Programa de Ingeniería Genómica, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.
Deparment of Developmental Biology, Washington University School of Medicine, St. Louis, MO, USA.
Microbiologyopen. 2018 Jun;7(3):e00573. doi: 10.1002/mbo3.573. Epub 2017 Dec 27.
Copper (Cu) is an essential micronutrient for all aerobic forms of life. Its oxidation states (Cu /Cu ) make this metal an important cofactor of enzymes catalyzing redox reactions in essential biological processes. In gram-negative bacteria, Cu uptake is an unexplored component of a finely regulated trafficking network, mediated by protein-protein interactions that deliver Cu to target proteins and efflux surplus metal to avoid toxicity. Rhizobium etliCFN42 is a facultative symbiotic diazotroph that must ensure its appropriate Cu supply for living either free in the soil or as an intracellular symbiont of leguminous plants. In crop fields, rhizobia have to contend with copper-based fungicides. A detailed deletion analysis of the pRet42e (505 kb) plasmid from an R. etli mutant with enhanced CuCl tolerance led us to the identification of the ropAe gene, predicted to encode an outer membrane protein (OMP) with a β-barrel channel structure that may be involved in Cu transport. In support of this hypothesis, the functional characterization of ropAe revealed that: (I) gene disruption increased copper tolerance of the mutant, and its complementation with the wild-type gene restored its wild-type copper sensitivity; (II) the ropAe gene maintains a low basal transcription level in copper overload, but is upregulated when copper is scarce; (III) disruption of ropAe in an actP (copA) mutant background, defective in copper efflux, partially reduced its copper sensitivity phenotype. Finally, BLASTP comparisons and a maximum likelihood phylogenetic analysis highlight the diversification of four RopA paralogs in members of the Rhizobiaceae family. Orthologs of RopAe are highly conserved in the Rhizobiales order, poorly conserved in other alpha proteobacteria and phylogenetically unrelated to characterized porins involved in Cu or Mn uptake.
铜（Cu）是所有需氧生命形式的必需微量元素。其氧化态（Cu/Cu ）使该金属成为催化重要生物学过程中氧化还原反应的酶的重要辅因子。在革兰氏阴性菌中，Cu 摄取是一个精细调控的运输网络中未被探索的组成部分，该网络通过蛋白质-蛋白质相互作用介导，将 Cu 递送到靶蛋白，并将多余的金属排出以避免毒性。根瘤菌 etliCFN42 是一种兼性共生固氮菌，必须确保其适当的 Cu 供应，以便在土壤中自由生存或作为豆科植物的细胞内共生体生存。在农田中，根瘤菌必须与铜基杀菌剂作斗争。对来自具有增强的 CuCl 耐受性的 R.etli 突变体的 pRet42e（505kb）质粒的详细缺失分析导致我们鉴定了 ropAe 基因，该基因预测编码一种具有β桶通道结构的外膜蛋白（OMP），可能参与 Cu 转运。支持这一假说，ropAe 的功能表征表明：（I）基因缺失增加了突变体的铜耐受性，并用野生型基因进行互补恢复了其野生型铜敏感性；（II）ropAe 基因在铜过载时保持低基础转录水平，但在铜缺乏时上调；（III）在铜外排缺陷的 actP（copA）突变体背景中缺失 ropAe 部分降低了其铜敏感性表型。最后，BLASTP 比较和最大似然系统发育分析突出了 Rhizobiaceae 家族成员中四个 RopA 旁系同源物的多样化。RopAe 的同源物在 Rhizobiales 目中高度保守，在其他α变形菌中保守性差，与参与 Cu 或 Mn 摄取的特征性孔蛋白在系统发育上没有关系。
