iProXProteomicsTao DongVibrio Cholerae O1 Biovar El Tor Str. N16961http://www.iprox.org/page/project.html?id=IPX0016964000dongt@sustech.edu.cnSouthern University of Science and TechnologyiProXWhile 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.Gene-in-gene coding generates dual-isoform Fha condensates to control type VI secretion system assembly.Pei Tong-Tong TT, Chen Qiao-Yu QY, Wang Xing-Yu XY, Ma Amy A, Liang Jia-Xin JX, Ye Zi-Yan ZY, Liu Yu-Zhao YZ, Su Jing-Tong JT, Liang Xiaoye X, An Ying Y, Zhu Jun J, Dong Tao TfalseIdentification and Analysis of Fha-Interacting Proteins in Vibrio choleraeThis study aimed to identify proteins interacting with Fha in Vibrio cholerae. A Strep-tagged Fha fusion protein (Fha-Strep) was expressed in Vibrio cholerae, and associated protein complexes were enriched using Strep-Tactin affinity purification following cell lysis. As a negative control, an sfGFP-Strep fusion protein was expressed and processed in parallel. The enriched protein samples were subjected to tryptic digestion and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using a timsTOF Pro mass spectrometer operated in dia-PASEF mode. This approach enabled comprehensive identification and quantification of Fha-associated proteins. Three biological replicates were included for each group to ensure reproducibility. The resulting dataset provides a resource for understanding the interaction network of Fha and its potential role in the Type VI Secretion System (T6SS) in Vibrio cholerae.Fri May 01 00:00:00 BST 2026PXD07804624327742191729