Optogenetic Control of Bacterial Expression by Red Light

In optogenetics, as in nature, sensory photoreceptors serve to control cellular processes by light. Bacteriophytochrome (BphP) photoreceptors sense red and farred light via a biliverdin chromophore and, in response, cycle between the spectroscopically, structurally, and functionally distinct Pr and Pfr states. BphPs commonly belong to two-component systems that control the phosphorylation of cognate response regulators and downstream gene expression through histidine kinase modules. We recently demonstrated that the paradigm BphP from Deinococcus radiodurans exclusively acts as a phosphatase but that its photosensory module can control the histidine kinase activity of homologous receptors. Here, we apply this insight to reprogram two widely used setups for bacterial gene expression from blue-light to red-light control. The resultant pREDusk and pREDawn systems allow gene expression to be regulated down and up, respectively, uniformly under red light by 100-fold or more. Both setups are realized as portable, single plasmids that encode all necessary components including the biliverdin-producing machinery. The triggering by red light affords high spatial resolution down to the single-cell level. As pREDusk and pREDawn respond sensitively to red light, they support multiplexing with optogenetic systems sensitive to other light colors. Owing to the superior tissue penetration of red light, the pREDawn system can be triggered at therapeutically safe light intensities through material layers, replicating the optical properties of the skin and skull. Given these advantages, pREDusk and pREDawn enable red-light-regulated expression for diverse use cases in bacteria.Peer reviewe

Femtosecond Stimulated Raman Study of the Photoactive Flavoprotein AppABLUF

Femtosecond stimulated Raman Spectroscopy (FSRS) is applied to study the photocycle of a blue light using flavin (BLUF) domain photoreceptor, AppABLUF. It is shown that FSRS spectra are sensitive to the light adapted state of the protein and probe its excited state dynamics. The dominant contribution to the most sensitive excited state Raman active modes is from flavin ring modes. However, TD-DFT calculations for excited state structures indicate that reproduction and assignment of the experimentally observed spectral shift will require high level calculations on the flavin in its specific protein environment

Zapomniane miasto, wieś i dwór Zwanowo. Przyczynek do badań nad późnośredniowiecznym i nowożytnym zespołem osadniczym w Niedźwiedzinach (przysiółek Dzwonowo), pow. wągrowiecki, woj. Wielkopolskie

A Forgotten Town, the Village and Manor Zwanowo. Contribution to the Studies into Late Mediaeval and Modern Settlement Complex in Niedźwiedziny (Hamlet Dzwonowo), Administrative District Wągrowiec, Great Poland. The article presents a preliminary interpretation of vegetation dissimilarities recognized due to the analysis of an orthophotomap published on the Internet site www.geoportal.gov.pl. The dissimilarities in question, visible in the grounds of the village of Niedźwiedziny (hamlet Dzwonowo), administrative district Wągrowiec, Great Poland (Wielkopolska), have been construed as the remains of a unique mediaeval settlement complex: oval-shaped village, chartered town and a mediaeval (?) and modern manor. During visits to the abovementioned sites, the area of the old churchyard cemetery was traced. On the basis of the written sources, it has been established that Dzwonowo was the main centre of the joint properties owned by Dobrogost – Poznań voivode, and subsequently his descendants

Full Length Structure of a Sensor Histidine Kinase Pinpoints Coaxial Coiled Coils as Signal Transducers and Modulators

SummaryTwo-component systems (TCSs), which comprise sensor histidine kinases (SHK) and response-regulator proteins, represent the predominant strategy by which prokaryotes sense and respond to a changing environment. Despite paramount biological importance, a dearth exists of intact SHK structures containing both sensor and effector modules. Here, we report the full-length crystal structure of the engineered, dimeric, blue-light-regulated SHK YF1 at 2.3 Å resolution, in which two N-terminal light-oxygen-voltage (LOV) photosensors are connected by a coiled coil to the C-terminal effector modules. A second coaxial coiled coil derived from the N-termini of the LOV photosensors and inserted between them crucially modulates light regulation: single mutations within this coiled coil attenuate or even invert the signal response of the TCS. Structural motifs identified in YF1 recur in signal receptors, and the underlying signaling principles and mechanisms may be widely shared between soluble and transmembrane, prokaryotic, and eukaryotic signal receptors of diverse biological activity

Engineering of temperature- and light-switchable Cas9 variants

Sensory photoreceptors have enabled non-invasive and spatiotemporal control of numerous biological processes. Photoreceptor engineering has expanded the repertoire beyond natural receptors, but to date no generally applicable strategy exists towards constructing light-regulated protein actuators of arbitrary function. We hence explored whether the homodimeric Rhodobacter sphaeroides light-oxygen-voltage (LOV) domain (RsLOV) that dissociates upon blue-light exposure can confer light sensitivity onto effector proteins, via amechanism of light-induced functional site release. We chose the RNA-guided programmable DNA endonuclease Cas9 as proof-of-principle effector, and constructed a comprehensive library of RsLOV inserted throughout the Cas9 protein. Screening with a high-throughput assay based on transcriptional repression in Es-cherichia coli yielded paRC9, a moderately light-activatable variant. As domain insertion can lead to protein destabilization, we also screened the library for temperature-sensitive variants and isolated tsRC9, a variant with robust activity at 29 degrees C but negligible activity at 37. C. Biochemical assays confirmed temperature-dependent DNA cleavage and binding for tsRC9, but indicated that the light sensitivity of paRC9 is specific to the cellular setting. Using tsRC9, the first temperature-sensitive Cas9 variant, we demonstrate temperature-dependent transcriptional control over ectopic and endogenous genetic loci. Taken together, RsLOV can confer light sensitivity onto an unrelated effector; unexpectedly, the same LOV domain can also impart strong temperature sensitivity

Optogenetic Control of Bacterial Expression by Red Light

In optogenetics, as in nature, sensory photoreceptors serve to control cellular processes by light. Bacteriophytochrome (BphP) photoreceptors sense red and farred light via a biliverdin chromophore and, in response, cycle between the spectroscopically, structurally, and functionally distinct Pr and Pfr states. BphPs commonly belong to two-component systems that control the phosphorylation of cognate response regulators and downstream gene expression through histidine kinase modules. We recently demonstrated that the paradigm BphP from Deinococcus radiodurans exclusively acts as a phosphatase but that its photosensory module can control the histidine kinase activity of homologous receptors. Here, we apply this insight to reprogram two widely used setups for bacterial gene expression from blue-light to red-light control. The resultant pREDusk and pREDawn systems allow gene expression to be regulated down and up, respectively, uniformly under red light by 100-fold or more. Both setups are realized as portable, single plasmids that encode all necessary components including the biliverdin-producing machinery. The triggering by red light affords high spatial resolution down to the single-cell level. As pREDusk and pREDawn respond sensitively to red light, they support multiplexing with optogenetic systems sensitive to other light colors. Owing to the superior tissue penetration of red light, the pREDawn system can be triggered at therapeutically safe light intensities through material layers, replicating the optical properties of the skin and skull. Given these advantages, pREDusk and pREDawn enable red-light-regulated expression for diverse use cases in bacteria.Peer reviewe