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

Jolanta Godziszewska,Anna Kulińska,Grażyna Jagura-Burdzy

doi:10.1186/s12866-014-0235-1

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.

Highlights

The IncU conjugative transfer module represents highly efficient promiscuous system widespread among conjugative plasmids of different incompatibility groups
The homologs of MobC from IncU plasmids (Figure 2B) have been found encoded in the promiscuous plasmids from PromA group [25], by loci positioned in the junction region between maintenance and conjugative transfer operons that display similar genetic organization as IncU plasmids [43]
A characteristic pattern of alternating hydrophilic-hydrophobic side chains is present along N-terminal β-strand with hydrophobic side chains at conserved positions 3, 5, and 7 (Figure 2A) while a positively charged residue usually occurs at position 2 or 6

Summary

Introduction

The IncU conjugative transfer module represents highly efficient promiscuous system widespread among conjugative plasmids of different incompatibility groups. We analyzed the putative transfer auxiliary protein MobC of the conjugative plasmid RA3 from the IncU incompatibility group. In Gram-negative bacteria the conjugation functions comprise processing of DNA for transfer (Dtr) nicking activity, stimulate ATPase activity of the coupling protein and act as transcriptional regulators for conjugative transfer operons [8,9,10,11,12,13,14,15,16,17,18,19]. The broad-host-range conjugative plasmid RA3, the archetype of the IncU incompatibility group, has been isolated from the fish pathogen Aeromonas hydrophila as the determinant of its antibiotic resistance [20]. It was confirmed that when an intergenic fragment of 417 bp containing putative oriTRA3 (Figure 1B) was cloned into pUC18 [23], it facilitated plasmid mobilization by the RA3 with a high frequency [21]

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Conclusion