Phage proteins block and trigger retron toxin/antitoxin systems

Abstract

18Bacteria carry dozens of Toxin/Antitoxin systems (TAs) in their chromosomes. Upon growth, 19 the antitoxin is co-expressed and neutralizes the toxin. TAs can be activated and inhibit growth, 20 but when and how this occurs has largely remained enigmatic, hindering our understanding of 21 their physiological roles. We developed TIC/TAC (Toxin Inhibition/Activation Conjugation), a 22 wide E. coli overexpression libraries, we identified dozens of triggers and blockers for the 68 retron-Sen2, enriched in phage-origin proteins. Phage proteins are sensed by the retron-TA 69 via multiple mechanisms, which are inherent to the tripartite architecture of the TA system. We 70 propose that the retron-TA acts as an anti-phage defense system, and we provide a method 71 that can be readily applied to uncover the physiological role of any TA system. 72 73 RESULTS 74A new systematic method for identifying TA blockers and triggers 75To survey for molecular cues of TA systems, we used genome-wide E. coli single-gene 76 overexpression libraries (MOB; p1 25 and TransBac; p2 26 ) in tandem with strains carrying 77 appropriate TA-expressing plasmids (Fig. 1A). We reasoned that this would allow us to identify 78 Fig. 1D). This inconsistency has many reasons: stringent cutoffs (hence false negatives), 105 quality of libraries (missing genes, plasmid mutations, or cloning errors), and importance of 106 trigger levels (e.g., MOB vectors are medium-copy 25 , while TransBac vectors are single-copy 107 26 ). 108 109To ensure that the identified triggers inhibit growth by specifically activating RcaT, we selected 110 15 triggers, sequenced the plasmid-inserts, and conjugated them again into E. coli strains 111 expressing either an empty vector, p-retron, p-retron-ΔrcaT, or p-rcaT. All 15 were benign 112 when co-expressed only with the antitoxin (p-retron-ΔrcaT plasmid), but inhibited growth when 113 expressed with the full retron (ED Fig. 2A), suggesting that they trigger RcaT. As in the screen, 114 different triggers displayed varying degrees of RcaT-activation, and required different IPTG 115 induction-levels to manifest their effect (ED Fig. 2B). We noticed that several triggers, 116 especially strong ones, were prophage-encoded genes (enrichment p-val = 0.01-0.025, 117 depending on library and induction level - Table S1) -recE (Rac-prophage), tfaP & ymfH (e14-118 prophage), retron RT-Eco1 (P2-like-prophage gene in E. coli BL21), and B21_03469 119 (prophage in E. coli BL21). Although dam is part of the E. coli core genome, Dam methylases 120 are also commonly found in phages 28 . Other triggers belonged to house-keeping processes, 121 such as translation (tufA, rplL, rplP, rplV), DNA-binding/transcription (rdgC, rraB, betI, bolA), 122 protein quality control and translocation (slyD, secB, yajC), chorismate-tetrahydrofolate 123 biosynthesis (folA, gcvR, aroF, aroK), but also orphan genes (ygfB, ydiE, ecpE, gnsA). 124 125Conversely to the TAC screen, we reasoned that strains able to grow upon p-rcaT induction, 126 carried library plasmi...

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.…”

Prophage-encoded hotspots of bacterial immune systems

“…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.…”

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

“…In the absence of this native operon, the ncRNA and RT can be expressed from a plasmid lacking the accessory protein. Since the accessory protein is a core component of the phage-defense conferred by retrons [10][11][12] , this reduced system would phage defense capacity, yet continues to produce abundant RT-DNA. We quantified this RT-DNA using a relative qPCR, comparing amplification by primers against the msd region, which can use both the RT-DNA and plasmid as a template, to amplification by primers that only amplify the plasmid (Fig 1c,d).…”

“…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.…”