Genomic resources for identification of the minimal N2-fixing symbiotic genome
Genomic resources for identification of the minimal N2-fixing symbiotic genome
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DOI：
10.1111/1462-2920.13221
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发表时间：
2016-08-01
影响因子：
5.1
通讯作者：
Finan, Turlough M.
中科院分区：
文献类型：
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作者：
diCenzo, George C.;Zamani, Maryam;Finan, Turlough M.
由于缺乏合适的基因组平台，无法使用功能增益方法来研究根瘤菌-豆科植物的共生，阻碍了共生固氮(SNF)所需和充分的基因的建立，并可能阻碍旨在设计这一过程的合成生物学方法。在这里，我们描述了通过对苜蓿中华根瘤菌进行反向工程来开发一个合适的系统。利用一种新的体内克隆方法，将对细胞存活和共生至关重要的EngA-tRNA-rmlC(ETR)区域从费氏中华根瘤菌转移到苜蓿链霉菌染色体上的祖先位置，使pSymB上的ETR区域变得多余。构建了该菌株的一个缺少两个大的共生复制子(pSymA和pSymB)的衍生物。将pSymA和pSymB重新转入该菌株，恢复了与紫花苜蓿的共生能力。为了确定SNF必需的单拷贝基因在这些复制子上的位置，我们筛选了一个苜蓿链霉菌缺失文库，代表了2900个共生复制子中95%的基因，用于研究它们与紫花苜蓿的表型。只有四个基因座，占
The lack of an appropriate genomic platform has precluded the use of gain-of-function approaches to study the rhizobium-legume symbiosis, preventing the establishment of the genes necessary and sufficient for symbiotic nitrogen fixation (SNF) and potentially hindering synthetic biology approaches aimed at engineering this process. Here, we describe the development of an appropriate system by reverse engineering Sinorhizobium meliloti. Using a novel in vivo cloning procedure, the engA-tRNA-rmlC (ETR) region, essential for cell viability and symbiosis, was transferred from Sinorhizobium fredii to the ancestral location on the S.meliloti chromosome, rendering the ETR region on pSymB redundant. A derivative of this strain lacking both the large symbiotic replicons (pSymA and pSymB) was constructed. Transfer of pSymA and pSymB back into this strain restored symbiotic capabilities with alfalfa. To delineate the location of the single-copy genes essential for SNF on these replicons, we screened a S.meliloti deletion library, representing >95% of the 2900 genes of the symbiotic replicons, for their phenotypes with alfalfa. Only four loci, accounting for