Phytochromobilin Binding and Specific Amino Acid Residues Near The Chromophore Contribute To Orange Light Perception By The Dualchrome Phytochrome Region.

A novel photoreceptor dualchrome 1 (DUC1), containing a fused structure of cryptochrome and phytochrome, was discovered in the marine green alga Pycnococcus provasolli. The DUC1 phytochrome region (PpDUC1-N) binds to the bilin (linear tetrapyrrole) chromophores, phytochromobilin (PΦB) or phycocyanobilin (PCB), and reversibly photoconverts between the orange-absorbing dark-adapted state and the far-red-absorbing photoproduct state. This contrasts with typical phytochromes, which photoconvert between the red-absorbing dark-adapted and far-red-absorbing photoproduct states. In this study, we examined the molecular mechanism of PpDUC1-N to sense orange light by identifying the chromophore species synthesized by P. provasolli and the amino acid residues within the PpDUC1-N responsible for sensing orange light in the dark-adapted state. We focused on the PcyA homolog of P. provasolli (PpPcyA). Coexpression with the photoreceptors followed by an enzymatic assay revealed that PpPcyA synthesized PCB. Next, we focused on the PpDUC1-N GAF domain responsible for chromophore binding and light sensing. Ten amino acid residues were selected as the mutagenesis target near the chromophore. Replacement of these residues with those conserved in typical phytochromes revealed that three mutations (F290Y/M304S/L353M) resulted in a 23-nm red-shift in the dark-adapted state. Finally, we combined these constructs to obtain the PΦB-binding F290Y/M304S/L353M mutant and a 38-nm red-shift was observed compared with the PCB-binding wild-type PpDUC1. The binding chromophore species and the key residues near the chromophore contribute to blue-shifted orange light sensing in the dark-adapted state of the PpDUC1-N.

Crucial Residue for Tuning Thermal Relaxation Kinetics in the Biliverdin-binding Cyanobacteriochrome Photoreceptor Revealed by Site-saturation Mutagenesis.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to the phytochromes sensing red and far-red light reversibly. Only the cGMP phosphodiesterase/Adenylate cyclase/FhlA (GAF) domain is needed for chromophore incorporation and proper photoconversion. The CBCR GAF domains covalently ligate linear tetrapyrrole chromophores and show reversible photoconversion between two light-absorbing states. In most cases, the two light-absorbing states are stable under dark conditions, but in some cases, the photoproduct state undergoes thermal relaxation back to the dark-adapted state during thermal relaxation. In this study, we examined the engineered CBCR GAF domain, AnPixJg2_BV4. AnPixJg2_BV4 covalently binds biliverdin IX-alpha (BV) and shows reversible photoconversion between a far-red-absorbing Pfr dark-adapted state and an orange-absorbing Po photoproduct state. Because the BV is an intrinsic chromophore of mammalian cells and absorbs far-red light penetrating into deep tissues, BV-binding CBCR molecules are useful for the development of optogenetic and bioimaging tools used in mammals. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis on the Phe319 position. We succeeded in obtaining variant molecules with higher chromophore-binding efficiency and higher molar extinction coefficient. Furthermore, we observed a wide variation in thermal relaxation kinetics, with an 81-fold difference between the slowest and fastest rates. Both molecules with relatively slow and fast thermal relaxation would be advantageous for optogenetic control.

Conformational change in an engineered biliverdin-binding cyanobacteriochrome during the photoconversion process.

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools.

Introduction of reversible cysteine ligation ability to the biliverdin-binding cyanobacteriochrome photoreceptor.

