Max-Planck-Institut für Züchtungsforschung, Abteilung Schell, D-5000 Köln 30, Federal Republic of Germany.
Proc Natl Acad Sci U S A. 1986 Dec;83(24):9581-5. doi: 10.1073/pnas.83.24.9581.
The nodA gene of Rhizobium meliloti encodes a 21.8-kDa protein, which is conserved in several Rhizobium species. We overproduced the nodA protein as a fusion product with a portion of the lambda cI repressor in Escherichia coli. This fusion protein was purified from inclusion bodies by gel and hydroxyapatite chromatography in the presence of NaDodSO(4). Monospecific polyclonal antibodies against the hybrid protein were used to detect the nodA protein in the cytosol of E. coli and R. meliloti by immunoblotting. In contrast to experiments with antibodies against the R. meliloti nodC membrane protein, the alfalfa-R. meliloti nodulation was not affected by the addition of anti-nodA antibodies to medium and inoculum. This suggests that the nodA protein is located within the cell and is therefore not accessible to antibodies. The expression of the nodA gene is induced in R. meliloti by various compounds present in the exudate of leguminous plants, particularly by the flavone luteolin. We show that the plant hormone trigonelline also has some inducing activity. The nodC protein was further localized in the membrane fraction of R. meliloti. Our experiments demonstrate that the nodC transmembrane protein is not necessary for the uptake of the compounds inducing the synthesis of the nodA protein. The nodA and the nodC proteins were also detected in mature nodules. During nodule development, the nodC protein may be processed to a 34-kDa protein.
根瘤菌 meliloti 的 nodA 基因编码一个 21.8kDa 的蛋白质，该蛋白质在几种根瘤菌中保守。我们在大肠杆菌中过量生产了 nodA 蛋白，该蛋白与 lambda cI 阻遏物的一部分融合。该融合蛋白通过凝胶和羟磷灰石层析在 NaDodSO4 存在下从包涵体中纯化。针对杂交蛋白的单特异性多克隆抗体用于通过免疫印迹检测大肠杆菌和根瘤菌 meliloti 胞质中的 nodA 蛋白。与针对根瘤菌 meliloti nodC 膜蛋白的抗体实验相反，向培养基和接种物中添加抗 nodA 抗体不会影响紫花苜蓿-根瘤菌的结瘤。这表明 nodA 蛋白位于细胞内，因此抗体无法接触到。nodA 基因的表达在根瘤菌 meliloti 中被豆科植物分泌物中存在的各种化合物诱导，特别是黄酮类芦丁。我们表明植物激素瓜氨酸也具有一些诱导活性。nodC 蛋白进一步定位于根瘤菌 meliloti 的膜部分。我们的实验表明，nodC 跨膜蛋白对于摄取诱导合成 nodA 蛋白的化合物不是必需的。nodA 和 nodC 蛋白也在成熟的根瘤中检测到。在根瘤发育过程中，nodC 蛋白可能被加工成 34kDa 的蛋白。
