Conjugative transfer of pRet42a from Rhizobium etli CFN42: Role of hypothetical conserved proteins (hcp) encoding genes located in the tra region, and influence of the recipient.

EUNICE LÓPEZ-FUENTES; LAURA CERVANTES; GONZALO A. TORRES TEJERIZO; SUSANA BROM

Cuatro Ciénegas, Coahuila

Congreso; III Congreso de Bioquímica y Biología Molecular de Bacterias; 2013

Sociedad Mexicana de Bioquímica

Plasmids pRet42a from Rhizobium etli CFN42 and pSfr64a from Sinorhizobium fredii GR64 are able to perform conjugative transfer at high frequency, and their transfer regions are very similar. Transfer of these plasmids is regulated through quorum-sensing (QS), involving Lux-type HSL-synthetases and transcriptional regulators. The plasmids differ in three genes encoding hcp, located between traH and traM: orfs147 (trrA), 148 and 149 in pSfr64a, and orfs163, 164 and 165 in pRet42a. Mutagenesis of orf147 (trrA) was shown to abolish transfer of pSfr64a. To determine if the hcp-encoding genes equivalently located in pRet42a also participate in conjugative transfer, we constructed mutant derivatives, and tested their effect on the conjugative transfer (CT) of this plasmid.             The pRet42a derivative with a mutation in orf163 showed a slight but reproducible increase in its CT compared to the wild type (wt). Unexpectedly, overexpression of the wt orf163, also led to an increase in CT. Transcriptional analysis performed with fusions to a reporter gene, showed that traI (HSL synthase) expression decreased 5 times in the mutant compared to the wt. This suggests that another HSL synthase, able to substitute TraI, is expressed in the absence of orf163. The sequence of the CFN42 genome has shown that it contains various HSLs synthases in addition to the pRet42a-encoded traI: cinI, located on the chromosome and raiI, located on pRet42f, next to raiR, a LuxR-type regulator. Transcriptional analysis of these genes indicated that orf163 acts as a repressor of raiI, cinI, and raiR, because their expression increased in the mutant background.             We conclude that orf163 functions as a dual regulator, ensuring pRet42a transfer, either by induction of traI, or allowing cinI and raiI expression when it is absent. Mutagenesis of orfs164 and 165 had no effect on CT under the conditions examined.             Another aspect addressed in this work was the role of the recipient in the CT of pRet42a. Previously, we defined pRet42a as self-transmissible because it was able to perform CT from Agrobacterium donors, however, the recipient used was always R. etli. In this work, we determined that the presence of pRet42f, possibly due to the raiI gene it carries, affects the CT from Agrobacterium donors. We propose a model for pRet42a transfer regulation, including new elements from pRet42a, as well as from other replicons, showing a more complex scheme than the one previously proposed.