Abstract
In bacteria, cyclic adenosine monophosphate (cAMP) signaling plays an essential regulatory role whose modulation via optogenetic tools would provide researchers an immense opportunity to control biological processes simply by illumination. The cAMP signaling in bacteria is a complex network of regulatory pathways, which utilizes distinct proteomic resources under different nutrient environments. We established an optogenetic modulation of cAMP and studied important cellular process of growth, biofilm formation, and virulence in the model bacterium E. coli using a light-gated adenylate cyclase (LgAC) from Beggiatoa sp. Blue light-induced activation of LgAC elevated the cAMP level in a blue light-dependent manner in E. coli. Quantitative proteomics revealed a decrease in the level of certain proteins governing growth (PTS, Adk, AckA, GlnA, and EFP), biofilm formation (IhfA, flagellin, YajQ, YeaG, and HlfC), and virulence (ClpP, YebC, KatE, BtuE, and Zur) in E. coli cells expressing LgAC upon blue light illumination. This optogenetic modulation of cAMP would be useful for deciphering cAMP-associated host–pathogen signaling of bacterial systems. Proteome knowledge established by this research work would also be useful for the scientific community while adapting LgAC-based optogenetic modulation for studying other relevant cAMP-driven bacterial physiology (e.g., energy metabolism). The systematic utilization of the established method and more extensively designed experiments regarding bacterial growth, biofilm, survival, and virulence might provide a road map for the identification of new targets for developing novel antibacterial drugs.
Highlights
Cyclic adenosine monophosphate is a secondary messenger, which plays a central role in a myriad of signaling processes and is synthesized by a specific membrane or soluble protein, adenylate cyclase
Was selected as the photoactivated adenylate cyclases (PACs) to be used as an optogenetic tool for the regulation of cyclic adenosine monophosphate (cAMP)-dependent bacterial physiological processes
To study the impact of light-gated adenylate cyclase on cAMP-dependent processes in bacteria, the heterologous expression of recombinant LgAC was induced in E. coli
Summary
Cyclic adenosine monophosphate (cAMP) is a secondary messenger, which plays a central role in a myriad of signaling processes and is synthesized by a specific membrane or soluble protein, adenylate cyclase This secondary messenger coordinates complex signaling by controlling intracellular metabolites like fructose 1,6-bisphosphate, phosphoenolpyruvate, acetyl-Coenzyme A, α-ketoglutaric acid, pyruvate, and oxaloacetate, known to be essential for enzyme-level regulation, transcriptional control, and nutrient homeostasis [1,2]. In 2007, Euglena PAC (ePAC) was expressed in a cell model system as well as in transgenic animals, and the cAMP level was manipulated by illumination with blue light [15,18] These large-sized (110 kDa) PACs showed significant cyclase activity in the dark. A label-free quantitative proteomic approach was adapted to analyze the light-induced cAMP-dependent variations in the proteome regulating growth, biofilm formation, and virulence in E. coli
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