Germ Warfare in a Microbial Mat Community: CRISPRs Provide Insights into the Co-Evolution of Host and Viral Genomes

CRISPR arrays and associated cas genes are widespread in bacteria and archaea and confer acquired resistance to viruses. To examine viral immunity in the context of naturally evolving microbial populations we analyzed genomic data from two thermophilic Synechococcus isolates (Syn OS-A and Syn OS-B′) as well as a prokaryotic metagenome and viral metagenome derived from microbial mats in hotsprings at Yellowstone National Park. Two distinct CRISPR types, distinguished by the repeat sequence, are found in both the Syn OS-A and Syn OS-B′ genomes. The genome of Syn OS-A contains a third CRISPR type with a distinct repeat sequence, which is not found in Syn OS-B′, but appears to be shared with other microorganisms that inhabit the mat. The CRISPR repeats identified in the microbial metagenome are highly conserved, while the spacer sequences (hereafter referred to as “viritopes” to emphasize their critical role in viral immunity) were mostly unique and had no high identity matches when searched against GenBank. Searching the viritopes against the viral metagenome, however, yielded several matches with high similarity some of which were within a gene identified as a likely viral lysozyme/lysin protein. Analysis of viral metagenome sequences corresponding to this lysozyme/lysin protein revealed several mutations all of which translate into silent or conservative mutations which are unlikely to affect protein function, but may help the virus evade the host CRISPR resistance mechanism. These results demonstrate the varied challenges presented by a natural virus population, and support the notion that the CRISPR/viritope system must be able to adapt quickly to provide host immunity. The ability of metagenomics to track population-level variation in viritope sequences allows for a culture-independent method for evaluating the fast co-evolution of host and viral genomes and its consequence on the structuring of complex microbial communities.     

Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus

Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.     

Viral biogeography revealed by signatures in Sulfolobus islandicus genomes

Viruses are a driving force of microbial evolution. Despite their importance, the evolutionary dynamics that shape diversity in viral populations are not well understood. One of the primary factors that define viral population structure is coevolution with microbial hosts. Experimental models predict that the trajectory of coevolution will be determined by the relative migration rates of viruses and their hosts; however, there are no natural microbial systems in which both have been examined. The biogeographic distribution of viruses that infect Sulfolobus islandicus is investigated using genome comparisons among four newly identified, integrated, Sulfolobus spindle-shaped viruses and previously sequenced viral strains. Core gene sequences show a biogeographic distribution where viral genomes are specifically associated with each local population. In addition, signatures of host–virus interactions recorded in the sequence-specific CRISPR (clustered regularly interspaced short palindromic repeats) system show that hosts have interacted with viral communities that are more closely related to local viral strains than to foreign ones. Together, both proviral and CRISPR sequences show a clear biogeographic structure for Sulfolobus viral populations. Our findings demonstrate that virus–microbe coevolution must be examined in a spatially explicit framework. The combination of host and virus biogeography suggests a model for viral diversification driven by host immunity and local adaptation.     

Metagenomic analyses of novel viruses and plasmids from a cultured environmental sample of hyperthermophilic neutrophiles

Two novel viral genomes and four plasmids were assembled from an environmental sample collected from a hot spring at Yellowstone National Park, USA, and maintained anaerobically in a bioreactor at 85°C and pH 6. The double‐stranded DNA viral genomes are linear (22.7 kb) and circular (17.7 kb), and derive apparently from archaeal viruses HAV1 and HAV2. Genomic DNA was obtained from samples enriched in filamentous and tadpole‐shaped virus‐like particles respectively. They yielded few significant matches in public sequence databases reinforcing, further, the wide diversity of archaeal viruses. Several variants of HAV1 exhibit major genomic alterations, presumed to arise from viral adaptation to different hosts. They include insertions up to 350 bp, deletions up to 1.5 kb, and genes with extensively altered sequences. Some result from recombination events occurring at low complexity direct repeats distributed along the genome. In addition, a 33.8 kb archaeal plasmid pHA1 was characterized, encoding a possible conjugative apparatus, as well as three cryptic plasmids of thermophilic bacterial origin, pHB1 of 2.1 kb and two closely related variants pHB2a and pHB2b, of 5.2 and 4.8 kb respectively. Strategies are considered for assembling genomes of smaller genetic elements from complex environmental samples, and for establishing possible host identities on the basis of sequence similarity to host CRISPR immune systems.     

