ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNA-binding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and site-directed modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria.

自然感受态细菌（naturally competent bacteria）的DNA摄取能力，此前被归因于类似IV型菌毛复合物（type IV pilus complexes）的DNA摄取机器的作用。然而，负责将DNA跨革兰氏阴性菌（Gram-negative bacteria）外膜转运的蛋白质仍未明确。本研究证实，感受态蛋白ComEA（ComEA）可在自然感受态霍乱弧菌（Vibrio cholerae）的周质空间（periplasm）中结合进入胞内的DNA，从而促进DNA摄取，其机制可能与ComEA结合DNA所介导的棘轮效应（ratcheting）及熵力（entropic forces）相关。本研究通过比较建模（comparative modeling）与分子模拟（molecular simulations），预测了ComEA的三维结构与DNA结合位点（DNA-binding site）。结合对野生型与定点突变ComEA变体的体内（in vivo）、体外（in vitro）验证，上述硅基实验（in silico）预测结果表明，ComEA并非仅作为DNA受体蛋白，而是直接参与DNA摄取过程。此外，本研究还发现，其他细菌（包括革兰氏阳性菌（Gram-positive bacteria）与革兰氏阴性菌）的ComEA同源蛋白可有效弥补霍乱弧菌中ComEA缺失的缺陷，这提示ComEA在DNA摄取过程中的作用可能在自然感受态细菌中具有保守性。