RESUMEN
Recognition of RNA from invading mobile genetic elements (MGE) prompts type III CRISPR systems to activate an HD nuclease domain and/or a nucleotide cyclase domain in the Cas10 subunit, eliciting an immune response. The cyclase domain can generate a range of nucleotide second messengers, which in turn activate a diverse family of ancillary effector proteins. These provide immunity by non-specific degradation of host and MGE nucleic acids or proteins, perturbation of membrane potentials, transcriptional responses, or the arrest of translation. The wide range of nucleotide activators and downstream effectors generates a complex picture that is gradually being resolved. Here, we carry out a global bioinformatic analysis of type III CRISPR loci in prokaryotic genomes, defining the relationships of Cas10 proteins and their ancillary effectors. Our study reveals that cyclic tetra-adenylate is by far the most common signalling molecule used and that many loci have multiple effectors. These typically share the same activator and may work synergistically to combat MGE. We propose four new candidate effector protein families and confirm experimentally that the Csm6-2 protein, a highly diverged, fused Csm6 effector, is a ribonuclease activated by cyclic hexa-adenylate.
Asunto(s)
Sistemas CRISPR-Cas , Biología Computacional , Biología Computacional/métodos , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Prokaryotic antiviral defence systems are frequently toxic for host cells and stringent regulation is required to ensure survival and fitness. These systems must be readily available in case of infection but tightly controlled to prevent activation of an unnecessary cellular response. Here we investigate how the bacterial cyclic oligonucleotide-based antiphage signalling system (CBASS) uses its intrinsic protein modification system to regulate the nucleotide cyclase. By integrating a type II CBASS system from Bacillus cereus into the model organism Bacillus subtilis, we show that the protein-conjugating Cap2 (CBASS associated protein 2) enzyme links the cyclase exclusively to the conserved phage shock protein A (PspA) in the absence of phage. The cyclase-PspA conjugation is reversed by the deconjugating isopeptidase Cap3 (CBASS associated protein 3). We propose a model in which the cyclase is held in an inactive state by conjugation to PspA in the absence of phage, with conjugation released upon infection, priming the cyclase for activation.
Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Bacillus subtilis/virología , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Bacillus cereus/virología , Bacillus cereus/enzimología , Bacillus cereus/genética , Bacillus cereus/inmunología , Transducción de Señal , Bacteriófagos/genética , Bacteriófagos/fisiología , Bacteriófagos/enzimología , Liasas de Fósforo-Oxígeno/metabolismo , Liasas de Fósforo-Oxígeno/genética , Regulación Bacteriana de la Expresión GénicaRESUMEN
Bacteria have evolved a variety of defence mechanisms to protect against mobile genetic elements, including restriction-modification systems and CRISPR-Cas. In recent years, dozens of previously unknown defence systems (DSs) have been discovered. Notably, diverse DSs often coexist within the same genome, and some co-occur at frequencies significantly higher than would be expected by chance, implying potential synergistic interactions. Recent studies have provided evidence of defence mechanisms that enhance or complement one another. Here, we review the interactions between DSs at the mechanistic, regulatory, ecological and evolutionary levels.
Asunto(s)
Bacteriófagos , Sistemas CRISPR-Cas , Bacterias/genética , Evolución Biológica , Bacteriófagos/genéticaRESUMEN
CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements1,2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against mobile genetic elements either directly, via the integral histidine-aspartate (HD) nuclease domain3-5 or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors6-9. A subset of type III CRISPR systems encode an uncharacterized CorA-family membrane protein and an associated NrN family phosphodiesterase that are predicted to function in antiviral defence. Here we demonstrate that the CorA-associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against mobile genetic elements when expressed in Escherichia coli. However, B. fragilis Cmr does not synthesize cyclic oligoadenylate species on activation, instead generating S-adenosyl methionine (SAM)-AMP (SAM is also known as AdoMet) by conjugating ATP to SAM via a phosphodiester bond. Once synthesized, SAM-AMP binds to the CorA effector, presumably leading to cell dormancy or death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR-associated phosphodiesterases or a SAM-AMP lyase, potentially providing an 'off switch' analogous to cyclic oligoadenylate-specific ring nucleases10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.
