Single-Molecule Detection of the Encounter and Productive Electron Transfer Complexes of a Photosynthetic Reaction Center.

Small, diffusible redox proteins play an essential role in electron transfer (ET) in respiration and photosynthesis, sustaining life on Earth by shuttling electrons between membrane-bound complexes via finely tuned and reversible interactions. Ensemble kinetic studies show transient ET complexes form in two distinct stages: an "encounter" complex largely mediated by electrostatic interactions, which subsequently, through subtle reorganization of the binding interface, forms a "productive" ET complex stabilized by additional hydrophobic interactions around the redox-active cofactors. Here, using single-molecule force spectroscopy (SMFS) we dissected the transient ET complexes formed between the photosynthetic reaction center-light harvesting complex 1 (RC-LH1) of Rhodobacter sphaeroides and its native electron donor cytochrome c2 (cyt c2). Importantly, SMFS resolves the distribution of interaction forces into low (∼150 pN) and high (∼330 pN) components, with the former more susceptible to salt concentration and to alteration of key charged residues on the RC. Thus, the low force component is suggested to reflect the contribution of electrostatic interactions in forming the initial encounter complex, whereas the high force component reflects the additional stabilization provided by hydrophobic interactions to the productive ET complex. Employing molecular dynamics simulations, we resolve five intermediate states that comprise the encounter, productive ET and leaving complexes, predicting a weak interaction between cyt c2 and the LH1 ring near the RC-L subunit that could lie along the exit path for oxidized cyt c2. The multimodal nature of the interactions of ET complexes captured here may have wider implications for ET in all domains of life.

Two pathways to understanding electron transfer in reaction centers from photosynthetic bacteria: A comparison of Rhodobacter sphaeroides and Rhodobacter capsulatus mutants.

The rates, yields, mechanisms and directionality of electron transfer (ET) are explored in twelve pairs of Rhodobacter (R.) sphaeroides and R. capsulatus mutant RCs designed to defeat ET from the excited primary donor (P*) to the A-side cofactors and re-direct ET to the normally inactive mirror-image B-side cofactors. In general, the R. sphaeroides variants have larger P+HB- yields (up to ∼90%) than their R. capsulatus analogs (up to ∼60%), where HB is the B-side bacteriopheophytin. Substitution of Tyr for Phe at L-polypeptide position L181 near BB primarily increases the contribution of fast P* â†’ P+BB- â†’ P+HB- two-step ET, where BB is the "bridging" B-side bacteriochlorophyll. The second step (∼6-8 ps) is slower than the first (∼3-4 ps), unlike A-side two-step ET (P* â†’ P+BA- â†’ P+HA-) where the second step (∼1 ps) is faster than the first (∼3-4 ps) in the native RC. Substitutions near HB, at L185 (Leu, Trp or Arg) and at M-polypeptide site M133/131 (Thr, Val or Glu), strongly affect the contribution of slower (20-50 ps) P* â†’ P+HB- one-step superexchange ET. Both ET mechanisms are effective in directing electrons "the wrong way" to HB and both compete with internal conversion of P* to the ground state (∼200 ps) and ET to the A-side cofactors. Collectively, the work demonstrates cooperative amino-acid control of rates, yields and mechanisms of ET in bacterial RCs and how A- vs. B-side charge separation can be tuned in both species.

Sulfoquinovosyl diacylglycerol is required for dimerisation of the Rhodobacter sphaeroides reaction centre-light harvesting 1 core complex.

The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer, in which 14 LH1 subunits form a C-shaped antenna around 1 RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding 2 RCs. Alongside the five RC and LH1 subunits, an additional polypeptide known as PufX provides an interface for dimerisation and also prevents LH1 ring closure, introducing a channel for quinone exchange that is essential for photoheterotrophic growth. Structures of Rba. sphaeroides RC-LH1 complexes revealed several new components; protein-Y, which helps to form the quinone channel; protein-Z, of unknown function and seemingly unique to dimers; and a tightly bound sulfoquinovosyl diacylglycerol (SQDG) lipid that interacts with two PufX arginine residues. This lipid lies at the dimer interface alongside weak density for a second molecule, previously proposed to be an ornithine lipid. In this work we have generated strains of Rba. sphaeroides lacking protein-Y, protein-Z, SQDG or ornithine lipids to assess the roles of these previously unknown components in the assembly and activity of RC-LH1. We show that whilst the removal of either protein-Y, protein-Z or ornithine lipids has only subtle effects, SQDG is essential for the formation of RC-LH1 dimers but its absence has no functional effect on the monomeric complex.

Light energy utilization and microbial catalysis for enhanced biohydrogen: Ternary coupling system of triethanolamine-mediated Fe@C-Rhodobacter sphaeroides.

