Mol Gen Genet (1989) 219:289-298 © Springer-Verlag 1989 Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: Nucleotide sequence and plant-inducible expression K. Goethals, M. Gao*, K. Tomekpe**, M. Van Montagu, and M. Holsters Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Gent, Belgium Summary. Azorhizobium caulinodans strain ORS571 induces nitrogen-fixing nodules on roots and stem-located root pri- mordia of Sesbania rostrata. Two essential Nod loci have been previously identified in the bacterial genome, one of which (Nod locus 1) shows weak homology with the com- mon nodC gene of Rhizobium meliloti. Here we present the nucleotide sequence of this region and show that it contains three contiguous open reading frames (ORFA, ORFB and ORFC) that are related to the nodABC genes of Rhizobium and Bradyrhizobium species. ORFC is followed by a fourth (ORF4) and probably a fifth (ORF5) open reading frame. ORF4 may be analogous to the nodI gene of R. legumino- sarum, whereas ORF5 could be similar to the rhizobial nodF genes. Coordinated expression of this set of five genes seems likely from the sequence organization. There is no typical nod promoter consensus sequence (nod box) in the region upstream of the first gene (ORFA) and there is no nodD-like gene. LacZ fusions constructed with ORFA, ORFB, ORFC, and ORF4 showed inducible /~-galactosidase ex- pression in the presence of S. rostrata seedlings as well as around stem-located root primordia. Among a series of phenolic compounds tested, the flavanone naringenin was the most efficient inducer of the expression of this ORS571 nod gene cluster. Key words: Azorhizobium caulinodans - Common nod genes DNA sequencing - Plant-inducible expression - Sesbania rostrata nodulation Introduction Bacteria belonging to the genera Rhizobium and Bradyrhizo- bium can cause the formation of nitrogen-fixing nodules on the roots of several leguminous plants. During the last decade a considerable amount of information has been gained on the bacterial genes that are important for the interaction with the plant (for recent reviews, see Djordjevic et al. 1987; Long 1989). Large Sym plasmids in rhizobia and the chromosome of bradyrhizobia have been shown to carry gene clusters that are involved in nodule induction * Present address: Institute for Application of Atomic Energy, P.O. Box 5109, Beijing, China ** Present address: Laboratoire de Biologie des Sols, O.R.S.T.O.M., P.B. 1386, Dakar, S6n6gal Offprint requests to: M. Holsters (nod genes). Three major types of nod genes have been iden- tified. The "common nod" genes are functionally and struc- turally conserved among all rhizobia and bradyrhizobia thus far studied. Although their exact biochemical role has not yet been elucidated, strong evidence has been obtained that the common nodA and nodB genes are essential for the production of a bacterial factor that can induce plant meristem formation, whereas the common nodC gene codes for an outer membrane protein that may function in signal exchange between bacteria and plant cells (John et al. 1988; Schmidt et al. 1988). The "host specificity" (hsn) genes function in determining the host range of a particular bacte- rial strain. Finally, the nodD genes encode regulatory pro- teins that in conjunction with plant-derived phenolic com- pounds - activate the transcription of the other nod operons (Mulligan and Long 1985; Rossen etal. 1985; Horvath et al. 1987). This transcriptional activation requires the presence of strongly conserved eis-acfing regulatory se- quences (nod boxes) upstream from the inducible nod genes (Rostas et al. 1986; Fisher et al. 1988). More recently, a novel bacterium, Azorhizobium caulino- dans gen. nov., sp. nov. strain ORS571, has been isolated from aerial nodules on the stem of the tropical leguminous plant Sesbania rostrata (Dreyfus et al. 1984, 1988). This strain differs from Bradyrhizobium and Rhizobium species by its taxonomic position and its unique capacity for both symbiotic and free-living nitrogen fixation. A. caulinodans induces effective nodules on the roots of its host and also on the stem, at the sites of dormant root primordia, the abundant occurrence of which is a characteristic feature of the S. rostrata species. We are interested in this particular symbiosis and have focused on the identification of bacteri- al genes involved in early interactions with the host. In a previous paper we have described the cloning of two ORS571 genomic loci that are essential for root and stem nodulation (Van den Eede et al. 1987). One of these (Nod locus 1) shows weak homology with the nodC gene of R. meliloti. Here, we report the further characterization of Nod locus 1. By DNA sequencing and laeZ-mediated expression studies we show the presence of genes related to the com- mon nodABC genes of Rhizobium and Bradyrhizobium. These A. eaulinodans genes seem to be organized in one operon together with a nodI-like and, unexpectedly, a nodF- like cistron. In all cases studied thus far, nodF has been described as the first gene of an hsn operon further contain- ing the nodE gene (Shearman et al. 1986; Schofield and Watson 1986; Debell6 and Sharma 1986). The expression