Conjugative transfer of p42a from rhizobium etli CFN42, which is required for mobilization of the symbiotic plasmid, is regulated by quorum sensing

Abstract

Rhizobium etli CFN42 contains six plasmids. Only one of them, p42a, is self-conjugative at high frequency. This plasmid is strictly required for mobilization of the symbiotic plasmid (pSym). To study the transfer mechanism of p42a, a self-transmissible cosmid clone containing its transfer region was isolated. Its sequence showed that most of the tra genes are highly similar to genes of Agrobacterium tumefaciens pTiC58 and other related plasmids. Four putative regulatory genes were identified; three of these (traI, traR, and cinR) belong to the LuxR-LuxI family. Mutagenesis of these genes confirmed their requirement for p42a transfer. We found that the conjugative transfer of p42a is dependent on quorum sensing, and consequently pSym transfer also was found to be similarly regulated, establishing a complex link between environmental conditions and pSym transfer. Although R. etli has been shown to produce different N-acyl-homoserine lactones, only one of them, a 3-oxo-C(8)-homoserine lactone encoded by the traI gene described here, was involved in transfer. Mutagenesis of the fourth regulatory gene, traM, had no effect on transfer. Analysis of transcriptional fusions of the regulatory genes to a reporter gene suggests a complex regulation scheme for p42a conjugative transfer. Conjugal transfer gene expression was found to be directly upregulated by TraR and the 3-oxo-C(8)-homoserine lactone synthesized by TraI. The traI gene was autoregulated by these elements and positively regulated by CinR, while cinR expression required traI. Finally, we did not detect expression of traM, indicating that in p42a TraM may be expressed so weakly that it cannot inhibit conjugal transfer, leading to the unrepressed transfer of p42a.

FIG. 1.

Physical map of pC-13, containing the transfer region of p42a. Arrows indicate the transcriptional directions of the identified genes. The regulator genes traI, traR, cinR, and traM are shown by black arrows. pCT11 contains the 143-bp mobilizable region, carrying the oriT of p42a. The 13 bp of the nic site sequence are shown. pCT53 and pCT64 contain the regions used to generate mutations in the traR and cinR genes. H, HindIII; E, EcoRI; X, XhoI; S, SalI.

FIG. 2.

Alignment of p42a transfer regulator proteins with the corresponding proteins of related systems. S. mel., S. meliloti; pNGRa, plasmid pNGR234a; R. leg., R. leguminosarum. Identical amino acids are boxed, and conserved amino acids are shown in the consensus. (A) TraI; (B) TraM; (C) TraR; (D) CinR.

FIG. 3.

Thin-layer chromatogram of the acyl-HSL produced by R. etli CFN42 and derivatives. Lane 1, A. tumefaciens NT1(pTiC58ΔaccR); lane 2, CFN42; lane 3, CFNX182 (cured of p42a); lane 4, CFNX671 (cinR::loxPSp); lane 5, CFNX670 (traR::loxPSp); lane 6, CFNX669 (traI::pSUPΩSp); lane 7, CFNX668 (traM::pSUPΩSp); lane 8, 3-oxo-C₈-HSL; lane 9, YM medium control.

FIG. 4.

Effect of culture density on the transfer of p42a and pSym from CFNX688. Transfer frequencies of p42a (triangles), of p42a with conditioned medium added before mating (squares), and of pSym (circles) are shown. Strain CFN2001 (cured of p42a and pSym) was always used as the recipient.

FIG. 5.

Model for the regulation of transfer gene expression in p42a. +, positive regulation; ?, unknown regulatory protein from the LuxR family.