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. TnS 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 genes 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.The interaction between leguminous plant species and soil bacteria belonging to the genera Bradyrhizobium and Rhizobium can lead to the development of nodules on the roots of the host plant. In these new plant organs, differentiated bacteria (bacteroids) encounter favorable conditions for symbiotic nitrogen fixation. Several bacterial and plant genes involved in the establishment of this symbiosis have been studied extensively during the last few years (for reviews, see references 23 and 26).More recently, Azorhizobium caulinodans ORS571 has been described as the archetypical species of a new genus of soil bacteria which, apart from being diazotrophic in freeliving conditions, is also able to nodulate effectively the tropical legume Sesbania rostrata (11). A unique feature of the host plant is the occurrence of dormant root primordia in vertical rows all along the stem. Upon infection by A. caulinodans, these primordia develop into N2-fixing aerial or stem nodules (9).To understand the factors that contribute to the formation of these N2-fixing aerial nodules, we started identifying and characterizing Azorhizobium genes involved in the nodulation process. In previous reports, we described the identification and characterization of common nodABC-related genes (14, 34) that are essential for the interaction with the host plant and are highly conserved among rhizobia, bradyrhizobia, and, to a lesser extent, the genus Azorhizobium.In (brady)rhizobia, the common nod genes are organized in an operon that is coregulated with other nod operons by a positive, regulatory function encoded by the nodD gene. The NodD proteins typicall...