Optogenetic Module for Dichromatic Control of c-di-GMP Signaling.
Many aspects of bacterial physiology and behavior, including motility, surface attachment, and the cell cycle, are controlled by cyclic di-GMP (c-di-GMP)-dependent signaling pathways on the scale of seconds to minutes. Interrogation of such processes in real time requires tools for introducing rapid and reversible changes in intracellular c-di-GMP levels. Inducing the expression of genes encoding c-di-GMP-synthetic (diguanylate cyclases) and -degrading (c-di-GMP phosphodiesterase) enzymes by chemicals may not provide adequate temporal control. In contrast, light-controlled diguanylate cyclases and phosphodiesterases can be quickly activated and inactivated. A red/near-infrared-light-regulated diguanylate cyclase, BphS, was engineered previously, yet a complementary light-activated c-di-GMP phosphodiesterase has been lacking. In search of such a phosphodiesterase, we investigated two homologous proteins fromAllochromatium vinosumandMagnetococcus marinus, designated BldP, which contain C-terminal EAL-BLUF modules, where EAL is a c-di-GMP phosphodiesterase domain and BLUF is a blue light sensory domain. Characterization of the BldP proteins inEscherichia coliandin vitroshowed that they possess light-activated c-di-GMP phosphodiesterase activities. Interestingly, light activation in both enzymes was dependent on oxygen levels. The truncated EAL-BLUF fragment fromA. vinosumBldP lacked phosphodiesterase activity, whereas a similar fragment fromM. marinusBldP, designated EB1, possessed such activity that was highly (>30-fold) upregulated by light. Following light withdrawal, EB1 reverted to the inactive ground state with a half-life of ∼6 min. Therefore, the blue-light-activated phosphodiesterase EB1 can be used in combination with the red/near-infrared-light-regulated diguanylate cyclase BphS for the bidirectional regulation of c-di-GMP-dependent processes inE. colias well as other bacterial and nonbacterial cells.IMPORTANCERegulation of motility, attachment to surfaces, the cell cycle, and other bacterial processes controlled by the c-di-GMP signaling pathways occur at a fast (seconds-to-minutes) pace. Interrogation of these processes at high temporal and spatial resolution using chemicals is difficult or impossible, while optogenetic approaches may prove useful. We identified and characterized a robust, blue-light-activated c-di-GMP phosphodiesterase (hydrolase) that complements a previously engineered red/near-infrared-light-regulated diguanylate cyclase (c-di-GMP synthase). These two enzymes form a dichromatic module for manipulating intracellular c-di-GMP levels in bacterial and nonbacterial cells.
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, USA.
Min-Hyung
MH
Ryu
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, USA.
Anastasia
A
Fomicheva
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, USA.
Oleg V
OV
Moskvin
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming, USA gomelsky@uwyo.edu.
Mark
M
Gomelsky
D020561:Alphaproteobacteria / Q000201:enzymology / Q000235:genetics; D002841:Chromatiaceae / Q000201:enzymology / Q000235:genetics; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D005827:Genetics, Microbial / Q000379:methods*; D008027:Light; D062308:Optogenetics / Q000379:methods*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D011994:Recombinant Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D010727:Phosphoric Diester Hydrolases; D006152:Cyclic GMP
biofilms; c-di-GMP; diguanylate cyclase; gene expression; motility; optogenetics; phosphodiesterase; photoreceptors; photosensory reception; signal transduction