Heliorhodopsin binds and regulates glutamine synthetase activity.

Photoreceptors are light-sensitive proteins found in various organisms that respond to light and relay signals into the cells. Heliorhodopsin, a retinal-binding membrane protein, has been recently discovered, however its function remains unknown. Herein, we investigated the relationship between Actinobacteria bacterium IMCC26103 heliorhodopsin (AbHeR) and an adjacent glutamine synthetase (AbGS) in the same operon. We demonstrate that AbHeR binds to AbGS and regulates AbGS activity. More specifically, the dissociation constant (Kd) value of the binding between AbHeR and AbGS is 6.06 µM. Moreover, the absence of positively charged residues within the intracellular loop of AbHeR impacted Kd value as they serve as critical binding sites for AbGS. We also confirm that AbHeR up-regulates the biosynthetic enzyme activity of AbGS both in vitro and in vivo in the presence of light. GS is a key enzyme involved in nitrogen assimilation that catalyzes the conversion of glutamate and ammonia to glutamine. Hence, the interaction between AbHeR and AbGS may be critical for nitrogen assimilation in Actinobacteria bacterium IMCC26103 as it survives in low-nutrient environments. Overall, the findings of our study describe, for the first time, to the best of our knowledge, a novel function of heliorhodopsin as a regulatory rhodopsin with the capacity to bind and regulate enzyme activity required for nitrogen assimilation.

AT-specific DNA visualization revisits the directionality of bacteriophage λ DNA ejection.

In this study, we specifically visualized DNA molecules at their AT base pairs after in vitro phage ejection. Our AT-specific visualization revealed that either end of the DNA molecule could be ejected first with a nearly 50% probability. This observation challenges the generally accepted theory of Last In First Out (LIFO), which states that the end of the phage λ DNA that enters the capsid last during phage packaging is the first to be ejected, and that both ends of the DNA are unable to move within the extremely condensed phage capsid. To support our observations, we conducted computer simulations that revealed that both ends of the DNA molecule are randomized, resulting in the observed near 50% probability. Additionally, we found that the length of the ejected DNA by LIFO was consistently longer than that by First In First Out (FIFO) during in vitro phage ejection. Our simulations attributed this difference in length to the stiffness difference of the remaining DNA within the phage capsid. In conclusion, this study demonstrates that a DNA molecule within an extremely dense phage capsid exhibits a degree of mobility, allowing it to switch ends during ejection.

Surgery-related anxiety on geriatric patients undergoing total knee arthroplasty: a retrospective observational study.

BACKGROUND: The prevalence of anxiety in patients undergoing total knee arthroplasty (TKA) and its association with postoperative functions are well known; however, the levels of anxiety or anxiety-related characteristics are unknown. This study aimed to investigate the prevalence of clinically significant state anxiety in geriatric patients undergoing TKA for osteoarthritis (OA) of the knee and to evaluate the anxiety-related characteristics experienced by these patients pre- and post-operatively. METHODS: This retrospective observational study recruited patients who had undergone TKA for knee OA using general anesthesia between February 2020 and August 2021. The study participants were geriatric patients older than 65 years who had moderate or severe OA. We evaluated patient characteristics including age, sex, body mass index, smoking status, hypertension, diabetes, and cancer. We assessed their levels of anxiety status using the STAI-X which comprises 20-item scales. Clinically meaningful state anxiety was defined as a total score of 52 or higher. An independent Student's t-test was used to determine differences of STAI score between subgroups in terms of patient characteristics. And patients were asked to complete questionnaires, which assessed four areas: (1) the main cause of anxiety; (2) the most helpful factor in overcoming anxiety before surgery; (3) the most helpful factor in reducing anxiety after surgery; and (4) the most anxious moment during the entire process. RESULTS: The mean STAI score of patients who underwent TKA was 43.0 points and 16.4% of patients experienced clinically significant state anxiety. The current smoking status affect STAI score and the proportion of patients with clinically meaningful state anxiety. The most common cause of preoperative anxiety was the surgery itself. Overall, 38% of patients reported that they experienced the greatest level of anxiety when the surgeon had recommended TKA in the outpatient clinic. The trust in the medical staff before surgery and the surgeon's explanations after surgery helped the most in reducing anxiety. CONCLUSIONS: One in six patients before TKA experience clinically meaningful state anxiety, and about 40% of patients experience anxiety from the time they are recommended for surgery. Patients tended to overcome anxiety before TKA through trust in the medical staff, and the surgeon's explanations after surgery was found to be helpful in reducing anxiety.

