Conjugative Transfer Of Rhizobial Plasmids Under Diverse Environmental Conditions

LUIS BAÑUELOS; GONZALO A. TORRES TEJERIZO; LAURA CERVANTES; SUSANA BROM

Congreso; 115th General Meeting - American Society for Microbiology; 2015

The rhizobia are Gram negative bacteria, with a great agricultural importance because they estabish symbiotic associations with leguminous plants, through the formation of nodules. Rhizobium etli CFN42 induces nitrogen fixing nodules in roots of Phaseolus vulgaris. The genome of R. etli CFN42 is constituted by one chromosome and six large plasmids, ranging in size from 184 to 642 kb. Among these, pRet42d has been identified as the symbiotic plasmid (pSym), because it carries most of the genes required for nodulation and nitrogen fixation. Most of the analysis of conjugal transfer of rhizobial plasmids has been carried out in laboratory conditions, so it is likely that other elements of the natural environment could affect conjugation, since their natural "habitat" is the rhizosphere.The aim of this work is to determine the effect of environmental conditions on the transfer of conjugative plasmids, including factors related to soil environment (changes in temperature, pH, nutrients) as well as elements related directly to the symbiosis (seed exudates, nodulation factors). To do these experiments, we used the strategy of double fluorescent labeling described by Nancharaiah, Wattiau et al. 2003 (Appl Environ Microbiol. 69: 4846-52). In this case, the donor cell has the red fluorescent protein (RFP) marker inserted in the chromosome, and the green fluorescent protein (GFP) marker in the plasmid. Thus, these cells have both fluorescences. When transferring the plasmid to another recipient, the transconjugants only express the GFP, allowing differential detection.We determined the transfer on the surface of the roots, and during the symbiosis, both in infection threads and in the interior of the nodules. We used assay tubes with hydroponic medium, where we inoculated donor and recipient strains in the presence and absence of plants. Our results showed that the frequency of conjugation seems to increase in the presence of the plant. Also, we were able to visualize transconjugants in infection threads, nodules, roots (biofilm formation) and rhizosphere (liquid medium) using multiphoton fluorescence microscopy and plate count.