Abstract
Cyclic nucleoside monophosphates (cNMP) serve as universal second messengers in signal transduction across prokaryotes and eukaryotes. As signaling often relies on transiently formed microdomains of elevated second messenger concentration, means to precisely perturb the spatiotemporal dynamics of cNMPs are uniquely poised for the interrogation of the underlying physiological processes. Optogenetics appears particularly suited as it affords light-dependent, accurate control in time and space of diverse cellular processes. Several sensory photoreceptors function as photoactivated adenylyl cyclases (PAC) and hence serve as light-regulated actuators for the control of intracellular levels of 3′, 5′-cyclic adenosine monophosphate. To characterize PACs and to refine their properties, we devised a test bed for the facile analysis of these photoreceptors. Cyclase activity is monitored in bacterial cells via expression of a fluorescent reporter, and programmable illumination allows the rapid exploration of multiple lighting regimes. We thus probed two PACs responding to blue and red light, respectively, and observed significant dark activity for both. We next engineered derivatives of the red-light-sensitive PAC with altered responses to light, with one variant, denoted DdPAC, showing enhanced response to light. These PAC variants stand to enrich the optogenetic toolkit and thus facilitate the detailed analysis of cNMP metabolism and signaling.
Acknowledgments
We thank members of the Möglich laboratory for discussions, Dr. Manuela Stierl for providing the pBADM-30-bPAC plasmid, Dr. Tilo Mathes for advice on generating the CmpX13 ΔcyaA knockout strain, Norbert Grillenbeck for technical support, and Dr. Markus Lippitz for advice on the design of the LED matrix. We are indebted to the electrical workshop at the University of Bayreuth for circuit-board design and overall stellar support. Funding through Boehringer-Ingelheim Fonds (R.O.), funder id: 10.13039/100005156, a Sofja-Kovalevskaya Award (to A.M.) by the Alexander-von-Humboldt Foundation, and Deutsche Forschungsgemeinschaft (funder id: 10.13039/501100001659, grant MO21921/4-1) is gratefully acknowledged.
References
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