Extracellular Polysaccharide Receptor and Receptor-Binding Proteins of the Rhodobacter capsulatus Bacteriophage-like Gene Transfer Agent RcGTA.

A variety of prokaryotes produce a bacteriophage-like gene transfer agent (GTA), and the alphaproteobacterial Rhodobacter capsulatus RcGTA is a model GTA. Some environmental isolates of R. capsulatus lack the ability to acquire genes transferred by the RcGTA (recipient capability). In this work, we investigated the reason why R. capsulatus strain 37b4 lacks recipient capability. The RcGTA head spike fiber and tail fiber proteins have been proposed to bind extracellular oligosaccharide receptors, and strain 37b4 lacks a capsular polysaccharide (CPS). The reason why strain 37b4 lacks a CPS was unknown, as was whether the provision of a CPS to 37b4 would result in recipient capability. To address these questions, we sequenced and annotated the strain 37b4 genome and used BLAST interrogations of this genome sequence to search for homologs of genes known to be needed for R. capsulatus recipient capability. We also created a cosmid-borne genome library from a wild-type strain, mobilized the library into 37b4, and used the cosmid-complemented strain 37b4 to identify genes needed for a gain of function, allowing for the acquisition of RcGTA-borne genes. The relative presence of CPS around a wild-type strain, 37b4, and cosmid-complemented 37b4 cells was visualized using light microscopy of stained cells. Fluorescently tagged head spike fiber and tail fiber proteins of the RcGTA particle were created and used to measure the relative binding to wild-type and 37b4 cells. We found that strain 37b4 lacks recipient capability because of an inability to bind RcGTA; the reason it is incapable of binding is that it lacks CPS, and the absence of CPS is due to the absence of genes previously shown to be needed for CPS production in another strain. In addition to the head spike fiber, we found that the tail fiber protein also binds to the CPS.

Intracytoplasmic-membrane development in alphaproteobacteria involves the homolog of the mitochondrial crista-developing protein Mic60.

Mitochondrial cristae expand the surface area of respiratory membranes and ultimately allow for the evolutionary scaling of respiration with cell volume across eukaryotes. The discovery of Mic60 homologs among alphaproteobacteria, the closest extant relatives of mitochondria, suggested that cristae might have evolved from bacterial intracytoplasmic membranes (ICMs). Here, we investigated the predicted structure and function of alphaproteobacterial Mic60, and a protein encoded by an adjacent gene Orf52, in two distantly related purple alphaproteobacteria, Rhodobacter sphaeroides and Rhodopseudomonas palustris. In addition, we assessed the potential physical interactors of Mic60 and Orf52 in R. sphaeroides. We show that the three α helices of mitochondrial Mic60's mitofilin domain, as well as its adjacent membrane-binding amphipathic helix, are present in alphaproteobacterial Mic60. The disruption of Mic60 and Orf52 caused photoheterotrophic growth defects, which are most severe under low light conditions, and both their disruption and overexpression led to enlarged ICMs in both studied alphaproteobacteria. We also found that alphaproteobacterial Mic60 physically interacts with BamA, the homolog of Sam50, one of the main physical interactors of eukaryotic Mic60. This interaction, responsible for making contact sites at mitochondrial envelopes, has been conserved in modern alphaproteobacteria despite more than a billion years of evolutionary divergence. Our results suggest a role for Mic60 in photosynthetic ICM development and contact site formation at alphaproteobacterial envelopes. Overall, we provide support for the hypothesis that mitochondrial cristae evolved from alphaproteobacterial ICMs and have therefore improved our understanding of the nature of the mitochondrial ancestor.

Formal recognition and classification of gene transfer agents as viriforms.

Morphological and genetic features strongly suggest that gene transfer agents (GTAs) are caudoviricete-derived entities that have evolved in concert with cellular genomes to such a degree that they should not be considered viruses. Indeed, GTA particles resemble caudoviricete virions, but, in contrast to caudoviricetes (or any viruses), GTAs can encapsidate at best only part of their own genomes, are induced solely in small subpopulations of prokaryotic host cells, and are transmitted vertically as part of cellular genomes during replication and division. Therefore, the lifecycles of GTAs are analogous to virus-derived entities found in the parasitoid wasps, which have recently been recognized as non-virus entities and therefore reclassified as viriforms. We evaluated three distinct, independently exapted GTA groups, for which the genetic basis for GTA particle production has been established. Based on the evidence, we outline a classification scheme for these viriforms.