The Sorcerer II Global Ocean Sampling Expedition: metagenomic characterization of viruses within aquatic microbial samples

Viruses are the most abundant biological entities on our planet. Interactions between viruses and their hosts impact several important biological processes in the world's oceans such as horizontal gene transfer, microbial diversity and biogeochemical cycling. Interrogation of microbial metagenomic sequence data collected as part of the Sorcerer II Global Ocean Expedition (GOS) revealed a high abundance of viral sequences, representing approximately 3% of the total predicted proteins. Cluster analyses of the viral sequences revealed hundreds to thousands of viral genes encoding various metabolic and cellular functions. Quantitative analyses of viral genes of host origin performed on the viral fraction of aquatic samples confirmed the viral nature of these sequences and suggested that significant portions of aquatic viral communities behave as reservoirs of such genetic material. Distributional and phylogenetic analyses of these host-derived viral sequences also suggested that viral acquisition of environmentally relevant genes of host origin is a more abundant and widespread phenomenon than previously appreciated. The predominant viral sequences identified within microbial fractions originated from tailed bacteriophages and exhibited varying global distributions according to viral family. Recruitment of GOS viral sequence fragments against 27 complete aquatic viral genomes revealed that only one reference bacteriophage genome was highly abundant and was closely related, but not identical, to the cyanomyovirus P-SSM4. The co-distribution across all sampling sites of P-SSM4-like sequences with the dominant ecotype of its host, Prochlorococcus supports the classification of the viral sequences as P-SSM4-like and suggests that this virus may influence the abundance, distribution and diversity of one of the most dominant components of picophytoplankton in oligotrophic oceans. In summary, the abundance and broad geographical distribution of viral sequences within microbial fractions, the prevalence of genes among viral sequences that encode microbial physiological function and their distinct phylogenetic distribution lend strong support to the notion that viral-mediated gene acquisition is a common and ongoing mechanism for generating microbial diversity in the marine environment.     

Microviridae goes temperate: microvirus-related proviruses reside in the genomes of Bacteroidetes

The Microviridae comprises icosahedral lytic viruses with circular single-stranded DNA genomes. The family is divided into two distinct groups based on genome characteristics and virion structure. Viruses infecting enterobacteria belong to the genus Microvirus, whereas those infecting obligate parasitic bacteria, such as Chlamydia, Spiroplasma and Bdellovibrio, are classified into a subfamily, the Gokushovirinae. Recent metagenomic studies suggest that members of the Microviridae might also play an important role in marine environments. In this study we present the identification and characterization of Microviridae-related prophages integrated in the genomes of species of the Bacteroidetes, a phylum not previously known to be associated with microviruses. Searches against metagenomic databases revealed the presence of highly similar sequences in the human gut. This is the first report indicating that viruses of the Microviridae lysogenize their hosts. Absence of associated integrase-coding genes and apparent recombination with dif-like sequences suggests that Bacteroidetes-associated microviruses are likely to rely on the cellular chromosome dimer resolution machinery. Phylogenetic analysis of the putative major capsid proteins places the identified proviruses into a group separate from the previously characterized microviruses and gokushoviruses, suggesting that the genetic diversity and host range of bacteriophages in the family Microviridae is wider than currently appreciated.     

