Reiteration of Nitrogen Fixation Gene Sequences and Specificity of Rhizobium in Nodulation and Nitrogen Fixation in Phaseolus vulgaris

Martínez, Esperanza; Pardo, Marco Aurelio; Palacios, Rafael; Miguel, Angel Cevallos

doi:10.1099/00221287-131-7-1779

Cited by 93 publications

(120 citation statements)

References 22 publications

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“…Reiteration of nitrogenase structural genes and other genomic regions is a common feature not only in R. phaseoli strains (9,22,23,33,34) but also in other Rhizobium species (3,9,13,14,16,18,28,32,35,42). Therefore, we believe that rearrangements similar to these may occur in other Rhizobium strains.…”

Section: Discussionmentioning

confidence: 95%

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

et al. 1991

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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...

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Section: Discussionmentioning

confidence: 95%

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

et al. 1991

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“…This strategy takes advantage of the fact that Agrobacterium transconjugants carrying pSym from R. leguminosarum bv. phaseoli strains are able to nodulate P. vulgaris (5,30). The unmarked plasmid from one Agrobacterium transconjugant was purified, labeled in vitro, and used as a hybridization probe against the set of cosmids covering pSym.…”

Section: Resultsmentioning

confidence: 99%

Structural complexity of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli

et al. 1991

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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.

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“…All Rhizobium etli (formerly Rhizobium phaseoli [53]) strains characterized to date have three copies of the nitrogenase reductase (nifH) gene (32,46). As in most of the nitrogenfixing eubacteria, in the type strain CE3, nifHa and nifHb are transcriptionally coupled with the nitrogenase structural genes, forming nifHDK operons, while no homologs of the latter genes have been found downstream of nifHc (47).…”

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Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes

et al. 1996

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In Rhizobium etli the nitrogenase reductase genes are reiterated. Strain CE3 has three copies; nifHa and nifHb form part of nifHDK operons with the nitrogenase structural genes, while nifHc is linked to a truncated nifD homolog. Their sequences are identical up to 6 residues upstream from a 54 -dependent promoter. A remarkable difference among them is the absence of canonical NifA binding sites upstream of nifHc while a canonical binding site is located 200 bp upstream of nifHa and nifHb. To evaluate the transcriptional regulation of the reiterated nifH genes, we constructed fusions of nifHa and nifHc with the lacZ gene of Escherichia coli. Both genes were expressed at maximum levels under 1% oxygen in free-living cultures, and their expression declined as the oxygen concentration was increased. This expression was dependent on the integrity of nifA, and nifHc was expressed at higher levels than nifHa. The same pattern was observed with root nodule bacteroids. Expression of both genes in E. coli required 54 in addition to NifA bound to the upstream activator sequence. In vivo dimethyl sulfate footprinting analyses showed that NifA binds to the canonical site upstream of nifHa and to a TGT half-site 6 nucleotides further upstream. NifA protected an imperfect binding site upstream of nifHc at position 85 from the promoter. The integration host factor stimulated each gene differently, nifHa being more dependent on this protein. The above results correlate the asymmetric arrangement of cis-acting elements with a differential expression of the reiterated nifH genes, both in culture and during symbiosis with bean plants.Eubacterial nitrogen fixation genes (nif and fix) are generally transcribed from 54 -dependent promoters located between positions Ϫ26 and Ϫ11 relative to the transcription start site and have the consensus sequence 5Ј-TGGCACN 5 TTGCA/T-3Ј (37). 54 (also known as RpoN, s N

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Reiteration of Nitrogen Fixation Gene Sequences and Specificity of Rhizobium in Nodulation and Nitrogen Fixation in Phaseolus vulgaris

Cited by 93 publications

References 22 publications

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

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

Structural complexity of the symbiotic plasmid of Rhizobium leguminosarum bv. phaseoli

Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes

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