Cyanobacteriochrome (CBCR) photoreceptors are distantly related to the canonical red/far-red reversible phytochrome photoreceptors. In the case of the CBCRs, only the GAF domain is required for chromophore incorporation and photoconversion. The GAF domains of CBCR are highly diversified into many lineages to sense various colors of light. These CBCR GAF domains are divided into two types: those possessing only the canonical Cys residue and those with both canonical and second Cys residues. The canonical Cys residue stably ligates to the chromophore in both cases. The second Cys residue mostly shows reversible adduct formation with the chromophore during photoconversion for spectral tuning. In this study, we focused on the CBCR GAF domain AnPixJg2_BV4, which possesses only the canonical Cys residue. AnPixJg2_BV4 covalently ligates to the biliverdin (BV) chromophore and shows far-red/orange reversible photoconversion. Because BV is a mammalian intrinsic chromophore, BV-binding molecules are advantageous for in vivo optogenetic and bioimaging tool development. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis and serendipitously obtained a unique variant molecule that showed far-red/blue reversible photoconversion, in which the Cys residue was introduced near the chromophore. This introduced Cys residue functioned as the second Cys residue that reversibly ligated with the chromophore. Because the position of the introduced Cys residue is distinct from the known second Cys residues, the variant molecule obtained in this study would expand our knowledge about the spectral tuning mechanism of CBCRs and contribute to tool development.

Role of hypoxanthine-guanine phosphoribosyltransferase in the metabolism of fairy chemicals in rice.

Fairy chemicals (FCs), 2-azahypoxanthine (AHX), imidazole-4-carboxamide (ICA), and 2-aza-8-oxohypoxanthine (AOH), are molecules with many diverse functions in plants. The defined biosynthetic pathway for FCs is a novel purine metabolism in which they are biosynthesized from 5-aminoimidazole-4-carboxamide. Here, we show that one of the purine salvage enzymes, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), recognizes AHX and AOH as substrates. Two novel compounds, AOH ribonucleotide and its ribonucleoside which are the derivatives of AOH, were enzymatically synthesized. The structures were determined by mass spectrometry, 1D and 2D NMR spectroscopy, and X-ray single-crystal diffraction analysis. This report demonstrates the function of HGPRT and the existence of novel purine metabolism associated with the biosynthesis of FCs in rice.

Evidence for an early green/red photocycle that precedes the diversification of GAF domain photoreceptor cyanobacteriochromes.

Phytochromes are linear tetrapyrrole-binding photoreceptors in eukaryotes and bacteria, primarily responding to red and far-red light signals reversibly. Among the GAF domain-based phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show various optical properties covering the entire visible region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here, we utilize ancestral sequence reconstruction and biochemical verification to show that the resurrected ancestral cyanobacteriochrome proteins reversibly respond to green and red light signals. pH titration analyses indicate that the deprotonation of the bound phycocyanobilin chromophore is crucial to perceive green light. The ancestral cyanobacteriochromes show only modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense the incident green/red light ratio. Many cyanobacteria can utilize green light for photosynthesis using phycobilisome light-harvesting complexes. The green/red sensing cyanobacteriochromes may have allowed better acclimation to changing light environments by rearranging the absorption capacity of the phycobilisome through chromatic acclimation.

Novel cyanobacteriochrome photoreceptor with the second Cys residue showing atypical orange/blue reversible photoconversion.

Cyanobacteriochromes (CBCRs) are cyanobacterial linear tetrapyrrole-binding photoreceptors distantly related to phytochromes. Only the GAF domain is needed for chromophore incorporation and proper photoconversion of the CBCRs. Most CBCR GAF domains possess the canonical Cys residue stably ligating to the chromophore. DXCF-type CBCR GAF domains also possess a second Cys residue within the DXCF motif. This second Cys residue reversibly ligates to the C10 of the chromophore. The Cys adduct formation is mostly observed for the dark-adapted state but not for the photoproduct state. In this study, we discovered novel CBCR GAF domains with a DXCI motif instead of the DXCF motif. Since these CBCR GAF domains are categorized into two subfamilies (DXCI-1 and DXCI-2), the GAF domains from each subfamily were analyzed. Although the CBCR GAF domain belonging to the DXCI-2 subfamily showed orange/green reversible photoconversion without transient Cys ligation, the CBCR GAF domain belonging to the DXCI-1 subfamily showed reversible photoconversion between an orange-absorbing dark-adapted state and a blue-absorbing photoproduct state. This indicates that the second Cys residue is covalently bound to the C10 of the chromophore in the photoproduct state but not in the dark-adapted state. Since the covalent bond formation in the photoproduct state is atypical, site-directed mutagenesis was conducted to understand the molecular mechanism of this GAF domain. The Ile residue within the DXCI motif may be key for covalent bond formation in the photoproduct state.