DNA Gyrase Inhibitors Increase the Frequency of Bacteriophage-like RcGTA-Mediated Gene Transfer in Rhodobacter capsulatus.

Rhodobacter capsulatus produces a bacteriophage-like particle called the gene transfer agent (RcGTA) that mediates horizontal gene transfer. RcGTA particles transfer random ~4.5-kb fragments of genomic DNA that integrate into recipient genomes by allelic replacement. This work addresses the effect of sub-inhibitory concentrations of antibiotics on gene transfer by RcGTA. A transduction assay was developed to test the effects of various substances on gene transfer. Using this assay, low concentrations of DNA gyrase inhibitors were found to increase the frequency of gene transfer. Novobiocin was studied in more detail, and it was found that this antibiotic did not influence the production or release of RcGTA but instead appeared to act on the recipient cells. The target of novobiocin in other species has been shown to be the GyrB subunit of DNA gyrase (a heterotetramer of 2GyrA and 2GyrB). R. capsulatus encodes GyrA and GyrB homologues, and a GyrB overexpression plasmid was created and found to confer resistance to novobiocin. The presence of the overexpression plasmid in recipient cells greatly diminished the novobiocin-mediated increase in gene transfer, confirming that this effect is due to the binding of novobiocin by GyrB. The results of this work show that antibiotics affect gene transfer in R. capsulatus and may be relevant to microbial genetic exchange in natural ecosystems.

Persulfide-Responsive Transcription Factor SqrR Regulates Gene Transfer and Biofilm Formation via the Metabolic Modulation of Cyclic di-GMP in Rhodobacter capsulatus.

Bacterial phage-like particles (gene transfer agents-GTAs) are widely employed as a crucial genetic vector in horizontal gene transfer. GTA-mediated gene transfer is induced in response to various stresses; however, regulatory mechanisms are poorly understood. We found that the persulfide-responsive transcription factor SqrR may repress the expression of several GTA-related genes in the photosynthetic bacterium Rhodobacter capsulatus. Here, we show that the sqrR deletion mutant (ΔsqrR) produces higher amounts of intra- and extracellular GTA and gene transfer activity than the wild type (WT). The transcript levels of GTA-related genes are also increased in ΔsqrR. In spite of the presumption that GTA-related genes are regulated in response to sulfide by SqrR, treatment with sulfide did not alter the transcript levels of these genes in the WT strain. Surprisingly, hydrogen peroxide increased the transcript levels of GTA-related genes in the WT, and this alteration was abolished in the ΔsqrR strain. Moreover, the absence of SqrR changed the intracellular cyclic dimeric GMP (c-di-GMP) levels, and the amount of c-di-GMP was correlated with GTA activity and biofilm formation. These results suggest that SqrR is related to the repression of GTA production and the activation of biofilm formation via control of the intracellular c-di-GMP levels.

Purification and preparation of Rhodobacter sphaeroides reaction centers for photocurrent measurements and atomic force microscopy characterization.

The formation of defined surfaces consisting of photosynthetic reaction centers (RCs) in biohybrid solar cells is challenging. Here, we start with the production of engineered RCs for oriented binding. RCs are deposited onto gold electrodes, and 6-mercapto-1-hexanol (MCH) is used to displace multilayers and non-specifically adsorbed RCs. The resulting electrode surfaces are analyzed for photocurrent generation using an intensity-modulated light and lock-in amplifier. Atomic force microscopy (AFM) is used to characterize the surface and the formation of RC structural assemblies. For complete details on the use and execution of this profile, please refer to Jun et al. (2021).

Genomic diversity of bacteriophages infecting Rhodobacter capsulatus and their relatedness to its gene transfer agent RcGTA.

