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Specificity in legume-Rhizobium symbiosis depends on plant and rhizobial genes. As our objective was to study broad host-range determinants of rhizobia, we sought a legume and a Rhizobium with the lowest possible specificity. By inoculating 12 different legumes with a heterogenous collection of 35 fast-growing rhizobia, we found Rhizobium sp. NGR234 to be the Rhizobium and Vigna unguiculata to be the plant with the lowest specificities. Transfer of cloned fragments of the Sym-plasmid pNGR234a into heterologous rhizobia, screening for extension of host-range of the transconjugants to include V. unguiculata, and restriction mapping of the Hsn- and overlapping clones, proved that there were at least three distinct Hsn-regions (HsnI, II, and III) on pNGR234a. HsnI is located next to nodD, HsnII is linked to nifKDH and HsnIII to nodC. In addition to nodulation of Vigna, HsnI conferred upon the transconjugants the ability to nodulate Glycine max, Macroptilium atropurpureum and Psophocarpus tetragonolobus. All three Hsn-regions, when transferred to the appropriate recipients, induced root-hair-curling on M. atropurpureum. Hsn-region III was able to complement a mutation in the host-range gene nodH of R. meliloti strain 2011. Homology to "nod-box"-sequences could be shown only for the sub-clones containing HsnII and HsnIII, thus suggesting different regulation mechanisms for HsnI and HsnII/III.

Abstract. Symbiotic DNA sequences involved in nodulation by Rhizobium must include genes responsible for recognizing homologous hosts. We sought these genes by mobilizing the symbiotic plasmid of a broad host-range Rhizobium MPIK3030 (= NGR234) that can nodulate Glycine max, Psophocarpus tetragonolobus, Vigna unguiculata, etc (4), and a number of Rhizobium genes involved in nodule development have been identified. These include genes that are required for synthesis of the enzyme nitrogenase (mf) j as well as some of the nodulation (nod) genes involved in root-hair curling (12, 21), an essential early nodulation step in many legumes (27). Nodulation genes are functionally conserved between R. leguminosarum and R. meliloti (2,11,23), and there is a 69-72% nucleotide sequence homology between the nod ABC genes of these two species (35, 41). In addition, DNA homology is demonstrable between the nod genes of Rhizobium spp and chromosome/plasmid DNAs of most other Rhizobium species (6). This DNA sequence homology between n/f and nod genes of different rhizobia allowed identification of the symbiotic plasmid in the broad-host range Rhizobium spp MPIK3030 (= NGR 234) and localization of the mfand nod gene clusters (1,8,29,33).As speciation within the Rhizobiaceae is defined by hostAbbreviations used in this paper." hsn, rhizobial genes involved in recognition of the host legume; fix, genes required for symbiotic nitrogen fixation; nil a class of fix genes required for synthesis of the enzyme nitrogenase; nod, genes required for nodulation; onc, oncogenic genes (ofAgrobacterium tumefaciens). range, the similarity in nod genes from different Rhizobium species suggests that they are not the sole determinant of hostspecificity. Rather, another set of genes must exist that allow specific rhizobia to recognize certain hosts. Indeed, Kondorosi et al. (21) have identified a region on the R. meliloti sym plasmid, located between nod and nil that appears to be involved in host-specificity. Mutations at this locus could not be complemented by the sym-plasmid from R. leguminosarum (21).In this study we attempted to identify loci on the 460 kbp sym-plasmid of MPIK3030 (MPIK3030 also contains a larger, cryptic plasmid), that contain hsn genes for the hosts Psophcu carpus tetragonolobus, Vigna unguiculata, and related legumes by mobilizing whole plasmids and cosmid-clones of the plasmids into rhizobia that cannot nodulate these hosts. Fully functional, wild-type rhizobia (i.e., can nodulate their homologous hosts) were chosen as the recipients on the assumption that their intact nod genes would be functional on heterologous hosts (2,11,23). Materials and Methods Bacterial StrainsAll strains used are listed in Table I. In general E. coli strains were grown on LB (37), and Rhizobium isolates on TY (3), although Rhizobium sp CB376 was grown on rhizobial minimal medium (in gl-~: mannitol, 10; K2HPO4,

