Rawool, S. R.; Sharma, G.

Chemosensory systems (CSSs) are multi-protein assemblies regulating bacterial motility and cellular functions. Despite extensive study of chemotaxis in Vibrio cholerae, a comprehensive evolutionary analysis of CSSs across order Vibrionales has been lacking. Using 116 curated representative genomes across 28 Vibrio clades and ~10,000 RefSeq/metagenome-assembled genomes, we characterized the chemosensory toolkit of Vibrionales. CheA-based phylogenetics, CSS architecture, sequence similarity networks, structural comparisons, and synteny analysis identified four discrete CSS types: F6, F7, F9, and a novel lineage, F8. F6 is universally conserved on chromosome I and essential for flagellar motility, while F7, F8, and F9 show patchy, replicon-flexible distributions reflecting lineage-specific retention or horizontal acquisition. F6, F7, and F8 were vertically inherited from Gammaproteobacteria; F9 was horizontally acquired from Alphaproteobacteria. Structural analysis reveals conserved CheA folds despite sequence divergence, with lineage-specific domain insertions in F8 and F9. Collectively, this study reveals a two-tier chemosensory architecture within order Vibrionales, 1) a chromosomally stable F6 motility core under purifying selection, 2) overlaid by dynamically evolving F7, F8, and F9 accessory systems, wherein multipartite genome organization itself serves as an evolutionary substrate for sensory innovation, enabling rapid niche adaptation without compromising core chemotactic fidelity.