Physical and genetic analysis of a symbiotic region of Rhizobium meliloti: Identification of nodulation genes

Abstract

A 135 kb long segment of the symbiotic region of the Rhizobium meliloti megaplasmid was mapped with the help of a Rhizobium meliloti gene library, made in the cosmid vehicle pJB8. A set of overlapping cosmid clones was used to identify the inserts in R-primes carrying megaplasmid sections, and to map 20 deletion mutations and 24 insertion mutations with Nod- or Fix- phenotypes. This led to the identification of DNA regions carrying nod or fix (nif) genes. The results of this study correlate well with transcription data of nodule-specific expression of plasmid sequences. The nod mutations were localized in two groups. Using directed Tn5 mutagenesis, correlated physical-genetic maps for these regions were established. One nod gene cluster is about 2.5–3.0 kb in size and carries genes involved in root hair curling, a very early step in nodule formation. Mutations in these genes can be complemented by sym plasmids of other Rhizobium species, such as Rhizobium leguminosarum. We designate these genes as “common” nod genes because mutations in them can be complemented by plasmids derived from different Rhizobium strains. The other nod gene cluster consists of a 2 kb and a 1 kb long DNA segment, separated by a 1 kb region nonessential for nodulation. These nod genes are probably involved in the host specificity of nodulation.