Transcriptional Regulation of ompC by Calcium Chloride May Involve envZ

The standard method of preparing chemically competent E. coli involves two steps: (1) resuspension and incubation of cells in ice-cold calcium chloride and (2) short heat-shock at 42oC. Exactly how DNA crosses the cell wall and enters the cytoplasm during this treatment is not understood. DNA entry may be expedited through lipid permeabilization and pore production in the outer membrane. The channel formed by the porin OmpC has also been proposed as a route of DNA entry. Differential expression of OmpC is regulated by transcription factor OmpR, which becomes phosphorylated by EnvZ in response to environmental changes, such as osmolarity. In high-osmolarity conditions, OmpC production is upregulated, while in low osmolarity, expression is suppressed. Overproduction of OmpC in CaCl2-mediated transformation has previously been reported. Based on this observation, we hypothesized that ompC transcription would also increase in response to increasing CaCl2 concentrations. To test this hypothesis, we used RT-qPCR to measure ompC transcription in E. coli treated with calcium chloride. Results showed a slight increase of ompC transcription following exposure to calcium chloride in E. coli strain BW 25113. In ΔenvZ strain JW 3367-3, ompC transcription did not increase following treatment with calcium chloride. This may indicate that the EnvZ-OmpR pathway is involved in the regulation of ompC in response to calcium chloride.