Rhizobium leguminosarum bv. phaseoli CFN42 contains six plasmids (pa to pf), and pd has been shown to be the symbiotic plasmid. To determine the participation of the other plasmids in cellular functions, we used a positive selection scheme to isolate derivatives cured of each plasmid. These were obtained for all except one (pe), of which only deleted derivatives were recovered. In regard to symbiosis, we found that in addition to pd, pb is also indispensable for nodulation, partly owing to the presence of genes involved in lipopolysaccharide synthesis. The positive contribution of pb, pc, pe, and pf to the symbiotic capacity of the strain was revealed in competition experiments. The strains that were cured (or deleted for pe) were significantly less competitive than the wild type. Analysis of the growth capacity of the cured strains showed the participation of the plasmids in free-living conditions: the pf- strain was unable to grow on minimal medium, while strains cured of any other plasmid had significantly reduced growth capacity in this medium. Even on rich medium, strains lacking pb or pc or deleted for pe had a diminished growth rate compared with the wild type. Complementation of the cured strains with the corresponding wild-type plasmid restored their original phenotypes, thus confirming that the effects seen were due only to loss of plasmids. The results indicate global participation of the Rhizobium genome in symbiotic and free-living functions.

High-frequency genomic rearrangements affecting the plasmids of Rhizobium leguminosarum bv. phaseoli CFN42 were analyzed. This strain contains six large plasmids ranging in size from 200 to 600 kb. In the absence of any selective pressure, we found 11 strains from 320 analyzed colonies that presented different kinds of plasmid-borne rearrangements, including sequence amplification, deletion, cointegration, and loss of plasmids. These data support the concept that the R. keguminosarum bv. phaseoli genome is a dynamic structure and imply that-strains we-mixtre of similar-but not identical cells.Bacteria of the genus Rhizobium interact with plant roots to form nitrogen-fixing nodules. Genetic information in Rhizobium spp. is usually distributed among the chromosome and different large plasmids. Rhizobium leguminosarum bv. phaseoli CFN42 contains six plasmids, ranging in size from 200 (p42a) to 600 (p42J) kb (Fig. 1A, lane 1). It has been shown that plasmid d corresponds to the symbiotic plasmid (pSym) (2,22). The occurrence of rearrangements in the genome of different strains of R. leguminosarum bv. phaseoli, including CFN42, has been shown through the hybridization of DNA isolated from different single colonies with recombinant plasmids carrying sequences from different reiterated families (9). These rearrangements were detected in nonstressing conditions and occurred at high frequencies. By using specific genetic constructions, it has been shown that the CFN42 pSym plasmid is subject to high-frequency amplification and deletion events, which involve recombination between nitrogenase (nif) gene reiterations (24).In this work, we determined the presence of rearrangements that occur at high frequency in the plasmids of R. leguminosarum bv. phaseoli CFN42. The rearrangements detected include sequence amplification, deletion, cointegration, and loss of plasmids.The plasmid profiles of 320 single colonies derived from strain CFN42 were analyzed by using the Eckhardt procedure (6). An overnight culture of strain CFN42 grown on PY medium (21) at 30°C was used to make dilutions in 10 mM MgSO4 with detergent (0.01% Tween 40) which were plated on PY medium and incubated at 30°C. Individual colonies (160) were isolated from these plates. Strain CFN42 was also used to inoculate Phaseolus vulgaris plants. P. vulgaris cv. Negro Jamapa seeds were surface sterilized as previously described (18) and grown in 250-ml Erlenmeyer flasks with the agar medium described by Fahraeus (7) without added nitrogen at 28°C. Plants were grown for 15 days. Surfacesterilized nodules (160) were crushed on PY plates, and a single colony was isolated from each nodule. The plasmid profiles that presented alterations, as well as 60 apparently normal, randomly selected profiles, were blotted onto n'itro- f as probes. These plasmids were purified from Agrobacterium transconjugants harboring each one of the plasmids (8) by the method of Hirsch et al. (14) and labeled by nick translation (23). Hybridization experiments were performed under high-stringency ...

RegulonDB version 2.0, a database on transcriptional regulation and operon organization in Escherichia coli, is now available on the web at the following URL: http://www.cifn.unam. mx/Computational_Biology/regulondb/. In this paper we describe the main computational changes to the database, which include migrating the database to Sybase, providing graphical descriptions of the internal organization of operons and regulons, and direct links to MEDLINE references. The web interface offers searching either by mechanisms of regulation or by operon organization. The results of a search (operon organization, or site collection) are displayed as hypertext, and can also be displayed graphically. In terms of its contents, RegulonDB contains a large number of operons, as well as the absolute position in the completed genome sequence of sites, promoters, and individual genes of E.coli.

The genes encoding malate synthase (glcB) and isocitrate lyase (aceA) and a 240-bp open reading frame (SMc00767) located downstream of aceA were isolated and functionally characterized in Sinorhizobium meliloti. Independent and double interposon mutants of each gene were constructed, and the corresponding phenotypes were analyzed. aceA mutants failed to grow on acetate, and mutants deficient in SMc00767 were also affected in acetate utilization. In contrast, mutants deficient in glcB grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed that aceA and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants. aceA-glcB, aceA-SMc00767, and glcB-SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-type aceA-glcB or aceA-SMc00767 loci were introduced into the double mutants. These data confirm the functional role of aceA and SMc00767 and show that glcB, in the absence of SMc00767, is required for acetate metabolism. Isocitrate lyase and malate synthase activities were measured in strain Rm5000, the mutant derivatives, and complemented strains. aceA and glcB were able to complement the enzymatic activity lacking in the corresponding single mutants. The enzymatic activities also showed that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirmed the repressor role of SMc00767, which regulates aceA expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant and wild-type strain Rm5000 showed that SMc00767 represses the expression of a moderate number of open reading frames, including aceA; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism in S. meliloti. Genetic and functional analyses indicated that aceA and SMc00767 constitute a functional two-gene operon, which is conserved in other ␣-proteobacteria. Alfalfa plants infected with the aceA and glcB mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in the Rhizobium-legume symbiosis.

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