IncC plasmids are a medically important group of broad-host-range plasmids that facilitate the spread of antibiotic resistance genes in human enteric pathogens. In particular, these plasmids are linked to the emergence of carbapenem-resistant Enterobacteriaceae and their carriage of genes encoding metallo-β-lactamases with carbapenemase activity.
Despite their impact on antibiotic resistance, our understanding of the genetics of IncC-conjugation is limited. Here, we utilised hyper-saturated transposon mutagenesis coupled with transposon directed insertion site sequencing (TraDIS) to determine the set of genes required for IncC-conjugation. Overall, 27 genes were identified, including all 17 predicted and known conjugation genes, two regulatory genes (acaDC) and eight genes not previously associated with conjugation. Targeted mutagenesis of the eight novel genes confirmed a significant role for five of these genes in conjugation.
We show that one gene, acaB, encodes a novel regulator that plays a central role in IncC regulation circuitry. AcaB activates conjugation via binding to a 13bp sequence upstream of the acaDC promoter and upregulates acaDC transcription, a controlling element that in-turn activates transcription of genes involved in IncC-conjugation. The crystal structure of AcaB revealed a new protein fold belonging to the ribbon-helix-helix (RHH) superfamily, based on two β-α-α units folding into a 4-helix bundle and an exposed two-stranded b-sheet. Functional analyses guided by these structural studies determined that the mechanism of AcaB binding to DNA occurs via the β-sheet region.
Overall, we have defined the specific regulatory mechanism of a novel element that is central to the control of conjugative transfer of IncC multidrug resistance plasmids.