High-frequency rearrangements in Rhizobium leguminosarum bv. phaseoli plasmids

Abstract

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 ...

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Classification of Austrian Rhizobia and the Mexican Isolate FL27 Obtained from Phaseolus vulgaris L. as Rhizobium gallicum

“…These plasmids may carry up to 25% of the genetic information in Rhizobium (29), and plasmids are prone to losses or alterations. High-frequency plasmid-borne rearrangements, including sequence amplification, deletion, cointegration, and loss, have been particularly observed in R. etli CFN 42T plasmids (4,33). In addition, exchange of plasmids among Rhizobium populations has been reported (31,36).…”

Rhizobial communities in symbiosis with legumes: genetic diversity, competition and interactions with host plants

“…Rhizobial plasmid replicons have less synteny than chromosomes [33], and it is possible that such mosaic plasmid structure results from frequent genetic rearrangements [34]. Moreover, some of them are selftransmissible or can be transferred to other bacterial cells in the presence of other plasmids [35,36].…”