We examined expression of the megaplasmid pRme41b of Rhizobium meliloti in two different Rhizobium sp. Strains and in Agrobacterium tumefaciens. Transfer of pRme41b into these bacteria was facilitated by insertion of a recombinant plasmid coding for mobilization functions of RP4 into the nil region (Kondorosi, A., E. Kondorosi, C. E. Pankhurst, W. J. Broughton, and Z. Banfalvi, 1982, Mol. Gen. Genet., 188:433-439). In all cases, transconjugants formed nodule-like structures on the roots of Medicago sativa. These structures were largely composed of meristematic cells but they were not invaded by bacteria. Bacteria were found only within infection threads in root hairs, and within intercellular spaces of the outermost cells of the structures.The donor strain of R. meliloti containing pAK11 or pAK12 in pRme41b initially produced nodules on M. sativa that did not fix nitrogen (Fix-). In these nodules, bacteria were released from infection threads into the host cells but they did not multiply appreciably. Any bacteroids formed degenerated prematurely. In some cases, however, reversion to a Fix + phenotype occured after 4 to 6 wk. Bacteria released into newly infected cells in these nodules showed normal development into bacteriods.The soil bacterium Rhizobium meliloti fixes molecular nitrogen in symbiotic association with the legume Medicago sativa. Development of this nitrogen-fixing symbiosis is a multistage process and is probably controlled by a large number of bacterial and plant genes.R. meliloti strains harbor very large plasmids (megaplasmids). Some of the genes required for nodulation (nod) ~ and for nitrogen fixation (fix) are carried by these plasmids (1, 16). Evidence for the presence of nod and fix genes on the megaplasmid pRme41 b of R. meliloti strains 41 comes from mapping mutations in symbiotically defective (Nod-and Fix-) mutants on pRme41 b (1, 5) and from hybridization of ~ Abbreviations used in this paper." nod, genes required for nodulation; fix, genes required for symbiotic nitrogen fixation; n/f, a class of fix genes required for the enzyme nitrogenase synthesis; and mob, genes required for plasmid mobilisation.cloned R. meliloti nifgenes to pRme41b (1). More recently, pRme41 b was made susceptible to mobilization by inserting a recombinant plasmid (pAK11 or pAK12) carrying RP4 mobilization and Tn5 functions into the nif region of pRme41b (9). Using another plasmid (pJB3JI) as a helper plasmid, and selecting for the kanamycin resistance marker of Tn5, pRme41b was transferred into other R. meliloti strains, into other Rhizobiurn sp. and into Agrobacterium tumefaciens. Upon transfer, pRme41b was able to restore nodulating ability to a nod n/f deletion mutant of R. meliloti and enabled the other Rhizobium sp. as well as A. tumefaciens to form nodule-like structures on the roots ofMedicago sativa. Not only does this confirm the presence of nod genes on pRme41b, it also demonstrates that some of these genes can be expressed in other bacteria (9).In this paper, we assess the extent to which...

The gene rpoN (ntrA, g1nF) encodes an alternative sigma factor, cr5'. In enteric bacteria, c&", also termed RPON, is a positive regulator of expression of glnA (encoding glutamine synthetase) and of the aut, hut, and put operons (encoding amino acid transport and degradative enzymes). The nitrogen fixation (nif) promoters of Klebsiella pneumoniae also require X54-RNA polymerase (RNAP). All promoters recognized by a54-RNAP are characterized by an invariant GG doublet at -24 and a GC doublet at -12 upstream of the transcriptional start site. Recognition complexes between a54-RNAP and the promoter are closed and nonproductive transcriptionally. Isomerization to the productive open complex requires binding of diverse activator proteins >100 nucleotides upstream of the transcription site. In the case of ginA and nifLA of K. pneumoniae, the activator is NTRC (34); in the case of other nif operons, it is NIFA (28, 30).In Rhizobium meliloti, the nitrogen-fixing endosymbiotic bacterium of Medicago sativa, the rpoN locus has been mutated and sequenced (36) and gene expression has been analyzed (2). A mutant formed Fix-alfalfa nodules and could not grow on C4-dicarboxylates, which are likely to be the plant-supplied substrate for nitrogen fixation in indeterminate nodules of alfalfa. The dicarboxylate permease gene (dctA) of R. meliloti has an RPON-RNAP consensus sequence 60 base pairs (bp) upstream of the first ATG and binding sites for the activator proteins DCTD (19) and NIFA (10).NGR (New Guinea Rhizobium) 234 is a broad-host-range strain (45). The rpoN (ntrA) gene of NGR234 has been cloned and mutated; its phenotype was pleiotropic and included a measurable effect on nodulation gene expression. Preliminary analysis of a site-directed interposon mutant, NGR234rnJ, and its minimally complementing subclone pSD23, was reported elsewhere (40). The rnl locus was mapped on the NGR234 chromosome between ade-J and * Corresponding author. his-2 (32). In the present report we provide the nucleotide sequence analysis of rpoN of NGR234. We compare the locus and encoded RPON protein with those of R. meliloti and Pseudomonas putida, which have rpoN genes respectively very homologous and dissimilar to that of NGR234. We also analyze the phenotype of NGR234rn3, a sitedirected mutant carrying an internal deletion in the rpoN coding sequence. We present evidence that the rpoN-encoded alternative sigma factor is required for symbiotic functions: normal nodulation kinetics and determinate nodule organogenesis as well as "late" nodule functions like dicarboxylate tranport and nitrogen fixation. We hypothesize that RPON is a primary coregulator of the endosymbiotic life-style of members of the genus Rhizobium. MATERIALS AND METHODSBacteriology and genetic techniques. Bacterial strains, plasmids, and bacteriophages are described in Table 1. Complex (LB, TY, or YM) and defined (RM) media, growth conditions for Rhizobium strain NGR234R and Escherichia coli, and antibiotic concentrations were as described previously (40). Azotobacter vinelandii...

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