Evidence that the nodulation regulatory gene nodD3 of Rhizobium meliloti is transcribed from two separate promoters.

The Bgl II fragment carried in plasmid pMH903, which covers the nodD3 region of Rhizobium meliloti, has been sequenced. By using both S1 nuclease mapping and primer extension, two transcription start sites were demonstrated in the sequence. After the first transcription start site, there were two open reading frames (ORF) followed by the nodD3 coding sequence which was also preceded by the second promoter. The nodD3 gene under the first promoter mediated high, constitutive expression of nodC-lacZ fusion, and the gene under the second promoter required the product of nodD1 and alfalfa (Medicago sativa) seed exudate for the activation of fusion. Nodulation experiments showed that the nodD3 gene under either promoter was functional in eliciting nodules on alfalfa. The deletion of part of the two ORFs after the first promoter or deletion of the second promoter did not block the constitutive expression of nodC-lacZ fusion, whereas the deletion of the first promoter region or a polar insertion mutation between the two promoters did cause nodD3 to activate nodC only in the presence of the inducer. It indicates that nodD3 can be transcribed from the first promoter as well as from a separate second promoter.