Enterococcal pheromone responsive conjugative plasmids like pCF10 promote horizontal spread of antibiotic resistance genes following induction of plasmid-containing cells by potential recipients. Transcription of conjugation genes from promoter PQ is inhibited by the master regulator PrgX, further repressed when PrgX is in complex with the inhibitory I peptide, and allowed when PrgX is in complex with the C inducing peptide. Single-cell analysis has shown that heterogeneity in the pheromone response is prevalent. Here, we systematically varied levels of regulatory molecules to better understand why some individual cells have increased propensity for induction. In this study, PrgX was confirmed to repress PQ in the absence of exogenous peptides in vivo, but cells with increased levels of PrgX were shown to be more prone to induction. Further, ablation of endogenous I reduced PrgX levels, resulting in reduced basal repression and loss of inducibility. Reduction of both endogenous peptides by washing increased the inducibility of cells. Together, these results show that endogenous PrgX, C, and I levels can impact the induction potential of a cell and establish the importance of basal I for regulation. These results also suggest that PrgX/C complexes may directly activate prgQ transcription, contrary to a long-standing working model.
Bibliographical noteFunding Information:
We thank Arpan Bandyopadhyay for valuable discussions and help building the model figures and Aaron Barnes for microscopy assistance. These studies were supported by NIH grant R35GM118079 (G.M.D.). R.J.B.E. was supported in part by predoctoral traineeships under T32-GM008347 and T90-DE0227232. Computational resources were provided by the Minnesota Supercomputing Institute (MSI) at the University of Minnesota (http://www.msi.umn.edu). The data that support the findings of this study are available from the corresponding author upon reasonable request.
© 2019 John Wiley & Sons Ltd