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1.  Casposons: a new superfamily of self-synthesizing DNA transposons at the origin of prokaryotic CRISPR-Cas immunity 
BMC Biology  2014;12:36.
Background
Diverse transposable elements are abundant in genomes of cellular organisms from all three domains of life. Although transposons are often regarded as junk DNA, a growing body of evidence indicates that they are behind some of the major evolutionary innovations. With the growth in the number and diversity of sequenced genomes, previously unnoticed mobile elements continue to be discovered.
Results
We describe a new superfamily of archaeal and bacterial mobile elements which we denote casposons because they encode Cas1 endonuclease, a key enzyme of the CRISPR-Cas adaptive immunity systems of archaea and bacteria. The casposons share several features with self-synthesizing eukaryotic DNA transposons of the Polinton/Maverick class, including terminal inverted repeats and genes for B family DNA polymerases. However, unlike any other known mobile elements, the casposons are predicted to rely on Cas1 for integration and excision, via a mechanism similar to the integration of new spacers into CRISPR loci. We identify three distinct families of casposons that differ in their gene repertoires and evolutionary provenance of the DNA polymerases. Deep branching of the casposon-encoded endonuclease in the Cas1 phylogeny suggests that casposons played a pivotal role in the emergence of CRISPR-Cas immunity.
Conclusions
The casposons are a novel superfamily of mobile elements, the first family of putative self-synthesizing transposons discovered in prokaryotes. The likely contribution of capsosons to the evolution of CRISPR-Cas parallels the involvement of the RAG1 transposase in vertebrate immunoglobulin gene rearrangement, suggesting that recruitment of endonucleases from mobile elements as ready-made tools for genome manipulation is a general route of evolution of adaptive immunity.
doi:10.1186/1741-7007-12-36
PMCID: PMC4046053  PMID: 24884953
Mobile genetic elements; CRISPR-Cas system; Adaptive immunity; Transposons; Archaea; DNA polymerases
2.  Enzymatic Synthesis of Bioinformatically Predicted Microcin C-Like Compounds Encoded by Diverse Bacteria 
mBio  2014;5(3):e01059-14.
ABSTRACT
The Trojan horse Escherichia coli antibiotic microcin C (McC) consists of a heptapeptide attached to adenosine through a phosphoramidate linkage. McC is synthesized by the MccB enzyme, which terminally adenylates the ribosomally synthesized heptapeptide precursor MccA. The peptide part is responsible for McC uptake; it is degraded inside the cell to release a toxic nonhydrolyzable aspartyl-adenylate. Bionformatic analysis reveals that diverse bacterial genomes encoding mccB homologues also contain adjacent short open reading frames that may encode MccA-like adenylation substrates. Using chemically synthesized predicted peptide substrates and recombinant cognate MccB protein homologs, adenylated products were obtained in vitro for predicted MccA peptide-MccB enzyme pairs from Helicobacter pylori, Streptococcus thermophilus, Lactococcus johnsonii, Bartonella washoensis, Yersinia pseudotuberculosis, and Synechococcus sp. Some adenylated products were shown to inhibit the growth of E. coli by targeting aspartyl-tRNA synthetase, the target of McC.
IMPORTANCE
Our results prove that McC-like adenylated peptides are widespread and are encoded by both Gram-negative and Gram-positive bacteria and by cyanobacteria, opening ways for analyses of physiological functions of these compounds and for creation of microcin C-like antibiotics targeting various bacteria.
doi:10.1128/mBio.01059-14
PMCID: PMC4010828  PMID: 24803518
3.  CRISPR-Cas 
RNA Biology  2013;10(5):679-686.
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.
doi:10.4161/rna.24022
PMCID: PMC3737325  PMID: 23439366
CRISPR-Cas; adaptive immunity; innate immunity; programmed cell death; dormancy; RRM domain
4.  CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems 
Frontiers in Genetics  2014;5:102.
