Schlüter A, Patschkowski T, Quandt J, Selinger L B, Weidner S, Krämer M, Zhou L, Hynes M F, Priefer U B
Okologie des Bodens, Botanisches Institut, RWTH Aachen, Germany.
Mol Plant Microbe Interact. 1997 Jul;10(5):605-16. doi: 10.1094/MPMI.1997.10.5.605.
DNA corresponding to two copies of the Rhizobium leguminosarum bv. viciae strain VF39 fixNOQP operon coding for a putative symbiotic terminal oxidase of the heme-copper oxidase superfamily was cloned, sequenced, and genetically analyzed. The first copy is located upstream of the fixK-fixL region on plasmid pRleVF39c, whereas the second copy resides on the nodulation plasmid pRleVF39d. Insertional mutagenesis with antibiotic resistance cassettes confirmed that both copies were functional, and that the presence of at least one functional copy was required for nitrogen fixation. The deduced amino acid sequences of both fixN genes are highly similar (95% identity) and contain 15 putative transmembrane helices, suggesting that the fixN gene products are integral membrane proteins. Furthermore, six histidine residues predicted to be the ligands for a heme-copper binuclear center and a low-spin heme b are conserved in both R. leguminosarum fixN proteins. The deduced fixO and fixP gene products show characteristics of membrane-bound monoheme and diheme cytochrome c, respectively. Upstream of both fixN copies putative Fnr-consensus binding sites (anaeroboxes) were found that differ in certain base pairs. As R. leguminosarum VF39 possesses two members of the Fnr/FixK regulator family, FnrN and FixK, the possible differential regulation of both fixN copies was analyzed with fixN-gusA reporter gene fusions. Both fixN fusions were induced under free-living microaerobic conditions and in the symbiotic zone of the root nodule. Induction of the expression of fixNc and fixNd was highly reduced in a fnrN mutant background and in a fixL mutant background, whereas fixK was only marginally involved in fixN regulation. Residual expression of fixN was observed in an fnrN/fixK double mutant.
与豆科根瘤菌蚕豆生物变种VF39菌株的fixNOQP操纵子两个拷贝相对应的DNA被克隆、测序并进行了遗传分析，该操纵子编码血红素-铜氧化酶超家族的一种假定共生末端氧化酶。第一个拷贝位于质粒pRleVF39c上fixK-fixL区域的上游，而第二个拷贝位于结瘤质粒pRleVF39d上。用抗生素抗性盒进行插入诱变证实两个拷贝都具有功能，并且固氮需要至少一个功能拷贝的存在。两个fixN基因推导的氨基酸序列高度相似（95%同一性），并包含15个假定的跨膜螺旋，这表明fixN基因产物是整合膜蛋白。此外，预测为血红素-铜双核中心和低自旋血红素b配体的六个组氨酸残基在两种豆科根瘤菌fixN蛋白中都保守。推导的fixO和fixP基因产物分别显示出膜结合单血红素和双血红素细胞色素c的特征。在两个fixN拷贝的上游发现了假定的Fnr一致结合位点（厌氧盒），它们在某些碱基对上有所不同。由于豆科根瘤菌VF39拥有Fnr/FixK调节家族的两个成员FnrN和FixK，因此用fixN-gusA报告基因融合分析了两个fixN拷贝可能的差异调节。两个fixN融合体在自由生活的微需氧条件下和根瘤的共生区域中都被诱导。在fnrN突变体背景和fixL突变体背景下，fixNc和fixNd表达的诱导显著降低，而FixK仅在fixN调节中起很小的作用。在fnrN/fixK双突变体中观察到fixN的残留表达。
