RetroN

Abstract: Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA The RT uses the non-coding RNA as a template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked Although retrons were discovered three decades ago, their function remained unknown In this study we report that retrons function as anti-phage defense systems The defensive unit is composed of three components: the RT, the non-coding RNA, and an effector protein Retron-containing systems are abundant in genomic "defense islands", suggesting a role for most retrons in phage resistance By cloning multiple retron systems into a retron-less Escherichia coli strain, we show that these systems confer defense against a broad range of phages, with different retrons defending against different phages Focusing on a single retron, Ec48, we show evidence that it is a "guardian" of RecBCD, a complex with central anti-phage functions in the bacterial cell Inhibition of RecBCD by dedicated phage proteins activates the retron, leading to abortive infection and cell death Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed Our results expose a new family of anti-phage defense systems abundant in bacteria

Abstract: Retrons are distinct DNA sequences that code for a reverse transcriptase (RT) similar to the RTs produced by retroviruses and other types of retroelements. Retron DNAs are commonly associated with pro

Abstract: Tremendous genetic variation exists in nature, but our ability to create and characterize individual genetic variants remains far more limited in scale. Likewise, engineering proteins and phenotypes requires the introduction of synthetic variants, but design of variants outpaces experimental measurement of variant effect. Here, we optimize efficient and continuous generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing efficiency can be obtained using this method, enabling multiplexed applications. We introduce Retron Library Recombineering (RLR), a system for high-throughput screens of variants, wherein the association of introduced edits with their retron elements enables a targeted deep sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus present new avenues for exploring variation, both designed and not, across the entire genome.

Abstract: The region (retron-Ec67) required for the biosynthesis of a branched-RNA-linked multicopy single-stranded DNA (msDNA-Ec67) from a clinical isolate of Escherichia coli was mapped at a position equivalent to 19 min on the K-12 chromosome. The element containing the retron consisted of a unique 34-kilobase sequence that was flanked by direct repeats of a 26-base-pair sequence found in the K-12 chromosomal DNA. This suggests that the 34-kilobase element was probably integrated into the E. coli genome by a mechanism related to transposition or phage integration. In the 34-kilobase sequence an open reading frame of 285 residues was found, which displays 44% sequence identity with the E. coli Dam methylase. Interestingly, there are three GATC sequences, the site of Dam methylation, in the promoter region of the gene for reverse transcriptase.