Plant Phytochrome Interactions Decode Light and Temperature Signals.

Plant phytochromes perceive red and far-red light to elicit adaptations to the changing environment. Downstream physiological responses revolve around red-light-induced interactions with phytochrome-interacting factors (PIF). Phytochromes double as thermoreceptors, owing to the pronounced temperature dependence of thermal reversion from the light-adapted Pfr to the dark-adapted Pr state. Here, we assess whether thermoreception may extend to the phytochrome:PIF interactions. While the association between Arabidopsis (Arabidopsis thaliana) PHYTOCHROME B (PhyB) and several PHYTOCHROME-INTERACTING FACTOR (PIF) variants moderately accelerates with temperature, the dissociation does more so, thus causing net destabilization of the phytochrome:PIF complex. Markedly different temperature profiles of PIF3 and PIF6 might underlie stratified temperature responses in plants. Accidentally, we identify a photoreception mechanism under strong continuous light, where the extent of phytochrome:PIF complexation decreases with red-light intensity rather than increases. Mathematical modeling rationalizes this attenuation mechanism and ties it to rapid red-light-driven Pr⇄Pfr interconversion and complex dissociation out of Pr. Varying phytochrome abundance, e.g., during diurnal and developmental cycles, and interaction dynamics, e.g., across different PIFs, modify the nature and extent of attenuation, thus permitting light-response profiles more malleable than possible for the phytochrome Pr⇄Pfr interconversion alone. Our data and analyses reveal a photoreception mechanism with implications for plant physiology, optogenetics, and biotechnological applications.

Simultaneous spectral illumination of microplates for high-throughput optogenetics and photobiology.

The biophysical characterization and engineering of optogenetic tools and photobiological systems has been hampered by the lack of efficient methods for spectral illumination of microplates for high-throughput analysis of action spectra. Current methods to determine action spectra only allow the sequential spectral illumination of individual wells. Here we present the open-source RainbowCap-system, which combines LEDs and optical filters in a standard 96-well microplate format for simultaneous and spectrally defined illumination. The RainbowCap provides equal photon flux for each wavelength, with the output of the LEDs narrowed by optical bandpass filters. We validated the RainbowCap for photoactivatable G protein-coupled receptors (opto-GPCRs) and enzymes for the control of intracellular downstream signaling. The simultaneous, spectrally defined illumination provides minimal interruption during time-series measurements, while resolving 10 nm differences in the action spectra of optogenetic proteins under identical experimental conditions. The RainbowCap is also suitable for studying the spectral dependence of light-regulated gene expression in bacteria, which requires illumination over several hours. In summary, the RainbowCap provides high-throughput spectral illumination of microplates, while its modular, customizable design allows easy adaptation to a wide range of optogenetic and photobiological applications.

Induction of bacterial expression at the mRNA level by light.

Vital organismal processes, including development, differentiation and adaptation, involve altered gene expression. Although expression is frequently controlled at the transcriptional stage, various regulation mechanisms operate at downstream levels. Here, we leverage the photoreceptor NmPAL to optogenetically induce RNA refolding and the translation of bacterial mRNAs. Blue-light-triggered NmPAL binding disrupts a cis-repressed mRNA state, thereby relieves obstruction of translation initiation, and upregulates gene expression. Iterative probing and optimization of the circuit, dubbed riboptoregulator, enhanced induction to 30-fold. Given action at the mRNA level, the riboptoregulator can differentially regulate individual structural genes within polycistronic operons. Moreover, it is orthogonal to and can be wed with other gene-regulatory circuits for nuanced and more stringent gene-expression control. We thus advance the pAurora2 circuit that combines transcriptional and translational mechanisms to optogenetically increase bacterial gene expression by >1000-fold. The riboptoregulator strategy stands to upgrade numerous regulatory circuits and widely applies to expression control in microbial biotechnology, synthetic biology and materials science.

Reduction midpoint potential of a paradigm light-oxygen-voltage receptor and its modulation by methionine residues.