Asunto(s)
Adenosina Trifosfato , Bacteroides fragilis , Sistemas CRISPR-Cas , Escherichia coli , S-Adenosilmetionina , Sistemas de Mensajero Secundario , Adenosina Trifosfato/metabolismo , Bacteroides fragilis/enzimología , Bacteroides fragilis/genética , Bacteroides fragilis/inmunología , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/inmunología , Sistemas CRISPR-Cas/fisiología , Endonucleasas/química , Endonucleasas/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Escherichia coli/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , ARN/inmunología , ARN/metabolismo , S-Adenosilmetionina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6' ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6' in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the 'jaws' of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.
Asunto(s)
Ribonucleasas , Streptococcus thermophilus , Dominio Catalítico , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Sistemas CRISPR-Cas , Nucleótidos Cíclicos , Ribonucleasas/química , Ribonucleasas/metabolismo , Sistemas de Mensajero Secundario , Streptococcus thermophilus/químicaRESUMEN
BACKGROUND AND AIMS: The Lythraceae are a mainly subtropical to tropical family of the order Myrtales with 28 currently accepted genera and approximately 600 species. There is currently no well-supported phylogenetic and biogeographical hypothesis of the Lythraceae incorporating all currently accepted genera, which we sought to provide. METHODS: Plastomes of representative species of 18 distinct Lythraceae genera were sequenced and annotated. Together with existing sequences, plastomes of all 28 currently accepted genera in the Lythraceae were brought together for the first time. The plastomes were aligned and a Bayesian phylogenetic hypothesis was produced. We then conducted a time-calibrated Bayesian analysis and a biogeographical analysis. KEY RESULTS: Plastome-based Bayesian and maximum-likelihood phylogenetic trees are generally congruent with recent nuclear phylogenomic data and resolve two deeply branching major clades in the Lythraceae. One major clade concentrates shrubby and arboreal South American and African genera that inhabit seasonally dry environments, with larger, often winged seeds, adapted to dispersal by the wind. The second major clade concentrates North American, Asian, African and several near-cosmopolitan herbaceous, shrubby and arboreal genera, often inhabiting humid or aquatic environments, with smaller seeds possessing structures that facilitate dispersal by water. CONCLUSIONS: We hypothesize that the Lythraceae dispersed early in the Late Cretaceous from South American to North American continents, with subsequent expansion in the Late Cretaceous of a North American lineage through Laurasia to Africa via a boreotropical route. Two later expansions of South American clades to Africa in the Palaeocene and Eocene, respectively, are also hypothesized. Transoceanic dispersal in the family is possibly facilitated by adaptations to aquatic environments that are common to many extant genera of the Lythraceae, where long-distance dispersal and vicariance may be invoked to explain several remarkable disjunct distributions in Lythraceae clades.
Asunto(s)
Lythraceae , Filogenia , Filogeografía , Teorema de Bayes , ÁfricaRESUMEN
CRISPR-Cas is a prokaryotic adaptive immune system, classified into six different types, each characterised by a signature protein. Type III systems, classified based on the presence of a Cas10 subunit, are rather diverse multi-subunit assemblies with a range of enzymatic activities and downstream ancillary effectors. The broad array of current biotechnological CRISPR applications is mainly based on proteins classified as Type II, however recent developments established the feasibility and efficacy of multi-protein Type III CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. The crenarchaeon Saccharolobus solfataricus has two type III system subtypes (III-B and III-D). Here, we report the cryo-EM structure of the Csm Type III-D complex from S. solfataricus (SsoCsm), which uses CRISPR RNA to bind target RNA molecules, activating the Cas10 subunit for antiviral defence. The structure reveals the complex organisation, subunit/subunit connectivity and protein/guide RNA interactions of the SsoCsm complex, one of the largest CRISPR effectors known.
