Molecular Microbiology (1990) 4(11), 1899-1910 Identification and cloning of nodulation genes and host specificity determinants of the broad host-range Rhizobium ieguminosarum biovar phaseo//strain CIAT899 C. Vargas,' L. J. Martinez^, M. Megias' and C. Quinto^* ' Departamento de Microbiologia y Parasitologia. Universidad de Seviila. Apartado Postal 874. 41003 Seville. Spain. ^Unidad de Biologia Molecular y Biotecnologia Vegetal. UNAM Apartado Postal 2-246. Cuernavaca, Morelos, Mexico. Summary Rhizobium Ieguminosarum biovar phaseoli type II strain CIAT899 nodulates a wide range of hosts: Phaseolus vulgaris (beans), Leucaena esculenta (leu- caena) and Macroptllium airopurpureum (siratro). A nodulation region from the symbiotic plasmid has been isolated and characterized. This region, which is contained in the overlapping cosmid clones pCV38 and pCV117, is able to induce nodutes in beans, teucaena and siratro roots when introduced in strains cured for the symbiotic plasmid, pSym. In addition, this cloned region extends the host range of Rhizobium melUoti and R. Ieguminosarum biovar (bv.) Mfolii wild-type strains to nodulate beans. Analysis of con- structed subclones indicates that a 6.4 kb HindWl fragment contains the essential genes required for nodule induction on all three hosts. Rhizobium Iegumi- nosarum bv. phaseoli type I strain CE3 nodulates only beans. However, CE3 transconjugants harbouring plasmid pCV3802 (which hybridized to a nodD heter- ologous probe), were capable of eliciting nodules on leucaena and siratro roots. Our results suggest that the CIAT899 DNA region hybridizing with the R. meiiloti nodD detector is involved in the extension of host specificity to promote nodule formation in P. vulgaris, L. esculenta and M. atropurpureum. introduction Symbiotic nitrogen-fixing bacteria have been classified into three genera: Rhizobium. Bradyrhizobium and Azorhi- Receiwed 4 Apnl, 1990; revised 1 June, 1990, 'For correspondence, Tel, (73) 131314; Fax |73) 175581, zobium. Establishing a nitrogen-fixing symbiosis between the bacteria and the leguminous plants is a complex developmental process that involves the expression of symbiotic genes in both the host and the symbiont. For Rhizobium species, the genes that control nodu- lation, host-range specificity and nitrogen fixation have been located on large indigenous plasmids called symbio- tic plasmids (pSym) (Banfalvi etai. 1981; Hombrecher ef a/.. 1981; Hooykaas et ai. 1981). In Bradyrhizobium and Azorhizobium species, the symbiotic genetic iniormation lies on the chromosome (Noti etal.. 1985; van den Eede ef al.. 1987). The genes required in the earliest stages of the infection process are classified according to their properties when transferred into other species of rhizobia or into the related bactena Agrobacterium (umefaciens. The nodABC genes are involved in root hair curling and nodule induction. These genes are conserved between species both func- tionally and at the nucleotide level (Banfalvi et a(., 1981; Rossen et ai. 1984; Torok et ai. 1984; Egelhoff et al.. 1985; Jacobs etai. 1985), Tn5 insertions in the nodABC genes abolish the nodulation capacity of rhizobia (Rossen et ai. 1984; Torok ef ai.. 1984; Djordjevic ef ai. 1985; Egelhoff ef al. 1985; Jacobs ef a/.. 1985; Debelle et ai. 1986). Downstream ot nodC. in Rhizobium Ieguminosarum bv. viciae (Evans and Downie, 1986), Rhizobium meiiloti (Long, 1989) and Bradyrhizobium japonicum (Nieuwkoop et ai. 1987; Hennecke et al., 1988), two other genes are found: nodi and nodJ. which are functionally inter- changeable with region II described for R. Ieguminosarum bv. fn/o/(y(DjOfdjevicefa/., 1985). Mutations in nod/Jgenes (region II) have no effect In B. japonicum (Nieuwkoop etai, 1987; Hennecke etai. 1988), However, in R. Ieguminosa- rum bv. viciae and bv. tntolii these mutations result in inefficient (delayed) nodulation on some hosts (Djordjevic etal.. 1985; Evans and Downie, 1986). The genes nocMB- CU are referred to as 'common' nod genes because they can be replaced by the corresponding genes ot other Rhizobium or Bradyrhizobium species without affecting the host range. Other nodulation genes appear to be involved in host- range specificity and nodulation efficiency: the nodFE