Schlüter A, Rüberg S, Krämer M, Weidner S, Priefer U B
Okologie des Bodens, Botanisches Institut, RWTH Aachen, Germany.
Mol Gen Genet. 1995 Apr 20;247(2):206-15. doi: 10.1007/BF00705651.
Hybridization analysis using the Rhizobium meliloti nitrogen fixation gene fixN as a probe revealed the presence of a homologous DNA region in the phytopathogenic bacterium Agrobacterium tumefaciens. Hybridization signals were also detected with total DNAs of Rhizobium leguminosarum bv. phaseoli, Rhodobacter capsulatus and Escherichia coli, but not those of Xanthomonas campestris pv. campestris and Pseudomonas putida. The hybridizing fragment from A. tumefaciens was cloned and sequenced. The predicted gene product of one of the two open reading frames identified on the sequenced fragment shows homology to FixN of different Rhizobiaceae as well as a low but significant similarity to subunit I of heme copper oxidases from various bacteria. The presence of five strictly conserved histidine residues previously implicated in forming ligands to heme and CuB in oxidases and the predicted membrane topology provide evidence that the A. tumefaciens fixN-like gene product is a component of the heme copper oxidase superfamily. The incomplete open reading frame starting only 8 nucleotides downstream of the fixN-like gene exhibits homology to Rhizobium fixO. Using an uidA (GUS) gene fusion it could be shown that the A. tumefaciens fixN-like gene is preferentially expressed under microaerobic conditions. Expression of the uidA fusion is abolished in R. meliloti fixJ and fixK mutants, indicating that an Fnr-like protein is involved in transcriptional regulation of the fixN-like gene in A. tumefaciens. The presence of an upstream DNA sequence motif identical to the Fnr-consensus binding site (anaerobox) further supports this hypothesis. A. tumefaciens mutated in the fixN-like gene shows decreased TMPD-specific oxidase activity under microaerobic conditions, indicating that the fixN-like gene or operon codes for proteins involved in respiration under reduced oxygen availability.
使用苜蓿根瘤菌固氮基因fixN作为探针进行杂交分析，结果显示在植物致病细菌根癌土壤杆菌中存在一个同源DNA区域。在菜豆根瘤菌、荚膜红细菌和大肠杆菌的总DNA中也检测到了杂交信号，但野油菜黄单胞菌野油菜致病变种和恶臭假单胞菌的总DNA中未检测到。对根癌土壤杆菌的杂交片段进行了克隆和测序。在测序片段上鉴定出的两个开放阅读框之一的预测基因产物，与不同根瘤菌科的FixN具有同源性，并且与各种细菌的血红素铜氧化酶亚基I有低但显著的相似性。先前涉及在氧化酶中形成血红素和CuB配体的五个严格保守的组氨酸残基的存在以及预测的膜拓扑结构，提供了证据表明根癌土壤杆菌fixN样基因产物是血红素铜氧化酶超家族的一个组成部分。仅在fixN样基因下游8个核苷酸处开始的不完整开放阅读框与根瘤菌fixO具有同源性。使用uidA（GUS）基因融合表明，根癌土壤杆菌fixN样基因在微需氧条件下优先表达。uidA融合的表达在苜蓿根瘤菌fixJ和fixK突变体中被消除，表明一种Fnr样蛋白参与了根癌土壤杆菌中fixN样基因的转录调控。与Fnr一致结合位点（厌氧盒）相同的上游DNA序列基序的存在进一步支持了这一假设。在fixN样基因中发生突变的根癌土壤杆菌在微需氧条件下显示出TMPD特异性氧化酶活性降低，表明fixN样基因或操纵子编码参与低氧条件下呼吸作用的蛋白质。
