Amplification and deletion of a nod-nif region in the symbiotic plasmid of Rhizobium phaseoli

Abstract

One remarkable characteristic of the genomes of some Rhizobium species is the frequent occurrence of rearrangements. In some instances these rearrangements alter the symbiotic properties of the strains. However, no detailed molecular mechanisms have been proposed for the generation of these rearrangements. To understand the mechanisms involved in the formation of rearrangements in the genome of Rhizobium phaseoli, we have designed a system which allows the positive selection for amplification and deletion events. We have applied this system to investigate the stability of the symbiotic plasmid of R. phaseoli. High-frequency amplification events were detected which increase the copy number of a 120-kb region carrying nodulation and nitrogen fixation genes two to eight times. Deletion events that affect the same region were also found, albeit at a lower frequency. Both kinds of rearrangements are generated by recombination between reiterated nitrogenase (nifHDK) operons flanking the 120-kb region.Rhizobium spp. are gram-negative soil bacteria studied primarily for their ability to establish nitrogen-fixing symbioses with leguminous plants. Intensive genetic analysis during the past decade has led to the identification of genes essential for the nodulation (nod genes) and nitrogen fixation (nif and fix genes) processes. In all the fast-growing Rhizobium species, these genes are carried on large plasmids, the so-called Sym plasmids or pSym (20,24).One interesting characteristic of the Rhizobium genome is the presence of a large number of reiterated DNA sequences. For Rhizobium phaseoli, the symbiont of the common bean (Phaseolus vulgaris L.), we have estimated the presence of about 700 reiterated elements, belonging to 200 different families (9). This high degree of reiteration is not restricted to R. phaseoli; other members of the Rhizobiaceae family, including the closely related bacteria Agrobacterium tumefaciens, possess a large amount of reiterated DNA (9, 24). For other organisms, it has been shown that recombination between pairs of repeated elements may lead to different kinds of genomic rearrangements, including additions, amplifications, deletions, and inversions (2, 31).Frequent genomic rearrangements (in the range of 10-2 to 10-3) have been commonly observed in different Rhizobium species, including Bradyrhizobium japonicum, Rhizobium trifolii, and R. phaseoli. These rearrangements, which are frequently deletions, may affect the symbiotic properties of the strain, either for nodulation or for nitrogen fixation (4,7,10,14,18,39,44). Unfortunately, the mechanisms involved in the generation of genetic instability in these species have been poorly analyzed.In order to understand the mechanisms involved in the genetic instability of R. phaseoli, we have developed an experimental approach for the general selection of genomic rearrangements. This system allows the positive selection of different kinds of rearrangements, including amplifications, deletions, insertions, and loss of plasmids. We have used this sys...

Molecular cloning and characterization of the recA gene of Rhizobium phaseoli and construction of recA mutants

“…The event was a deletion in the pSym of about 120 kb that occurs with a cumulative frequency of 1.7 x 10-2 and was recA dependent. This rearrangement was similar to others previously found in our laboratory (37).…”

“…Moreover, a high frequency of rearrangements has been found to be associated with the presence of repeated DNA (12, 38). Genomic instability and rearrangements that affect nodulation or nitrogen fixation abilities have been observed in several Rhizobium species (25,27,37,42).…”

Random amplified polymorphic DNA analysis of effective Rhizobium sp. associated with beans cultivated in brazilian cerrado soils

“…In general, strains within the Rhizobium genus are fast growing, such as there studied here that nodulate beans. They appear to be more taxonomically diverse and ecologically adaptable than the slower growing strains from other genera like Bradyrhizobium and Azorhizobium These differences probable occur because in the slow growing strains the genetic information, in general, is contained in the chromossomal DNA, while in fast growing strains many genes, including those related to biological nitrogen fixation are located on plasmids which confers dynamic qualities to the genome (5,12). These characteristics favor recombination between genotypes of the Rhizobium population present in soils (14).…”

“…PCA also verified that the R. leguminosarum bv. phaseoli strains had more variation among themselves than the R. tropici did, probably due to the formers higher genetic plasticity (12,13). In addition, this analysis identified adequate primers for strain identification.…”

Rhizobium meliloti lacking mosA synthesizes the rhizopine scyllo-inosamine in place of 3-O-methyl-scyllo-inosamine

“…As deletions in symbiotic plasmids are common (Romero et al, 1991), we consider it most likely that the Rm220-3 mos locus is the result of a deletion. Consistent with this suggestion a recent survey of the frequency of inositol rhizopines has shown that only one strain (Rm220-30) out of 22 rhizopine strains isolated produced SI (Wexler e t al., 1995).…”