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To contend with the diversity and ubiquity of bacteriophages and other mobile genetic elements, bacteria have developed an arsenal of immune defence mechanisms. Bacterial defences include CRISPR–Cas, restriction–modification and a growing list of mechanistically diverse systems, which constitute the bacterial ‘immune system’. As a response, bacteriophages and mobile genetic elements have evolved direct and indirect mechanisms to circumvent or block bacterial defence pathways and ensure successful infection. Recent advances in methodological and computational approaches, as well as the increasing availability of genome sequences, have boosted the discovery of direct inhibitors of bacterial defence systems. In this Review, we discuss methods for the discovery of direct inhibitors, their diverse mechanisms of action and perspectives on their emerging applications in biotechnology and beyond., To contend with the diversity and ubiquity of bacteriophages and other mobile genetic elements, bacteria have developed an arsenal of immune defence mechanisms. Bacterial defences include CRISPR–Cas, restriction–modification and a growing list of mechanistically diverse systems, which constitute the bacterial ‘immune system’. As a response, bacteriophages and mobile genetic elements have evolved direct and indirect mechanisms to circumvent or block bacterial defence pathways and ensure successful infection. Recent advances in methodological and computational approaches, as well as the increasing availability of genome sequences, have boosted the discovery of direct inhibitors of bacterial defence systems. In this Review, we discuss methods for the discovery of direct inhibitors, their diverse mechanisms of action and perspectives on their emerging applications in biotechnology and beyond.

Prokaryotic adaptation is strongly influenced by the horizontal acquisition of beneficial traits via mobile genetic elements (MGEs), such as viruses/bacteriophages and plasmids. However, MGEs can also impose a fitness cost due to their often parasitic nature and differing evolutionary trajectories. In response, prokaryotes have evolved diverse immune mechanisms against MGEs. Recently, our understanding of the abundance and diversity of prokaryotic immune systems has greatly expanded. These defense systems can degrade the invading genetic material, inhibit genome replication, or trigger abortive infection, leading to population protection. In this review, we highlight these strategies, focusing on the most recent discoveries. The study of prokaryotic defenses not only sheds light on microbial evolution but also uncovers novel enzymatic activities with promising biotechnological applications.

Many prokaryotes encode CRISPR-Cas systems as immune protection against mobile genetic elements (MGEs), yet a number of MGEs also harbor CRISPR-Cas components. With a few exceptions, CRISPR-Cas loci encoded on MGEs are uncharted and a comprehensive analysis of their distribution, prevalence, diversity, and function is lacking. Here, we systematically investigated CRISPR-Cas loci across the largest curated collection of natural bacterial and archaeal plasmids. CRISPR-Cas loci are widely but heterogeneously distributed across plasmids and, in comparison to host chromosomes, their mean prevalence per Mbp is higher and their distribution is distinct. Furthermore, the spacer content of plasmid CRISPRs exhibits a strong targeting bias towards other plasmids, while chromosomal arrays are enriched with virus-targeting spacers. These contrasting targeting preferences highlight the genetic independence of plasmids and suggest a major role for mediating plasmid-plasmid conflicts. Altogether, CRISPR-Cas are frequent accessory components of many plasmids, which is an overlooked phenomenon that possibly facilitates their dissemination across microbiomes.

CRISPR-Cas systems provide prokaryotes with adaptive immune functions against viruses and other genetic parasites. In contrast to all other types of CRISPR-Cas systems, type IV has remained largely overlooked. Here, we describe a previously uncharted diversity of type IV gene cassettes, primarily encoded by plasmid-like elements from diverse prokaryotic taxa. Remarkably, via a comprehensive analysis of their CRISPR spacer content, these systems were found to exhibit a strong bias towards the targeting of other plasmids. Our data indicate that the functions of type IV systems have diverged from those of other host-related CRISPR-Cas immune systems to adopt a role in mediating conflicts between plasmids. Furthermore, we find evidence for cross-talk between certain type IV and type I CRISPR-Cas systems that co-exist intracellularly, thus providing a simple answer to the enigmatic absence of type IV adaptation modules. Collectively, our results lead to the expansion and reclassification of type IV systems and provide novel insights into the biological function and evolution of these elusive systems.

