1.
Using Light-Activated Enzymes for Modulating Intracellular c-di-GMP Levels in Bacteria.
Abstract:
Signaling pathways involving second messenger c-di-GMP regulate various aspects of bacterial physiology and behavior. We describe the use of a red light-activated diguanylate cyclase (c-di-GMP synthase) and a blue light-activated c-di-GMP phosphodiesterase (hydrolase) for manipulating intracellular c-di-GMP levels in bacterial cells. We illustrate the application of these enzymes in regulating several c-di-GMP-dependent phenotypes, i.e., motility and biofilm phenotypes in E. coli and chemotactic behavior in the alphaproteobacterium Azospirillum brasilense. We expect these light-activated enzymes to be also useful in regulating c-di-GMP-dependent processes occurring at the fast timescale, for spatial control of bacterial populations, as well as for analyzing c-di-GMP-dependent phenomena at the single-cell level.
2.
Optogenetic manipulation of c-di-GMP levels reveals the role of c-di-GMP in regulating aerotaxis receptor activity in Azospirillum brasilense.
Abstract:
Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger, c-di-GMP, as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense, c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared light-regulated diguanylate cyclase and a blue-light regulated c-di-GMP phosphodiesterase. It allows generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the timescale of chemotaxis signaling. We provide experimental evidence that c-di-GMP binding to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense changes with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state.IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and modulates their contribution to the overall chemotaxis response. Here, we used an optogenetic system to perturb intracellular concentrations of the bacterial second messenger, c-di-GMP, to show that in some chemotaxis receptors, c-di-GMP functions in a similar feedback loop to connect metabolic status of the cells to sensory activity of chemotaxis receptors.
