A new Rhizobium species that nodulates PhaseoZus vulgaris L. and Leucaena spp. is proposed on the basis of the results of multilocus enzyme electrophoresis, DNA-DNA hybridization, an analysis of ribosomal DNA organization, a sequence analysis of 16s rDNA, and an analysis of phenotypic characteristics. This taxon, Rhizobium tropici sp. nov., was previously named Rhizobium Zeguminosarum biovar phaseoli (type I1 strains) and was recognized by its host range (which includes Leucaena spp.) and nifgene organization. In contrast to R. Zeguminosarum biovar phaseoli, R. tropici strains tolerate high temperatures and high levels of acidity in culture and are symbiotically more stable. We identified two subgroups within R . tropici and describe them in this paper.Members of the genus Rhizobium nodulate the roots of leguminous plants. The rhizobia that infect peas, clovers, and beans (Phaseolus vulgaris L.) are clustered in a single species, Rhizobium leguminosarum (29), which has three biovars (Rhizobium leguminosarum biovar viciae, Rhizobium leguminosarum biovar trifolii, and Rhizobium leguminosarum biovar phaseoli); these biovars contain different symbiotic plasmids that encode distinct nodulation specificities. Nevertheless, heterogeneity in Rhizobium leguminosarum biovar phaseoli has been identified by using such different criteria as protein pattern (50), antibiotic resistance (2), serological type (49), multilocus enzyme electrophoresis behavior (45), DNA-DNA hybridization data (10, 26, 54), plasmid profile (37) , and exopolysaccharide structure (70).We previously distinguished two different types among isolates obtained from bean nodules and found differences in their symbiotic plasmids (36, 38, 39). Type I strains have multiple copies of nitrogenase n i m genes (39, 46), a narrow nodulation host range, and hybridize with the psi (polysaccharide inhibition) gene (3). Type I1 strains have single copies of nifgenes, nodulate Leucaena spp., and do not hybridize with the psi gene (36, 39).Type I1 strains have received attention because their symbiotic plasmids promote an effective and completely differentiated symbiotic process in Agrobacterium tumefaciens recipients (5, 38). They are genetically stable, retaining their symbiotic plasmid after prolonged incubation at 37°C. Some are heat tolerant (31) or acid and aluminum resistant (12, 25, 30, 62). The nodulation genes from one of these strains have been cloned (64). The chemical composition and structure of the extracellular polysaccharides from one type I1 strain differ from the chemical composition and structure of the extracellular polysaccharides from type I isolates (23).Type I1 strains have been less successful in competition for bean nodule occupancy than the type I strains used (41). The former have been reported to occur less frequently in * Corresponding author bean nodules (39). Nodule occupancy by type I1 strains can be enhanced under acid conditions (47, 63).To define the taxonomic position and the genetic relatedness of type I1 strains, we analyzed 64 typ...

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The complete coding sequence of the nitro-

Bacteria of the genus Rhizobium interact with the roots of leguminous plants, eliciting a symbiotic process. During symbiosis, a new organ is formed, the nodule, product of the differentiation of both bacteria and plant cells. In the nodule atmospheric nitrogen is reduced to ammonia, which in turn is assimilated by the plant.It is a common observation that Rhizobium strains can generate variability in colony morphology and symbiotic properties. Such observations have led to certain practices that diminish the possibilities of losing some important characteristics. Preservation of strains and its use for inoculation are frequently performed without the isolation of single colonies. Strains that are supposed to be homogeneous are commonly passed through symbiotic cycles to select clones that present the desired properties.Several reports indicate that, when exposed to certain stress conditions or genetic manipulations, Rhizobium cells can undergo genomic rearrangements (1-4, 6, 9, 20, 23, 25, 26). In some cases, such rearrangements alter the symbiotic properties of the strains.We have performed experiments to analyze genomic rearrangements that occur at high frequency under commonly used laboratory conditions that are not considered to cause stress in bacterial populations. The experimental approach was similar to that reported by Sapienza et al. (21) (10), were used. Escherichia coli S17-1(pSUP5011) was used to introduce TnS-mob into R. phaseoli (22). Recombinant plasmids pMF9 and pMF18 were obtained from a gene library of strain CFN-42; pMF1l1 and pMF122 were from a gene library of strain CFN-285. Both libraries were made in the EcoRI site of pBR329. They contain single EcoRI fragments that detect repeated DNA families in R. phaseoli (7). The sizes of the inserts were 5, 0.8, 2.0, and 3.0 kilobases for pMF9, pMF18, pMF1l1, and pMF102, respectively. Other recombinant plasmids utilized were pCQ15 (15, 16), which carries a 4.7-kilobase EcoRI insert with nitrogenase structural genes, and pSUP5011 (22)

Rhizobium species elicit the formation of nitrogen-fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen-fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen-fixing nodules on the roots of beans (Phaseolus vulgaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl-CoA and oxaloacetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5-mob insertion causes the bacteria to form fewer nodules (30-50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented all the phenotypic alterations of the pcsA mutant, and conferred Rhizobium leguminosarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.