Bladergroen M R, Badelt K, Spaink H P
Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.
Mol Plant Microbe Interact. 2003 Jan;16(1):53-64. doi: 10.1094/MPMI.2003.16.1.53.
Rhizobium leguminosarum strain RBL5523 is able to form nodules on pea, but these nodules are ineffective for nitrogen fixation. The impairment in nitrogen fixation appears to be caused by a defective infection of the host plant and is host specific for pea. A Tn5 mutant of this strain, RBL5787, is able to form effective nodules on pea. We have sequenced a 33-kb region around the phage-transductable Tn5 insertion. The Tn5 insertion was localized to the 10th gene of a putative operon of 14 genes that was called the imp (impaired in nitrogen fixation) locus. Several highly similar gene clusters of unknown function are present in Pseudomonas aeruginosa, Vibrio cholerae, Edwardsiella ictaluri, and several other animal pathogens. Homology studies indicate that several genes of the imp locus are involved in protein phosphorylation, either as a kinase or dephosphorylase, or contain a phosphoprotein-binding module called a forkhead-associated domain. Other proteins show similarity to proteins involved in type III protein secretion. Two dimensional gel electrophoretic analysis of the secreted proteins in the supernatant fluid of cultures of RBL5523 and RBL5787 showed the absence in the mutant strain of at least four proteins with molecular masses of approximately 27 kDa and pIs between 5.5 and 6.5. The production of these proteins in the wild-type strain is temperature dependent. Sequencing of two of these proteins revealed that their first 20 amino acids are identical. This sequence showed homology to that of secreted ribose binding proteins (RbsB) from Bacilus subtilis and V. cholerae. Based on this protein sequence, the corresponding gene encoding a close homologue of RbsB was cloned that contains a N-terminal signal sequence that is recognized by type I secretion systems. Inoculation of RBL5787 on pea plants in the presence of supernatant of RBL5523 caused a reduced ability of RBL5787 to nodulate pea and fix nitrogen. Boiling of this supernatant before inoculation restored the formation of effective nodules to the original values, indicating that secreted proteins are indeed responsible for the impaired phenotype. These data suggest that the imp locus is involved in the secretion to the environment of proteins, including periplasmic RbsB protein, that cause blocking of infection specifically in pea plants.
豌豆根瘤菌菌株RBL5523能够在豌豆上形成根瘤，但这些根瘤对于固氮是无效的。固氮作用的损害似乎是由宿主植物感染缺陷引起的，并且对豌豆具有宿主特异性。该菌株的一个Tn5突变体RBL5787能够在豌豆上形成有效的根瘤。我们对噬菌体可转导的Tn5插入位点周围的一个33kb区域进行了测序。Tn5插入定位于一个由14个基因组成的假定操纵子的第10个基因，该操纵子被称为imp（固氮受损）位点。铜绿假单胞菌、霍乱弧菌、鮰爱德华氏菌和其他几种动物病原体中存在几个功能未知的高度相似的基因簇。同源性研究表明，imp位点的几个基因参与蛋白质磷酸化，要么作为激酶或去磷酸化酶，要么包含一个称为叉头相关结构域的磷蛋白结合模块。其他蛋白质与参与III型蛋白质分泌的蛋白质相似。对RBL5523和RBL5787培养物上清液中分泌蛋白的二维凝胶电泳分析表明，突变菌株中至少四种分子量约为27kDa、pI在5.5至6.5之间的蛋白质缺失。野生型菌株中这些蛋白质的产生是温度依赖性的。对其中两种蛋白质进行测序后发现，它们的前20个氨基酸是相同的。该序列与枯草芽孢杆菌和霍乱弧菌分泌的核糖结合蛋白（RbsB）的序列具有同源性。基于该蛋白质序列，克隆了编码RbsB紧密同源物的相应基因，该基因包含一个被I型分泌系统识别的N端信号序列。在RBL5523上清液存在的情况下，用RBL5787接种豌豆植株会导致RBL5787结瘤和固氮能力下降。接种前将该上清液煮沸可使有效根瘤的形成恢复到原始值，这表明分泌蛋白确实是导致表型受损的原因。这些数据表明，imp位点参与了包括周质RbsB蛋白在内的蛋白质向环境的分泌，这些蛋白质特异性地导致豌豆植株感染受阻。
