Identification and characterization of a functional nodD gene in Azorhizobium caulinodans ORS571

Azorhizobium caulinodans ORS571, a bacterium capable of nodulating roots and stems of the tropical legume Sesbania rostrata, has been shown to have no nodD-like gene located immediately upstream from its common nodABC locus. A clone carrying a functional nodD gene of strain ORS571 has now been isolated from a pLAFR1 gene library by screening for naringenin-induced expression of the common nod genes in an Agrobacterium background. Tn5 mutagenesis of the cloned insert DNA delimited the inducing activity to a +/- 0.8-kilobase-pair fragment. One of the Tn5 insertions in the activator locus was homogenotized in the ORS571 genome. This resulted in a mutant strain (ORS571-3) that was unable to induce common nod gene expression in the presence of host plant exudate or the flavanone naringenin and that had lost the capacity to nodulate the roots and stems of S. rostrata. Complementation of both mutant phenotypes was achieved upon introduction of the cloned nodD gene. Sequencing of the nodD locus indicated the presence of a single, 942-base-pair-long open reading frame (ORFD) with significant homology to the nodD gene of (brady)rhizobia. The level of homology, however, is the lowest thus far reported for this kind of gene. ORFD most likely initiates translation with a TTG start codon. Upstream from ORFD, a divergently oriented nod box-like sequence is present, the function of which remains to be determined.