Light-dependent adaptations of organismal physiology, development, and behavior abound in nature and depend on sensory photoreceptors. As one class, light-oxygen-voltage (LOV) photoreceptors harness flavin-nucleotide chromophores to sense blue light. Photon absorption drives the LOV receptor to its signaling state, characterized by a metastable thioadduct between the flavin and a conserved cysteine residue. With this cysteine absent, LOV receptors instead undergo photoreduction to the flavin semiquinone which however can still elicit downstream physiological responses. Irrespective of the cysteine presence, the LOV photochemical response thus entails a formal reduction of the flavin. Against this backdrop, we here investigate the reduction midpoint potential E 0 in the paradigmatic LOV2 domain from Avena sativa phototropin 1 (AsLOV2), and how it can be deliberately varied. Replacements of residues at different sites near the flavin by methionine consistently increase E 0 from its value of around -280 mV by up to 40 mV. Moreover, methionine introduction invariably impairs photoactivation efficiency and thus renders the resultant AsLOV2 variants less light-sensitive. Although individual methionine substitutions also affect the stability of the signaling state and downstream allosteric responses, no clear-cut correlation with the redox properties emerges. With a reduction midpoint potential near -280 mV, AsLOV2 and, by inference, other LOV receptors may be partially reduced inside cells which directly affects their light responsiveness. The targeted modification of the chromophore environment, as presently demonstrated, may mitigate this effect and enables the design of LOV receptors with stratified redox sensitivities.

Leveraging the histidine kinase-phosphatase duality to sculpt two-component signaling.

Bacteria must constantly probe their environment for rapid adaptation, a crucial need most frequently served by two-component systems (TCS). As one component, sensor histidine kinases (SHK) control the phosphorylation of the second component, the response regulator (RR). Downstream responses hinge on RR phosphorylation and can be highly stringent, acute, and sensitive because SHKs commonly exert both kinase and phosphatase activity. With a bacteriophytochrome TCS as a paradigm, we here interrogate how this catalytic duality underlies signal responses. Derivative systems exhibit tenfold higher red-light sensitivity, owing to an altered kinase-phosphatase balance. Modifications of the linker intervening the SHK sensor and catalytic entities likewise tilt this balance and provide TCSs with inverted output that increases under red light. These TCSs expand synthetic biology and showcase how deliberate perturbations of the kinase-phosphatase duality unlock altered signal-response regimes. Arguably, these aspects equally pertain to the engineering and the natural evolution of TCSs.

Multimodal Control of Bacterial Gene Expression by Red and Blue Light.

By applying sensory photoreceptors, optogenetics realizes the light-dependent control of cellular events and state. Given reversibility, noninvasiveness, and exquisite spatiotemporal precision, optogenetic approaches enable innovative use cases in cell biology, synthetic biology, and biotechnology. In this chapter, we detail the implementation of the pREDusk, pREDawn, pCrepusculo, and pAurora optogenetic circuits for controlling bacterial gene expression by red and blue light, respectively. The protocols provided here guide the practical use and multiplexing of these circuits, thereby enabling graded protein production in bacteria at analytical and semi-preparative scales.

An RNA Motif That Enables Optozyme Control and Light-Dependent Gene Expression in Bacteria and Mammalian Cells.

The regulation of gene expression by light enables the versatile, spatiotemporal manipulation of biological function in bacterial and mammalian cells. Optoribogenetics extends this principle by molecular RNA devices acting on the RNA level whose functions are controlled by the photoinduced interaction of a light-oxygen-voltage photoreceptor with cognate RNA aptamers. Here light-responsive ribozymes, denoted optozymes, which undergo light-dependent self-cleavage and thereby control gene expression are described. This approach transcends existing aptamer-ribozyme chimera strategies that predominantly rely on aptamers binding to small molecules. The optozyme method thus stands to enable the graded, non-invasive, and spatiotemporally resolved control of gene expression. Optozymes are found efficient in bacteria and mammalian cells and usher in hitherto inaccessible optoribogenetic modalities with broad applicability in synthetic and systems biology.

Engineering Bacteriophytochrome-coupled Photoactivated Adenylyl Cyclases for Enhanced Optogenetic cAMP Modulation.

Sensory photoreceptors abound in nature and enable organisms to adapt behavior, development, and physiology to environmental light. In optogenetics, photoreceptors allow spatiotemporally precise, reversible, and non-invasive control by light of cellular processes. Notwithstanding the development of numerous optogenetic circuits, an unmet demand exists for efficient systems sensitive to red light, given its superior penetration of biological tissue. Bacteriophytochrome photoreceptors sense the ratio of red and far-red light to regulate the activity of enzymatic effector modules. The recombination of bacteriophytochrome photosensor modules with cyclase effectors underlies photoactivated adenylyl cyclases (PAC) that catalyze the synthesis of the ubiquitous second messenger 3', 5'-cyclic adenosine monophosphate (cAMP). Via homologous exchanges of the photosensor unit, we devised novel PACs, with the variant DmPAC exhibiting 40-fold activation of cyclase activity under red light, thus surpassing previous red-light-responsive PACs. Modifications of the PHY tongue modulated the responses to red and far-red light. Exchanges of the cyclase effector offer an avenue to further enhancing PACs but require optimization of the linker to the photosensor. DmPAC and a derivative for 3', 5'-cyclic guanosine monophosphate allow the manipulation of cyclic-nucleotide-dependent processes in mammalian cells by red light. Taken together, we advance the optogenetic control of second-messenger signaling and provide insight into the signaling and design of bacteriophytochrome receptors.