The diversity of bacteriophages is likely unparalleled in the biome due to the immense variety of hosts and the multitude of viruses that infect them. Recent efforts have led to description at the genomic level of numerous bacteriophages that infect the Actinobacteria, but relatively little is known about those infecting other prokaryotic phyla, such as the purple non-sulfur photosynthetic α-proteobacterium Rhodobacter capsulatus. This species is a common inhabitant of freshwater ecosystems and has been an important model system for the study of photosynthesis. Additionally, it is notable for its utilization of a unique form of horizontal gene transfer via a bacteriophage-like element known as the gene transfer agent (RcGTA). Only three bacteriophages of R. capsulatus had been sequenced prior to this report. Isolation and characterization at the genomic level of 26 new bacteriophages infecting this host advances the understanding of bacteriophage diversity and the origins of RcGTA. These newly discovered isolates can be grouped along with three that were previously sequenced to form six clusters with four remaining as single representatives. These bacteriophages share genes with RcGTA that seem to be related to host recognition. One isolate was found to cause lysis of a marine bacterium when exposed to high-titer lysate. Although some clusters are more highly represented in the sequenced genomes, it is evident that many more bacteriophage types that infect R. capsulatus are likely to be found in the future.

Correlating structural assemblies of photosynthetic reaction centers on a gold electrode and the photocurrent - potential response.

The use of biomacromolecules is a nascent development in clean alternative energies. In applications of biosensors and biophotovoltaic devices, the bacterial photosynthetic reaction center (RC) is a protein-pigment complex that has been commonly interfaced with electrodes, in large part to take advantage of the long-lived and high efficiency of charge separation. We investigated assemblies of RCs on an electrode that range from monolayer to multilayers by measuring the photocurrent produced when illuminated by an intensity-modulated excitation light source. In addition, atomic force microscopy and modeling of the photocurrent with the Marcus-Hush-Chidsey theory detailed the reorganization energy for the electron transfer process, which also revealed changes in the RC local environment due to the adsorbed conformations. The local environment in which the RCs are embedded significantly influenced photocurrent generation, which has implications for electron transfer of other biomacromolecules deposited on a surface in sensor and photovoltaic applications employing a redox electrolyte.

The CckA-ChpT-CtrA Phosphorelay Controlling Rhodobacter capsulatus Gene Transfer Agent Production Is Bidirectional and Regulated by Cyclic di-GMP.

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus, these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads.IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

Introduction of the Menaquinone Biosynthetic Pathway into Rhodobacter sphaeroides and de Novo Synthesis of Menaquinone for Incorporation into Heterologously Expressed Integral Membrane Proteins.

Quinones are redox-active molecules that transport electrons and protons in organelles and cell membranes during respiration and photosynthesis. In addition to the fundamental importance of these processes in supporting life, there has been considerable interest in exploiting their mechanisms for diverse applications ranging from medical advances to innovative biotechnologies. Such applications include novel treatments to target pathogenic bacterial infections and fabricating biohybrid solar cells as an alternative renewable energy source. Ubiquinone (UQ) is the predominant charge-transfer mediator in both respiration and photosynthesis. Other quinones, such as menaquinone (MK), are additional or alternative redox mediators, for example in bacterial photosynthesis of species such as Thermochromatium tepidum and Chloroflexus aurantiacus. Rhodobacter sphaeroides has been used extensively to study electron transfer processes, and recently as a platform to produce integral membrane proteins from other species. To expand the diversity of redox mediators in R. sphaeroides, nine Escherichia coli genes encoding the synthesis of MK from chorismate and polyprenyl diphosphate were assembled into a synthetic operon in a newly designed expression plasmid. We show that the menFDHBCE, menI, menA, and ubiE genes are sufficient for MK synthesis when expressed in R. sphaeroides cells, on the basis of high performance liquid chromatography and mass spectrometry. The T. tepidum and C. aurantiacus photosynthetic reaction centers produced in R. sphaeroides were found to contain MK. We also measured in vitro charge recombination kinetics of the T. tepidum reaction center to demonstrate that the MK is redox-active and incorporated into the QA pocket of this heterologously expressed reaction center.

Induction of Rhodobacter capsulatus Gene Transfer Agent Gene Expression Is a Bistable Stochastic Process Repressed by an Extracellular Calcium-Binding RTX Protein Homologue.

