Optogenetics initially used plant photoreceptors to monitor neural circuits, later it has expanded to include engineered plant photoreceptors. Recently photoreceptors from bacteria, algae and cyanobacteria have been used as an optogenetic tool. Bilin-based photoreceptors are common light-sensitive photoswitches in plants, algae, bacteria and cyanobacteria. Here we discuss the photoreceptors from cyanobacteria. Several new photoreceptors have been explored in cyanobacteria which are now proposed as cyanobacteriochrome. The domains in the cyanobacteriochrome, light-induced signaling transduction, photoconversion, are the most attractive features for the optogenetic system. The wider spectral feature of cyanobacteriochrome from UV to visible radiation makes it a light potential sensitive optogenetic tool. Besides, cyanobacterial phytochrome responses to yellow, orange and blue light have more application in optogenetics. This chapter summarizes the photoconversion, phototaxis, cell aggregation, cell signaling mediated by cyanobacteriochrome and cyanophytochrome. As there is a wide range of cyanobacteriochrome and its combination delivers a varied light-sensitive response. Besides coordination among cyanobacteriochromes in cell signaling reduces the engineering of photoreceptors for the optogenetic system.
- cell signaling transduction
Photoreceptors in cyanobacteria are diverse in their spectral character from ultraviolet to visible wavelength. Plant photoreceptors were widely used in optogenetics, but their responses to specific wavelengths need more revision. When compared to these photoreceptors cyanobacteriochromes (CBCRs) receive more attention as a versatile optogenetic tool. Several photoreceptors respond to a wide range of light, photoconversion ability and photoswitches for dual light are new approaches and powerful tools for optogenetics . Engineering of these photoreceptors will develop more versatile CBCR to alleviate the conventional methods like mutation and recombination . Optogenetics in mammalian tissue adopted far-red illumination and adjacent infra-red radiance to visualize and activate responses in the cell. The CBCRs with linear tetrapyrrole is very sensitive to red and far-red light. Utilization of these infra-red sensitive and red light responsive CBCRs raised their application in optogenetics. So far phytochromobilin is used in mammalian cells recently cyanobacterial phytochrome 1 (CPH1) has been applied in mammalian cells proven its benefit in synthetic biology .
Cyanobacteria are evolutionarily ancient phototrophic Gram-negative bacteria widely distributed in terrestrial, freshwater and marine environments. They are oxygenic photosynthesizers having major photosynthetic pigment chlorophyll-a and light-harvesting pigments phycobiliproteins. They survive in many extreme environments, such as hot and cold deserts, hot springs, and hypersaline environments .
Light is an important factor for their nutrition and growth, therefore, it has a multitude photosensory complex that responds to a wide array of illumination. Each chromophore is a response to a particular wavelength based on the incident light it changes the arrangement and composition of pigments in the photon capturing antenna. This rearrangement of pigments to the incident light is the process of complementary chromatic acclimation. Cyanobacteria possess phototaxis movements it means they can move towards or away from specific light. Photoreceptors in cyanobacteria are commonly referred to as CBCRs .
Generally, Phytochromes are photoreceptors that have been found in plants, algae, and bacteria. These photoreceptors are broadly utilized in biosensors and optogenetics to screen and regulate diverse intracellular cycles like phosphorylation, gene activation, degradation of protein and change of calcium ions .
1.4 Phytochromes from cyanobacteria
Phytochromes are photochromic photoreceptors, generally responding to red and far-red radiation in the visible spectrum. Bilin is the most important portion in the chromophore and it is distributed in three different forms. Phytochrome in plants made of phytochromobilin, whereas in cyanobacteria it is in the form of Phycocyanobilin. Further Phytochromes in plants, algae and cyanobacteria constitute linear tetrapyrrole biliverdin . The chromophore part in plant phytochrome has cysteine at the N terminal site of the protein. The phytochrome in plants differs from cyanobacteria by having biliverdin in the chromophore part. Evolutionary development in cyanobacteria brings out cysteine linked with biliverdin in the GAF domain and formed as phycocyanobilin also referred to as phytochromobilin. The transformation of phytochrome into CBCR is due to changes in the molecular level.
