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

Abstract

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.

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

“…We found the type I event in strain CFNX102 only; therefore the loss of the pSym was recA independent. In pSym, several families of reiterated sequences have been found (14); some of these sequences (those that are in a direct orientation) may participate in homologous recombination to generate a deleted pSym. A different approach is required to stabilize the symbiotic phenotype in those strains that lose the pSym.…”

Conjugative Transfer of p42a from Rhizobium etli CFN42, Which Is Required for Mobilization of the Symbiotic Plasmid, Is Regulated by Quorum Sensing

“…Seven bands purified from several vectors containing the transfer genes of pTiC58 (Table 1) were labeled with [␣-32 P]dCTP by random priming with Amersham's Rediprime system and used as probes. Hybridizations were done as previously described (17).…”

New Native Rhizobia Strains for Inoculation of Common Bean in the Brazilian Savanna

“…phaseoli) (Amarger et al, 1997), R. leucaenae (Ribeiro et al, 2012), R. freirei (Dall'Agnol et al, 2013), and R. paranaense (Dall'Agnol et al, 2014). An important feature of the bean microsymbiont rhizobia genome is the presence of DNA repeated sequences found in the symbiotic plasmid regions, pSym containing the structural nitrogenase gene, nif, or the nodulation gene, nod (Girard et al, 1991;Segovia et al, 1993). These copies are usually needed for expression of the BNF process and effectiveness, but they also represent recombination sites where genomic rearrangements occur, which can lead to loss of properties in these strains (Girard et al, 1991), providing the explanation for the frequent occurrence of loss of ability to create nodules and to fix N 2 in various strains.…”