A red light-responsive photoswitch for deep tissue optogenetics.

Red light penetrates deep into mammalian tissues and has low phototoxicity, but few optogenetic tools that use red light have been developed. Here we present MagRed, a red light-activatable photoswitch that consists of a red light-absorbing bacterial phytochrome incorporating a mammalian endogenous chromophore, biliverdin and a photo-state-specific binder that we developed using Affibody library selection. Red light illumination triggers the binding of the two components of MagRed and the assembly of split-proteins fused to them. Using MagRed, we developed a red light-activatable Cre recombinase, which enables light-activatable DNA recombination deep in mammalian tissues. We also created red light-inducible transcriptional regulators based on CRISPR-Cas9 that enable an up to 378-fold activation (average, 135-fold induction) of multiple endogenous target genes. MagRed will facilitate optogenetic applications deep in mammalian organisms in a variety of biological research areas.

Identification of significant residues for intermediate accumulation in phycocyanobilin synthesis.

Phycocyanobilin, the primary pigment of both light perception and light-harvesting in cyanobacteria, is synthesized from biliverdin IXα (BV) through intermediate 181, 182-dihydrobiliverdin (181, 182-DHBV) by a phycocyanobilin:ferredoxin oxidoreductase (PcyA). In our previous study, we discovered two PcyA homologs (AmPcyAc and AmPcyAp) derived from Acaryochloris marina MBIC 11017 (A. marina) that exceptionally uses chlorophyll d as the primary photosynthetic pigment, absorbing longer wavelength far-red light than chlorophyll a, the photosynthetic pigment found in most cyanobacteria. Biochemical characterization of the two PcyA homologs identified functional diversification of these two enzymes: AmPcyAc provides 181, 182-DHBV, and PCB to the cyanobacteriochrome (CBCR) photoreceptors, whereas, AmPcyAp specifically provides PCB to the light-harvesting phycobilisome subunit. In this study, we focused on the residues necessary for 181, 182-DHBV supply to the CBCR photoreceptors by AmPcyAc. Based on the SyPcyA structure, we concentrated on the 30 residues that constitute the substrate-binding pocket. Among them, we discovered that Leu151 and Val225 in AmPcyAc were both substituted with isoleucine. During the enzymatic reaction, the SyPcyA variant molecule, possessing V225I and L151I replacements, accumulates the 181, 182-DHBV and supplies it to a CBCR molecule derived from A. marina. It is worth noting that the substitution of Val225 with isoleucine was specifically conserved among the Acaryochloris genus. Collectively, we propose that the specific evolution of PcyA among the Acaryochloris genus may correlate with the acquisition of Chl. d synthetic ability and growth in long-wavelength far-red light environments.

An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway.

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm-1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.

Identification of a dual orange/far-red and blue light photoreceptor from an oceanic green picoplankton.

Photoreceptors are conserved in green algae to land plants and regulate various developmental stages. In the ocean, blue light penetrates deeper than red light, and blue-light sensing is key to adapting to marine environments. Here, a search for blue-light photoreceptors in the marine metagenome uncover a chimeric gene composed of a phytochrome and a cryptochrome (Dualchrome1, DUC1) in a prasinophyte, Pycnococcus provasolii. DUC1 detects light within the orange/far-red and blue spectra, and acts as a dual photoreceptor. Analyses of its genome reveal the possible mechanisms of light adaptation. Genes for the light-harvesting complex (LHC) are duplicated and transcriptionally regulated under monochromatic orange/blue light, suggesting P. provasolii has acquired environmental adaptability to a wide range of light spectra and intensities.

Unusual ring D fixation by three crucial residues promotes phycoviolobilin formation in the DXCF-type cyanobacteriochrome without the second Cys.

