Role of plant compounds in the modulation of the conjugative transfer of pRet42a

One of the most studied mechanisms involved in bacterial evolution and diversification is conjugative transfer (CT) of plasmids. Plasmids able to transfer by CT often encode beneficial traits for bacterial survival under specific environmental conditions. Rhizobium etli CFN42 is a Gram-negative bacterium of agricultural relevance due to its symbiotic association with Phaseolus vulgaris through the formation of Nitrogen-fixing nodules. The genome of R. etli CFN42 consists of one chromosome and six large plasmids. Among these, pRet42a has been identified as a conjugative plasmid. The expression of the transfer genes is regulated by a quorum sensing (QS) system that includes a traI gene, which encodes an acyl-homoserine lactone (AHL) synthase and two transcriptional regulators (TraR and CinR). Recently, we have shown that pRet42a can perform CT on the root surface and inside nodules. The aim of this work was to determine the role of plant-related compounds in the CT of pRet42a. We found that bean root exudates or root and nodule extracts induce the CT of pRet42a in the plant rhizosphere. One possibility is that these compounds are used as nutrients, allowing the bacteria to increase their growth rate and reach the population density leading to the activation of the QS system in a shorter time. We tested if P. vulgaris compounds could substitute the bacterial AHL synthesized by TraI, to activate the conjugation machinery. The results showed that the transfer of pRet42a in the presence of the plant is dependent on the bacterial QS system, which cannot be substituted by plant compounds. Additionally, individual compounds of the plant exudates were evaluated; among these, some increased and others decreased the CT. With these results, we suggest that the plant could participate at different levels to modulate the CT, and that some compounds could be activating genes in the conjugation machinery.Fil: Bañuelos Vazquez, Luis Alfredo. Universidad Nacional Autónoma de México; MéxicoFil: Castellani, Lucas Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Luchetti, Abril. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Romero, David. Universidad Nacional Autónoma de México; MéxicoFil: Torres Tejerizo, Gonzalo Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Brom, Susana. Universidad Nacional Autónoma de México; Méxic

Transfer of the symbiotic plasmid of rhizobium etli CFN42 to endophytic bacteria inside nodules

Conjugative transfer is one of the mechanisms allowing diversification and evolution ofbacteria. Rhizobium etli CFN42 is a bacterial strain whose habitat is the rhizosphere andis able to form nodules as a result of the nitrogen-fixing symbiotic relationship it mayestablish with the roots of Phaseolus vulgaris. R. etli CFN42 contains one chromosomeand six large plasmids (pRet42a ? pRet42f). Most of the genetic information involvedin the establishment of the symbiosis is localized on plasmid pRet42d, named asthe symbiotic plasmid (pSym). This plasmid is able to perform conjugation, usingpSym encoded transfer genes controlled by the RctA/RctB system. Another plasmidof CFN42, pRet42a, has been shown to perform conjugative transfer not only in vitro,but also on the surface of roots and inside nodules, using other rhizobia as recipients.In addition to the rhizobia involved in the formation of nodules, these structures havebeen shown to contain endophytic bacteria from different genera and species. Inthis work, we have explored the conjugative transfer of the pSym (pRet42d) fromR. etli CFN42 to endophytic bacteria as putative recipients, using as donor a CFN42derivative labeled with GFP in the pRet42d and RFP in the chromosome. We wereable to isolate some transconjugants, which inherit the GFP, but not the RFP marker.Some of them were identified, analyzed and evaluated for their ability to nodulate.We found transconjugants from genera such as Stenotrophomonas, Achromobacter,and Bacillus, among others. Although all the transconjugants carried the GFP marker,and nod, fix, and nif genes from pRet42d, not all were able to nodulate. Ultrastructuremicroscopy analysis showed some differences in the structure of the nodules of one ofthe transconjugants. A replicon of the size of pRet42d (371 Kb) could not be visualizedin the transconjugants, suggesting that the pSym or a segment of the plasmid isintegrated in the chromosome of the recipients. These findings strengthen the proposalthat nodules constitute a propitious environment for exchange of genetic informationamong bacteria, in addition to their function as structures where nitrogen fixation andassimilation takes placeFil: Bañuelos Vazquez, Luis Alfredo. Universidad Nacional Autónoma de México; MéxicoFil: Cazares, Daniel. Universidad Nacional Autónoma de México; MéxicoFil: Rodríguez, Susana. Universidad Nacional Autónoma de México; MéxicoFil: Cervantes De la Luz, Laura. Universidad Nacional Autónoma de México; MéxicoFil: Sánchez López, Rosana. Universidad Nacional Autónoma de México; MéxicoFil: Castellani, Lucas Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Torres Tejerizo, Gonzalo Arturo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Brom, Susana. Universidad Nacional Autónoma de México; Méxic