Cloning of Rhizobium meliloti nodulation genes by direct complementation of Nod− mutants
Cloning of Rhizobium meliloti nodulation genes by direct complementation of Nod− mutants
复制标题
通过直接互补Nod−突变体克隆苜蓿根瘤菌结瘤基因
作者：
S. Long;W. Buikema;F. Ausubel
固氮根瘤是由根瘤菌属内共生细菌和豆科植物宿主的多步相互作用形成的。鉴定参与这一过程的每一步的细菌和宿主基因对于阐明共生的分子基础是必要的，并且是改善现有共生协会或扩展共生范围的重要先决条件。鉴定这些基因的一种方法是通过分离和表征影响共生的突变体2 -7。我们之前已经描述了苜蓿根瘤菌结瘤缺陷（Nod-）突变体的分离6，8。然而，由这些突变定义的结瘤（nod）基因的功能尚不清楚。作为nod基因的分子遗传学特征的第一步，我们在这里描述了克隆，通过直接选择，nod基因，补充了Nod− R的结晶缺陷。苜蓿突变体我们用一个广寄主范围的粘粒作为大R.苜蓿DNA插入物。该系统可广泛应用于其它不易转化的革兰氏阴性菌的遗传学研究，结合直接选择策略，尤其适用于致病菌毒力基因的研究。利用克隆的R.通过对用此方法获得的苜蓿根瘤形成基因和从nif位点延伸的粘粒克隆库的一系列重叠成员的比较，我们发现苜蓿根瘤形成基因位于离nif位点30个酶对（kbp）的范围内。苜蓿nifK的三个结构基因为固氮酶。
Nitrogen-fixing root nodules are formed by the multi-step interaction of an endosymbiotic bacterium in the genus Rhizobium and a plant host of the legume family1. Identifying the bacterial and host genes involved in each step of this process is necessary to elucidate the molecular basis of the symbiosis, and is an important prerequisite for improving existing symbiotic associations or extending the range of symbioses. One way of identifying such genes is through the isolation and characterization of mutants that affect the symbiosis2–7. We have previously described the isolation of nodulation defective (Nod−) mutants of Rhizobium meliloti6,8. However, the functions of the nodulation (nod) genes defined by these mutations are not understood. As a first step in the molecular genetic characterization of nod genes, we describe here the cloning, by direct selection, of nod genes which complement the nodulation defect of a Nod− R. meliloti mutant. We have used a broad host range cosmid as a vector for large R. meliloti DNA inserts. This system should be generally useful for genetic studies of other Gram-negative bacteria which cannot easily be transformed; combined with the strategy of direct selection, it may be especially applicable to the study of virulence genes in pathogenic bacteria. Using the cloned R. meliloti nodulation gene obtained by this method, and a series of overlapping members of a cosmid clone bank extending from the nif locus, we have found that the gene(s) for nodulation are located within 30 kilobase pairs (kbp) of the nif loci, on the R. meliloti nifK side of the three structural genes for nitrogenase.