Does spiked tibial cement spacer reduce spacer-related problems in two-stage revision total knee arthroplasty for infection?

PURPOSE: Articulating cement spacers are frequently used in staged approaches for infected total knee arthroplasty (TKA). This study investigated whether a tibial cement spacer (TCS) with spikes could reduce spacer-related problems in two-stage revision TKA (R-TKA). METHODS: A total of 27 patients (27 knees; 10 men and 17 women) who underwent two-stage R-TKA for infected TKA were retrospectively analyzed. Group A comprised 12 patients who used TCS with spikes added to the bottom surface, whereas group B consisted of 15 patients who used conventional TCS with a flat bottom. For each group, plain radiographs were obtained after cement spacer implantation and before R-TKA to measure mediolateral (ML) translation and TCS's tilting angle. Patients' demographic data, ML translation of the TCS, and changes in the TCS's tilting angle between the groups were analyzed. RESULTS: The mean ML translation was significantly lower in group A than that in group B (1.7 mm vs. 5.4 mm, p = 0.04). The mean change in the tilting angle was significantly lower in group A than that in group B (4.5° vs. 19.4°, p = 0.047). CONCLUSION: The spiked TCS in two-stage R-TKA provides superior stability compared to the TCS with a conventional design.

Fluorescence Enhancement of a Microbial Rhodopsin via Electronic Reprogramming.

The engineering of microbial rhodopsins with enhanced fluorescence is of great importance in the expanding field of optogenetics. Here we report the discovery of two mutants (W76S/Y179F and L83Q) of a sensory rhodopsin from the cyanobacterium Anabaena PCC7120 with opposite fluorescence behavior. In fact, while W76S/Y179F displays, with respect to the wild-type protein, a nearly 10-fold increase in red-light emission, the second is not emissive. Thus, the W76S/Y179F, L83Q pair offers an unprecedented opportunity for the investigation of fluorescence enhancement in microbial rhodopsins, which is pursued by combining transient absorption spectroscopy and multiconfigurational quantum chemistry. The results of such an investigation point to an isomerization-blocking electronic effect as the direct cause of instantaneous (subpicosecond) fluorescence enhancement.

Biosynthetic production of fully carbon-13 labeled retinal in E. coli for structural and functional studies of rhodopsins.

The isomerization of a covalently bound retinal is an integral part of both microbial and animal rhodopsin function. As such, detailed structure and conformational changes in the retinal binding pocket are of significant interest and are studied in various NMR, FTIR, and Raman spectroscopy experiments, which commonly require isotopic labeling of retinal. Unfortunately, the de novo organic synthesis of an isotopically-labeled retinal is complex and often cost-prohibitive, especially for large scale expression required for solid-state NMR. We present the novel protocol for biosynthetic production of an isotopically labeled retinal ligand concurrently with an apoprotein in E. coli as a cost-effective alternative to the de novo organic synthesis. Previously, the biosynthesis of a retinal precursor, ß-carotene, has been introduced into many different organisms. We extended this system to the prototrophic E. coli expression strain BL21 in conjunction with the inducible expression of a ß-dioxygenase and proteo-opsin. To demonstrate the applicability of this system, we were able to assign several new carbon resonances for proteorhodopsin-bound retinal by using fully 13C-labeled glucose as the sole carbon source. Furthermore, we demonstrated that this biosynthetically produced retinal can be extracted from E. coli cells by applying a hydrophobic solvent layer to the growth medium and reconstituted into an externally produced opsin of any desired labeling pattern.

Protein conformational alterations induced by the retinal excited state in proton and sodium pumping rhodopsins.