RESUMEN
Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3' end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.
Asunto(s)
Proteínas Bacterianas , Proteínas Asociadas a CRISPR , Ectothiorhodospiraceae , Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Microscopía por Crioelectrón , ADN/química , Proteínas Bacterianas/químicaRESUMEN
Cyclic nucleotide signalling is a key component of antiviral defence in all domains of life. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway1, which originated in bacteria2. These defence systems require a sensor domain to bind the cyclic nucleotide and are often coupled with an effector domain that, when activated, causes cell death by destroying essential biomolecules3. One example is the Toll/interleukin-1 receptor (TIR) domain, which degrades the essential cofactor NAD+ when activated in response to infection in plants and bacteria2,4,5 or during programmed nerve cell death6. Here we show that a bacterial antiviral defence system generates a cyclic tri-adenylate that binds to a TIR-SAVED effector, acting as the 'glue' to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organize and complete a composite active site, allowing NAD+ degradation. Activation requires extended filament formation, both in vitro and in vivo. Our study highlights an example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.
Asunto(s)
Bacterias , Nucleótidos Cíclicos , Receptores de Interleucina-1 , Receptores Toll-Like , Animales , Antivirales/inmunología , Antivirales/metabolismo , Bacterias/inmunología , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , NAD/metabolismo , Nucleótidos Cíclicos/química , Nucleótidos Cíclicos/inmunología , Nucleótidos Cíclicos/metabolismo , Receptores de Interleucina-1/química , Receptores de Interleucina-1/inmunología , Receptores de Interleucina-1/metabolismo , Sistemas de Mensajero Secundario , Receptores Toll-Like/química , Receptores Toll-Like/inmunología , Receptores Toll-Like/metabolismoRESUMEN
PREMISE: To further advance the understanding of the species-rich, economically and ecologically important angiosperm order Myrtales in the rosid clade, comprising nine families, approximately 400 genera and almost 14,000 species occurring on all continents (except Antarctica), we tested the Angiosperms353 probe kit. METHODS: We combined high-throughput sequencing and target enrichment with the Angiosperms353 probe kit to evaluate a sample of 485 species across 305 genera (76% of all genera in the order). RESULTS: Results provide the most comprehensive phylogenetic hypothesis for the order to date. Relationships at all ranks, such as the relationship of the early-diverging families, often reflect previous studies, but gene conflict is evident, and relationships previously found to be uncertain often remain so. Technical considerations for processing HTS data are also discussed. CONCLUSIONS: High-throughput sequencing and the Angiosperms353 probe kit are powerful tools for phylogenomic analysis, but better understanding of the genetic data available is required to identify genes and gene trees that account for likely incomplete lineage sorting and/or hybridization events.
Asunto(s)
Magnoliopsida , Myrtales , Núcleo Celular , Magnoliopsida/genética , FilogeniaRESUMEN
Fossil pollen believed to be related to extant Hagenia abyssinica were discovered in the early Miocene (21.73 Ma) Mush Valley paleoflora, Ethiopia, Africa. Both the fossil and extant pollen grains of H. abyssinica were examined with combined light microscopy, scanning electron microscopy, and transmission electron microscopy to compare the pollen and establish their relationships. Based on this, the fossil pollen grains were attributed to Hagenia. The presence of Hagenia in the fossil assemblage raises the questions if its habitat has changed over time, and if the plants are/were wind pollinated. To shed light on these questions, the morphology of extant anthers was also studied, revealing specialized hairs inside the anthers, believed to aid in insect pollination. Pollen and anther morphology are discussed in relation to the age and origin of the genus within a molecular dated phylogenetic framework, the establishment of complex topography in East Africa, other evidence regarding pollination modes, and the palynological record. The evidence presented herein, and compiled from the literature, suggests that Hagenia was an insect-pollinated lowland rainforest element during the early Miocene of the Mush Valley. The current Afromontane habitat and ambophilous (insect and wind) pollination must have evolved in post-mid-Miocene times.