Bacteriophage-like gene transfer agents (GTAs) have been discovered in both of the prokaryotic branches of the three-domain phylogenetic tree of life. The production of a GTA (RcGTA) by the phototrophic alphaproteobacterium Rhodobacter capsulatus is regulated by quorum sensing and a phosphorelay homologous to systems in other species that control essential functions such as the initiation of chromosome replication and cell division. In wild-type strains, RcGTA is produced in <3% of cells in laboratory cultures. Mutants of R. capsulatus that exhibit greatly elevated production of RcGTA were created decades ago by chemical mutagenesis, but the nature and molecular consequences of the mutation were unknown. We show that the number of cells in a population that go on to express RcGTA genes is controlled by a stochastic process, in contrast to a genetic process. We used transposon mutagenesis along with a fluorescent protein reporter system and genome sequence data to identify a gene, rcc00280, that encodes an RTX family calcium-binding protein homologue. The Rc280 protein acts as an extracellular repressor of RcGTA gene expression by decreasing the percentage of cells that induce the production of RcGTA.IMPORTANCE GTAs catalyze horizontal gene transfer (HGT), which is important for genomic evolution because the majority of genes found in bacterial genomes have undergone HGT at some point in their evolution. Therefore, it is important to determine how the production of GTAs is regulated to understand the factors that modulate the frequency of gene transfer and thereby specify the tempo of evolution. This work describes a new type of genetic regulation in which an extracellular calcium-binding protein homologue represses the induction of the Rhodobacter capsulatus GTA, RcGTA.

Porphyrin Excretion Resulting From Mutation of a Gene Encoding a Class I Fructose 1,6-Bisphosphate Aldolase in Rhodobacter capsulatus.

This paper describes a mutant (called SB1707) of the Rhodobacter capsulatus wild type strain SB1003 in which a transposon-disrupted rcc01707 gene resulted in a ∼25-fold increase in the accumulation of coproporphyrin III in the medium of phototrophic (anaerobic) cultures grown in a yeast extract/peptone medium. There was little or no stimulation of pigment accumulation in aerobic cultures. Therefore, this effect of rcc01707 mutation appears to be specific for the anaerobic coproporphyrinogen III oxidase HemN as opposed to the aerobic enzyme HemF. The protein encoded by rcc01707 is homologous to Class I fructose 1,6-bisphosphate aldolases, which catalyze a glycolytic reaction that converts fructose 1, 6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, precursors of pyruvate. There were significant differences in coproporphyrin III accumulation using defined media with individual organic acids and sugars as the sole carbon source: pyruvate, succinate and glutamate stimulated accumulation the most, whereas glucose suppressed coproporphyrin III accumulation to 10% of that of succinate. However, although quantitatively lesser, similar effects of carbon source on the amount of accumulated pigment in the culture medium were seen in a wild type control. Therefore, this mutation appears to exaggerate effects also seen in the wild type strain. It is possible that mutation of rcc01707 causes a metabolic bottleneck or imbalance that was not rectified during growth on the several carbon sources tested. However, we speculate that, analogous to other fructose 1,6-bisphosphate aldolases, the rcc01707 gene product has a "moonlighting" activity that in this case is needed for the maximal expression of the hemN gene. Indeed, it was found that the rcc01707 gene is needed for maximal expression of a hemN promoter-lacZ reporter. With the decrease in hemN expression due to the absence of the rcc01707 gene product, coproporphyrinogen III accumulates and is released from the cell, yielding the spontaneous oxidation product coproporphyrin III.

The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution.

Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this 'one size fits all' method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during 'on-column', 'in-gel', and 'on-bead' reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.

The Protease ClpXP and the PAS Domain Protein DivL Regulate CtrA and Gene Transfer Agent Production in Rhodobacter capsulatus.

Several members of the Rhodobacterales (Alphaproteobacteria) produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is the Rhodobacter capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in R. capsulatus and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the RhodobacteralesIMPORTANCE Members of the Rhodobacterales are abundant in ocean and freshwater environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles.

A physiological perspective on the origin and evolution of photosynthesis.

