Structure And Function Of New Naturally Generated pSYMs Of Rhizobium Etli

Programa de Genética Molecular de Plásmidos Bacterianos CIFN. UNAM. Apdo. Postal 565-A, Cuernavaca, Mor., México

The pSym of Rhizobium etli strain CFN42 iss subject to conjugative transfer, depending on the presence of p42a, another endogenous, self-transmissible plasmid (Brom et al. 2000). We have analyzed transconjugants generated during pSym transfer. This allowed the identification of new pSyms, which we have named pSyms*. The pSyms* contain deletions of the pSym and integrations of p42a. The pSyms* have acquired some features of p42a, such as self-transfer, and compatibility with the wild-type pSym, thus allowing the construction of derivatives containing both, a pSym* and a wild-type pSym.

Regarding their structure, we have determined that most of the pSyms* (7/10), share one of the endpoints for the recombination between the pSym and p42a. The sequence analysis of the p42a border shows homology to a fragment of pRleVF39b, a plasmid from R. leguminosarum strain VF39, subject to a rearrangement with the endogenous pSym (Zhang et al. 2001), and to a sequence encoding a prophage lambda integrase. Also, we have seen that the generation of pSym* is independent of RecA, suggesting the involvement of a very specific recombination system.

As to their symbiotic performance, derivatives containing a pSym*, showed approximately a two-fold increase in nitrogen fixation, measured through acetylene reduction. This increase could imply that the pSym region deleted in these strains contains a repressor for nitrogen fixation. Alternatively, it could be due to an increase in the plasmid's copy number, provided by the new oriV. The increased nitrogen fixation is abolished in the presence of a wild-type pSym, furthermore, in one case the effect observed is the reverse, a drastic reduction in nitrogen fixation. The strains containing a pSym*, and the derivatives containing two pSyms showed a decrease in competitivity for nodulation. The strains containing two pSyms are very stable in vitro, however, the derivatives isolated from nodules usually lost the wild-type pSym.

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