Abstract:Autoinducer-2 (AI-2), a quorum sensing signal molecule ubiquitous in bacteria, influences a variety of bacterial physiological processes including biofilm formation and motility. However, the role of AI-2 in regulation of these phenotypes and the underlying mechanism have not been reported for Plesiomonas shigelloides. [Objective] To reveal the mechanism through which AI-2 regulates biofilm formation and motility of P.shigelloides by regulating the intracellular cyclodiguanosine monophosphate (c-di-GMP) level, and provide a new idea for the prophylaxis and treatment of P.shigelloides infections. [Methods] Firstly, we constructed the luxS gene knockout strain (ΔluxS) by homologous recombination method. Soft agar plate assays and crystal violet staining assays were employed to compare the swimming motility and biofilm formation between ΔluxS and the wild type. Then, we identified the potential receptor protein DosC (SAMEA2665130_2180) of AI-2 by sequence alignment.Vibrio harveyi MM32 bioluminescence and isothermal titration calorimetry (ITC) were employed to test the binding affinity of the ligand-binding domain of DosC (DosC-LBD) to AI-2. Afterwards, we studied the effect of AI-2 on DosC activity by in vitro enzyme activity assay and intracellular c-di-GMP quantitative assay. Finally, we constructed the dosC knockout strain (ΔdosC) according to the above method and compared its swimming motility and biofilm formation with those of the wild type. [Results] AI-2 bound to DosC-LBD with high affinity. Compared with the wild type, ΔluxS and ΔdosC showed significantly weakened swimming motility and biofilm formation and dramatically increased intracellular c-di-GMP level. HPLC demonstrated that AI-2 enhanced the phosphodiesterase (PDE) activity of DosC. [Conclusion] The c-di-GMP-metabolizing enzyme DosC enhances its PDE activity in response to AI-2, thus affecting the intracellular c-di-GMP level and regulating the biofilm formation and swimming motility of P.shigelloides.
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