Inoculation of alfalfa plants with either incompatible Rhizobium or a Rhizobium mutant blocked in Nod factor synthesis led to an accumulation of salicylic acid in roots, in contrast to plants inoculated with a wild-type (compatible) R. meliloti strain. When salicylic acid was exogenously applied prior to inoculation of alfalfa plants with either purified Nod factor or compatible Rhizobium strains, a significant inhibition of nodule primordia formation and a reduction of the number of emerging nodules, respectively, as well as a delay in nodule visualization, were observed. These results suggest an involvement of Rhizobium-synthesized Nod factors in the inhibition of salicylic acid-mediated defense in legumes.

An analysis of the conjugative transfer of pRetCFN42d, the symbiotic plasmid (pSym) of Rhizobium etli , has revealed a novel gene, rctA , as an essential element of a regulatory system for silencing the conjugative transfer of R. etli pSym by repressing the transcription of conjugal transfer genes in standard laboratory media. The rctA gene product lacks sequence conservation with other proteins of known function but may belong to the winged-helix DNA-binding subfamily of transcriptional regulators. Similar to that of many transcriptional repressors, rctA transcription seems to be positively autoregulated. rctA expression is greatly reduced upon overexpression of another gene, rctB , previously identified as a putative activator of R. etli pSym conjugal transfer. Thus, rctB seems to counteract the repressive action of rctA. rctA homologs are present in at least three other bacterial genomes within the order Rhizobiales , where they are invariably located adjacent to and divergently transcribed from putative virB -like operons. We show that similar to that of R. etli pSym, conjugative transfer of the 1.35-Mb symbiotic megaplasmid A of Sinorhizobium meliloti is also subjected to the inhibitory action of rctA . Our data provide strong evidence that the R. etli and S. meliloti pSym plasmids are indeed self-conjugative plasmids and that this property would only be expressed under optimal, as yet unknown conditions that entail inactivation of the rctA function. The rctA gene seems to represent novel but probably widespread regulatory systems controlling the transfer of conjugative elements within the order Rhizobiales .

An approach originally designed to identify functional origins of conjugative transfer ( oriT or mob ) in a bacterial genome (J. A. Herrera-Cervera, J. M. Sanjuán-Pinilla, J. Olivares, and J. Sanjuán, J. Bacteriol. 180:4583-4590, 1998) was modified to improve its reliability and prevent selection of undesired false mob clones. By following this modified approach, we were able to identify two functional mob regions in the genome of Rhizobium etli CFN42. One corresponds to the recently characterized transfer region of the nonsymbiotic, self-transmissible plasmid pRetCFN42a (C. Tun-Garrido, P. Bustos, V. González, and S. Brom, J. Bacteriol. 185:1681-1692, 2003), whereas the second mob region belongs to the symbiotic plasmid pRetCFN42d. The new transfer region identified contains a putative oriT and a typical conjugative ( tra ) gene cluster organization. Although pRetCFN42d had not previously been shown to be self-transmissible, mobilization of cosmids containing this tra region required the presence of a wild-type pRetCFN42d in the donor cell; the presence of multiple copies of this mob region in CFN42 also promoted conjugal transfer of the Sym plasmid pRetCFN42d. The overexpression of a small open reading frame, named yp028 , located downstream of the putative relaxase gene traA , appeared to be responsible for promoting the conjugal transfer of the R. etli pSym under laboratory conditions. This yp028 -dependent conjugal transfer required a wild-type pRetCFN42d traA gene. Our results suggest for the first time that the R. etli symbiotic plasmid is self-transmissible and that its transfer is subject to regulation. In wild-type CFN42, pRetCFN42d tra gene expression appears to be insufficient to promote plasmid transfer under standard laboratory conditions; gene yp028 may play some role in the activation of conjugal transfer in response to as-yet-unknown environmental conditions.