Gutiérrez D, Hernando Y, Palacios J M, Imperial J, Ruiz-Argüeso T
Laboratorio de Microbiología, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, Spain.
J Bacteriol. 1997 Sep;179(17):5264-70. doi: 10.1128/jb.179.17.5264-5270.1997.
Rhizobium leguminosarum bv. viciae UPM791 contains a second copy of the fnrN gene, which encodes a redox-sensitive transcriptional activator functionally homologous to Escherichia coli Fnr. This second copy (fnrN2) is located in the symbiotic plasmid, while fnrN1 is in the chromosome. Isolation and sequencing of the fnrN2 gene revealed that the deduced amino acid sequence of FnrN2 is 87.5% identical to the sequence of FnrN1, including a conserved cysteine-rich motif characteristic of Fnr-like proteins. Individual R. leguminosarum fnrN1 and fnrN2 mutants exhibited a Fix+ phenotype and near wild-type levels of nitrogenase and hydrogenase activities in pea (Pisum sativum L.) nodules. In contrast, an fnrN1 fnrN2 double mutant formed ineffective nodules lacking both nitrogenase and hydrogenase activities. Unlike the wild-type strain and single fnrN1 or fnrN2 mutants, the fnrN1 fnrN2 double mutant was unable to induce micro-oxic or bacteroid activation of the hypBFCDEX operon, which encodes proteins essential for hydrogenase synthesis. In the search for symbiotic genes that could be controlled by FnrN, a fixNOQP operon, putatively encoding a micro-oxically induced, bacteroid-specific cbb3-type terminal cytochrome oxidase, was isolated from strain UPM791 and partially sequenced. The fixNOQP operon was present in a single copy located in the symbiotic plasmid, and an anaerobox was identified in the fixN promoter region. Consistent with this, a fixNOQP'-lacZ fusion was shown to be highly induced in micro-oxic cells of the wild-type strain. A high level of micro-oxic induction was also observed in single fnrN1 and fnrN2 mutants, but no detectable induction was observed in the fnrN1 fnrN2 double mutant. The lack of expression of fixNOQP in the fnrN1 fnrN2 double mutant is likely to cause the observed Fix- phenotype. These data demonstrate that, contrary to the situation in other rhizobia, FnrN controls both hydrogenase and nitrogenase activities of R. leguminosarum bv. viciae UPM791 in the nodule and suggest that this strain lacks a functional fixK gene.
豌豆根瘤菌蚕豆生物变种UPM791含有fnrN基因的第二个拷贝，该基因编码一种对氧化还原敏感的转录激活因子，其功能与大肠杆菌Fnr同源。这个第二个拷贝（fnrN2）位于共生质粒中，而fnrN1位于染色体上。对fnrN2基因的分离和测序表明，推导的FnrN2氨基酸序列与FnrN1的序列有87.5%的同一性，包括一个类似Fnr蛋白特有的保守富半胱氨酸基序。单独的豌豆根瘤菌fnrN1和fnrN2突变体在豌豆（Pisum sativum L.）根瘤中表现出Fix+表型以及接近野生型水平的固氮酶和氢化酶活性。相比之下，fnrN1 fnrN2双突变体形成了缺乏固氮酶和氢化酶活性的无效根瘤。与野生型菌株和单个fnrN1或fnrN2突变体不同，fnrN1 fnrN2双突变体无法诱导hypBFCDEX操纵子的微氧或类菌体激活，该操纵子编码氢化酶合成所必需的蛋白质。在寻找可能受FnrN控制的共生基因时，从菌株UPM791中分离出一个推测编码微氧诱导的、类菌体特异性cbb3型末端细胞色素氧化酶的fixNOQP操纵子，并进行了部分测序。fixNOQP操纵子以单拷贝形式存在于共生质粒中，并且在fixN启动子区域鉴定出一个厌氧盒。与此一致的是，一个fixNOQP'-lacZ融合体在野生型菌株的微氧细胞中被高度诱导。在单个fnrN1和fnrN2突变体中也观察到高水平的微氧诱导，但在fnrN1 fnrN2双突变体中未观察到可检测的诱导。fnrN1 fnrN2双突变体中fixNOQP表达的缺乏可能导致观察到的Fix-表型。这些数据表明，与其他根瘤菌的情况相反，FnrN控制豌豆根瘤菌蚕豆生物变种UPM791在根瘤中的氢化酶和固氮酶活性，并表明该菌株缺乏功能性的fixK基因。