Cyanobacteriochromes are linear tetrapyrrole-binding photoreceptors produced by cyanobacteria. Their chromophore-binding GAF domains are categorized into many lineages. Among them, dual Cys-type cyanobacteriochrome GAF domains possessing not only a highly conserved 'first Cys' but also a 'second Cys' are found from multiple lineages. The first Cys stably attaches to C31 of the A-ring, while the second Cys mostly shows reversible ligation to the C10 of the chromophore. Notably, the position of the second Cys in the primary sequence is diversified, and the most abundant dual Cys-type GAF domains have a 'second Cys' within the DXCF motif, which are called DXCF GAF domains. It has been long known that the second Cys in the DXCF GAF domains not only shows the reversible ligation but also is involved in isomerization activity (reduction in C4=C5 double bond) from the initially incorporated phycocyanobilin to phycoviolobilin. However, comprehensive site-directed mutagenesis on the DXCF GAF domains, AM1_6305g1 and AM1_1499g1, revealed that the second Cys is dispensable for isomerization activity, in which three residues participate by fixing the C- and D-rings. Fixation of the chromophore on both sides of the C5 bridge is necessary, even though one side of the fixation site is far from this bridge, with the other side at C31 fixed by the first Cys.

Transient electronic and vibrational signatures during reversible photoswitching of a cyanobacteriochrome photoreceptor.

Cyanobacteriochromes (CBCRs) are an emerging class of photoreceptors that are distant relatives of the phytochromes family. Unlike phytochromes, CBCRs have gained popularity in optogenetics due to their highly diverse spectral properties spanning the UV to near-IR region and only needing a single compact binding domain. AnPixJg2 is a CBCR that can reversibly photoswitch between its red-absorbing (15ZPr) and green-absorbing (15EPg) forms of the phycocyanobilin (PCB) cofactor. To reveal primary events of photoconversion, we implemented femtosecond transient absorption spectroscopy with a homemade LED box and a miniature peristaltic pump flow cell to track transient electronic responses of the photoexcited AnPixJg2 on molecular time scales. The 525 nm laser-induced Pg-to-Pr reverse conversion exhibits a ~3 ps excited-state lifetime before reaching the conical intersection (CI) and undergoing further relaxation on the 30 ps time scale to generate a long-lived Lumi-G ground state intermediate en route to Pr. The 650 nm laser-induced Pr-to-Pg forward conversion is less efficient than reverse conversion, showing a longer-lived excited state which requires two steps with ~13 and 217 ps time constants to enter the CI region. Furthermore, using a tunable ps Raman pump with broadband Raman probe on both the Stokes and anti-Stokes sides, we collected the pre-resonance ground-state femtosecond stimulated Raman spectroscopy (GS-FSRS) data with mode assignments aided by quantum calculations. Key vibrational marker bands at ~850, 1050, 1615, and 1649 cm-1 of the Pr conformer exhibit a notable blueshift to those of the Pg conformer inside AnPixJg2, reflecting the PCB chromophore terminal D (major) and A (minor) ring twist along the primary photoswitching reaction coordinate. This integrated ultrafast spectroscopy and computational platform has the potential to elucidate photochemistry and photophysics of more CBCRs and photoactive proteins in general, providing the highly desirable mechanistic insights to facilitate the rational design of functional molecular sensors and devices.

Comparison of the Forward and Reverse Photocycle Dynamics of Two Highly Similar Canonical Red/Green Cyanobacteriochromes Reveals Unexpected Differences.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15Z and 15E configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (∼68%) and similar chromophore environments: AnPixJg2 from Anabaena sp. PCC 7120 and NpR6012g4 from Nostoc punctiforme. Using broadband transient absorption spectroscopy on the primary (125 fs to 7 ns) and secondary (7 ns to 10 ms) time scales together with global analysis modeling, our studies revealed that AnPixJg2 and NpR6012g4 have comparable quantum yields for initiating the forward (15ZPr → 15EPg) and reverse (15EPg → 15ZPr) reactions, which proceed through monotonic and nonmonotonic mechanisms, respectively. In addition to small discrepancies in the kinetics, the secondary reverse dynamics resolved unique features for each domain: intermediate shunts in NpR6012g4 and a Meta-Gf intermediate red-shifted from the 15ZPr photoproduct in AnPixJg2. Overall, this study supports the conclusion that sequence similarity is a useful criterion for predicting pathways of the light-induced evolution and quantum yield of generating primary intermediate Φp within subfamilies of CBCRs, but more studies are still needed to develop a comprehensive molecular level understanding of these processes.