Genome-resolved viral and cellular metagenomes revealed potential key virus-host interactions in a deep freshwater lake

Metagenomics has dramatically expanded the known virosphere, but freshwater viral diversity and their ecological interaction with hosts remain poorly understood. Here, we conducted a metagenomic exploration of planktonic dsDNA prokaryotic viruses by sequencing both virion (<0.22 μm) and cellular (0.22–5.0 μm) fractions collected spatiotemporally from a deep freshwater lake (Lake Biwa, Japan). This simultaneously reconstructed 183 complete (i.e., circular) viral genomes and 57 bacterioplankton metagenome-assembled genomes. Analysis of metagenomic read coverage revealed vertical partitioning of the viral community analogous to the vertically stratified bacterioplankton community. The hypolimnetic community was generally stable during stratification, but occasionally shifted abruptly, presumably due to lysogenic induction. Genes involved in assimilatory sulfate reduction were encoded in 20 (10.9%) viral genomes, including those of dominant viruses, and may aid viral propagation in sulfur-limited freshwater systems. Hosts were predicted for 40 (21.9%) viral genomes, encompassing 10 phyla (or classes of Proteobacteria) including ubiquitous freshwater bacterioplankton lineages (e.g., Ca. Fonsibacter and Ca. Nitrosoarchaeum). Comparison with viral genomes derived from published metagenomes revealed viral phylogeographic connectivity in geographically isolated habitats. Notably, analogous to their hosts, actinobacterial viruses were among the most diverse, ubiquitous and abundant viral groups in freshwater systems, with potential high lytic activity in surface waters.     

Phage-bacteria relationships and CRISPR elements revealed by a metagenomic survey of the rumen microbiome

Viruses are the most abundant biological entities on the planet and play an important role in balancing microbes within an ecosystem and facilitating horizontal gene transfer. Although bacteriophages are abundant in rumen environments, little is known about the types of viruses present or their interaction with the rumen microbiome. We undertook random pyrosequencing of virus-enriched metagenomes (viromes) isolated from bovine rumen fluid and analysed the resulting data using comparative metagenomics. A high level of diversity was observed with up to 28,000 different viral genotypes obtained from each environment. The majority (~78%) of sequences did not match any previously described virus. Prophages outnumbered lytic phages approximately 2:1 with the most abundant bacteriophage and prophage types being associated with members of the dominant rumen phyla (Firmicutes and Proteobacteria). Metabolic profiling based on SEED subsystems revealed an enrichment of sequences with putative functional roles in DNA and protein metabolism, but a surprisingly low proportion of sequences assigned to carbohydrate and amino acid metabolism. We expanded our analysis to include previously described metagenomic data and 14 reference genomes. Clustered regularly interspaced short palindromic repeats (CRISPR) were detected in most of the microbial genomes, suggesting previous interactions between viral and microbial communities.     

RNA Viral Metagenome of Whiteflies Leads to the Discovery and Characterization of a Whitefly-Transmitted Carlavirus in North America

Whiteflies from the Bemisia tabaci species complex have the ability to transmit a large number of plant viruses and are some of the most detrimental pests in agriculture. Although whiteflies are known to transmit both DNA and RNA viruses, most of the diversity has been recorded for the former, specifically for the Begomovirus genus. This study investigated the total diversity of DNA and RNA viruses found in whiteflies collected from a single site in Florida to evaluate if there are additional, previously undetected viral types within the B. tabaci vector. Metagenomic analysis of viral DNA extracted from the whiteflies only resulted in the detection of begomoviruses. In contrast, whiteflies contained sequences similar to RNA viruses from divergent groups, with a diversity that extends beyond currently described viruses. The metagenomic analysis of whiteflies also led to the first report of a whitefly-transmitted RNA virus similar to Cowpea mild mottle virus (CpMMV Florida) (genus Carlavirus) in North America. Further investigation resulted in the detection of CpMMV Florida in native and cultivated plants growing near the original field site of whitefly collection and determination of its experimental host range. Analysis of complete CpMMV Florida genomes recovered from whiteflies and plants suggests that the current classification criteria for carlaviruses need to be reevaluated. Overall, metagenomic analysis supports that DNA plant viruses carried by B. tabaci are dominated by begomoviruses, whereas significantly less is known about RNA viruses present in this damaging insect vector.     

