Structure and mechanism of the CMR complex for CRISPR-Mediated antiviral immunity

The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse "payload" of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endo-nucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5' sequence in the crRNA, but not on the presence of a proto-spacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets.Publisher PDFPeer reviewe

Characterisation of proteins involved in CRISPR-mediated antiviral defence in Sulfolobus solfataricus

One of the most surprising realisations to emerge from metagenomics studies in the early ‘00s was that the population of viruses and phages in nature is about 10 times larger than the population of prokaryotic organisms. Thus, bacteria and archaea are under constant pressure to develop resistance methods against a population of viruses with extremely high turnover and evolution rates, in what has been described as an evolutionary “arms race”. A novel, adaptive and heritable immune system encoded by prokaryotic genomes is the CRISPR/Cas system. Arrays of clustered regularly interspersed short palindromic repeats (CRISPR) are able to incorporate viral or plasmid sequences which are then used to inactivate the corresponding invader element via an RNA interference mechanism. A number of CRISPR-associated (Cas) protein families are responsible for the maintenance, expansion and function of the CRISPR loci. This system can be classified in a number of types and subtypes that differ widely in their gene composition and mode of action. This thesis describes the biochemical characteristics of CRISPR-mediated defense in the crenarchaeon Sulfolobus solfataricus. The process of CRISPR loci transcription and their subsequent maturation into small guide crRNA units by the processing endonuclease of the system (Cas6) is investigated. After this step, different pathways and effector proteins are involved in the recognition and silencing of DNA or RNA exogenous nucleic acids. This thesis reports the identification and purification of a native multiprotein complex from S. solfataricus P2, the Cmr complex, a homologue of which has been found to recognise and cleave RNA targets in P. furiosus. The recognition and silencing of DNA targets in E. coli has been shown to involve a multiprotein complex termed CASCADE as well as Cas3, a putative helicase-HD nuclease. S. solfataricus encodes orthologues for the core proteins of this complex, and the formation and function of an archaeal CASCADE is investigated in this thesis

Ancestral Reconstructions Decipher Major Adaptations of Ammonia-Oxidizing Archaea upon Radiation into Moderate Terrestrial and Marine Environments

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Genome wide transcriptomic analysis of the soil ammonia oxidizing archaeon Nitrososphaera viennensis upon exposure to copper limitation

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Ammonia Oxidation by the Arctic Terrestrial Thaumarchaeote Candidatus Nitrosocosmicus arcticus Is Stimulated by Increasing Temperatures

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Proteomics and comparative genomics of Nitrososphaera viennensis reveal the core genome and adaptations of archaeal ammonia oxidizers

International audienceAmmonia-oxidizing archaea (AOA) are among the most abundant microorganisms and key players in the global nitrogen and carbon cycles. They share a common energy metabolism but represent a heterogeneous group with respect to their environmental distribution and adaptions, growth requirements, and genome contents. We report here the genome and proteome of Nitrososphaera viennensis EN76, the type species of the archaeal class Nitrososphaeria of the phylum Thaumarchaeota encompassing all known AOA. N. viennensis is a soil organism with a 2.52-Mb genome and 3,123 predicted protein-coding genes. Proteomic analysis revealed that nearly 50% of the predicted genes were translated under standard laboratory growth conditions. Comparison with genomes of closely related species of the predominantly terrestrial Nitrososphaerales as well as the more streamlined marine Nitrosopumilales [ Candidatus ( Ca. ) order] and the acidophile “ Ca. Nitrosotalea devanaterra” revealed a core genome of AOA comprising 860 genes, which allowed for the reconstruction of central metabolic pathways common to all known AOA and expressed in the N. viennensis and “ Ca . Nitrosopelagicus brevis” proteomes. Concomitantly, we were able to identify candidate proteins for as yet unidentified crucial steps in central metabolisms. In addition to unraveling aspects of core AOA metabolism, we identified specific metabolic innovations associated with the Nitrososphaerales mediating growth and survival in the soil milieu, including the capacity for biofilm formation, cell surface modifications and cell adhesion, and carbohydrate conversions as well as detoxification of aromatic compounds and drugs

Candidatus Nitrosocaldus cavascurensis, an Ammonia Oxidizing, Extremely Thermophilic Archaeon with a Highly Mobile Genome

Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first enrichment of a thermophilic representative with a sequenced genome, which facilitates the search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota. Candidatus Nitrosocaldus cavascurensis has been enriched from a hot spring in Ischia, Italy. It grows optimally at 68°C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 23 h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome of Ca. N. cavascurensis harbors an amoAXCB gene cluster encoding ammonia monooxygenase and genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although a bona fide gene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the potential importance of this compound for AOA metabolism. Ca. N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits

A Dimeric Rep Protein Initiates Replication of a Linear Archaeal Virus Genome: Implications for the Rep Mechanism and Viral Replication ▿ †

The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between an active-site tyrosine and the 5′ end of the DNA, releasing a 3′ DNA end as a primer for DNA synthesis. The enzyme can also catalyze the joining reaction that is necessary to reseal the DNA hairpin and terminate replication. The dimeric structure points to a simple mechanism through which two closely positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral replication are discussed