Retinal proteins' biological activity is triggered by the retinal chromophore's light absorption, which initiates a photocycle. However, the mechanism by which retinal light excitation induces the protein's response is not completely understood. Recently, two new retinal proteins were discovered, namely, King Sejong 1-2 (KS1-2) and Nonlabens (Donghaeana) dokdonensis (DDR2), which exhibit H+ and Na+ pumping activities, respectively. To pinpoint whether protein conformation alterations can be achieved without light-induced retinal C13[double bond, length as m-dash]C14 double-bond isomerization, we utilized the hydroxylamine reaction, which cleaves the protonated Schiff base bond through which the retinal chromophore is covalently bound to the protein. The reaction is accelerated by light even though the cleavage is not a photochemical reaction. Therefore, the cleavage reaction may serve as a tool to detect protein conformation alterations. We discovered that in both KS1-2 and DDR2, the hydroxylamine reaction is light accelerated, even in artificial pigments derived from synthetic retinal in which the crucial C13[double bond, length as m-dash]C14 double-bond isomerization is prevented. Therefore, we propose that in both proteins the light-induced retinal charge redistribution taking place in the retinal excited state polarizes the protein, which, in turn, triggers protein conformation alterations. A further general possible application of the present finding is associated with other photoreceptor proteins having retinal or other non-retinal chromophores whose light excitation may affect the protein conformation.

Iatrogenic Medial Collateral Ligament Injury by Valgus Stress During Arthroscopic Surgery of the Knee.

PURPOSE: To evaluate the radiographic and clinical follow-up results of iatrogenic medial collateral ligament (MCL) injuries caused by valgus stress during arthroscopic surgery of the knee. METHODS: This study retrospectively evaluated 15 knees in 15 patients (8 female and 7 male patients), with a mean age of 58 years (range, 45-66 years), with iatrogenic MCL injuries caused by valgus stress during arthroscopic surgery of the knee. All patients were treated conservatively without an immobilizer or brace. The mean follow-up period was 24 months (range, 18-51 months). Evaluations included magnetic resonance imaging immediately postoperatively, as well as physical examinations and valgus stress radiographs (at 0° and 30° of knee flexion) 6 weeks after surgery and at final follow-up. RESULTS: Postoperative magnetic resonance imaging in all patients showed increased signal intensity, swelling, and partial loss of continuity at the meniscofemoral portion of the MCL. Physical examination showed mild tenderness in only 1 patient after 6 weeks and none at final follow-up. Valgus stress tests and valgus stress radiographs showed no significant differences between the injured and uninjured knees at 6 weeks postoperatively and at final follow-up (P > .05). CONCLUSIONS: Iatrogenic MCL injuries during arthroscopic knee surgery could be treated successfully without a splint or brace. LEVEL OF EVIDENCE: Level IV, prognostic case series.

Expression of Anabaena sensory rhodopsin is influenced by different codons of seven residues at the N-terminal region.

Microbial rhodopsins are well-known seven-transmembrane proteins that have been extensively studied for their structure and function. These retinal-binding proteins can be divided into two types. Type I is microbial rhodopsin, and type II (visual pigment) is expressed mostly in mammalian eyes. The two primary functions of type I rhodopsin are ion pumping activity and sensory transduction. Anabaena sensory rhodopsin (ASR) is a microbial rhodopsin with a specific function of photosensory transduction. ASR is expressed at moderate levels in Escherichia coli, but its expression level is lower compared to the general green light absorbing proteorhodopsin (GPR). In this study, full-length ASR was used to test the influence of codon usage on expression E. coli. Seven amino acids at the N-terminal region of ASR after the Met start codon were changed randomly using designed primers, which allowed for 8192 different nucleotide combinations. The codon changes were screened for the preferable codons that resulted in higher expression yield. Among the 57 selected mutations, 24 color-enhanced E. coli colonies contained ASR proteins, and they expressed ASR at a higher level than the bacteria with wild-type ASR codon usage. This result strongly suggests that the specific codon usage of only the N-terminal portion of a protein can increase the expression level of the entire protein.

An evolutionary optimization of a rhodopsin-based phototrophic metabolism in Escherichia coli.

BACKGROUND: The expression of the Gloeobacter rhodopsin (GR) in a chemotrophic Escherichia coli enables the light-driven phototrophic energy generation. Adaptive laboratory evolution has been used for acquiring desired phenotype of microbial cells and for the elucidation of basic mechanism of molecular evolution. To develop an optimized strain for the artificially acquired phototrophic metabolism, an ancestral E. coli expressing GR was adaptively evolved in a chemostat reactor with constant illumination and limited glucose conditions. This study was emphasized at an unexpected genomic mutation contributed to the improvement of microbial performance. RESULTS: During the chemostat culture, increase of cell size was observed, which were distinguished from that of the typical rod-shaped ancestral cells. A descendant ET5 strain was randomly isolated from the chemostat culture at 88-days. The phototrophic growth and the light-induced proton pumping of the ET5 strain were twofold and eightfold greater, respectively, than those of the ancestral E. coli strain. Single point mutation of C1082A at dgcQ gene (encoding diguanylate cyclase, also known as the yedQ gene) in the chromosome of ET5 strain was identified from whole genome sequencing analysis. An ancestral E. coli complemented with the same dgcQ mutation from the ET5 was repeated the subsequently enhancements of light-driven phototrophic growth and proton pumping. Intracellular c-di-GMP, the product of the diguanylate cyclase (dgcQ), of the descendant ET5 strain was suddenly increased while that of the ancestral strain was negligible. CONCLUSIONS: Newly acquired phototrophic metabolism of E. coli was further improved via adaptive laboratory evolution by the rise of a point mutation on a transmembrane cell signaling protein followed by increase of signal molecule that eventually led an increase proton pumping and phototrophic growth.