CRISPR-Cas adaptive immunity systems of bacteria and archaea insert fragments of virus or plasmid DNA as spacer sequences into CRISPR repeat loci. Processed transcripts encompassing these spacers guide the cleavage of the cognate foreign DNA or RNA. Most CRISPR-Cas loci, in addition to recognized cas genes, also include genes that are not directly implicated in spacer acquisition, CRISPR transcript processing or interference. Here we comprehensively analyze sequences, structures and genomic neighborhoods of one of the most widespread groups of such genes that encode proteins containing a predicted nucleotide-binding domain with a Rossmann-like fold, which we denote CARF (CRISPR-associated Rossmann fold). Several CARF protein structures have been determined but functional characterization of these proteins is lacking. The CARF domain is most frequently combined with a C-terminal winged helix-turn-helix DNA-binding domain and “effector” domains most of which are predicted to possess DNase or RNase activity. Divergent CARF domains are also found in RtcR proteins, sigma-54 dependent regulators of the rtc RNA repair operon. CARF genes frequently co-occur with those coding for proteins containing the WYL domain with the Sm-like SH3 β-barrel fold, which is also predicted to bind ligands. CRISPR-Cas and possibly other defense systems are predicted to be transcriptionally regulated by multiple ligand-binding proteins containing WYL and CARF domains which sense modified nucleotides and nucleotide derivatives generated during virus infection. We hypothesize that CARF domains also transmit the signal from the bound ligand to the fused effector domains which attack either alien or self nucleic acids, resulting, respectively, in immunity complementing the CRISPR-Cas action or in dormancy/programmed cell death.
doi:10.3389/fgene.2014.00102
PMCID: PMC4012209  PMID: 24817877
CRISPR; Rossmann fold; beta barrel; DNA-binding proteins; phage defense
5.  Classification and evolution of type II CRISPR-Cas systems 
Nucleic Acids Research  2014;42(10):6091-6105.
The CRISPR-Cas systems of archaeal and bacterial adaptive immunity are classified into three types that differ by the repertoires of CRISPR-associated (cas) genes, the organization of cas operons and the structure of repeats in the CRISPR arrays. The simplest among the CRISPR-Cas systems is type II in which the endonuclease activities required for the interference with foreign deoxyribonucleic acid (DNA) are concentrated in a single multidomain protein, Cas9, and are guided by a co-processed dual-tracrRNA:crRNA molecule. This compact enzymatic machinery and readily programmable site-specific DNA targeting make type II systems top candidates for a new generation of powerful tools for genomic engineering. Here we report an updated census of CRISPR-Cas systems in bacterial and archaeal genomes. Type II systems are the rarest, missing in archaea, and represented in ∼5% of bacterial genomes, with an over-representation among pathogens and commensals. Phylogenomic analysis suggests that at least three cas genes, cas1, cas2 and cas4, and the CRISPR repeats of the type II-B system were acquired via recombination with a type I CRISPR-Cas locus. Distant homologs of Cas9 were identified among proteins encoded by diverse transposons, suggesting that type II CRISPR-Cas evolved via recombination of mobile nuclease genes with type I loci.
doi:10.1093/nar/gku241
PMCID: PMC4041416  PMID: 24728998
6.  Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems 
Nucleic Acids Research  2013;42(4):2577-2590.
The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool.
doi:10.1093/nar/gkt1074
PMCID: PMC3936727  PMID: 24270795
7.  Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis 
Journal of molecular biology  2012;420(0):366-383.
Microcin C (McC) is heptapeptide-adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine-peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its AMP-complex reveal specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance.
doi:10.1016/j.jmb.2012.04.011
PMCID: PMC3690760  PMID: 22516613
MccF; serine peptidase; nucleophilic elbow; catalytic triad (Ser-His-Glu); substrate binding loop
8.  Comprehensive analysis of the HEPN superfamily: identification of novel roles in intra-genomic conflicts, defense, pathogenesis and RNA processing 
Biology Direct  2013;8:15.
Background
The major role of enzymatic toxins that target nucleic acids in biological conflicts at all levels has become increasingly apparent thanks in large part to the advances of comparative genomics. Typically, toxins evolve rapidly hampering the identification of these proteins by sequence analysis. Here we analyze an unexpectedly widespread superfamily of toxin domains most of which possess RNase activity.