RESUMEN
Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.
Asunto(s)
Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas , Desoxirribonucleasa I/química , Ribonucleasas/química , Clostridiales/enzimología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/química , Desoxirribonucleasa I/metabolismo , Activación Enzimática , Escherichia coli/virología , Secuencias Repetitivas Esparcidas , Metales/química , Modelos Moleculares , Dominios Proteicos , Ribonucleasas/metabolismoRESUMEN
Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.
Bacteria protect themselves from infections using a system called CRISPR-Cas, which helps the cells to detect and destroy invading threats. The type III CRISPR-Cas system, in particular, is one of the most widespread and efficient at killing viruses. When a bacterium is infected, the CRISPR-Cas system takes a fragment of the genetic material of the virus, and copies it into a molecule. These molecular 'police mugshots' are then loaded into a complex of Cas proteins that patrol the cell, looking for a match and destroying any virus that can be identified. Some Cas proteins also produce alarm signals, called cyclic oligoadenylates (cOAs), which can trigger additional defences. However, this process can damage the genetic material of the bacterium, harming or even killing the cell. Enzymes known as ring nucleases can promptly degrade cOAs and turn off this defence system before it causes harm. However, ring nucleases have only been found in a few species to date; how most bacteria deal with cOA toxicity has remained unknown. Here, Athukoralage et al. set out to determine whether a widespread enzyme known as Csx3, which is often associated with type III CRISPR-Cas systems, could be an alternative off switch for cOA triggered defences. Initial 'test tube' experiments with purified Csx3 proteins confirmed that the enzyme could indeed break down cOAs. A careful dissection of Csx3's molecular structure, using biochemical and biophysical techniques, revealed that it worked by 'sandwiching' a cOA molecule between two co-operating portions of the enzyme. As a final test, Csx3 was introduced into strains of bacteria genetically engineered to have a fully functional Type III CRISPR-Cas system. In these cells, Csx3 successfully turned off the Type III immune response. These results reveal a new way that bacteria avoid the toxic side effects of their own immune defences. Ultimately, this could pave the way for the development of anti-bacterial drugs that work by blocking Csx3 or similar proteins.
Asunto(s)
Archaeoglobus fulgidus/enzimología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ribonucleasas/metabolismo , Archaeoglobus fulgidus/genética , Proteínas Asociadas a CRISPR/metabolismo , Catálisis , Dominio Catalítico , Endonucleasas/metabolismo , Escherichia coli/metabolismo , Cinética , Methanosarcina , Modelos Moleculares , Oligonucleótidos/química , Multimerización de Proteína , ARN/metabolismo , Ribonucleasas/genética , Sistemas de Mensajero Secundario , Transducción de SeñalRESUMEN
Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. Some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The cA4 binding affinities and activities of the two constituent enzymes in the fusion protein may have evolved to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ribonucleasas/metabolismo , Sitios de Unión , Modelos Moleculares , Dominios Proteicos , ARN/metabolismo , Estabilidad del ARN , Sistemas de Mensajero SecundarioRESUMEN
Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an 'off-switch' for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.
Asunto(s)
Sistemas CRISPR-Cas , Interacciones Microbiota-Huesped , Nucleótidos Cíclicos/metabolismo , Transducción de Señal , Virus/enzimología , Virus/genética , Escherichia coli/enzimología , Escherichia coli/genética , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismoRESUMEN
The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3-5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7-10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host-pathogen interactions11-13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.
