Sequencing approach and characterization of the symbiotic plasmid of the novel specie Rhizobium grahamii sp

M. J. ALTHABEGOITI; G. TORRES TEJERIZO; M.A. ROGEL-HERNÁNDEZ; E. ORMEÑO-ORRILLO; S. BROM ; E. MARTINEZ ROMERO

Bilbao

Congreso; International Plasmid Biology Conference; 2012

International Plasmid Biology Conference

Rhizobia are nitrogen-fixing bacteria that form nodules in legumes. Genetic determinants that allow this interaction are called symbiotic genes and are located in plasmids in Rhizobium and Sinorhizobium. One of the most important crops in Latin America is common bean (Phaseolus vulgaris), which is mainly nodulated by Rhizobium etli or R. phaseoli. However, beans are promiscuous hosts and may form nodules with different rhizobial species, such as Rhizobium grahamii sp., recently proposed by Lopez-Lopez as a novel specie (1). It was isolated from Dalea leporina in bean fields and is capable of nodulating Leucaena leucocephala and P. vulgaris. However, in presence of R. etli it has little opportunity to compete for nodule formation in bean. In Eckhardt-gels we observed two plasmids of 1.4 Mbp and 483 kbp in R. grahamii sp. CCGE502. A draft sequence of CCGE502 genome was obtained and the subsequent assembly by Newbler generated three large scaffolds, two of them correlated with the size of the plasmids. We identify the smaller as the symbiotic plasmid (pRg502a), and 183 ORFs were classified in Clusters of Orthologous Groups. A cluster of genes involved in conjugal transfer was found similar to the one present in pCFN42a, which is self-transmissible and its conjugation is regulated by Quorum Sensing. This suggested that the transfer of the symbiotic plasmid of R. grahamii could be regulated by population density. In regard to replication systems, repC genes of pRg502a and pCFN42a were found closely related. This evidence correlated with the incompatibility that we observed in conjugation assays. Conjugal experiments showed that pRg502a is mobilizable from its genomic background to Agrobacterium tumefaciens GMI 9023 (plasmid free) at a frequency of ca.10-6 transconjugants per recipient cell. Nevertheless, we were unable to obtain retransfer from A. tumefaciens GMI 9023 (pRg502a) suggesting that some traits presents in the chromosome or in pRg502b are necessary for conjugal transfer of pRg502a. Plasmid pRg502a carries the nodulation and nitrogen fixation genes. When pRg502a is present in a derivative of CFN42 lacking its symbiotic plasmid, it restored nodulation of the strain. A. tumefaciens GMI 9023 transconjugants carrying pRg502a were able to form full nodules in bean. Experiments that cross-combine pSym determinants in different genomic background will be helpful to further study the mechanisms that contribute to rhizobial effectiveness and competition.