Construction and application of a dual fluorescence reporter plasmid for measuring c-di-GMP levels in Escherichia coli

Abstract:

Bacteria modulate intracellular concentrations of the second messenger cyclic dimeric guanosine monophosphate (c-di-GMP), which facilitates the adaptation to environments, survival, and infection. [Objective] To establish an effective method for measuring the c-di-GMP levels in Escherichia coli. [Methods] We designed the primers for the dual fluorescence reporter plasmid based on the regulation pattern of the riboswitch receptor by c-di-GMP and the fluorescence reporter genes. The dual fluorescence reporter plasmid pAmCherry-Vc2EGFP (pACVcE) was constructed by overlap polymerase chain reaction (overlap PCR) and homologous recombination. Then, we constructed the mutants overexpressing and lacking the genes involved in c-di-GMP metabolism, and used pACVcE to measure the c-di-GMP levels in Escherichia coli. [Results] The targeted genes were successfully amplified with correct sequences. The intracellular c-di-GMP levels in Escherichia coli expressing the c-di-GMP synthase DgcZ were significantly increased, while the intracellular c-di-GMP levels expressing the c-di-GMP phosphodiesterase PdeK were significantly decreased. The deletion of the pdeK gene encoding c-di-GMP phosphodiesterase elevated the level of c-di-GMP in avian pathogenic Escherichia coli. [Conclusion] We constructed a dual fluorescence reporter plasmid based on riboswitch, which can facilitate the rapid determination of c-di-GMP levels in Escherichia coli.