The betaproteobacterium Castellaniella defragrans 65Phen grows on monoterpenes at concentrations toxic to many bacteria. Tolerance mechanisms include modifications of the membrane fatty acid composition and the mineralization of monoterpenes. In this study, we characterized an efflux transporter associated to the monoterpene metabolism. The inner-membrane transporter AmeD (apolar monoterpene efflux) affiliated to the HAE3 (hydrophobe/amphiphile efflux) family within the Resistance-Nodulation-Division (RND) superfamily. RND pumps of the HAE3 family are known for transporting substrates into the periplasm. AmeD is co-expressed with the outer membrane protein AmeA and the periplasmic proteins AmeB and AmeC, suggesting an export channel into the environment similar to HAE1-type RND exporters. Proteins AmeABCD are encoded within a genetic island involved in the metabolism of acyclic and cyclic monoterpenes. The deletion of ameABCD translated into a decrease in tolerance to monoterpenes in liquid cultures. The addition of acetate as cosubstrate in limonene-containing cultures partially alleviated monoterpene toxicity in the deletion mutant. Accumulation of Nile Red in cells of C. defragrans required dissipation of the proton motive force with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells lacking AmeABCD accumulated more Nile Red, suggesting an export function of the proteins. Our observations suggest that the tetrapartite RND transporter AmeABCD acts as an exporter during monoterpene detoxification in C. defragrans.

中文翻译：

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RND转运蛋白在小菜板栗单萜代谢中的作用。

该betaproteobacterium Castellaniella defragrans65Phen在单萜类化合物上生长，其浓度对许多细菌有毒。耐受机制包括膜脂肪酸成分的修饰和单萜的矿化。在这项研究中，我们表征了与单萜代谢相关的外排转运蛋白。内膜转运蛋白AmeD（非极性单萜外排）隶属于抗药性结节（RND）超家族中的HAE3（疏水/两亲外排）家族。众所周知，HAE3系列的RND泵可将底物转运到周质中。AmeD与外膜蛋白AmeA以及周质蛋白AmeB和AmeC共表达，这表明向环境的出口渠道类似于HAE1型RND出口商。蛋白质AmeABCD在涉及无环和环状单萜代谢的遗传岛内编码。删除ameABCD转化为液体培养物中对单萜的耐受性降低。在含柠檬烯的培养物中添加乙酸盐作为共底物可部分减轻缺失突变体中单萜的毒性。在的细胞尼罗红的积累C. defragrans所需的质子动力的耗散与羰基氰化物米-chlorophenylhydrazone（CCCP）。缺乏AmeABCD的细胞会积累更多的尼罗红，表明该蛋白质具有输出功能。我们的观察结果表明，四分体RND转运蛋白AmeABCD可以在C. defragrans的单萜解毒过程中充当出口者的角色。