Expanding the catalog of cas genes with metagenomes

The CRISPR (clusters of regularly interspaced short palindromic repeats)–Cas adaptive immune system is an important defense system in bacteria, providing targeted defense against invasions of foreign nucleic acids. CRISPR–Cas systems consist of CRISPR loci and cas (CRISPR-associated) genes: sequence segments of invaders are incorporated into host genomes at CRISPR loci to generate specificity, while adjacent cas genes encode proteins that mediate the defense process. We pursued an integrated approach to identifying putative cas genes from genomes and metagenomes, combining similarity searches with genomic neighborhood analysis. Application of our approach to bacterial genomes and human microbiome datasets allowed us to significantly expand the collection of cas genes: the sequence space of the Cas9 family, the key player in the recently engineered RNA-guided platforms for genome editing in eukaryotes, is expanded by at least two-fold with metagenomic datasets. We found genes in cas loci encoding other functions, for example, toxins and antitoxins, confirming the recently discovered potential of coupling between adaptive immunity and the dormancy/suicide systems. We further identified 24 novel Cas families; one novel family contains 20 proteins, all identified from the human microbiome datasets, illustrating the importance of metagenomics projects in expanding the diversity of cas genes.     

NAR Breakthrough Article: The casposon-encoded Cas1 protein from Aciduliprofundum boonei is a DNA integrase that generates target site duplications

Many archaea and bacteria have an adaptive immune system known as CRISPR which allows them to recognize and destroy foreign nucleic acid that they have previously encountered. Two CRISPR-associated proteins, Cas1 and Cas2, are required for the acquisition step of adaptation, in which fragments of foreign DNA are incorporated into the host CRISPR locus. Cas1 genes have also been found scattered in several archaeal and bacterial genomes, unassociated with CRISPR loci or other cas proteins. Rather, they are flanked by nearly identical inverted repeats and enclosed within direct repeats, suggesting that these genetic regions might be mobile elements (‘casposons’). To investigate this possibility, we have characterized the in vitro activities of the putative Cas1 transposase (‘casposase’) from Aciduliprofundum boonei. The purified Cas1 casposase can integrate both short oligonucleotides with inverted repeat sequences and a 2.8 kb excised mini-casposon into target DNA. Casposon integration occurs without target specificity and generates 14–15 basepair target site duplications, consistent with those found in casposon host genomes. Thus, Cas1 casposases carry out similar biochemical reactions as the CRISPR Cas1-Cas2 complex but with opposite substrate specificities: casposases integrate specific sequences into random target sites, whereas CRISPR Cas1-Cas2 integrates essentially random sequences into a specific site in the CRISPR locus.     

Extraordinary diversity of viruses in deep-sea sediments as revealed by metagenomics without prior virion separation

Our current knowledge of the virosphere in deep-sea sediments remains rudimentary. Here we investigated viral diversity at both gene and genomic levels in deep-sea sediments of Southwest Indian Ocean. Analysis of 19 676 106 non-redundant genes from the metagenomic DNA sequences revealed a large number of unclassified viral groups in these samples. A total of 1106 high-confidence viral contigs were obtained after two runs of assemblies, and 217 of these contigs with sizes up to ~120 kb were shown to represent complete viral genomes. These contigs are clustered with no known viral genomes, and over 2/3 of the ORFs on the viral contigs encode no known functions. Furthermore, most of the complete viral contigs show limited similarity to known viral genomes in genome organization. Most of the classified viral contigs are derived from dsDNA viruses belonging to the order Caudovirales, including primarily members of the families Myoviridae, Podoviridae and Siphoviridae. Most of these viruses infect Proteobacteria and, less frequently, Planctomycetes, Firmicutes, Chloroflexi, etc. Auxiliary metabolic genes (AMGs), present in abundance on the viral contigs, appear to function in modulating the host ability to sense environmental gradients and community changes, and to uptake and metabolize nutrients.     