1.5 Phytochrome classification
Phytochromes were primarily arranged into three subfamilies dependent on the number of domains in their photosensory core module (PCM). Phytochrome has three domains in their core structure, for example, PHY - phytochrome-explicit area, PAS - Per-Arnt-Sim, and GAF - cGMP phosphodiesterase-adenylate cyclase-FhlA. Even though the amino acid groupings of these domains have a dissimilar sequence, their structures were similar. Further subfamilies are cyanobacterial phytochromes (Cph), lack an N-terminal PAS area, and CBCRs, which contain a solitary GAF domain . The domain proteins of PAS, GAF and PHY were interconnected to form homo and heterodimers .
1.6 Features of cyanobacteriochrome
Phytochrome in plants and algae has the sensitivity to the different light spectrum. Plant phytochromes are sensitive to red radiance furthermore, it performs red and far-red photoreversible photocycle. The phytochrome with bilin photoreceptors in eukaryotic green algae and prokaryotic cyanobacteria are sensitive to the visible spectrum [10, 11, 12].
The CBCRs are photoreceptors involved in the regulation of phototaxis. The photoreceptors SyCcaS, SyPixJ1, TePixJ, AnPixJ, SyCikA are now proposed to be CBCRs due to the presence of chromophore binding GAF domain.
The domain GAF is enough for photoconversion
chromophore in GAF domain varies from phytochrome GAF
The GAF domain binds to linear tetrapyrrole pigments like phycoviolobilin or phycocyanobilin
The chromes are responsive to a wide range of light from ultraviolet to the red region
2. CBCR in cyanobacteria
The cyanobacterial genomes of Anabaena and Nostoc harbor
Chromatic acclimation is an adaptive mechanism in some cyanobacteria capable of modifying their photosynthetic system reaction to the incident radiance. The phycocyanin content in
The chromophore-binding GAF domain of CikA in
3. Functions of CBCR
3.1 Coordination of the cyanobacteriochromes
The photo biochemical properties of SesA holoprotein from the cyanobacterium
SesB has GGDEF- type DGC (Diguanylate cyclase) domain (Figure 3B) and SesC has EAL- type PDE domain to deliver the c-di-GMP signal (Figure 3C). The SesB DGC for c-di-GMP signal degraded under teal light, in contrast, expressed higher in blue light. In Ses, A c-di-GMP is higher under blue light and lowered in teal blue light. SesC DGF activity is maximum in blue light and minimum in green light. This is a chrome-responsive cyanobacterial c-di-GMP signaling coordination of (Ses –A, B and C) CBCRs.
SesA a blue light-responsive DGC
SesB a teal light-responsive and GTP sensitive PDE
SesC, a dual-active CBCR having blue light-sensitive DGC and green light-responsive PDE activity
3.2 Cyanobacteriochrome in cell aggregation
The cell aggregation signaling molecule Cyclic dimeric guanosine monophosphate (c-di-GMP) is unique to cyanobacteria and bacteria . Light is a key factor in controlling c-di-GMP signaling [25, 26]. The domain (GGDEF) for the synthesis and (EAL/HD-GYP) (Figure 3A and B) destruction of the c-di-GMP is higher in the CBCR GAF structure of freshwater cyanobacterial genomes. The CBCR induces the c-di-GMP signaling pathway. The CBCR—GAF domain of SesA (Figure 3A) from the thermophilic cyanobacterium
3.3 Cyanobacterial photobiological responses
Prokaryotic photosynthetic organisms, cyanobacteria, depend on bilin-linked phytochromes (Cphs) and CBCRs, photoreceptors which are structurally and functionally vary from plant photoreceptors. The CBCRs are made of light-absorbing domains with various color-tuning and signal transmission processes, that make cyanobacteria capture a wide wavelength of light from UV–visible to far-red lights. The genome of filamentous cyanobacteria has a different type of CBCRs with wide chromophore-linked selectivity and photocycle protochromicity. The Cph lineage can absorb a wide range of light from blue-violet to yellow-orange light. This chapter also emphasized the color-sensitive diversity [29, 30] and signal transmission process of Cphs and CBCRs, concerning optogenetic.
Bilin-linked phytochrome Cphs and plant phytochromes (Phys) are similar in structure, with an N-terminal photosensory core module (PCM) and a C-terminal output regulatory module. The PCM contains the following domains PAS (Period/Arnt/Single-minded), GAF(C-GMP phosphodiesterase/Adenylylcyclase/FhlA), and PHY (phytochrome-specific). The GAF domain is necessary for forming the bilin cross-linking; PAS and PHY structures are involved in bilin lyase activity . Cyanobacteria have two types of bilin-linked photoreceptors Cphs, and CBCRs. In contrast to Cphs with PAS and PHY domain, CBCRs (lack PAS and PHY) absorb a wide array of light, by the GAF structure . This wide array of light absorption by CBCR is called a color or spectral tuning mechanism.
