Bacterial retrons encode tripartite toxin/antitoxin systems

Abstract

19Retrons are genetic retroelements, commonly found in bacterial genomes and recently 20 repurposed as genome editing tools. Their encoded reverse transcriptase (RT) produces a 21 multi-copy single-stranded DNA (msDNA). Despite our understanding of their complex 22 biosynthesis, the function of msDNAs and therefore, the physiological role of retrons has 23 remained elusive. We establish that the retron-Sen2 in Salmonella Typhimurium encodes a 24 toxin, which we have renamed as RcaT (Retron cold-anaerobic Toxin). RcaT is activated when 25 msDNA biosynthesis is perturbed and its toxicity is higher at ambient temperatures or during 26 anaerobiosis. The RT and msDNA form together the antitoxin unit, with the RT binding RcaT, 27 and the msDNA enabling the antitoxin activity. Using another E. coli retron, we establish that 28 this toxin/antitoxin function is conserved, and that RT-toxin interactions are cognate. 29Altogether, retrons constitute a novel family of tripartite toxin/antitoxin systems. 30 31 1A), do not affect msDNA biosynthesis 13,18 , and neither their sequence nor the position in 54 retron elements are conserved. 55 56The bottleneck to understand the natural function of retrons has been the absence of 57 phenotypes associated with retron deletions. The first retron-deletion phenotype was reported 58 for retron-Sen2 of Salmonella enterica subsp. enterica ser. Typhimurium str. 14028s (STm), 59wherein the RT-Sen2 was found to be essential for STm survival in calves 18 . This was because 60 it allows STm to grow in anaerobic conditions, present in calf intestines 19 . Here, we report that 61 retron-Sen2 deletion mutants are also unable to grow at lower temperatures. By exploiting the 62 retron cold-sensitivity phenotype, we show that the retron-Sen2 accessory gene STM14_4640 63 (rcaT) encodes a bona fide toxin. Perturbing msDNA biosynthesis at any stage results in toxin 64 activation, and thereby, growth inhibition in anaerobic conditions and cold. Although 65 reminiscent of Toxin/Antitoxin (TA) systems, which are composed of a protein or RNA antitoxin 66 and cognate toxin 20 , retron-Sen2 forms a novel tripartite TA system: RcaT is the toxin, and 67 the RT-msDNA complex is the antitoxin. Using another retron encoded by E. coli NILS-16 21 , 68 3 retron-Eco9, we demonstrate that this TA function is conserved and that the RT provides 69 specificity to the TA system. We propose that bacterial retrons function as TA systems, where 70 the RT-msDNA antitoxins directly inhibit retron-encoded toxins, by forming inactive msDNA-71 RT-toxin complexes. 72 73 RESULTS 74Perturbations in msDNA-Sen2 biosynthesis inhibit STm growth in cold 75As part of a larger chemical-genetics effort, we profiled the fitness of a single-gene deletion 76STm library 22 across hundreds of conditions (unpublished data). The two gene deletions that 77 led to the highest growth sensitivity at room temperature (cold-sensitivity) were ΔrrtT (ΔRT-78 Sen2) and ΔxseA (Fig. 1C). Both RT-Sen2 and Exo VII are involved in msDNA-Sen2 126Retron-Sen2 i...

Prophage-encoded hotspots of bacterial immune systems

“…This suggests that most of the diverse genetic systems encoded at this hotspot are involved in anti-phage defense. A retron from E. coli E101 associated with a transmembrane effector protected against P1vir and 186cIts, as expected from previous findings (7,8,18,37). Among unknown systems with known defense domains, we identified a HEPN-family protein as well as other single-protein systems that comprise either an AAA+ ATPase, a sirtuin (SIR2) or a toll/interleukin-1-receptor-like (TIR) domain.…”

“…1B, Table S2). They can be broadly grouped into three categories : (i) known defense systems such as a type-III restriction enzyme, the type-II EcoO109I RM system (39), retrons (7,8,18,37), Gabija (17) or Septu (17); (ii) uncharacterized systems with protein domains that were previously associated with bacterial immunity such as SIR2 (17,41), TIR (17) and HEPN (18,42); (iii) systems encoding proteins with no annotated domain or whose domain is currently not associated with anti-phage defense, such as NACHT and various domains of unknown function (DUFs). Based on these observations, we hypothesized that the P4-encoded locus between psu and int may constitute a reservoir of bacterial immune systems that likely participate in interviral competition.…”

Systematic prediction of genes functionally associated with bacterial retrons and classification of the encoded tripartite systems

“…Recently, it was reported that some retrons members of the type systems I-A (Retron+ATPase+HNH), II-A3 (Nuc_deoxy+Retron), I-C1 (Retron-Toprim) and XII (Retron-TIR) defined here mediate antiphage defense and that the additional proteins or RT-fused domains are also required for activity ( 81 ). Moreover, a recent report ( 82 ) and two pre-prints ( 83 , 84 ) have also suggested that retrons may be novel anti-phage defense systems and that they may function as a toxin/antitoxin system. The first of these roles is thought to be mediated by the RT and msDNA, with the linked effector protein responsible for toxin/antitoxin functions.…”

Improved architectures for flexible DNA production using retrons across kingdoms of life

“…This reverse transcribed DNA (RT-DNA) is produced in cells from a universally delivered cassette in high abundance such that it overcomes inefficiencies and increases rates of genome editing. One retroelement class that has been useful in this regard are bacterial retrons 6,8,9 , which are elements involved in phage defense [10][11][12][13] . Retrons are attractive as a biotechnology due to their compact size, tightly defined sites of RT initiation and termination, lack of known host factor requirements, and lack of transposable elements.…”