The First Endogenous Herpesvirus,Identified in the Tarsier Genome,and Novel Sequences from Primate Rhadinoviruses and Lymphocryptoviruses

Herpesviridae is a diverse family of large and complex pathogens whose genomes are extremely difficult to sequence. This is particularly true for clinical samples, and if the virus, host, or both genomes are being sequenced for the first time. Although herpesviruses are known to occasionally integrate in host genomes, and can also be inherited in a Mendelian fashion, they are notably absent from the genomic fossil record comprised of endogenous viral elements (EVEs). Here, we combine paleovirological and metagenomic approaches to both explore the constituent viral diversity of mammalian genomes and search for endogenous herpesviruses. We describe the first endogenous herpesvirus from the genome of the Philippine tarsier, belonging to the Roseolovirus genus, and characterize its highly defective genome that is integrated and flanked by unambiguous host DNA. From a draft assembly of the aye-aye genome, we use bioinformatic tools to reveal over 100,000 bp of a novel rhadinovirus that is the first lemur gammaherpesvirus, closely related to Kaposi''s sarcoma-associated virus. We also identify 58 genes of Pan paniscus lymphocryptovirus 1, the bonobo equivalent of human Epstein-Barr virus. For each of the viruses, we postulate gene function via comparative analysis to known viral relatives. Most notably, the evidence from gene content and phylogenetics suggests that the aye-aye sequences represent the most basal known rhadinovirus, and indicates that tumorigenic herpesviruses have been infecting primates since their emergence in the late Cretaceous. Overall, these data show that a genomic fossil record of herpesviruses exists despite their extremely large genomes, and expands the known diversity of Herpesviridae, which will aid the characterization of pathogenesis. Our analytical approach illustrates the benefit of intersecting evolutionary approaches with metagenomics, genetics and paleovirology.     

Persisting viral sequences shape microbial CRISPR-based immunity

Well-studied innate immune systems exist throughout bacteria and archaea, but a more recently discovered genomic locus may offer prokaryotes surprising immunological adaptability. Mediated by a cassette-like genomic locus termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), the microbial adaptive immune system differs from its eukaryotic immune analogues by incorporating new immunities unidirectionally. CRISPR thus stores genomically recoverable timelines of virus-host coevolution in natural organisms refractory to laboratory cultivation. Here we combined a population genetic mathematical model of CRISPR-virus coevolution with six years of metagenomic sequencing to link the recoverable genomic dynamics of CRISPR loci to the unknown population dynamics of virus and host in natural communities. Metagenomic reconstructions in an acid-mine drainage system document CRISPR loci conserving ancestral immune elements to the base-pair across thousands of microbial generations. This 'trailer-end conservation' occurs despite rapid viral mutation and despite rapid prokaryotic genomic deletion. The trailer-ends of many reconstructed CRISPR loci are also largely identical across a population. 'Trailer-end clonality' occurs despite predictions of host immunological diversity due to negative frequency dependent selection (kill the winner dynamics). Statistical clustering and model simulations explain this lack of diversity by capturing rapid selective sweeps by highly immune CRISPR lineages. Potentially explaining 'trailer-end conservation,' we record the first example of a viral bloom overwhelming a CRISPR system. The polyclonal viruses bloom even though they share sequences previously targeted by host CRISPR loci. Simulations show how increasing random genomic deletions in CRISPR loci purges immunological controls on long-lived viral sequences, allowing polyclonal viruses to bloom and depressing host fitness. Our results thus link documented patterns of genomic conservation in CRISPR loci to an evolutionary advantage against persistent viruses. By maintaining old immunities, selection may be tuning CRISPR-mediated immunity against viruses reemerging from lysogeny or migration.     