Seeing the light brings more food in the deep sea.

Cyclic di-GMP signaling regulates sessile-to-motile lifestyle transition and associated physiological and metabolic features in bacteria. The presence of potential cyclic di-GMP turnover proteins in deepest branching bacteria indicates that cyclic di-GMP is an ancient signaling molecule. In this issue of The EMBO Journal, Cai et al (2023) describe light-induced activation of a thiosulfate oxidation pathway in the deep-sea cold-seep bacterium Qipengyuania flava, thus coupling cyclic di-GMP with the regulation of the global abiotic sulfur cycle.

Investigation of the Thermal Stability of Proteinase K for the Melt Processing of Poly(l-lactide).

The enzymatic degradation of aliphatic polyesters offers unique opportunities for various use cases in materials science. Although evidently desirable, the implementation of enzymes in technical applications of polyesters is generally challenging due to the thermal lability of enzymes. To prospectively overcome this intrinsic limitation, we here explored the thermal stability of proteinase K at conditions applicable for polymer melt processing, given that this hydrolytic enzyme is well established for its ability to degrade poly(l-lactide) (PLLA). Using assorted spectroscopic methods and enzymatic assays, we investigated the effects of high temperatures on the structure and specific activity of proteinase K. Whereas in solution, irreversible unfolding occurred at temperatures above 75-80 °C, in the dry, bulk state, proteinase K withstood prolonged incubation at elevated temperatures. Unexpectedly little activity loss occurred during incubation at up to 130 °C, and intermediate levels of catalytic activity were preserved at up to 150 °C. The resistance of bulk proteinase K to thermal treatment was slightly enhanced by absorption into polyacrylamide (PAM) particles. Under these conditions, after 5 min at a temperature of 200 °C, which is required for the melt processing of PLLA, proteinase K was not completely denatured but retained around 2% enzymatic activity. Our findings reveal that the thermal processing of proteinase K in the dry state is principally feasible, but equally, they also identify needs and prospects for improvement. The experimental pipeline we establish for proteinase K analysis stands to benefit efforts directed to this end. More broadly, our work sheds light on enzymatically degradable polymers and the thermal processing of enzymes, which are of increasing economical and societal relevance.

Light-regulated gene expression in Bacteria: Fundamentals, advances, and perspectives.

Numerous photoreceptors and genetic circuits emerged over the past two decades and now enable the light-dependent i.e., optogenetic, regulation of gene expression in bacteria. Prompted by light cues in the near-ultraviolet to near-infrared region of the electromagnetic spectrum, gene expression can be up- or downregulated stringently, reversibly, non-invasively, and with precision in space and time. Here, we survey the underlying principles, available options, and prominent examples of optogenetically regulated gene expression in bacteria. While transcription initiation and elongation remain most important for optogenetic intervention, other processes e.g., translation and downstream events, were also rendered light-dependent. The optogenetic control of bacterial expression predominantly employs but three fundamental strategies: light-sensitive two-component systems, oligomerization reactions, and second-messenger signaling. Certain optogenetic circuits moved beyond the proof-of-principle and stood the test of practice. They enable unprecedented applications in three major areas. First, light-dependent expression underpins novel concepts and strategies for enhanced yields in microbial production processes. Second, light-responsive bacteria can be optogenetically stimulated while residing within the bodies of animals, thus prompting the secretion of compounds that grant health benefits to the animal host. Third, optogenetics allows the generation of precisely structured, novel biomaterials. These applications jointly testify to the maturity of the optogenetic approach and serve as blueprints bound to inspire and template innovative use cases of light-regulated gene expression in bacteria. Researchers pursuing these lines can choose from an ever-growing, versatile, and efficient toolkit of optogenetic circuits.

Light-Dependent Control of Bacterial Expression at the mRNA Level.

