Assorted elements influence conjugative transfer of Sinorhizobium fredii plasmid pSfr64a

LAURA CERVANTES; YAXAL SOTO; GONZALO TORRES TEJERIZO; DAVID ROMERO; SUSANA BROM

Santander

Conferencia; International Plasmid Biology Conference 2012; 2012

ISPB

Among the soil inhabitants, rhizobia are interesting because of their ability to form nitrogen fixing nodules in symbiosis with plant roots. Different types of evidence suggest that conjugative transfer has impacted on the diversity of rhizobial strains. Plasmid pSfrGR64 from Sinorhizobium fredii GR64 is a chimeric transmissible plasmid, whose conjugation related genes are highly similar to those of the conjugative plasmid pRet42a from Rhizobium etli CFN42. In spite of this similarity, pSfr64a and plasmid pRet42a are unable to transfer from the genomic background of R. etli CFN42 and GR64, respectively.We have analyzed the regulatory mechanism of the conjugative transfer of plasmid pSfr64a from S. fredii GR64, by determining the transfer ability of derivatives with mutations in the different quorum-sensing-related genes, and in some other genes, encoding hypothetical conserved proteins (hcp) localized in the transfer region. Also, we constructed transcriptional  fusions of the different promoter regions to a ß-glucuronidase (GUS) reporter gene, and analyzed its expression in the wild type and mutant backgrounds. Furthermore, using thin-layer chromatography we determined the production of homoserine-lactone molecules in the wild type GR64 and in the different mutant derivatives. Altogether, the results indicate that conjugative transfer is regulated by quorum-sensing through elements encoded in plasmid pSfr64a (TraRa, TraIa), in the symbiotic plasmid (TraIb, TraRb) and in the chromosome (NgrI). Additionally, orf 147, one of the hcp encoding genes, was found to be required for transfer. We have named orf 147 as trrA, for transfer regulator. QS and trrA regulators are independent of each other, not bound in a hierarchical structure. We have shown that the relaxase of pRet42a is able to process the oriT of pSfr64a, and viceversa. This agrees with the fact that the oriT nick-site of pSfr64a is identical to that of pRet42a, indicating that the host-specific behaviour for conjugative transfer is due to other factors, presumably those encoded in the the symbiotic plasmid and in the chromosome. So, we propose that, in addition to the external environmental signals, the "cytoplasmic environment"  may pose a barrier to plasmid dissemination, even if the plasmid may be functional in other aspects.