Cloning of Rhizobium meliloti nodulation genes by direct complementation of Nod− mutants

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.