Transfer of the Symbiotic Plasmid of Rhizobium etli CFN42 to Endophytic Bacteria Inside Nodules

Abstract

Conjugative transfer is one of the mechanisms allowing diversification and evolution of bacteria. Rhizobium etli CFN42 is a bacterial strain whose habitat is the rhizosphere and is able to form nodules as a result of the nitrogen-fixing symbiotic relationship it may establish with the roots of Phaseolus vulgaris . R. etli CFN42 contains one chromosome and six large plasmids (pRet42a – pRet42f). Most of the genetic information involved in the establishment of the symbiosis is localized on plasmid pRet42d, named as the symbiotic plasmid (pSym). This plasmid is able to perform conjugation, using pSym encoded transfer genes controlled by the RctA/RctB system. Another plasmid of 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 have been shown to contain endophytic bacteria from different genera and species. In this work, we have explored the conjugative transfer of the pSym (pRet42d) from R. etli CFN42 to endophytic bacteria as putative recipients, using as donor a CFN42 derivative labeled with GFP in the pRet42d and RFP in the chromosome. We were able 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. Ultrastructure microscopy analysis showed some differences in the structure of the nodules of one of the transconjugants. A replicon of the size of pRet42d (371 Kb) could not be visualized in the transconjugants, suggesting that the pSym or a segment of the plasmid is integrated in the chromosome of the recipients. These findings strengthen the proposal that nodules constitute a propitious environment for exchange of genetic information among bacteria, in addition to their function as structures where nitrogen fixation and assimilation takes place.

Genetic characterization at the species and symbiovar level of indigenous rhizobial isolates nodulating Phaseolus vulgaris in Greece

“…phaseoli evolved with common beans in America 39 , 86 and probably disseminated worldwide along with bean seeds 2 , 87 , it is possible that native rhizobia in various countries have acquired symbiotic genes typical of sv. phaseoli through horizontal gene transfer in the rhizosphere or within nodules 88 , 89 .…”

Exopolysaccharide Characterization of Rhizobium favelukesii LPU83 and Its Role in the Symbiosis With Alfalfa

“…A similar event was observed in soils of the Brazilian Cerrados ( Batista et al, 2007 ), where evidence of HGT from inoculated Bradyrhizobium strains to soil bacteria was detected. Recently, it was shown that the symbiotic plasmid of Rhizobium etli CFN42, or at least fragments of it, can be transferred to bean-endophytic bacteria, generating new strains with the ability to nodulate and fix nitrogen ( Bañuelos-Vazquez et al, 2020 ). In our group, we showed that the gene cluster involved in the synthesis of the nodulation factors (NFs) of LPU83 has a structure similar to that of E. meliloti , with whom it also shares a close phylogenetic relationship ( Del Papa et al, 2007 ; Torres Tejerizo et al, 2011 ).…”

Why are rhizobial symbiosis genes mobile?

“… [ 45 ] This plasmid can also exploit other transfer machineries—mobilization has been shown to occur via integration and mobilization of the class I QS-induced plasmid p42a. [ 46 , 47 ] pSymA in Ensifer/Sinorhizobium melliloti strain 1021 P Large (1354 kb) conjugative plasmid. Transfer has yet to be observed in the laboratory although there is evidence for transfer within nodules.…”

“…For example, in pSym pRetCFN42d ( Rhizobium etli) RctA repression can be experimentally relieved and transfer induced, showing that transmission is active, but the exact trigger cannot be identified [46,51]. However, recent work has demonstrated that pRetCFN42d transfer occurs within root nodules [47], suggesting that conjugation is tuned in yet unknown ways to the root environment.Type III elements, such as R. leguminosarum sv. viceae pSym pRL10JI, are not able to self-mobilize as they lack genes required for mate pair formation, but have retained the genes required for DNA transfer and replication.…”