Phytochromes and Cyanobacteriochromes: Photoreceptor Molecules Incorporating a Linear Tetrapyrrole Chromophore.

In this chapter, we summarize the molecular mechanisms of the linear tetrapyrrole-binding photoreceptors, phytochromes, and cyanobacteriochromes. We especially focus on the color-tuning mechanisms and conformational changes during the photoconversion process. Furthermore, we introduce current status of development of the optogenetic tools based on these molecules. Huge repertoire of these photoreceptors with diverse spectral properties would contribute to development of multiplex optogenetic regulation. Among them, the photoreceptors incorporating the biliverdin IXα chromophore is advantageous for in vivo optogenetics because this is intrinsic in the mammalian cells, and absorbs far-red light penetrating into deep mammalian tissues.

The Cruciality of Single Amino Acid Replacement for the Spectral Tuning of Biliverdin-Binding Cyanobacteriochromes.

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms.

A photoproduct of DXCF cyanobacteriochromes without reversible Cys ligation is destabilized by rotating ring twist of the chromophore.

Cyanobacteriochrome photoreceptors (CBCRs) ligate linear tetrapyrrole chromophores via their first (canonical) Cys residue and show reversible photoconversion triggered by light-dependent Z/E isomerization of the chromophore. Among the huge repertoire of CBCRs, DXCF CBCRs contain a second Cys residue within the highly conserved Asp-Xaa-Cys-Phe (DXCF) motif. In the typical receptors, the second Cys covalently attaches to the 15Z-chromophore in the dark state and detaches from the 15E-chromophore in the photoproduct state, whereas atypical ones that lack reversible ligation activity show red-shifted absorption in the dark state due to a more extended π-conjugated system. Moreover, some DXCF CBCRs show blue-shifted absorption in the photoproduct state due to the twisted geometry of the rotating ring. During the process of rational color tuning of a certain DXCF CBCR, we unexpectedly found that twisted photoproducts of some variant molecules showed dark reversion to the dark state, which prompted us to hypothesize that the photoproduct is destabilized by the twisted geometry of the rotating ring. In this study, we comprehensively examined the photoproduct stability of the twisted and relaxed molecules derived from the same CBCR scaffolds under dark conditions. In the DXCF CBCRs lacking reversible ligation activity, the twisted photoproducts showed faster dark reversion than the relaxed ones, supporting our hypothesis. By contrast, in the DXCF CBCRs exhibiting reversible ligation activity, the twisted photoproducts showed no detectable photoconversion. Reversible Cys adduct formation thus results in drastic rearrangement of the protein-chromophore interaction in the photoproduct state, which would contribute to the previously unknown photoproduct stability.

Evolution-inspired design of multicolored photoswitches from a single cyanobacteriochrome scaffold.

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.

Tlr0485 is a cAMP-activated c-di-GMP phosphodiesterase in a cyanobacterium Thermosynechococcus.

Second messenger molecules are crucial components of environmental signaling systems to integrate multiple inputs and elicit physiological responses. Among various kinds of second messengers, cyclic nucleotides cAMP and cyclic di-GMP (c-di-GMP) play pivotal roles in bacterial environmental responses. However, how these signaling systems are interconnected for a concerted regulation of cellular physiology remains elusive. In a thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN, incident light color is sensed by cyanobacteriochrome photoreceptors to transduce the light information to the levels of c-di-GMP, which induces cellular aggregation probably via cellulose synthase activation. Herein, we identified that Tlr0485, which is composed of a cGMP-specific phosphodiesterases, adenylate cyclases, and FhlA (GAF) domain and an HD-GYP domain, is a cAMP-activated c-di-GMP phosphodiesterase. We also show biochemical evidence that the two class-III nucleotide cyclases, Cya1 and Cya2, are both adenylate cyclases to produce cAMP in T. vulcanus. The prevalence of cAMP-activated c-di-GMP phosphodiesterase genes in cyanobacterial genomes suggests that the direct crosstalk between cAMP and c-di-GMP signaling systems may be crucial for cyanobacterial environmental responses.