Funbtional analysis of the nifQdctA1y4vGHIJ operon of Sinorhizobium fredii strain NGR234 using a transposon with a NifA-dependent read-out promoter
Funbtional analysis of the nifQdctA1y4vGHIJ operon of Sinorhizobium fredii strain NGR234 using a transposon with a NifA-dependent read-out promoter
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DOI：
10.1099/mic.0.049999-0
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发表时间：
2011-10-01
期刊：
影响因子：
2.8
通讯作者：
Perret, Xavier
中科院分区：
文献类型：
--
作者：
Fumeaux, Coralie;Bakkou, Nadia;Perret, Xavier
根瘤菌是一组不同的土壤细菌，能够与豆科植物共生(Fix表型)，降低大气中的氮含量。固氮酶及其附属成分的合成受关键调控因子NIFA的转录控制，通常限于被称为类杆菌的根瘤菌的内生共生形式。在所研究的根瘤菌中，费氏中华根瘤菌NGR234菌株具有显著的固氮能力，它与73个豆科属130多种植物形成了确定的、不确定的或针叶状根瘤。因此，NGR234是研究多种豆科植物固氮作用的模式生物。共生质粒pSfrNGR234a携带50多个受NIFA转录控制的基因。为了便于NIFA调控基因的功能分析，构建了一个新的转座元件TnEKm-PWA。该转座子结合了克隆DNA片段的体外诱变和NGR234(PWA)的条件读出启动子的优点，该启动子重新启动转座位点下游依赖NIFA的转录。为了测试新转座子的特性，用Omega内插子或TnEKm-PWA突变了nifQdctA1y4vGHIJ操纵子。详细分析了这些突变体在不同寄主上的共生表型和转录特征，并与缺乏NIFA功能拷贝的菌株NGR Delta NIFA的无效(Fix(-))表型进行了比较。QRT-PCR证实插入的TnEKm-PWA在类杆菌中重新转录。出人意料的是，dctA1和nifQ中的极性突变体在所有测试的寄主上都是Fix(+)，这表明NGR234的nifQ操纵子的六个基因中没有一个对形成确定或不确定类型根瘤的植物共生固氮是必需的。
Rhizobia are a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes (Fix phenotype). Synthesis of the nitrogenase and its accessory components is under the transcriptional control of the key regulator NifA and is generally restricted to the endosymbiotic forms of rhizobia known as bacteroids. Amongst studied rhizobia, Sinorhizobium fredii strain NGR234 has the remarkable ability to fix nitrogen in association with more than 130 species in 73 legume genera that form either determinate, indeterminate or aeschynomenoid nodules. Hence, NGR234 is a model organism to study nitrogen fixation in association with a variety of legumes. The symbiotic plasmid pSfrNGR234a carries more than 50 genes that are under the transcriptional control of NifA. To facilitate the functional analysis of NifA-regulated genes a new transposable element, TnEKm-PwA, was constructed. This transposon combines the advantages of in vitro mutagenesis of cloned DNA fragments with a conditional read-out promoter from NGR234 (PwA) that reinitiates NifA-dependent transcription downstream of transposition sites. To test the characteristics of the new transposon, the nifQdctA1y4vGHIJ operon was mutated using either the Omega interposon or TnEKm-PwA. The symbiotic phenotypes on various hosts as well as the transcriptional characteristics of these mutants were analysed in detail and compared with the ineffective (Fix(-)) phenotype of strain NGR Delta nifA, which lacks a functional copy of nifA. De novo transcription from inserted copies of TnEKm-PwA inside bacteroids was confirmed by qRT-PCR. Unexpectedly, polar mutants in dctA1 and nifQ were Fix(+) on all of the hosts tested, indicating that none of the six genes of the nifQ operon of NGR234 is essential for symbiotic nitrogen fixation on plants that form nodules of either determinate or indeterminate types.