Rhizobium meliloti Nodulation Genes: Identification of nodDABC

A set of conserved, or common, bacterial nodulation (nod) loci is required for host plant infection by Rhizobium melioti and other Rhizobium species. Four such genes, nodDABC, have beeii indicated in R. meliloti 021'by genetic analysis and DNA sequencing. An essential step toward Iunderstanding the function of these genes is to characterize their protein products. We used in vitro and maxicell Escherichia coli expression systems, together with gel electrophoresis and autoradiography, to detect proteins encoded by nodDABC. We facilitated expression of genes on these DNA fragments by inserting them downstream of the Salmonella typhimurium trp promoter, both in colEl and incP plasmid-based vectors. Use pf'the incP trp promoter plasmid allowed overetpression of a nodABC gene fragment in R. meliloti. We fqund that nodA encodes a protein of 21 kilodaltons (kDa), and nodB encodes one 'of 28 kDa; the nodC'product appears as two polypeptide bands at 44 and 45 kDa. Expression of the divergently read nodD yields a single polype tide of 33 kDa. Whether these represent true Rhizobium gene products must be demonstrated by corrqlatipg these proteins with genetically defined Rhizobium locj. We purified the 21-kDa putative nodA protein product'by gel electrophoresis, selective precipitation, and ion-exchange chromatography and generated 4ntierum to the purified gene product. This permitted the immunological demonstration that the 21-kla protein is present in wild4ype cells and in nodBor nodC-defective strains, but is absent from nod4::TnS mutants, which confirms that the product expressed in E. coli is identical to that produced by R. meliloti nodA. Using antisera detection, we found that the level of nodA protein is increased by exposure of R. melilo#i cells to plant exudate, indicating regulation of the bacterial nod genes by the plant host.