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Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid‐mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. Here, we structurally and kinetically characterized threeVibrioCAT proteins from a nonpathogenic species (Aliivibrio fisheri) and two important human pathogens (Vibrio choleraeandVibrio vulnificus). We found all three proteins, including one in a superintegron (V. cholerae), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that theV. choleraeandV. vulnificusCATs belong to the Type B class and theA. fisheriCAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation inVibrioand other species.

Self-discrimination, a critical but ill-defined molecular process programmed during thymocyte development, requires myriad pre–T cell receptors (preTCRs) and αβTCRs. Using x-ray crystallography, we show how a preTCR applies the concave β-sheet surface of its single variable domain (Vβ) to “horizontally” grab the protruding MHC α2-helix. By contrast, αβTCRs purpose all six complementarity-determining region (CDR) loops of their paired VαVβ module to recognize peptides bound to major histocompatibility complex molecules (pMHCs) in “vertical” head-to-head binding. The preTCR topological fit ensures that CDR3β reaches the peptide’s featured C-terminal segment for pMHC sampling, establishing the subsequent αβTCR canonical docking mode. “Horizontal” docking precludes germline CDR1β- and CDR2β-MHC binding to broaden β-chain repertoire diversification before αβTCR-mediated selection refinement. Thus, one subunit successively attunes the recognition logic of related multicomponent receptors.