Engelke T, Jording D, Kapp D, Pühler A
Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Federal Republic of Germany.
J Bacteriol. 1989 Oct;171(10):5551-60. doi: 10.1128/jb.171.10.5551-5560.1989.
Transposon Tn5-induced C4-dicarboxylate transport mutants of Rhizobium meliloti 2011 which could be complemented by cosmid pRmSC121 were subdivided into two classes. Class I mutants (RMS37 and RMS938) were defective in symbiotic C4-dicarboxylate transport and in nitrogen fixation. They were mutated in the structural gene dctA, which codes for the C4-dicarboxylate carrier. Class II mutants (RMS11, RMS16, RMS17, RMS24, and RMS31) expressed reduced activity in symbiotic C4-dicarboxylate transport and in nitrogen fixation. These mutants were mutated in regulatory dct genes which do not play an essential role in the symbiotic state. Thin sections of alfalfa nodules induced by the wild type and class I and class II mutants were analyzed by light microscopy. Class mutants induced typical Fix- nodules, showing a large senescent zone, whereas nodules induced by class II mutants only differed in an enhanced content of starch granules compared with wild-type nodules. Class I mutants could be complemented by a 2.1-kilobase SalI-HindIII subfragment of cosmid pRmSC121. DNA sequencing of this fragment resulted in the identification of an open reading frame, which was designated dctA because Tn5 insertion sites of the class I mutants mapped within this coding region. The dctA gene was preceded by a nif consensus promoter and an upstream NifA-binding element. Upstream of the dctA promoter, the 5' end of the R. meliloti dctB gene could be localized. The amino acid sequence of the N-terminal part of the R. meliloti DctB protein shared 49% homology with the corresponding part of the R. leguminosarum DctB protein. The DctA protein consisted of 441 or 453 amino acids due to two possible ATG start codons, with calculated molecular masses of 46.1 and 47.6 kilodaltons, respectively. The hydrophobicity plot suggests that DctA is a membrane protein with several membrane passages. The amino acid sequences of the R. meliloti and the R. leguminosarum DctA proteins were highly conserved (82%).
转座子Tn5诱导的苜蓿中华根瘤菌2011的C4 - 二羧酸转运突变体，可被黏粒pRmSC121互补，这些突变体被分为两类。I类突变体（RMS37和RMS938）在共生C4 - 二羧酸转运和固氮方面存在缺陷。它们在编码C4 - 二羧酸载体的结构基因dctA中发生了突变。II类突变体（RMS11、RMS16、RMS17、RMS24和RMS31）在共生C4 - 二羧酸转运和固氮方面表现出活性降低。这些突变体在调控dct基因中发生了突变，这些基因在共生状态下不发挥 essential作用。通过光学显微镜分析了由野生型以及I类和II类突变体诱导的苜蓿根瘤的薄片。I类突变体诱导出典型的Fix - 根瘤，显示出一个大的衰老区，而II类突变体诱导的根瘤与野生型根瘤相比，仅在淀粉颗粒含量增加方面有所不同。I类突变体可被黏粒pRmSC121的一个2.1千碱基的SalI - HindIII亚片段互补。对该片段进行DNA测序后鉴定出一个开放阅读框，因其I类突变体的Tn5插入位点位于该编码区域内，故将其命名为dctA。dctA基因之前有一个nif共有启动子和一个上游NifA结合元件。在dctA启动子的上游，可以定位苜蓿中华根瘤菌dctB基因的5'端。苜蓿中华根瘤菌DctB蛋白N端部分的氨基酸序列与豌豆根瘤菌DctB蛋白的相应部分有49%的同源性。由于两个可能的ATG起始密码子，DctA蛋白由441或453个氨基酸组成，计算分子量分别为46.1和47.6千道尔顿。疏水性图谱表明DctA是一种具有多个跨膜通道的膜蛋白。苜蓿中华根瘤菌和豌豆根瘤菌DctA蛋白的氨基酸序列高度保守（82%）。