3.4 CBCR in photobiological responses
Growth of the cyanobacterium
3.5 Dual light system
The CBCR response to two different light systems is mediated by the histidine (His) kinase domain. In
The non-flagellated cyanobacteria adapt phototaxis in response to light. In
In some cyanobacteria, photoinhibition light conditions trigger the synthesis of photoprotective pigments. For example, intense radiation or UV radiation, accumulate mycosporine-like amino acids and scytonemins [30, 42]. Cyanobacteria, like
3.8 Circadian clock
Cyanobacteria are responsive to diurnal photoperiods by adjusting their photosynthesis and respiration. In
Cyanobacteria form biofilms, which favor attachment on a surface to grow and produce extracellular polymers. This biofilm development in
4. Photosensitive features of CBCR
4.1 Color sensing by Cphs and CBCRS
Cyanobacterial proteins contain the accompanying regions PAS-GAF-PHY . Entire genome sequencing of cyanobacterial species, for example,
4.2 Dual cays residues in CBCR for dual photocycle
Some CBCRs with exceptionally unchanged DXCF motif or the feebly rationed CXXR/K motif have extra Cys amino acids in the insertion loop (embed - Cys) via second thioether bond at the C10 atom under dark phase . This sort of double Cys CBCRs, with a second thioether bond, is fragile and light-labile. These CBCRs are extremely responsive to capture violet or blue light in dark phase but it absorbs green, yellow, orange or greenish-blue light in the light phase. The cyanobacterial CBCRs are primarily linked to PCB yet some may link to phycoviolobilin (PVB) like Cphs [53, 54]. The change of PCB into PVB is unique to the DXCF-CBCRs subfamily . The color tuning systems of CBCRs for far-red to orange (Fr/O) remain unidentified .
4.3 Signal transmission by CBCR
Cyanobacterial photoreceptors associated with signal transmission through phosphor transfer or c-di-GMP. Phosphorelay is a signal transmission process engaged with the autophosphorylation of His amino acid residue by His kinases, continued by phosphotransfer in association with reaction controllers. A film bound His kinase CBCR-UirS in
4.4 Autolyase and autoisomerase in CBCR
Cyanobacterial photoreceptors are also called CBCRs that are similar to phytochromes . PixJ GAF, from a thermophilic cyanobacterium
The primary CBCR for the phototaxis controller was recognized as PixJ. The CBCR SyPixJl of
At the point when His6-TePixJ_GAF was digested with acidic urea in the dark phase, the Pb peak (433 nm) was changed from native form to a peak at 594 nm with a shoulder at 565 nm. The PVB in TePixJ_GAF captures a shorter wavelength of light than PCB. In any case, it ought to be noticed that the Pb absorb at 433 nm in native form is extraordinarily smaller than the urea-denatured PVB absorb at 594 nm. PVB is an isomer of PCB with a similar atomic mass, yet conjugated double bonds are detached at the C5 position. PVB with the apoprotein is accountable for the extraordinary blue capturing structure Pb and photoreversible modifications. The PCB transformation to PVB is because of the PecE and PecF proteins which are fundamental for ligation and isomerization.
5. Color-tuning mechanisms of cyanobacteriochromes
The term cyanobacteriochrome was first reported in 2004 by Dr. Ikeuchi in his paper about photoreceptor SypixJ1 . This photoreceptor covalently binds a linear tetrapyrrole chromophore and performed reversible photoconversion between a blue-absorbing form (Pb) and a green-absorbing form (Pg). This protein has a cGMP-phosphodiesterase/adenylate cyclase/FhlA (GAF) similar to those of the Phytochromes. The GAF in cyanobacterial signals transduction proteins is identified as CBCRs. The GAF s of cyanobacteria are covalently linked to tetrapyrrole chromophore. This chromophore can sense different light (UV–visible spectra). In some cyanobacteria, this GAF regulate phototactic motility, chromatic acclimation and light-dependent cell aggregation.
5.1 CBCR structure and diversity
In CBCRs, the GAF is essential for chromophore incorporation and photoconversion  CBCRs have N and C terminals in which GAF s are located at the N-terminus. Signal output s viz. (HisKA + HATPase_c), Methyl-accepting (MA), GGDEF, and EAL s located at the C- terminus. His kinase is frequently detected as signal output s.
