The complete coding sequence of the nitro-

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...

The complete nucleotide sequence of the genomic RNA of a French isolate of Pepino mosaic virus (PepMV) was determined. With a length of 6,425 nucleotides excluding the poly(A) tail at the 3' terminus, the PepMV genome contains five major open reading frames encoding a 164 kDa replicase, triple gene block proteins of 26 kDa, 14 kDa and 9 kDa, and a 25 kDa coat protein. This genome organization, as well as characteristic consensus motifs and high degrees of similarity of PepMV proteins with those of other potexviruses confirmed that PepMV belongs to the Potexvirus genus. Phylogenetic analyses carried out on replicase, triple gene block protein 1 and coat protein amino acid sequences revealed that PepMV is closely related to species Narcissus mosaic virus (NMV), Scallion virus X (SVX), Cymbidium mosaic virus (CymMV), and Potato aucuba mosaic virus (PAMV).

The complete physical map of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli strain CFN42 was established. The data support the concept that Rhizobium symbiotic genes are part of a complex genomic structure which contains a large amount of reiterated DNA sequences. This plasmid is a circular structure of 390 kb with approximately 10 families of internally reiterated DNA sequences of two to three elements each. One family includes two directly oriented nitrogenase operons situated 120 kb apart. We also found several stretches of pSym that are reiterated in other replicons of the cell. Localization of symbiotic gene sequences by heterologous hybridization revealed that nodABC sequences are separated in two regions, each of which contains a nod boxlike element, and it also suggested the presence of two copies of the nifA and nodD gene sequences. We propose that the complex structure of the symbiotic plasmid allows interactions between repeated DNA sequences which, in turn, might result in frequent rearrangements.Bacteria of the genus Rhizobium interact with the roots of leguminous plants to develop a highly specialized structure, the nitrogen-fixing nodule. Many genetic determinants of the symbionts are responsible for this differentiation process. The Rhizobium genome is organized in two different types of replicons, the chromosome and large plasmids. Usually, the genes involved in nodulation and nitrogen fixation processes are located on a single large plasmid. This plasmid is referred to as the symbiotic plasmid (pSym) (22,34,46).A general characteristic of the Rhizobium genome is the presence of a large number of reiterated DNA sequences (15,31,40). It has been established that genomic rearrangements in Rhizobium leguminosarum bv. phaseoli, the symbiont of the common bean plant, Phaseolus vulgaris, occur at high frequency (16,31). DNA rearrangements caused by recombination between repeated sequences might participate in the generation of genomic diversity.Establishment of the physical structure of the Rhizobium genome, in particular, the locations and orientations of reiterated DNA sequences, is of primary importance in elucidating the molecular mechanisms involved in genomic dynamics.As a model structure, we selected pSym of R. leguminosarum bv. phaseoli. Several physical maps of symbiotic regions of different Rhizobium species have been published, including large portions of the R. meliloti and Sinorhizobium fredii symbiotic plasmids (25, 32), as well as the complete physical map of the 240-kb pSym of an R. leguminosarum bv. viciae strain (38). A genetic map of R. meliloti megaplasmid pRmeSU47b has also been reported recently (8). In such studies, emphasis has been put on localization of specific genes and regulatory sequences. The studies reported here were focussed on the underlying structural complexity of pSym with respect to the presence of reiterated DNA Tn5-Mob element and its insertion site are also shown.

A genetic element that allows the positive selection of different genomic rearrangements was used to analyze DNA amplification in Rhizobium leguminosarum biovar phaseoli. Discrete amplifiable DNA regions (amplicons) were detected in different regions of the genome of the model strain CFN42, including the chromosome and several large plasmids. Amplicons were mobilized into Escherichia coli using a genetic approach that involves the introduction of an origin of replication active in E. coli and an origin of conjugal transfer into the amplifiable DNA regions ofthe Rhizobium genome. The strategy can be a valuable tool for studies on genome organization and function. We propose that amplicons define a structural characteristic of the genome that may play an important biological role.Rhizobium species have stimulated scientific interest due to their ability to interact with plants establishing nitrogenfixing symbioses. The genome ofdifferent Rhizobium species contains a large amount of reiterated DNA sequences that include complete operons, specific genes, regulatory sequences, and insertion sequences (for review, see ref. 1). Presumably due to the presence of reiterated DNA sequences, the genome of some Rhizobium strains is subjected to frequent genomic rearrangements (2-5).We have recently constructed a genetic element (the GDYN1 cassette) that allows the positive selection of different types of genomic rearrangements (5). This element contains the kanamycin/gentamycin and spectinomycin/ streptomycin resistance markers from plasmid pSa. In Rhizobium leguminosarum biovar phaseoli (R. phaseoli), the symbiont of the common bean plant Phaseolus vulgaris, these genes confer resistance to high levels of spectinomycin and to low levels of kanamycin. Because the level of kanamycin resistance increases with gene dosage, the element provides a positive selection system to detect variants with amplified DNA regions. The GDYN1 cassette was used to demonstrate high-frequency amplification and deletion events of a 120-kb region located in the symbiotic plasmid of R. phaseoli (5).In the present study we used a GDYN1 derivative to analyze gene amplification events in different regions of the genome ofR. phaseoli strain CFN42, including the chromosome and several large plasmids. Discrete amplifiable DNA regions, referred to here as amplicons, were identified and mobilized from R. phaseoli to Escherichia coli by an in vivo genetic procedure. MATERIALS AND METHODSConstruction of TnS-GDYNl. This transposon derivative was constructed by in vitro manipulation of the TnS insert in pSUP2021, which is a mobilizable pBR325 derivative (6). The central Bgl II-Bgl II fragment of TnS in pSUP2021 was replaced by a 5.7-kb BamHI-BamHI fragment of pGUS3 (5), carrying the GDYN1 cassette. After ligation, the mixture was transformed into E. coli HB101, selecting for recombinant plasmids in LB medium containing chloramphenicol and spectinomycin. Colonies resistant to chloramphenicol and spectinomycin that showed sensitivity to sucrose were scre...