Mistic-fused expression of algal rhodopsins in Escherichia coli and its photochemical properties.

BACKGROUND: Since algal rhodopsins, the eukaryotic seven-transmembrane proteins, are generally difficult to express in Escherichia coli, eukaryotic cells have been used for heterologous expression. Mistic, a membrane-associated protein that was originally discovered in Bacillus subtilis, has been shown to improve the expression levels of many foreign integral membrane proteins in E. coli when used as a fusion partner linked to the N-terminus of cargo proteins. METHODS: Here, we expressed two algal rhodopsins with N- and C-terminal Mistic domains in E. coli-Acetabularia rhodopsin I (ARI) and Chlamydomonas sensory rhodopsin B (CSRB, channel rhodopsin 2). UV/VIS spectroscopy, pH titration of proton acceptor residue, laser-induced photolysis and electrophysiological measurement were used for investigating important residues in proton transport and spectroscopic characters of the proteins. RESULTS: Protein yield of two algal rhodopsins was enhanced, obtaining 0.12mg of Mistic-ARI and 0.04mg of Mistic-CSRB per liter of culture. Spheroplast expression Mistic-ARI had outward proton-pumping activity, indicating protein functionality. Asp89 of ARI changed its protonation state by light absorption, and Asp100 was important for O(600) formation. Electrophysiology revealed that both residues took part in proton transport. The spectroscopic analyses of Mistic-CSRB revealed its characteristics. CONCLUSIONS: Fusion to the membrane-integrating protein Mistic can enhance overexpression of eukaryotic type I rhodopsins in E. coli. GENERAL SIGNIFICANCE: These findings indicate that Mistic fusion and E. coli expression method could be an effective, low cost technique for studying eukaryotic membrane proteins. This may have useful implications, for example, in studying structural characteristics and optogenetics for rhodopsins.

Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein.

Determination of structure of integral membrane proteins, especially in their native environment, is a formidable challenge in structural biology. Here we demonstrate that magic angle spinning solid-state NMR spectroscopy can be used to determine structures of membrane proteins reconstituted in synthetic lipids, an environment similar to the natural membrane. We combined a large number of experimentally determined interatomic distances and local torsional restraints to solve the structure of an oligomeric membrane protein of common seven-helical fold, Anabaena sensory rhodopsin (ASR). We determined the atomic resolution detail of the oligomerization interface of the ASR trimer, and the arrangement of helices, side chains and the retinal cofactor in the monomer.

Structural basis for the slow photocycle and late proton release in Acetabularia rhodopsin I from the marine plant Acetabularia acetabulum.

Although many crystal structures of microbial rhodopsins have been solved, those with sufficient resolution to identify the functional water molecules are very limited. In this study, the Acetabularia rhodopsin I (ARI) protein derived from the marine alga A. acetabulum was synthesized on a large scale by the Escherichia coli cell-free membrane-protein production method, and crystal structures of ARI were determined at the second highest (1.52-1.80 Å) resolution for a microbial rhodopsin, following bacteriorhodopsin (BR). Examinations of the photochemical properties of ARI revealed that the photocycle of ARI is slower than that of BR and that its proton-transfer reactions are different from those of BR. In the present structures, a large cavity containing numerous water molecules exists on the extracellular side of ARI, explaining the relatively low pKa of Glu206(ARI), which cannot function as an initial proton-releasing residue at any pH. An interhelical hydrogen bond exists between Leu97(ARI) and Tyr221(ARI) on the cytoplasmic side, which facilitates the slow photocycle and regulates the pKa of Asp100(ARI), a potential proton donor to the Schiff base, in the dark state.