5.2 Chromophore variation in color-tuning mechanism
Generally, four types of linear tetrapyrrole chromophores, phycocyanobilin (PCB), biliverdin (BV), phytochromobilin (PFB) and phycoviolobilin (PVB), have been identified in the CBCR GAF domain. The mixture of these chromophores in the GAF domain results in broad and diverse spectral features [54, 62]. These chromophores are arranged in an order from a longer wavelength absorbing system to a shorter wavelength absorber (BV > PFB > PCB > PVB). The longer wavelength of light is absorbed by the longer length chromophores. Sometimes, the PCB isomerizes to PVB at the GAF region. This sort of PVB linked GAF area in CBCR has been distinguished from the cyanobacterium
The absorption of the Cys-free form is highly affected by the linked species of chromophores. The chromophore PCB in AM1_1186g2 revealed a reversible photoconversion between a Cys-free red-absorber (Pr) and a Cys-dependent blue absorber Pb , while covalently linked PVB in TePixJg showed reversible photoconversion between a Cys-free Pg absorber to a Cys-linked Pb absorber [62, 64]. The Cys-linked Pb absorber in the CBCR GAF s is the same as the blue to green reversible, but the Cys-free teal-absorber (Pt) is often shifted to blue absorber in association with the typical green absorber Pg. A twist in the D ring of the conserved Phe residues in the Pt absorber contributes to the blue-shift [65, 66]. The conversion of the Pt form into the typical red-shifted Pg form is mediated by the loss of these Phe residues . Likewise in the XRG lineage, CBCR GAF s have red to green reversible photoconversion. Blue-shift of the Pg form to red absorbing Pr caused a small twist in the A and D ring . This photocycle is mediated by the proton donor and the acceptor is Glu amino acid.
5.3 Dark reversion
The two distinctive light-harvesting types of the CBCR GAF domain are generally constant under the dark phase, thus these CBCR GAFs can detect the proportion of two wavelengths. Further, in some CBCR GAF showed unidirectional photoconversion and rapid dark reversion. This can identify the concentration of certain colors of light . Some XRG CBCR GAF s are unidirectional photoconversion from Pr-to-Pg in dark conditions, and after 4–25 s it rapidly undergoes dark reversion from Pg-to-Pr . The kinetics of these GAF s of the dark reversion is highly dependent on higher temperature [53, 69, 70]. Light and temperature were indulged in the regulation of these GAFs. These characteristics may be physiologically relevant to sense light intensity for efficient photosynthesis because the same light intensity with lower temperatures severely inhibited photosynthesis.
Several CBCRs have been designed to change the color-tuning interaction and output activity. Inclusion of the second Cys residue and modifications of PCB-binding in the GAF s caused reversible photoconversion from red/green into blue/green. This is due to the isomerization of PCB to PVB by the incorporation of the second Cys residue which attaches to the chromophore in the reversible form [70, 71]. The twisted geometry of the D ring can also be removed . The output activity of the native GAF was modified with other lineage s by adenylate cyclases [72, 73, 74] that respond to various light. Changing the length of the CBCR GAF linker region in CcaS and HisKA turns the light receptiveness of the green to red lineage .
Optogenetics, is a new branch of synthetic biology, is generally defined as the engineering of particular light-induced cellular reactions. This study was initiated with light-sensitive Phytochromes and bacteriochromes experimented in bacteria and mammalian neurons. Recently this research field is sensational due to the CBCRs from cyanobacteria are widely used as signaling components. These CBCRs in optogenetic systems performed the regulation of cellular responses spatially and temporally by precisely applying and removing light. A cyanobacterial photoreceptor-based optogenetic system was implemented to study the protein interaction and cell signaling in cyanobacteria, bacteria and mammalian cells. The application of CBCRs in optogenetic systems extends their usage in developing potential new therapeutics. Smaller size photosensory regions and autocatalytic activity of CBCRs are more advantageous than other photoreceptors.
The author S. Sivasankari thanks the DST-Government of India for financial support under the grant number DST/INSPIRE FELLOWSHIP/2014/IF140666.
Conflicts of interest
The author declares that there is no conflict of interest.
Cyanobacterial phytochrome 1
Photosensory core module
cGMP phosphodiesterase-adenylate cyclase-FhlA