Reversible switching and stability of the epigenetic memory system in bacteria

Abstract

In previous work, we have developed a DNA methylation-based epigenetic memory system that operates in Escherichia coli to detect environmental signals, trigger a phenotypic switch of the cells and store the information in DNA methylation. The system is based on the CcrM DNA methyltransferase and a synthetic zinc finger (ZnF4), which binds DNA in a CcrM methylation-dependent manner and functions as a repressor for a ccrM gene expressed together with an egfp reporter gene. Here, we developed a reversible reset for this memory system by adding an increased concentration of ZnSO 4 to the bacterial cultivation medium and demonstrate that one bacterial culture could be reversibly switched ON and OFF in several cycles. We show that a previously developed differential equation model of the memory system can also describe the new data. Then, we studied the long-term stability of the ON-state of the system over approximately 100 cell divisions showing a gradual loss of ON-state signal starting after 4 days of cultivation that is caused by individual cells switching from an ON-into the OFF-state. Over time, the methylation of the ZnF4-binding sites is not fully maintained leading to an increased OFF switching probability of cells, because stronger binding of ZnF4 to partially demethylated operator sites leads to further reductions in the cellular concentrations of CcrM. These data will support future design to further stabilize the ONstate and enforce the binary switching behaviour of the system. Together with the development of a reversible OFF switch, our new findings strongly increase the capabilities of bacterial epigenetic biosensors. AbbreviationsCcrM, Caulobacter crescentus cell-cycle-regulated methyltransferase; ZnF, synthetic zinc finger protein; ZnF4, homotetramer of the ZnF protein.

Microbial Synthetic Epigenetic Tools Design and Applications

“…However, after 4 days of culture, a progressive loss of signal stability appeared, demonstrating that the stability issue still existed over longterm signal suppression. 72 To achieve controllable cellular phenotypic variations in the future, computer-aided modeling can predict the time scale of memory propagation and memory loss. 12 In addition, small molecule-inducible tools can save considerable effort to isolate tools functioning from the host machinery and restrict crosstalk with existing components throughout their development, allowing for the design of orthogonal epigenetic memory systems and stable regulatory tools.…”

“…An upgraded version of this memory switch that provides a reversible reset for this memory system and demonstrates how one bacterial culture may be reversibly switched ON and OFF across approximately 100 cell divisions was made by adding a greater concentration of ZnSO 4 to the bacterial growth medium. However, after 4 days of culture, a progressive loss of signal stability appeared, demonstrating that the stability issue still existed over long-term signal suppression …”