Abstract
We describe a portable epigenetic switch based on opvAB, a Salmonella enterica operon that undergoes bistable expression under DNA methylation control. A DNA fragment containing the opvAB promoter and the opvAB upstream regulatory region confers bistability to heterologous genes, yielding OFF and ON subpopulations. Bistable expression under opvAB control is reproducible in Escherichia coli, showing that the opvAB switch can be functional in a heterologous host. Subpopulations of different sizes can be produced at will using engineered opvAB variants. Controlled formation of antibiotic-resistant and antibiotic-susceptible subpopulations may allow use of the opvAB switch in the study of bacterial heteroresistance to antibiotics.
Highlights
We describe a portable epigenetic switch based on opvAB, a Salmonella enterica operon that undergoes bistable expression under DNA methylation control
Www.nature.com/scientificreports promoter and regulatory region, with the GATC sites outlined. (B) Diagram of the wild type opvAB operon and the PopvAB::lacZY construct. (C) Colonies formed on lysogeny broth (LB) + X-gal by a S. enterica strain carrying the lacZY operon under the control of the wild type opvAB control region (SV9700, left), and by S. enterica strains carrying the lacZY operon under the control of mutant opvAB control regions (SV9701, PopvAB GATC1,2::lacZY::gfp, center; SV9702, PopvAB GATC-less::lacZY::gfp, right). (D) Flow cytometry analysis of PopvAB::lacZY expression in strains SV9700, SV9701 and SV9702
The ability of the opvAB epigenetic switch to confer bistable expression to a heterologous locus was tested by engineering a strain that harbored the E. coli lacZY operon downstream of the opvAB promoter and its upstream regulatory region (PopvAB - Fig. 1A,B)
Summary
We describe a portable epigenetic switch based on opvAB, a Salmonella enterica operon that undergoes bistable expression under DNA methylation control. A common type of microbial biosensor is an engineered strain that responds to physical or chemical inputs generating electrochemical or optical signals Sensors of this type often employ a promoter sensitive to a specific input and a reporter gene that produces a detectable signal[1,3]. We show that a cassette of 689 nucleotides containing the opvAB promoter and the upstream regulatory region confers bistability to heterologous genes, and describe examples of opvAB-based constructs that produce bacterial subpopulations with distinct phenotypes. One of the examples involves formation of an antibiotic-resistant subpopulation upon cloning of an antibiotic resistance gene downstream of the opvAB promoter This construct may provide an experimental system to study bacterial heteroresistance (HR) to antibiotics under highly controlled conditions[12]. Error bars represent the standard error of the mean from 3 independent replicates. (F) Reversible formation of LacOFF and LacON subpopulations under opvAB control in strain SV9700
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