Results
The HEPN superfamily is comprised of all α-helical domains that were first identified as being associated with DNA polymerase β-type nucleotidyltransferases in prokaryotes and animal Sacsin proteins. Using sensitive sequence and structure comparison methods, we vastly extend the HEPN superfamily by identifying numerous novel families and by detecting diverged HEPN domains in several known protein families. The new HEPN families include the RNase LS and LsoA catalytic domains, KEN domains (e.g. RNaseL and Ire1) and the RNase domains of RloC and PrrC. The majority of HEPN domains contain conserved motifs that constitute a metal-independent endoRNase active site. Some HEPN domains lacking this motif probably function as non-catalytic RNA-binding domains, such as in the case of the mannitol repressor MtlR. Our analysis shows that HEPN domains function as toxins that are shared by numerous systems implicated in intra-genomic, inter-genomic and intra-organismal conflicts across the three domains of cellular life. In prokaryotes HEPN domains are essential components of numerous toxin-antitoxin (TA) and abortive infection (Abi) systems and in addition are tightly associated with many restriction-modification (R-M) and CRISPR-Cas systems, and occasionally with other defense systems such as Pgl and Ter. We present evidence of multiple modes of action of HEPN domains in these systems, which include direct attack on viral RNAs (e.g. LsoA and RNase LS) in conjunction with other RNase domains (e.g. a novel RNase H fold domain, NamA), suicidal or dormancy-inducing attack on self RNAs (RM systems and possibly CRISPR-Cas systems), and suicidal attack coupled with direct interaction with phage components (Abi systems). These findings are compatible with the hypothesis on coupling of pathogen-targeting (immunity) and self-directed (programmed cell death and dormancy induction) responses in the evolution of robust antiviral strategies. We propose that altruistic cell suicide mediated by HEPN domains and other functionally similar RNases was essential for the evolution of kin and group selection and cell cooperation. HEPN domains were repeatedly acquired by eukaryotes and incorporated into several core functions such as endonucleolytic processing of the 5.8S-25S/28S rRNA precursor (Las1), a novel ER membrane-associated RNA degradation system (C6orf70), sensing of unprocessed transcripts at the nuclear periphery (Swt1). Multiple lines of evidence suggest that, similar to prokaryotes, HEPN proteins were recruited to antiviral, antitransposon, apoptotic systems or RNA-level response to unfolded proteins (Sacsin and KEN domains) in several groups of eukaryotes.
Conclusions
Extensive sequence and structure comparisons reveal unexpectedly broad presence of the HEPN domain in an enormous variety of defense and stress response systems across the tree of life. In addition, HEPN domains have been recruited to perform essential functions, in particular in eukaryotic rRNA processing. These findings are expected to stimulate experiments that could shed light on diverse cellular processes across the three domains of life.
Reviewers
This article was reviewed by Martijn Huynen, Igor Zhulin and Nick Grishin
doi:10.1186/1745-6150-8-15
PMCID: PMC3710099  PMID: 23768067
9.  Insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from Obsidian Pool, Yellowstone National Park 
Biology Direct  2013;8:9.
Background
A single cultured marine organism, Nanoarchaeum equitans, represents the Nanoarchaeota branch of symbiotic Archaea, with a highly reduced genome and unusual features such as multiple split genes.
Results
The first terrestrial hyperthermophilic member of the Nanoarchaeota was collected from Obsidian Pool, a thermal feature in Yellowstone National Park, separated by single cell isolation, and sequenced together with its putative host, a Sulfolobales archaeon. Both the new Nanoarchaeota (Nst1) and N. equitans lack most biosynthetic capabilities, and phylogenetic analysis of ribosomal RNA and protein sequences indicates that the two form a deep-branching archaeal lineage. However, the Nst1 genome is more than 20% larger, and encodes a complete gluconeogenesis pathway as well as the full complement of archaeal flagellum proteins. With a larger genome, a smaller repertoire of split protein encoding genes and no split non-contiguous tRNAs, Nst1 appears to have experienced less severe genome reduction than N. equitans. These findings imply that, rather than representing ancestral characters, the extremely compact genomes and multiple split genes of Nanoarchaeota are derived characters associated with their symbiotic or parasitic lifestyle. The inferred host of Nst1 is potentially autotrophic, with a streamlined genome and simplified central and energetic metabolism as compared to other Sulfolobales.
