A region of 16.8 kb of the Sym(biosis) plasmid pRL1JI of Rhizobium leguminosarum, consisting of the established 9.7 kb nodulation region which confers nodulation ability on Vicia hirsuta and a region of 7.1 kb which appeared to be necessary for nodulation on V. sativa and Trifolium subterraneum, was subcloned as fragments of maximally 2.5 kb in a newly developed IncQ transcriptional fusion vector. The expression of these fragments was studied in Rhizobium. One constitutive promoter, pr.nodD, and three plant-exudate inducible promoters were found, namely the known pr.nodA and pr.nodF as well as a new promoter designated pr.nodM. The latter promoters were localized within 114 bp, 330 bp and 630 bp respectively and they regulate the transcription of the operons nodA, B, C, I, J, nodF, E and of an operon of at least 2.5 kb located in the 7.1 kb region. Induction of the three inducible operons required plant exudate and a functional nodD product. The flavanone naringenin could replace plant exudate. Each of the three inducible promoters contained a nod-box. A consensus for the nod-box sequence, based on known sequences, is proposed. The 114 bp fragment which contains pr.nodA activity was used to localize pr.nodA by means of deletion mapping. The fragment which appeared necessary for complete pr.nodA activity is 72 bp in size, contains the complete nod-box and in addition a region of 21 bp downstream of the nod-box, in which the loosely conserved sequence AT(T)AG appears to be important for promoter activity.

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In a previous paper it was shown that cocultivation of Rhizobium leguminosarum with the plant Vicia sativa subsp. nigra on solid medium causes a changed mode of growth of the plant roots, resulting in thick and short roots (Tsr). The Sym plasmid present in the bacterium appeared to be essential for causing Tsr (A. A. N. van Brussel, T. Tak, A. Wetselaar, E. Pees, and C. A. Wiffelman, Plant Sci. Lett. 27:317-325, 1982 Root nodule formation by fast-growing Rhizobium bacteria on leguminous plants requires the presence of a symbiosis plasmid (Sym plasmid) in the bacterium (6, 10). These plasmids harbor genes involved in host-specific nodulation (hsn), specific root hair curling (hac), and root hair deformation (had) as has been shown after TnS mutagenesis (5, 14, 17, 21a

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