Garcia-Rodriguez, F. M.; Martinez-Abarca, F.; Molina, R.; Toro, N.

Type VI retrons are bacterial defense systems that couple reverse transcription to phage immunity through dedicated toxin-antitoxin modules, yet the mechanisms linking ssDNA synthesis to toxin activation remain unclear. Here we characterize Retron-Sen3, a Type VI retron from Salmonella enterica encoding a small protein (SP) toxin, an HTH antitoxin, and a reverse transcriptase (RT). Unlike previously examined Type VI retrons, Retron-Sen3 constitutively produces RT-dependent ssDNA species in the absence of phage infection, and these levels remain unchanged during infection, establishing that defense activation is uncoupled from ssDNA synthesis. Comparative analyses revealed a conserved ncRNA architecture across the cl2075 lineage, including a previously undescribed stem-loop essential for defense, and an extended transcript encompassing the SP coding region within a structurally conserved RNA framework. SP is constitutively produced and intrinsically toxic, whereas HTH neutralizes SP toxicity, is required for basal viability, and physically associates with RT in vivo independently of catalytic activity; notably, the HTH-SP toxin-antitoxin module operates independently of RT and ncRNA. Phage escape mutants and genetic analyses establish that inhibition of the host RecBCD complex triggers defense activation, identifying RecBCD surveillance as the signal that couples phage infection to toxin activation. Together, these findings reveal a post-translational regulatory mechanism distinct from the msDNA-dependent translational control described for Retron-Vpa2, establishing mechanistic diversity within this retron family.