Conclusions
Comparison of the N. equitans and Nst1 genomes suggests that the marine and terrestrial lineages of Nanoarchaeota share a common ancestor that was already a symbiont of another archaeon. The two distinct Nanoarchaeota-host genomic data sets offer novel insights into the evolution of archaeal symbiosis and parasitism, enabling further studies of the cellular and molecular mechanisms of these relationships.
Reviewers
This article was reviewed by Patrick Forterre, Bettina Siebers (nominated by Michael Galperin) and Purification Lopez-Garcia
doi:10.1186/1745-6150-8-9
PMCID: PMC3655853  PMID: 23607440
Archaea evolution; Single cell genomics; Symbiosis; Hyperthermophiles; Split genes
10.  Comparative genomics of defense systems in archaea and bacteria 
Nucleic Acids Research  2013;41(8):4360-4377.
Our knowledge of prokaryotic defense systems has vastly expanded as the result of comparative genomic analysis, followed by experimental validation. This expansion is both quantitative, including the discovery of diverse new examples of known types of defense systems, such as restriction-modification or toxin-antitoxin systems, and qualitative, including the discovery of fundamentally new defense mechanisms, such as the CRISPR-Cas immunity system. Large-scale statistical analysis reveals that the distribution of different defense systems in bacterial and archaeal taxa is non-uniform, with four groups of organisms distinguishable with respect to the overall abundance and the balance between specific types of defense systems. The genes encoding defense system components in bacterial and archaea typically cluster in defense islands. In addition to genes encoding known defense systems, these islands contain numerous uncharacterized genes, which are candidates for new types of defense systems. The tight association of the genes encoding immunity systems and dormancy- or cell death-inducing defense systems in prokaryotic genomes suggests that these two major types of defense are functionally coupled, providing for effective protection at the population level.
doi:10.1093/nar/gkt157
PMCID: PMC3632139  PMID: 23470997
11.  Updated clusters of orthologous genes for Archaea: a complex ancestor of the Archaea and the byways of horizontal gene transfer 
Biology Direct  2012;7:46.
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).
doi:10.1186/1745-6150-7-46
PMCID: PMC3534625  PMID: 23241446
Archaea; Orthologs; Horizontal gene transfer
12.  Live virus-free or die: coupling of antivirus immunity and programmed suicide or dormancy in prokaryotes 
Biology Direct  2012;7:40.
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.
doi:10.1186/1745-6150-7-40
PMCID: PMC3506569  PMID: 23151069
13.  Novel Bacteriophages Containing a Genome of Another Bacteriophage within Their Genomes 
PLoS ONE  2012;7(7):e40683.
A novel bacteriophage infecting Staphylococus pasteuri was isolated during a screen for phages in Antarctic soils. The phage named SpaA1 is morphologically similar to phages of the family Siphoviridae. The 42,784 bp genome of SpaA1 is a linear, double-stranded DNA molecule with 3′ protruding cohesive ends. The SpaA1 genome encompasses 63 predicted protein-coding genes which cluster within three regions of the genome, each of apparently different origin, in a mosaic pattern. In two of these regions, the gene sets resemble those in prophages of Bacillus thuringiensis kurstaki str. T03a001 (genes involved in DNA replication/transcription, cell entry and exit) and B. cereus AH676 (additional regulatory and recombination genes), respectively. The third region represents an almost complete genome (except for the short terminal segments) of a distinct bacteriophage, MZTP02. Nearly the same gene module was identified in prophages of B. thuringiensis serovar monterrey BGSC 4AJ1 and B. cereus Rock4-2. These findings suggest that MZTP02 can be shuttled between genomes of other bacteriophages and prophages, leading to the formation of chimeric genomes. The presence of a complete phage genome in the genome of other phages apparently has not been described previously and might represent a ‘fast track’ route of virus evolution and horizontal gene transfer. Another phage (BceA1) nearly identical in sequence to SpaA1, and also including the almost complete MZTP02 genome within its own genome, was isolated from a bacterium of the B. cereus/B. thuringiensis group. Remarkably, both SpaA1 and BceA1 phages can infect B. cereus and B. thuringiensis, but only one of them, SpaA1, can infect S. pasteuri. This finding is best compatible with a scenario in which MZTP02 was originally contained in BceA1 infecting Bacillus spp, the common hosts for these two phages, followed by emergence of SpaA1 infecting S. pasteuri.
