Gene-in-gene coding generates dual-isoform Fha condensates to control type VI secretion system assembly

While eukaryotes employ alternative splicing to diversify protein functions, analogous strategies in bacteria remain underexplored. Here we identify a conserved intragenic coding mechanism in Vibrio cholerae that generates two isoforms of the essential scaffold Fha and show that these isoforms cooperate through liquid-liquid phase separation to promote the assembly of the type VI secretion system (T6SS). The full-length isoform, Fha^L, seeds assembly by engaging the membrane complex, whereas an internally translated isoform, Fha^S, enhances secretion efficiency by strengthening specific interactions with baseplate components. This isoform partitioning is ecologically critical; a mutant producing only Fha^L is impaired in bacterial competition, susceptible to eukaryotic predation, and defective in host colonization. Both isoforms form condensates, and a single residue change within a C-terminal helix abolishes condensate formation and significantly reduces T6SS activities. The internal translation and condensate-forming residues are strictly conserved across >10,000 V. cholerae isolates and active in diverse Vibrio species. These findings define a translational-biophysical mechanism that tunes a widespread contractile protein nanomachine for ecological success.