (cas) genes that have been previously implicated in a novel form of DNA repair on the basis of comparative analysis of their protein product sequences. However, the proximity of CRISPR and cas genes strongly suggests that they have related functions which is hard to reconcile with the repair hypothesis. Results The protein sequences of the numerous cas gene products were classified into ~25 distinct protein families; several new functional and structural predictions are described. Comparative-genomic analysis of CRISPR and cas genes leads to the hypothesis that the CRISPR-Cas system (CASS) is a mechanism of defense against invading phages and plasmids that functions analogously to the eukaryotic RNA interference (RNAi) systems. Specific functional analogies are drawn between several components of CASS and proteins involved in eukaryotic RNAi, including the double-stranded RNA-specific helicase-nuclease (dicer), the endonuclease cleaving target mRNAs (slicer), and the RNA-dependent RNA polymerase. However, none of the CASS components is orthologous to its apparent eukaryotic functional counterpart. It is proposed that unique inserts of CRISPR, some of which are homologous to fragments of bacteriophage and plasmid genes, function as prokaryotic siRNAs (psiRNA), by base-pairing with the target mRNAs and promoting their degradation or translation shutdown. Specific hypothetical schemes are developed for the functioning of the predicted prokaryotic siRNA system and for the formation of new CRISPR units with unique inserts encoding psiRNA conferring immunity to the respective newly encountered phages or plasmids. The unique inserts in CRISPR show virtually no similarity even between closely related bacterial strains which suggests their rapid turnover, on evolutionary scale. Corollaries of this finding are that, even among closely related prokaryotes, the most commonly encountered phages and plasmids are different and/or that the dominant phages and plasmids turn over rapidly. Conclusion We proposed previously that Cas proteins comprise a novel DNA repair system. The association of the cas genes with CRISPR and, especially, the presence, in CRISPR units, of unique inserts homologous to phage and plasmid genes make us abandon this hypothesis. It appears most likely that CASS is a prokaryotic system of defense against phages and plasmids that functions via the RNAi mechanism. The functioning of this system seems to involve integration of fragments of foreign genes into archaeal and bacterial chromosomes yielding heritable immunity to the respective agents. However, it appears that this inheritance is extremely unstable on the evolutionary scale such that the repertoires of unique psiRNAs are completely replaced even in closely related prokaryotes, presumably, in response to rapidly changing repertoires of dominant phages and plasmids. This article was reviewed by: Eric Bapteste, Patrick Forterre, and Martijn Huynen. Open peer review Reviewed by Eric Bapteste, Patrick Forterre, and Martijn Huynen. For the full reviews, please go to the Reviewers' comments section.

We report a comprehensive computational dissection of the domain architecture of the SAMD9 family proteins that are involved in antivirus and antitumor response in humans. We show that the SAMD9 protein family is represented in most animals and also, unexpectedly, in bacteria, in particular actinomycetes. From the N to C terminus, the core SAMD9 family architecture includes DNA/RNA-binding AlbA domain, a variant Sir2-like domain, a STAND-like P-loop NTPase, an array of TPR repeats and an OB-fold domain with predicted RNA-binding properties. Vertebrate SAMD9 family proteins contain the eponymous SAM domain capable of polymerization, whereas some family members from other animals instead contain homotypic adaptor domains of the DEATH superfamily, known as dedicated components of apoptosis networks. Such complex domain architecture is reminiscent of the STAND superfamily NTPases that are involved in various signaling processes, including programmed cell death, in both eukaryotes and prokaryotes. These findings suggest that SAMD9 is a hub of a novel, evolutionarily conserved defense network that remains to be characterized. [ABSTRACT FROM AUTHOR]

Several recent discoveries reveal unexpected versatility of the bacterial and archaeal cytoskeleton systems that are involved in cell division and other processes based on membrane remodeling. Here we apply methods for distant protein sequence similarity detection, phylogenetic approaches, and genome context analysis to described two previously unnoticed families of the FtsZ-tubulin superfamily. One of these families is limited in its spread to Proteobacteria whereas the other is represented in diverse bacteria and archaea, and might be the key component of a novel, multicomponent membrane remodeling system that also includes a Von Willebrand A domain-containing protein, a distinct GTPase and membrane transport proteins of the OmpA family. This article was reviewed by Purificación López-García and Gáspár Jékely; for complete reviews, see the Reviewers Reports section. [ABSTRACT FROM AUTHOR]

: Although most of the key components of the transcription apparatus, and in particular, RNA polymerase (RNAP) subunits, are conserved between archaea and eukaryotes, no archaeal homologs of the small RPB8 subunit of eukaryotic RNAP have been detected. We report that orthologs of RPB8 are encoded in all sequenced genomes of hyperthermophilic Crenarchaeota and a recently sequenced "korarchaeal" genome, but not in Euryarchaeota or the mesophilic crenarchaeon Cenarchaeum symbiosum. These findings suggest that all 12 core subunits of eukaryotic RNAPs were already present in the last common ancestor of the extant archaea. [ABSTRACT FROM AUTHOR]

Background: Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved., Results: We performed a comparative analysis of archaeal, eukaryotic, and bacterial B-family DNA polymerases, which are the main replicative polymerases in archaea and eukaryotes, combined with an analysis of domain architectures. Surprisingly, we found that eukaryotic Polymerase epsilon consists of two tandem exonuclease-polymerase modules, the active N-terminal module and a C-terminal module in which both enzymatic domains are inactivated. The two modules are only distantly related to each other, an observation that suggests the possibility that Pol epsilon evolved as a result of insertion and subsequent inactivation of a distinct polymerase, possibly, of bacterial descent, upstream of the C-terminal Zn-fingers, rather than by tandem duplication. The presence of an inactivated exonuclease-polymerase module in Pol epsilon parallels a similar inactivation of both enzymatic domains in a distinct family of archaeal B-family polymerases. The results of phylogenetic analysis indicate that eukaryotic B-family polymerases, most likely, originate from two distantly related archaeal B-family polymerases, one form giving rise to Pol epsilon, and the other one to the common ancestor of Pol alpha, Pol delta, and Pol zeta. The C-terminal Zn-fingers that are present in all eukaryotic B-family polymerases, unexpectedly, are homologous to the Zn-finger of archaeal D-family DNA polymerases that are otherwise unrelated to the B family. The Zn-finger of Polepsilon shows a markedly greater similarity to the counterpart in archaeal PolD than the Zn-fingers of other eukaryotic B-family polymerases., Conclusion: Evolution of eukaryotic DNA polymerases seems to have involved previously unnoticed complex events. We hypothesize that the archaeal ancestor of eukaryotes encoded three DNA polymerases, namely, two distinct B-family polymerases and a D-family polymerase all of which contributed to the evolution of the eukaryotic replication machinery. The Zn-finger might have been acquired from PolD by the B-family form that gave rise to Pol epsilon prior to or in the course of eukaryogenesis, and subsequently, was captured by the ancestor of the other B-family eukaryotic polymerases. The inactivated polymerase-exonuclease module of Pol epsilon might have evolved by fusion with a distinct polymerase, rather than by duplication of the active module of Pol epsilon, and is likely to play an important role in the assembly of eukaryotic replication and repair complexes.