Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion
Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion
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DOI：
10.1094/mpmi.2003.16.1.53
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发表时间：
2003-01-01
影响因子：
3.5
通讯作者：
Spaink, HP
中科院分区：
文献类型：
--
作者：
Bladergroen, MR;Badelt, K;Spaink, HP
豆科根瘤菌RBL5523能在豌豆上形成根瘤，但这些根瘤对固氮不起作用。固氮损伤似乎是由寄主植物的缺陷感染引起的，并且是豌豆特有的寄主。该菌株的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插入物周围33kb的区域进行了测序。Tn5插入定位于14个被称为imp（氮固定受损）位点的假定操纵子的第10个基因。铜绿假单胞菌、霍乱弧菌、爱德华氏菌和其他几种动物病原体中存在一些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磷酸化，或者作为激酶或去磷酸化酶，或者包含一个称为叉头相关结构域的磷酸化蛋白结合模块。其他蛋白质显示与III型蛋白分泌相关的蛋白质相似。对RBL5523和RBL5787培养的上清液中分泌的蛋白进行二维凝胶电泳分析，发现突变株中至少缺少4种蛋白，分子量约为27 kDa， pi在5.5 ~ 6.5之间。在野生型菌株中，这些蛋白质的产生与温度有关。对其中两种蛋白质的测序显示，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核糖结合蛋白（RbsB）序列同源。基于该蛋白序列，克隆了RbsB的同源基因，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，将RBL5787接种在豌豆植株上，导致RBL5787结瘤能力和固氮能力下降。在接种前将这种上清煮沸，使有效结节的形成恢复到原来的值，这表明分泌的蛋白质确实是造成表型受损的原因。这些数据表明，imp位点参与向环境中分泌蛋白质，包括质周RbsB蛋白，从而导致豌豆植物特异性的感染阻断。
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.