Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA.

Bacterial abortive-infection systems limit the spread of foreign invaders by shutting down or killing infected cells before the invaders can replicate1,2. Several RNA-targeting CRISPR-Cas systems (that is, types III and VI) cause abortive-infection phenotypes by activating indiscriminate nucleases3-5. However, a CRISPR-mediated abortive mechanism that leverages indiscriminate DNase activity of an RNA-guided single-effector nuclease has yet to be observed. Here we report that RNA targeting by the type V single-effector nuclease Cas12a2 drives abortive infection through non-specific cleavage of double-stranded DNA (dsDNA). After recognizing an RNA target with an activating protospacer-flanking sequence, Cas12a2 efficiently degrades single-stranded RNA (ssRNA), single-stranded DNA (ssDNA) and dsDNA. Within cells, the activation of Cas12a2 induces an SOS DNA-damage response and impairs growth, preventing the dissemination of the invader. Finally, we harnessed the collateral activity of Cas12a2 for direct RNA detection, demonstrating that Cas12a2 can be repurposed as an RNA-guided RNA-targeting tool. These findings expand the known defensive abilities of CRISPR-Cas systems and create additional opportunities for CRISPR technologies.

The genome of the intracellular bacterium of the coastal bivalve, Solemya velum: a blueprint for thriving in and out of symbiosis.

BACKGROUND: Symbioses between chemoautotrophic bacteria and marine invertebrates are rare examples of living systems that are virtually independent of photosynthetic primary production. These associations have evolved multiple times in marine habitats, such as deep-sea hydrothermal vents and reducing sediments, characterized by steep gradients of oxygen and reduced chemicals. Due to difficulties associated with maintaining these symbioses in the laboratory and culturing the symbiotic bacteria, studies of chemosynthetic symbioses rely heavily on culture independent methods. The symbiosis between the coastal bivalve, Solemya velum, and its intracellular symbiont is a model for chemosynthetic symbioses given its accessibility in intertidal environments and the ability to maintain it under laboratory conditions. To better understand this symbiosis, the genome of the S. velum endosymbiont was sequenced. RESULTS: Relative to the genomes of obligate symbiotic bacteria, which commonly undergo erosion and reduction, the S. velum symbiont genome was large (2.7 Mb), GC-rich (51%), and contained a large number (78) of mobile genetic elements. Comparative genomics identified sets of genes specific to the chemosynthetic lifestyle and necessary to sustain the symbiosis. In addition, a number of inferred metabolic pathways and cellular processes, including heterotrophy, branched electron transport, and motility, suggested that besides the ability to function as an endosymbiont, the bacterium may have the capacity to live outside the host. CONCLUSIONS: The physiological dexterity indicated by the genome substantially improves our understanding of the genetic and metabolic capabilities of the S. velum symbiont and the breadth of niches the partners may inhabit during their lifecycle.

Metatranscriptomic analysis of sulfur oxidation genes in the endosymbiont of solemya velum.

Thioautotrophic endosymbionts in the Domain Bacteria mediate key sulfur transformations in marine reducing environments. However, the molecular pathways underlying symbiont metabolism and the extent to which these pathways are expressed in situ are poorly characterized for almost all symbioses. This is largely due to the difficulty of culturing symbionts apart from their hosts. Here, we use pyrosequencing of community RNA transcripts (i.e., the metatranscriptome) to characterize enzymes of dissimilatory sulfur metabolism in the model symbiosis between the coastal bivalve Solemya velum and its intracellular thioautotrophic symbionts. High-throughput sequencing of total RNA from the symbiont-containing gill of a single host individual generated 1.6 million sequence reads (500 Mbp). Of these, 43,735 matched Bacteria protein-coding genes in BLASTX searches of the NCBI database. The taxonomic identities of the matched genes indicated relatedness to diverse species of sulfur-oxidizing Gammaproteobacteria, including other thioautotrophic symbionts and the purple sulfur bacterium Allochromatium vinosum. Manual querying of these data identified 28 genes from diverse pathways of sulfur energy metabolism, including the dissimilatory sulfite reductase (Dsr) pathway for sulfur oxidation to sulfite, the APS pathway for sulfite oxidation, and the Sox pathway for thiosulfate oxidation. In total, reads matching sulfur energy metabolism genes represented 7% of the Bacteria mRNA pool. Together, these data highlight the dominance of thioautotrophy in the context of symbiont community metabolism, identify the likely pathways mediating sulfur oxidation, and illustrate the utility of metatranscriptome sequencing for characterizing community gene transcription of uncultured symbionts.