The origin and early evolution of photosynthesis are reviewed from an ecophysiological perspective. Earth's first ecosystems were chemotrophic, fueled by geological H2 at hydrothermal vents and, required flavin-based electron bifurcation to reduce ferredoxin for CO2 fixation. Chlorophyll-based phototrophy (chlorophototrophy) allowed autotrophs to generate reduced ferredoxin without electron bifurcation, providing them access to reductants other than H2. Because high-intensity, short-wavelength electromagnetic radiation at Earth's surface would have been damaging for the first chlorophyll (Chl)-containing cells, photosynthesis probably arose at hydrothermal vents under low-intensity, long-wavelength geothermal light. The first photochemically active pigments were possibly Zn-tetrapyrroles. We suggest that (i) after the evolution of red-absorbing Chl-like pigments, the first light-driven electron transport chains reduced ferredoxin via a type-1 reaction center (RC) progenitor with electrons from H2S; (ii) photothioautotrophy, first with one RC and then with two, was the bridge between H2-dependent chemolithoautotrophy and water-splitting photosynthesis; (iii) photothiotrophy sustained primary production in the photic zone of Archean oceans; (iv) photosynthesis arose in an anoxygenic cyanobacterial progenitor; (v) Chl a is the ancestral Chl; and (vi), anoxygenic chlorophototrophic lineages characterized so far acquired, by horizontal gene transfer, RCs and Chl biosynthesis with or without autotrophy, from the architects of chlorophototrophy-the cyanobacterial lineage.

The Rhodobacter capsulatus gene transfer agent is induced by nutrient depletion and the RNAP omega subunit.

Small bacteriophage-like particles called gene transfer agents (GTAs) that mediate DNA transfer between cells are produced by a variety of prokaryotes. The model GTA, produced by the alphaproteobacterium Rhodobacter capsulatus (RcGTA), is controlled by several cellular regulators, and production is induced upon entry into the stationary phase. We report that RcGTA production and gene transfer are stimulated by nutrient depletion. Cells depleted of organic carbon or blocked for amino acid biosynthesis increased RcGTA production and release from cells. Furthermore, cells lacking the sole RelA-SpoT homologue produced decreased levels of RcGTA, and the RNA polymerase omega (ω) subunit was required for appreciable production of RcGTA.

The Distribution, Evolution, and Roles of Gene Transfer Agents in Prokaryotic Genetic Exchange.

Diverse prokaryotes produce gene transfer agents (GTAs), which are bacteriophage-like particles that exclusively package pieces of the producing cell's genome and transfer them to other cells. There are clear evolutionary connections between GTAs and phages, but GTAs have properties that lead us to suggest they are more than simply defective phages and instead provide a selective advantage for the producing organisms. The five types of currently known GTAs are genetically distinct, indicating multiple instances of convergent evolution. GTA production can be regulated by the producing organism and coordinated to coincide with development of the capability to receive DNA from GTAs. Recent discoveries of the genetic basis of GTA production in the bacterium Rhodobacter capsulatus and characterization of novel phages that possess homologs of this GTA's structural and regulatory genes have provided important new connections among these elements and highlight the tangled evolutionary relationships within the phageome.

Guaranteeing a captive audience: coordinated regulation of gene transfer agent (GTA) production and recipient capability by cellular regulators.

Gene transfer agents (GTAs) are bacteriophage-like particles produced by many prokaryotes. Several members of the Alphaproteobacteria produce a class of genetically-related GTAs that is best studied in Rhodobacter capsulatus. DNA transfer by the R. capsulatus GTA (RcGTA) combines aspects of both transduction and natural transformation, as recipient cells require a natural transformation-like system to incorporate donated DNA. The genes involved in RcGTA production and recipient capability are located at multiple loci in the bacterial genome; however, a conserved phosphorelay containing the response regulator CtrA and a quorum sensing system regulate both RcGTA production and recipient capability. This review highlights recent discoveries in RcGTA biology, and focuses on the co-regulation of genes involved in RcGTA production and recipient capability.

Electrochemical Field-Effect Transistor Utilization to Study the Coupling Success Rate of Photosynthetic Protein Complexes to Cytochrome c.

Due to the high internal quantum efficiency, reaction center (RC) proteins from photosynthetic organisms have been studied in various bio-photoelectrochemical devices for solar energy harvesting. In vivo, RC and cytochrome c (cyt c; a component of the biological electron transport chain) can form a cocomplex via interprotein docking. This mechanism can be used in vitro for efficient electron transfer from an electrode to the RC in a bio-photoelectrochemical device. Hence, the success rate in coupling RCs to cyt c is of great importance for practical applications in the future. In this work, we use an electrochemical transistor to study the binding of the RC to cytochrome. The shift in the transistor threshold voltage was measured in the dark and under illumination to estimate the density of cytochrome and coupled RCs on the gate of the transistor. The results show that ~33% of the cyt cs on the transistor gate were able to effectively couple with RCs. Due to the high sensitivity of the transistor, the approach can be used to make photosensors for detecting low light intensities.