Asunto(s)
Sistemas CRISPR-Cas/inmunología , Endonucleasas/metabolismo , Interacciones Microbiota-Huesped/inmunología , Sulfolobus/virología , Proteínas Virales/metabolismo , Virus/enzimología , Nucleótidos de Adenina/química , Nucleótidos de Adenina/metabolismo , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , ADN Viral/metabolismo , Endonucleasas/química , Modelos Moleculares , Nucleótidos Cíclicos/química , Nucleótidos Cíclicos/metabolismo , Oligorribonucleótidos/química , Oligorribonucleótidos/metabolismo , Filogenia , Transducción de Señal , Sulfolobus/genética , Sulfolobus/inmunología , Sulfolobus/metabolismo , Proteínas Virales/química , Proteínas Virales/clasificación , Virus/inmunologíaRESUMEN
The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes. On binding invading RNA species, Type III CRISPR systems generate cyclic oligoadenylate (cOA) signalling molecules, potentiating a powerful immune response by activating downstream effector proteins, leading to viral clearance, cell dormancy or death. Here we describe the structure and mechanism of a cOA-activated CRISPR defence DNA endonuclease, CRISPR ancillary nuclease 1 (Can1). Can1 has a unique monomeric structure with two CRISPR associated Rossman fold (CARF) domains and two DNA nuclease-like domains. The crystal structure of the enzyme has been captured in the activated state, with a cyclic tetra-adenylate (cA4) molecule bound at the core of the protein. cA4 binding reorganises the structure to license a metal-dependent DNA nuclease activity specific for nicking of supercoiled DNA. DNA nicking by Can1 is predicted to slow down viral replication kinetics by leading to the collapse of DNA replication forks.
Asunto(s)
Nucleótidos de Adenina/farmacología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Endonucleasas/química , Endonucleasas/metabolismo , Oligorribonucleótidos/farmacología , Sitios de Unión , ADN/metabolismo , Modelos Moleculares , Plásmidos/genética , Dominios Proteicos , Homología Estructural de Proteína , Thermus thermophilus/genéticaRESUMEN
The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.
Asunto(s)
Nucleótidos de Adenina/genética , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Mycobacterium tuberculosis/genética , Oligorribonucleótidos/genética , Inmunidad Adaptativa/inmunología , Nucleótidos de Adenina/biosíntesis , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/inmunología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/inmunología , Secuencias Repetitivas Esparcidas/genética , Secuencias Repetitivas Esparcidas/inmunología , Mycobacterium tuberculosis/inmunología , Oligorribonucleótidos/biosíntesis , Células Procariotas/inmunología , División del ARN/genética , División del ARN/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
The pantropical Picrodendraceae produce mostly spheroidal to slightly oblate, echinate pollen grains equipped with narrow circular to elliptic pori that can be hard to identify to family level in both extant and fossil material using light microscopy only. Fossil pollen of the family have been described from the Paleogene of America, Antarctica, Australia, New Zealand, and Europe, but until now none have been reported from Afro-India. Extant pollen described here include representatives from all recent Picrodendraceae genera naturally occurring in Africa and/or Madagascar and south India and selected closely related tropical American taxa. Our analyses, using combined light microscopy and scanning electron microscopy, show that pollen of the Afro-Indian genera encompass three morphological types: Type 1, comprising only Hyaenanche; Type 2, including Aristogeitonia, Mischodon, Oldfieldia and Voatamalo; Type 3, comprising the remaining two genera, Androstachys and Stachyandra. Based on the pollen morphology presented here it is evident that some previous light microscopic accounts of spherical and echinate fossil pollen affiliated with Arecaceae, Asteraceae, Malvaceae, and Myristicaceae from the African continent could belong to Picrodendraceae. The pollen morphology of Picrodendraceae, fossil pollen records, a dated intra-familial phylogeny, seed dispersal modes, and the regional Late Cretaceous to early Cenozoic paleogeography, together suggest the family originated in the Americas and dispersed from southern America across Antarctica and into Australasia. A second dispersal route is believed to have occurred from the Americas into continental Africa via the North Atlantic Land Bridge and Europe.