A role of Anabaena sensory rhodopsin transducer (ASRT) in photosensory transduction.

In 2003, Anabaena sensory rhodopsin (ASR), a membrane-bound light sensor protein, was discovered in cyanobacteria. Since then, a large number of functions have been described for ASR, based on protein biochemical and biophysical studies. However, no study has determined the in vivo mechanism of photosensory transduction for ASR and its transducer protein (ASRT). Here, we aimed to determine the role of ASRT in physiological photo-regulation. ASRT is known to be related to photochromism, because it regulates the expression of phycocyanin (cpc-gene) and phycoerythrocyanin (pec gene), two major proteins of the phycobilisome in cyanobacteria. By examining wild type and knockout mutant Anabaena cells, we showed that ASRT repressed the expression of these two genes. We also demonstrated physical interactions between ASRT, ASR, and the promoter regions of cpc, pec, kaiABC (circadian clock gene) and the asr operon, both in vitro and in vivo. Binding assays indicated that ASRT had different sites of interaction for binding to ASR and DNA promoter regions. ASRT also influenced the retinal re-isomerization rate in dark through a physical interaction with ASR, and it regulated reporter gene expression in vivo. These results suggested that ASRT relayed the photosignal from ASR and directly regulated gene expression.

100 fs photo-isomerization with vibrational coherences but low quantum yield in Anabaena Sensory Rhodopsin.

Anabaena Sensory Rhodopsin (ASR) stands out among the microbial retinal proteins in that, under light-adaptation (LA) conditions, it binds both the 13-cis isomer and the all-trans isomer of the protonated Schiff base of retinal (PSBR). In the dark-adapted (DA) state, more than 95% of the proteins bear all-trans PSBR, and the protein environment adopts a different equilibrium state. We report the excited state and photo-isomerization kinetics of ASR under different LA conditions. The full data set allows confirming that the photoisomerization of the 13C isomer occurs within 100 fs and indications of an excited and ground state wavepacket launched by the ultrafast non-adiabatic reaction are reported. Even though this recalls the record isomerization time and the coherent reaction scenario of 11-cis PSBR in rhodopsin, the photoisomerization quantum yield (QY) is much lower, actually the lowest value ever reported for retinal proteins (<15%). Noticeably, in ASR the excited state lifetime (ESL) is at least five times larger and the QY is more than twice as large for AT PSBR as compared to 13C PSBR. We argue that ESL and QY cannot be expected to be correlated at all, but that the latter is decided on, as often anticipated, by the wavepacket pathways leading to the conical intersection seam.

DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy.

Anabaena sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by Anabaena sensory rhodopsin. Here, we characterized DNA binding activity of ASRT probed by using fluorescence correlation spectroscopy. We observed clear decrease of diffusion coefficient of DNA upon binding of ASRT. The dissociation constant, K(D), of ASRT to 20 bp-long DNA fragments lied in micro-molar range and varied moderately with DNA sequence. Our results suggest that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of several genes that need to be activated depending on the light illumination.

Growth retardation of Escherichia coli by artificial increase of intracellular ATP.

Overexpression of phosphoenolpyruvate carboxykinase (PCK) was reported to cause the harboring of higher intracellular ATP concentration in Escherichia coli, accompanied with a slower growth rate. For systematic determination of the relationship between the artificial increase of ATP and growth retardation, PCKWT enzyme was directly evolved in vitro and further overexpressed. The evolved PCK67 showed a 60% greater catalytic efficiency than that of PCKWT. Consequently, the PCK67-overexpressing E. coli showed the highest ATP concentration at the log phase of 1.45 µmol/gcell, with the slowest growth rate of 0.66 h(-1), while the PCKWT-overexpressing cells displayed 1.00 µmol/gcell ATP concentration with the growth rate of 0.84 h(-1) and the control had 0.28 µmol/gcell with 1.03 h(-1). To find a plausible reason, PCK-overexpressing cells in a steady state during chemostat growth were applied to monitor intracellular reactive oxygen species (ROS). Higher amount of intracellular ROS were observed as the ATP levels increased. To confirm the hypothesis of slower growth rate without perturbation of the carbon flux by PCK-overexpression, phototrophic Gloeobacter rhodopsin (GR) was expressed. The GR-expressing strain under illumination harbored 81% more ATP concentration along with 82% higher ROS, with a 54% slower maximum growth rate than the control, while both the GR-expressing strain under dark and dicarboxylate transporter (a control membrane protein)-expressing strain showed a lower ATP and increased ROS, and slower growth rate. Regardless of carbon flux changes, the artificial ATP increase was related to the ROS increase and it was reciprocally correlated to the maximum growth rate. To verify that the accumulated intracellular ROS were responsible for the growth retardation, glutathione was added to the medium to reduce the ROS. As a result, the growth retardation was restored by the addition of 0.1 mM glutathione. Anaerobic culture even enabled the artificial ATP-increased E. coli to grow faster than control. Collectively, it was concluded that artificial ATP increases inhibit the growth of E. coli due to the overproduction of ROS.