Reconstructing viral genomes from the environment using fosmid clones: the case of haloviruses

Background

Metaviriomes, the viral genomes present in an environment, have been studied by direct sequencing of the viral DNA or by cloning in small insert libraries. The short reads generated by both approaches make it very difficult to assemble and annotate such flexible genomic entities. Many environmental viruses belong to unknown groups or prey on uncultured and little known cellular lineages, and hence might not be present in databases.

Methodology and Principal Findings

Here we have used a different approach, the cloning of viral DNA into fosmids before sequencing, to obtain natural contigs that are close to the size of a viral genome. We have studied a relatively low diversity extreme environment: saturated NaCl brines, which simplifies the analysis and interpretation of the data. Forty-two different viral genomes were retrieved, and some of these were almost complete, and could be tentatively identified as head-tail phages (Caudovirales).

Conclusions and Significance

We found a cluster of phage genomes that most likely infect Haloquadratum walsbyi, the square archaeon and major component of the community in these hypersaline habitats. The identity of the prey could be confirmed by the presence of CRISPR spacer sequences shared by the virus and one of the available strain genomes. Other viral clusters detected appeared to prey on the Nanohaloarchaea and on the bacterium Salinibacter ruber, covering most of the diversity of microbes found in this type of environment. This approach appears then as a viable alternative to describe metaviriomes in a much more detailed and reliable way than by the more common approaches based on direct sequencing. An example of transfer of a CRISPR cluster including repeats and spacers was accidentally found supporting the dynamic nature and frequent transfer of this peculiar prokaryotic mechanism of cell protection.     

Comparative genomic analysis of hyperthermophilic archaeal Fuselloviridae viruses

The complete genome sequences of two Sulfolobus spindle-shaped viruses (SSVs) from acidic hot springs in Kamchatka (Russia) and Yellowstone National Park (United States) have been determined. These nonlytic temperate viruses were isolated from hyperthermophilic Sulfolobus hosts, and both viruses share the spindle-shaped morphology characteristic of the Fuselloviridae family. These two genomes, in combination with the previously determined SSV1 genome from Japan and the SSV2 genome from Iceland, have allowed us to carry out a phylogenetic comparison of these geographically distributed hyperthermal viruses. Each virus contains a circular double-stranded DNA genome of approximately 15 kbp with approximately 34 open reading frames (ORFs). These Fusellovirus ORFs show little or no similarity to genes in the public databases. In contrast, 18 ORFs are common to all four isolates and may represent the minimal gene set defining this viral group. In general, ORFs on one half of the genome are colinear and highly conserved, while ORFs on the other half are not. One shared ORF among all four genomes is an integrase of the tyrosine recombinase family. All four viral genomes integrate into their host tRNA genes. The specific tRNA gene used for integration varies, and one genome integrates into multiple loci. Several unique ORFs are found in the genome of each isolate.     

Long-run bacteria-phage coexistence dynamics under natural habitat conditions in an environmental biotechnology system

Bacterial viruses are widespread and abundant across natural and engineered habitats. They influence ecosystem functioning through interactions with their hosts. Laboratory studies of phage–host pairs have advanced our understanding of phenotypic and genetic diversification in bacteria and phages. However, the dynamics of phage–host interactions have been seldom recorded in complex natural environments. We conducted an observational metagenomic study of the dynamics of interaction between Gordonia and their phages using a three-year data series of samples collected from a full-scale wastewater treatment plant. The aim was to obtain a comprehensive picture of the coevolution dynamics in naturally evolving populations at relatively high time resolution. Coevolution was followed by monitoring changes over time in the CRISPR loci of Gordonia metagenome-assembled genome, and reciprocal changes in the viral genome. Genome-wide analysis indicated low strain variability of Gordonia, and almost clonal conservation of the trailer end of the CRISPR loci. Incorporation of newer spacers gave rise to multiple coexisting bacterial populations. The host population carrying a shorter CRISPR locus that contain only ancestral spacers, which has not acquired newer spacers against the coexisting phages, accounted for more than half of the total host abundance in the majority of samples. Phages genome co-evolved by introducing directional changes, with no preference for mutations within the protospacer and PAM regions. Metagenomic reconstruction of time-resolved variants of host and viral genomes revealed how the complexity at the population level has important consequences for bacteria-phage coexistence.Subject terms: Microbial ecology, Metagenomics, Bacteriophages     

Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon

The 2 465 177 bp genome of Sulfolobus islandicus LAL14/1, host of the model rudivirus SIRV2, was sequenced. Exhaustive comparative genomic analysis of S. islandicus LAL14/1 and the nine other completely sequenced S. islandicus strains isolated from Iceland, Russia and USA revealed a highly syntenic common core genome of approximately 2 Mb and a long hyperplastic region containing most of the strain-specific genes. In LAL14/1, the latter region is enriched in insertion sequences, CRISPR (clustered regularly interspaced short palindromic repeats), glycosyl transferase genes, toxin–antitoxin genes and MITE (miniature inverted-repeat transposable elements). The tRNA genes of LAL14/1 are preferential targets for the integration of mobile elements but clusters of atypical genes (CAG) are also integrated elsewhere in the genome. LAL14/1 carries five CRISPR loci with 10 per cent of spacers matching perfectly or imperfectly the genomes of archaeal viruses and plasmids found in the Icelandic hot springs. Strikingly, the CRISPR_2 region of LAL14/1 carries an unusually long 1.9 kb spacer interspersed between two repeat regions and displays a high similarity to pING1-like conjugative plasmids. Finally, we have developed a genetic system for S. islandicus LAL14/1 and created ΔpyrEF and ΔCRISPR_1 mutants using double cross-over and pop-in/pop-out approaches, respectively. Thus, LAL14/1 is a promising model to study virus–host interactions and the CRISPR/Cas defence mechanism in Archaea.     

Intricate interactions between the bloom-forming cyanobacterium Microcystis aeruginosa and foreign genetic elements, revealed by diversified clustered regularly interspaced short palindromic repeat (CRISPR) signatures

Clustered regularly interspaced short palindromic repeats (CRISPR) confer sequence-dependent, adaptive resistance in prokaryotes against viruses and plasmids via incorporation of short sequences, called spacers, derived from foreign genetic elements. CRISPR loci are thus considered to provide records of past infections. To describe the host-parasite (i.e., cyanophages and plasmids) interactions involving the bloom-forming freshwater cyanobacterium Microcystis aeruginosa, we investigated CRISPR in four M. aeruginosa strains and in two previously sequenced genomes. The number of spacers in each locus was larger than the average among prokaryotes. All spacers were strain specific, except for a string of 11 spacers shared in two closely related strains, suggesting diversification of the loci. Using CRISPR repeat-based PCR, 24 CRISPR genotypes were identified in a natural cyanobacterial community. Among 995 unique spacers obtained, only 10 sequences showed similarity to M. aeruginosa phage Ma-LMM01. Of these, six spacers showed only silent or conservative nucleotide mutations compared to Ma-LMM01 sequences, suggesting a strategy by the cyanophage to avert CRISPR immunity dependent on nucleotide identity. These results imply that host-phage interactions can be divided into M. aeruginosa-cyanophage combinations rather than pandemics of population-wide infectious cyanophages. Spacer similarity also showed frequent exposure of M. aeruginosa to small cryptic plasmids that were observed only in a few strains. Thus, the diversification of CRISPR implies that M. aeruginosa has been challenged by diverse communities (almost entirely uncharacterized) of cyanophages and plasmids.