MobC of conjugative RA3 plasmid from IncU group autoregulates the expression of bicistronic mobC-nic operon and stimulates conjugative transfer

Abstract

BackgroundThe IncU conjugative transfer module represents highly efficient promiscuous system widespread among conjugative plasmids of different incompatibility groups. Despite its frequent occurrence the mechanisms of relaxosome formation/action are far from understood. Here we analyzed the putative transfer auxiliary protein MobC of the conjugative plasmid RA3 from the IncU incompatibility group.ResultsMobC is a protein of 176 amino acids encoded in the bicistronic operon mobC-nic adjacent to oriT. MobC is homologous to prokaryotic transcription factors of the ribbon-helix-helix (RHH) superfamily. Conserved LxxugxNlNQiaxxLn motif clusters MobC with the clade of conjugative transfer auxilliary proteins of MobP relaxases. MobC forms dimers in solution and autoregulates the expression of mobCp by binding to an imperfect palindromic sequence (OM) located between putative -35 and -10 motifs of the promoter. Medium-copy number test plasmid containing the oriT-mobCp region is mobilized with a high frequency by the RA3 conjugative system. The mutations introduced into OM that abolished MobC binding in vitro decreased 2-3 fold the frequency of mobilization of the test plasmids. The deletion of OM within the RA3 conjugative module had no effect on transfer if the mobC-nic operon was expressed from the heterologous promoter. If only nic was expressed from the heterologous promoter (no mobC) the conjugative transfer frequency of such plasmid was 1000-fold lower.ConclusionThe MobC is an auxiliary transfer protein of dual function. It autoregulates the expression of mobC-nic operon while its presence significantly stimulates transfer efficiency.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0235-1) contains supplementary material, which is available to authorized users.

Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family)

“…The first one, IR-SnaBI-1 (RA3 coordinates 2075 to 2090) is located in the divergent orf02p/orf02prev promoter region (20) between the replication module and orf02, the first ORF of unknown function in the maintenance region (10). The previously described IRSnaBI-2 palindrome (RA3 coordinates 9707 to 9722), which is integral to parS, is adjacent to the promoter of the mobC-nic operon (conjugative module) (19) and close to the nick site in the oriT region. The third conserved motif, IR-SnaBI-3 (RA3 coordinates 23579 to 23594), localizes within orf25, of unknown function, in the conjugative transfer module (18).…”

“…2A). The promoter activity of those constructs together with the three transcriptional fusions in pPDB11.18 (orf02p-xylE), pMWB6.6 (orf02prev-xylE), and pJSB7.9 (mobCp-xylE) (19) was studied in exponentially growing cultures. The three previously identified promoters showed fairly diverse activity (orf02p, 5.5 U of XylE activity; orf02prev, 1.6 U; and mobCp, 0.5 U), and the putative orf23 promoter showed an intermediate activity of 1.8 U.…”

“…2A) and overlapping the predicted transcription start site (TSS) for orf02prev (20). The mobC promoter has been shown to be shut off by MobC binding to an imperfect palindrome O M overlapping both the Ϫ35 and Ϫ10 motifs of mobCp (19). Such a strong regulation of those promoters by dedicated repressors precluded direct studies of their cooperativity with KorB, a weak repressor.…”

“…Its DNA sequence has been established (GenBank accession no. DQ401103) (10), and individual functional modules have been analyzed (18,19). The plasmid encodes a number of autoregulators, i.e., RepA and RepB in the replication module (10), KfrA and KorA in the maintenance module (18,20), and MobC in the conjugative transfer unit (19).…”

“…DQ401103) (10), and individual functional modules have been analyzed (18,19). The plasmid encodes a number of autoregulators, i.e., RepA and RepB in the replication module (10), KfrA and KorA in the maintenance module (18,20), and MobC in the conjugative transfer unit (19). KorC, encoded in the stability module, plays the role of a global transcriptional regulator controlling replication, maintenance, and conjugative transfer functions (10,20).…”