Identification of Functional mob Regions in Rhizobium etli : Evidence for Self-Transmissibility of the Symbiotic Plasmid pRetCFN42d

Abstract

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.

Identification of the rctA Gene, Which Is Required for Repression of Conjugative Transfer of Rhizobial Symbiotic Megaplasmids

“…The conjugal transfer frequencies from the individual R. etli CFNX218Spc donors were similar to those previously obtained from the A. tumefaciens GMI9023 donors (data no shown). These results confirmed that Tn5 insertions harbored by pRetCFN42d and selected with our strategy promoted conjugative transfer of this plasmid in standard laboratory media without the help of any other R. etli plasmid, in contrast to the wild-type plasmid, which shows no detectable self-transfer under these conditions (10,45).…”

“…yp028 is located downstream of traA, the hypothetical plasmid relaxase gene. Recently, yp028 was described as a gene which may act in the activation of conjugal transfer of the R. etli symbiotic plasmid (45).…”

“…The symbiotic plasmid of R. etli CFN42, pRetCFN42d, has never been shown to be self-transmissible. However, data from a previous work indicated that conjugative transfer of this pSym may be silent under laboratory conditions, suggesting the possibility of an active repression system in this plasmid (45). In order to identify putative genes participating in the repression of R. etli pSym conjugal transfer, strain CFNX182 of R. etli (pRetCFN42a Ϫ ) was subjected to random Tn5 mutagenesis using the suicide vector pSUP2021 (55).…”

“…However, Pérez-Mendoza and coworkers have recently identified a gene (yp028) involved in the promotion of R. etli pSym transfer at relatively high frequencies in standard media in the absence of pRetCFN42a (45). The identification of this gene opened the possibility that conjugal transfer functions encoded by the R. etli pSym might be silent under laboratory conditions and that transfer could be activated with the participation of yp028 under as yet unknown conditions.…”

Transcriptional Interference and Repression Modulate the Conjugative Ability of the Symbiotic Plasmid of Rhizobium etli

“…Our previous work suggested that the pSym has an intrinsic ability for conjugal transfer, independent of pRetCFN42a, although this ability is tightly repressed (27). By various genetic strategies, two genes that participate in the regulation of the pRetCFN42d conjugational transfer were identified previously (28).…”