doi:10.1371/journal.pone.0040683
PMCID: PMC3398947  PMID: 22815791
14.  Evolution and classification of the CRISPR-Cas systems 
Nature Reviews. Microbiology  2011;9(6):467-477.
The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a `polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci.
doi:10.1038/nrmicro2577
PMCID: PMC3380444  PMID: 21552286
15.  Evolution of diverse cell division and vesicle formation systems in Archaea 
Nature Reviews. Microbiology  2010;8(10):731-741.
Recently a novel cell division system comprised of homologues of eukaryotic ESCRT-III (endosomal sorting complex required for transport III) proteins was discovered in the hyperthermophilic crenarchaeote Sulfolobus acidocaldarius. On the basis of this discovery, we undertook a comparative genomic analysis of the machineries for cell division and vesicle formation in Archaea. Archaea possess at least three distinct membrane remodelling systems: the FtsZ-based bacterial-type system, the ESCRT-III-based eukaryote-like system and a putative novel system that uses an archaeal actin-related protein. Many archaeal genomes encode assortments of components from different systems. Evolutionary reconstruction from these findings suggests that the last common ancestor of the extant Archaea possessed a complex membrane remodelling apparatus, different components of which were lost during subsequent evolution of archaeal lineages. By contrast, eukaryotes seem to have inherited all three ancestral systems.
doi:10.1038/nrmicro2406
PMCID: PMC3293450  PMID: 20818414
16.  Nature and Intensity of Selection Pressure on CRISPR-Associated Genes 
Journal of Bacteriology  2012;194(5):1216-1225.
The recently discovered CRISPR-Cas adaptive immune system is present in almost all archaea and many bacteria. It consists of cassettes of CRISPR repeats that incorporate spacers homologous to fragments of viral or plasmid genomes that are employed as guide RNAs in the immune response, along with numerous CRISPR-associated (cas) genes that encode proteins possessing diverse, only partially characterized activities required for the action of the system. Here, we investigate the evolution of the cas genes and show that they evolve under purifying selection that is typically much weaker than the median strength of purifying selection affecting genes in the respective genomes. The exceptions are the cas1 and cas2 genes that typically evolve at levels of purifying selection close to the genomic median. Thus, although these genes are implicated in the acquisition of spacers from alien genomes, they do not appear to be directly involved in an arms race between bacterial and archaeal hosts and infectious agents. These genes might possess functions distinct from and additional to their role in the CRISPR-Cas-mediated immune response. Taken together with evidence of the frequent horizontal transfer of cas genes reported previously and with the wide-spread microscale recombination within these genes detected in this work, these findings reveal the highly dynamic evolution of cas genes. This conclusion is in line with the involvement of CRISPR-Cas in antiviral immunity that is likely to entail a coevolutionary arms race with rapidly evolving viruses. However, we failed to detect evidence of strong positive selection in any of the cas genes.
doi:10.1128/JB.06521-11
PMCID: PMC3294813  PMID: 22178975
17.  The CMG (CDC45/RecJ, MCM, GINS) complex is a conserved component of the DNA replication system in all archaea and eukaryotes 
Biology Direct  2012;7:7.
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.
doi:10.1186/1745-6150-7-7
PMCID: PMC3307487  PMID: 22329974
18.  Francisella RNA polymerase contains a heterodimer of non-identical α subunits 
BMC Molecular Biology  2011;12:50.
