The LonA (or Lon) protease is a central post-translational regulator in diverse bacterial species. In Vibrio cholerae, LonA regulates a broad range of behaviors including cell division, biofilm formation, flagellar motility, c-di-GMP levels, the type VI secretion system (T6SS), virulence gene expression, and host colonization. Despite LonA’s role in cellular processes critical for V. cholerae’s aquatic and infectious life cycles, relatively few LonA substrates have been identified. LonA protease substrates were therefore identified through comparison of the proteomes of wild-type and ΔlonA strains following translational inhibition. The most significantly enriched LonA-dependent protein was TfoY, a known regulator of motility and the T6SS in V. cholerae. Experiments showed that TfoY was required for LonA-mediated repression of motility and T6SS-dependent killing. In addition, TfoY was stabilized under high c-di-GMP conditions and biochemical analysis determined direct binding of c-di-GMP to LonA results in inhibition of its protease activity. The work presented here adds to the list of LonA substrates, identifies LonA as a c-di-GMP receptor, demonstrates that c-di-GMP regulates LonA activity and TfoY protein stability, and helps elucidate the mechanisms by which LonA controls important V. cholerae behaviors.

LonA（或 Lon）蛋白酶是多种细菌物种的中心翻译后调节剂。在霍乱弧菌中，LonA 调节范围广泛的行为，包括细胞分裂、生物膜形成、鞭毛运动、c-di-GMP 水平、VI 型分泌系统 (T6SS)、毒力基因表达和宿主定植。尽管 LonA 在对V至关重要的细胞过程中发挥作用。霍乱的水生和传染性生命周期，已确定的 LonA 底物相对较少。因此，通过比较野生型和ΔlonA的蛋白质组，鉴定了 LonA 蛋白酶底物翻译抑制后的菌株。最显着富集的 LonA 依赖性蛋白是 TfoY，一种已知的运动调节器和V 中的 T6SS 。霍乱。实验表明，TfoY 是 LonA 介导的运动抑制和 T6SS 依赖性杀伤所必需的。此外，TfoY 在高 c-di-GMP 条件下稳定，生化分析确定 c-di-GMP 与 LonA 的直接结合会抑制其蛋白酶活性。此处介绍的工作添加到 LonA 底物列表中，将 LonA 鉴定为 c-di-GMP 受体，证明 c-di-GMP 调节 LonA 活性和 TfoY 蛋白质稳定性，并有助于阐明 LonA 控制重要V的机制。霍乱行为。