Sensory photoreceptors mediate numerous light-dependent adaptations across organisms. In optogenetics, photoreceptors achieve the reversible, non-invasive, and spatiotemporally precise control by light of gene expression and other cellular processes. The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination. By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest, their expression can be downregulated by light. We developed the pCrepusculo and pAurora optogenetic systems that are based on PAL and allow to down- and upregulate, respectively, bacterial gene expression using blue light. Both systems are realized as compact, single plasmids that exhibit stringent blue-light responses with low basal activity and up to several 10-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that control transcription initiation. By integrating regulatory mechanisms operating at the DNA and mRNA levels, optogenetic circuits with emergent properties can thus be devised. As a case in point, the pEnumbra setup permits to upregulate gene expression under moderate blue light whereas strong blue light shuts off expression again. Beyond providing novel signal-responsive expression systems for diverse applications in biotechnology and synthetic biology, our work also illustrates how the light-dependent PAL-aptamer interaction can be harnessed for the control and interrogation of RNA-based processes.

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 far-red 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.

Signal transduction in light-oxygen-voltage receptors lacking the active-site glutamine.

In nature as in biotechnology, light-oxygen-voltage photoreceptors perceive blue light to elicit spatiotemporally defined cellular responses. Photon absorption drives thioadduct formation between a conserved cysteine and the flavin chromophore. An equally conserved, proximal glutamine processes the resultant flavin protonation into downstream hydrogen-bond rearrangements. Here, we report that this glutamine, long deemed essential, is generally dispensable. In its absence, several light-oxygen-voltage receptors invariably retained productive, if often attenuated, signaling responses. Structures of a light-oxygen-voltage paradigm at around 1 Å resolution revealed highly similar light-induced conformational changes, irrespective of whether the glutamine is present. Naturally occurring, glutamine-deficient light-oxygen-voltage receptors likely serve as bona fide photoreceptors, as we showcase for a diguanylate cyclase. We propose that without the glutamine, water molecules transiently approach the chromophore and thus propagate flavin protonation downstream. Signaling without glutamine appears intrinsic to light-oxygen-voltage receptors, which pertains to biotechnological applications and suggests evolutionary descendance from redox-active flavoproteins.

Comparative analysis of two paradigm bacteriophytochromes reveals opposite functionalities in two-component signaling.

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics.

Identification of an atypical interaction site in the BTB domain of the MYC-interacting zinc-finger protein 1.

The repurposing of structurally conserved protein domains in different functional contexts is thought to be a driving force in the evolution of complex protein interaction networks. The BTB/POZ domain is such a versatile binding module that occurs over 200 times in the human proteome with diverse protein-specific adaptations. In BTB-zinc-finger transcription factors, the BTB domain drives homo- and heterodimerization as well as interactions with non-BTB-domain-containing proteins. Which mechanisms encode specificity in these interactions at a structural level is incompletely understood. Here, we uncover an atypical peptide-binding site in the BTB domain of the MYC-interacting zinc-finger protein 1 (MIZ1) that arises from local flexibility of the core BTB fold and may provide a target site for MIZ1-directed therapeutic approaches. Intriguingly, the identified binding mode requires the BTB domain to be in a homodimeric state, thus holding opportunities for functional discrimination between homo- and heterodimers of MIZ1 in the cell.

A Light-Oxygen-Voltage Receptor Integrates Light and Temperature.

Sensory photoreceptors enable organisms to adjust their physiology, behavior, and development in response to light, generally with spatiotemporal acuity and reversibility. These traits underlie the use of photoreceptors as genetically encoded actuators to alter by light the state and properties of heterologous organisms. Subsumed as optogenetics, pertinent approaches enable regulating diverse cellular processes, not least gene expression. Here, we controlled the widely used Tet repressor by coupling to light-oxygen-voltage (LOV) modules that either homodimerize or dissociate under blue light. Repression could thus be elevated or relieved, and consequently protein expression was modulated by light. Strikingly, the homodimeric RsLOV module from Rhodobacter sphaeroides not only dissociated under light but intrinsically reacted to temperature. The limited light responses of wild-type RsLOV at 37 °C were enhanced in two variants that exhibited closely similar photochemistry and structure. One variant improved the weak homodimerization affinity of 40 µM by two-fold and thus also bestowed light sensitivity on a receptor tyrosine kinase. Certain photoreceptors, exemplified by RsLOV, can evidently moonlight as temperature sensors which immediately bears on their application in optogenetics and biotechnology. Properly accounted for, the temperature sensitivity can be leveraged for the construction of signal-responsive cellular circuits.