Light-driven Na(+) pump from Gillisia limnaea: a high-affinity Na(+) binding site is formed transiently in the photocycle.

A group of microbial retinal proteins most closely related to the proton pump xanthorhodopsin has a novel sequence motif and a novel function. Instead of, or in addition to, proton transport, they perform light-driven sodium ion transport, as reported for one representative of this group (KR2) from Krokinobacter. In this paper, we examine a similar protein, GLR from Gillisia limnaea, expressed in Escherichia coli, which shares some properties with KR2 but transports only Na(+). The absorption spectrum of GLR is insensitive to Na(+) at concentrations of ≤3 M. However, very low concentrations of Na(+) cause profound differences in the decay and rise time of photocycle intermediates, consistent with a switch from a "Na(+)-independent" to a "Na(+)-dependent" photocycle (or photocycle branch) at ∼60 µM Na(+). The rates of photocycle steps in the latter, but not the former, are linearly dependent on Na(+) concentration. This suggests that a high-affinity Na(+) binding site is created transiently after photoexcitation, and entry of Na(+) from the bulk to this site redirects the course of events in the remainder of the cycle. A greater concentration of Na(+) is needed for switching the reaction path at lower pH. The data suggest therefore competition between H(+) and Na(+) to determine the two alternative pathways. The idea that a Na(+) binding site can be created at the Schiff base counterion is supported by the finding that upon perturbation of this region in the D251E mutant, Na(+) binds without photoexcitation. Binding of Na(+) to the mutant shifts the chromophore maximum to the red like that of H(+), which occurs in the photocycle of the wild type.

Real-time Visualization of Transcribed mRNA via Click Chemistry in a Liposomal Space.

We present a CuAAC (Copper-Catalyzed Azide-Alkyne Cycloaddition) reaction protocol designed for the visualization of mRNA. To achieve this, we synthesized stable mRNA molecules incorporating the modified nucleoside analog, EU, a crucial element for fluorophore attachment. Leveraging this modified mRNA, we successfully executed the CuAAC reaction, wherein the pro-fluorophore, coumarin, was conjugated to EU on the mRNA through our meticulously designed CuAAC process. This innovative approach resulted in the emission of fluorescence, enabling both precise quantification and visual observation of mRNA. Furthermore, we demonstrated the feasibility of concurrent mRNA synthesis and visualization by seamlessly integrating the CuAAC reaction mix into the mRNA transcription process. Additionally, our novel methodology opens avenues for prospective real-time monitoring of mRNA transcription within artificial cells. These advancements hold significant promise for expanding our comprehension of fundamental cellular processes and finding applications across diverse biological contexts in the future.

Proton-pumping photoreceptor controls expression of ABC transporter by regulating transcription factor through light.

Light is a significant factor for living organisms with photosystems, like microbial rhodopsin-a retinal protein that functions as an ion pump, channel, and sensory transduction. Gloeobacter violaceus PCC7421, has a proton-pumping rhodopsin gene, the Gloeobacter rhodopsin (GR). The helix-turn-helix family of transcriptional regulators has various motifs, and they regulate gene expression in the presence of various metal ions. Here, we report that active proton outward pumping rhodopsin interacted with the helix-turn-helix transcription regulator and regulated gene expression. This interaction is confirmed using ITC analysis (KD of 8 µM) and determined the charged residues required. During in vitro experiments using fluorescent and luciferase reporter systems, ATP-binding cassette (ABC) transporters and the self-regulation of G. violaceus transcriptional regulator (GvTcR) are regulated by light, and gene regulation is observed in G. violaceus using the real-time polymerase chain reaction. These results expand our understanding of the natural potential and limitations of microbial rhodopsin function.