Background
All sequenced genomes of representatives of the Francisella genus contain two rpoA genes, which encode non-identical RNA polymerase (RNAP) subunits, α1 and α2. In all other bacteria studied to date, a dimer of identical α subunits initiates the assembly of the catalytically proficient RNAP core (subunit composition α2ββ'). Based on an observation that both α1 and α2 are incorporated into Francisella RNAP, Charity et al. (2007) previously suggested that up to four different species of RNAP core enzyme might form in the same Francisella cell.
Results
By in vitro assembly from fully denatured state, we determined that both Francisella α subunits are required for efficient dimerization; no homodimer formation was detected. Bacterial two-hybrid system analysis likewise indicated strong interactions between the α1 and α2 N-terminal domains (NTDs, responsible for dimerization). NTDs of α2 did not interact detectably, while weak interaction between α1 NTDs was observed. This weak homotypic interaction may explain low-level transcription activity observed in in vitro RNAP reconstitution reactions containing Francisella large subunits (β', β) and α1. No activity was observed with RNAP reconstitution reactions containing α2, while robust transcription activity was detected in reactions containing α1 and α2. Phylogenetic analysis based on RpoA resulted in a tree compatible with standard bacterial taxonomy with both Francisella RpoA branches positioned within γ-proteobacteria. The observed phylogeny and analysis of constrained trees are compatible with Francisella lineage-specific rpoA duplication followed by acceleration of evolutionary rate and subfunctionalization.
Conclusions
The results strongly suggest that most Francisella RNAP contains α heterodimer with a minor subfraction possibly containing α1 homodimer. Comparative sequence analysis suggests that this heterodimer is oriented, in a sense that only one monomer, α1, interacts with the β subunit during the α2β RNAP subassembly formation. Most likely the two rpoA copies in Francisella have emerged through a lineage-specific duplication followed by subfunctionalization of interacting paralogs.
doi:10.1186/1471-2199-12-50
PMCID: PMC3294249  PMID: 22108176
19.  Defense Islands in Bacterial and Archaeal Genomes and Prediction of Novel Defense Systems ▿†‡ 
Journal of Bacteriology  2011;193(21):6039-6056.
The arms race between cellular life forms and viruses is a major driving force of evolution. A substantial fraction of bacterial and archaeal genomes is dedicated to antivirus defense. We analyzed the distribution of defense genes and typical mobilome components (such as viral and transposon genes) in bacterial and archaeal genomes and demonstrated statistically significant clustering of antivirus defense systems and mobile genes and elements in genomic islands. The defense islands are enriched in putative operons and contain numerous overrepresented gene families. A detailed sequence analysis of the proteins encoded by genes in these families shows that many of them are diverged variants of known defense system components, whereas others show features, such as characteristic operonic organization, that are suggestive of novel defense systems. Thus, genomic islands provide abundant material for the experimental study of bacterial and archaeal antivirus defense. Except for the CRISPR-Cas systems, different classes of defense systems, in particular toxin-antitoxin and restriction-modification systems, show nonrandom clustering in defense islands. It remains unclear to what extent these associations reflect functional cooperation between different defense systems and to what extent the islands are genomic “sinks” that accumulate diverse nonessential genes, particularly those acquired via horizontal gene transfer. The characteristics of defense islands resemble those of mobilome islands. Defense and mobilome genes are nonrandomly associated in islands, suggesting nonadaptive evolution of the islands via a preferential attachment-like mechanism underpinned by the addictive properties of defense systems such as toxins-antitoxins and an important role of horizontal mobility in the evolution of these islands.
doi:10.1128/JB.05535-11
PMCID: PMC3194920  PMID: 21908672
20.  The Complete Genome Sequence of Thermoproteus tenax: A Physiologically Versatile Member of the Crenarchaeota 
PLoS ONE  2011;6(10):e24222.