Automated classification of CRISPR-Cas systems has been challenged by their dynamic nature and expanding classification. Here, we developed CRISPRCasTyper, an automated tool with improved capabilities for identifying and typing CRISPR arrays and cas loci based on the latest nomenclature (44 subtypes/variants). As a novel feature, CRISPRCasTyper uses a machine learning approach to subtype CRISPR arrays based on the sequences of the repeats, which allows the typing of orphan and distant arrays. CRISPRCasTyper provides a graphical output, where CRISPRs and cas operons are visualized as gene maps, thus aiding annotation of partial and novel systems through synteny. CRISPRCasTyper was benchmarked against a manually curated set of 31 subtypes with a median accuracy of 98.6% and used to explore CRISPR-Cas diversity across >3,000 metagenomes. Altogether, we present an up-to-date software for improved automated prediction of CRISPR-Cas loci. CRISPRCasTyper is available through conda and as a web server (cctyper.crispr.dk).

Genetic engineering of viruses has generally been challenging. This is also true for archaeal rod-shaped viruses, which carry linear double-stranded DNA genomes with hairpin ends. In this paper, we describe two different genome editing approaches to mutate the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) using the archaeon Sulfolobus islandicus LAL14/1 and its derivatives as hosts. The anti-CRISPR (Acr) gene acrID1, which inhibits CRISPR-Cas subtype I-D immunity, was first used as a selection marker to knock out genes from SIRV2M, an acrID1-null mutant of SIRV2. Moreover, we harnessed the endogenous CRISPR-Cas systems of the host to knock out the accessory genes consecutively, which resulted in a genome comprised solely of core genes of the 11 SIRV members. Furthermore, infection of this series of knockout mutants in the CRISPR-null host of LAL14/1 (∆arrays) confirmed the non-essentiality of the deleted genes and all except the last deletion mutant propagated as efficiently as the WT SIRV2. This suggested that the last gene deleted, SIRV2 gp37, is important for the efficient viral propagation. The generated viral mutants will be useful for future functional studies including searching for new Acrs and the approaches described in this case are applicable to other viruses.

[ES] A pesar de la importante función de la microbiota en el tracto gastrointestinal y su impacto en la salud durante toda la vida, el proceso de sucesión a través del cual se desarrolla esta comunidad microbiana durante la infancia es aún poco conocido. Especialmente, se sabe poco sobre como la cantidad y tipo de virus, es decir, el viroma, varía a través de este periodo y sobre la función que este conjunto de virus puede desarrollar en el ensamblaje de la microbiota del tracto gastrointestinal. En este estudio se analizaron los patrones de cambio taxonómico en la comunidad viral del tracto gastrointestinal en una cohorte de niños sanos durante el primer año de vida. Para ello, se utilizaron muestras fecales que fueron recogidas a diferentes tiempos para los niños y sus madres. El ADN viral fue aislado, amplificado aleatoriamente utilizando la polimerasa phi29 y secuenciado por pirosecuenciación 454. A continuación, las secuencias virales obtenidas fueron taxonómicamente asignadas por medio de análisis bioinformáticos para establecer la composición del viroma presente en cada muestra. Finalmente, se realizaron análisis estadísticos para evaluar el proceso de la dinámica viral a través del tiempo. A través del análisis tanto de la riqueza y diversidad taxonómicas como del clustering de muestras en base a la composición y la abundancia viral, se ha observado que la dinámica viral en el intestino es distinta en cada individuo y no se han podido identificar patrones claros. Sin embargo, se han observado tendencias generales en el desarrollo del viroma, como el decrecimiento de la riqueza y la diversidad taxonómicas durante el primer año de vida, contrariamente al comportamiento de la microbiota bacteriana, y un cambio direccional a través del tiempo hacia la composición taxonómica del viroma materno., [EN] Despite the important role of the microbiota in the human gastrointestinal tract (GIT) and its impact on life-long health, the successional process through which this microbial community develops during infancy is still poorly understood. Specially, little is known about how the amount and type of viruses present in the GIT, i. e. the virome, varies throughout this period and about the role this collection of viruses may play in the assembly of the GIT microbiota. In this study, the patterns of taxonomic change of the GIT viral community were analyzed in a birth cohort of healthy infants during the first year of life. Fecal samples were collected at different timepoints for the infants and their mothers. Viral DNA was isolated, randomly amplified using phi29 polymerase and sequenced through high-throughput 454 pyrosequencing. Then, the obtained viral sequences were taxonomically assigned through bioinformatic analyses to establish the composition of the virome present in each sample. Finally, statistical analyses were performed in order to assess the process of viral dynamics through time. Through the analysis of taxonomic richness and diversity, as well as clustering based on viral composition and abundance, it has been observed that individual instances of viral dynamics in the gut are distinct, and clear patterns have not been identified. However, general trends have been observed in virome development, such as the decrease of viral taxonomic richness and diversity during the first year of life, contrary to the behaviour of the bacterial microbiota, and a directionality of change through time towards the taxonomic composition of the maternal virome.