Here, we report on the complete genome sequence of the hyperthermophilic Crenarchaeum Thermoproteus tenax (strain Kra1, DSM 2078T) a type strain of the crenarchaeotal order Thermoproteales. Its circular 1.84-megabase genome harbors no extrachromosomal elements and 2,051 open reading frames are identified, covering 90.6% of the complete sequence, which represents a high coding density. Derived from the gene content, T. tenax is a representative member of the Crenarchaeota. The organism is strictly anaerobic and sulfur-dependent with optimal growth at 86°C and pH 5.6. One particular feature is the great metabolic versatility, which is not accompanied by a distinct increase of genome size or information density as compared to other Crenarchaeota. T. tenax is able to grow chemolithoautotrophically (CO2/H2) as well as chemoorganoheterotrophically in presence of various organic substrates. All pathways for synthesizing the 20 proteinogenic amino acids are present. In addition, two presumably complete gene sets for NADH:quinone oxidoreductase (complex I) were identified in the genome and there is evidence that either NADH or reduced ferredoxin might serve as electron donor. Beside the typical archaeal A0A1-ATP synthase, a membrane-bound pyrophosphatase is found, which might contribute to energy conservation. Surprisingly, all genes required for dissimilatory sulfate reduction are present, which is confirmed by growth experiments. Mentionable is furthermore, the presence of two proteins (ParA family ATPase, actin-like protein) that might be involved in cell division in Thermoproteales, where the ESCRT system is absent, and of genes involved in genetic competence (DprA, ComF) that is so far unique within Archaea.
doi:10.1371/journal.pone.0024222
PMCID: PMC3189178  PMID: 22003381
21.  Phylogenomics of prokaryotic ribosomal proteins 
Genome Biology  2011;12(Suppl 1):P30.
doi:10.1186/gb-2011-12-s1-p30
PMCID: PMC3439054
22.  Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems 
Biology Direct  2011;6:38.
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.
doi:10.1186/1745-6150-6-38
PMCID: PMC3150331  PMID: 21756346
23.  CRISPR-Cas: an adaptive immunity system in prokaryotes 
Most of the archaea and numerous bacteria possess an elaborate system of adaptive immunity to mobile genetic elements known as the CRISPR (clustered regularly interspaced short palindromic repeats)-associated system (CRISPR-Cas), which consists of arrays of short repeats interspersed with unique DNA spacers and adjacent operons encompassing CRISPR-associated (cas) genes with predicted and, in some cases, experimentally validated nuclease, helicase, and polymerase activities. The system functions by integrating fragments of alien DNA between the repeats and employing their transcripts to degrade the DNA of the respective invading elements via an RNA interference-like mechanism. The CRISPR-Cas system is a case of apparent Lamarckian inheritance.
doi:10.3410/B1-95
PMCID: PMC2884157  PMID: 20556198
24.  Archaeal Ubiquitin-Like Proteins: Functional Versatility and Putative Ancestral Involvement in tRNA Modification Revealed by Comparative Genomic Analysis 
Archaea  2010;2010:710303.
The recent discovery of protein modification by SAMPs, ubiquitin-like (Ubl) proteins from the archaeon Haloferax volcanii, prompted a comprehensive comparative-genomic analysis of archaeal Ubl protein genes and the genes for enzymes thought to be functionally associated with Ubl proteins. This analysis showed that most archaea encode members of two major groups of Ubl proteins with the β-grasp fold, the ThiS and MoaD families, and indicated that the ThiS family genes are rarely linked to genes for thiamine or Mo/W cofactor metabolism enzymes but instead are most often associated with genes for enzymes of tRNA modification. Therefore it is hypothesized that the ancestral function of the archaeal Ubl proteins is sulfur insertion into modified nucleotides in tRNAs, an activity analogous to that of the URM1 protein in eukaryotes. Together with additional, previously described genomic associations, these findings indicate that systems for protein quality control operating at different levels, including tRNA modification that controls translation fidelity, protein ubiquitination that regulates protein degradation, and, possibly, mRNA degradation by the exosome, are functionally and evolutionarily linked.
doi:10.1155/2010/710303
PMCID: PMC2948915  PMID: 20936112
25.  Two new families of the FtsZ-tubulin protein superfamily implicated in membrane remodeling in diverse bacteria and archaea 
Biology Direct  2010;5:33.
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.
doi:10.1186/1745-6150-5-33
PMCID: PMC2875224  PMID: 20459678

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