This study investigated the mediating effect of Triethanolamine on Fe@C-Rhodobacter sphaeroides hybrid photosynthetic system to achieve efficient biohydrogen production. The biocompatible Fe@C generates excited electrons upon exposure to light, releasing ferrum for nitrogenase synthesis, and regulating the pH of the fermentation environment. Triethanolamine was introduced to optimize the electron transfer chain, thereby improving system stability, prolonging electron lifespan, and facilitating ferrum corrosion. This, in turn, stimulated the lactic acid synthetic metabolic pathway of Rhodobacter sphaeroides, resulting in increased reducing power in the biohybrid system. The ternary coupling system was analyzed through the regulation of concentration, initial pH, and light intensity. The system achieved the highest total H2 production of 5410.9 mL/L, 1.29 times higher than the control (2360.5 mL/L). This research provides a valuable strategy for constructing ferrum-carbon-based composite-cellular biohybrid systems for photo-fermentation H2 production.

Modulation of Electron Transfer Branches by Atrazine and Triazine Herbicides in Photosynthetic Reaction Centers.

Quinone analogue molecules, functioning as herbicides, bind to the secondary quinone site, QB, in type-II photosynthetic reaction centers, including those from purple bacteria (PbRC). Here, we investigated the impact of herbicide binding on electron transfer branches, using herbicide-bound PbRC crystal structures and employing the linear Poisson-Boltzmann equation. In contrast to urea and phenolic herbicides [Fufezan, C. Biochemistry 2005, 44, 12780-12789], binding of atrazine and triazine did not cause significant changes in the redox-potential (Em) values of the primary quinone (QA) in these crystal structures. However, a slight Em difference at the bacteriopheophytin in the electron transfer inactive branch (HM) was observed between the S(-)- and R(+)-triazine-bound PbRC structures. This discrepancy is linked to variations in the protonation pattern of the tightly coupled Glu-L212 and Glu-H177 pairs, crucial components of the proton uptake pathway in native PbRC. These findings suggest the existence of a QB-mediated link between the electron transfer inactive HM and the proton uptake pathway in PbRCs.

Rhodobacter sphaeroides supplementation improves defense ability of Chinese mitten crab Eriocheir sinensis against Shewanella putrefaciens infection via intestinal flora and metabolism regulation.

Shewanella putrefaciens is a vital bacterial pathogen implicated in serious diseases in Chinese mitten crab Eriocheir sinensis. Yet the use of probiotics to improve the defense ability of E. sinensis against S. putrefaciens infection remains poorly understood. In the present study, the protective effect of dietary R. sphaeroides against S. putrefaciens infection in E. sinensis was evaluated through antioxidant capability, immune response, and survival under bacterial challenge assays, and its protective mechanism was further explored using a combination of intestinal flora and metabolome assays. Our results indicated that dietary R. sphaeroides could significantly improve immunity and antioxidant ability of Chinese mitten crabs, thereby strengthening their disease resistance with the relative percentage survival of 81.09% against S. putrefaciens. In addition, dietary R. sphaeroides could significantly alter the intestinal microbial composition and intestinal metabolism of crabs, causing not only the reduction of potential threatening pathogen load but also the increase of differential metabolites in tryptophan metabolism, pyrimidine metabolism, and glycerophospholipid metabolism. Furthermore, the regulation of differential metabolites such as N-Acetylserotonin positively correlated with beneficial Rhodobacter could be a potential protection strategy for Shewanella infection. To the best of our knowledge, this is the first study to illustrate the protective effect and mechanism of R. sphaeroides supplementation to protect E. sinensis against S. putrefaciens infection.

The potential of Rhodobacter sphaeroides extract as an alternative supplement for cell culture systems.

It is essential to identify suitable supplements that enhance cell growth, viability, and functional development in cell culture systems. The use of fetal bovine serum (FBS) has been common, but it has limitations, such as batch-to-batch variability, ethical concerns, and risks of environmental contamination. In this study, we explore the potential of Rhodobacter sphaeroides extract, derived from a probiotic photosynthetic bacterium, as an alternative supplement. Our results demonstrate that the extract from R. sphaeroides significantly improves various aspects of cell behavior compared to serum-free conditions. It enhances cell growth and viability to a greater extent than FBS supplementation. Additionally, the extract alleviates oxidative stress by reducing intracellular levels of reactive oxygen species and stimulates lysosomal activity, contributing to cellular processes. The presence of abundant amino acids, glycine and arginine, in the extract may play a role in promoting cell growth. These findings emphasize the potential of R. sphaeroides extract as a valuable supplement for cell culture, offering advantages over the use of FBS.IMPORTANCEThe choice of supplements for cell culture is crucial in biomedical research, but the widely used fetal bovine serum (FBS) has limitations in terms of variability, ethics, and environmental risks. This study explores the potential of an extract from Rhodobacter sphaeroides, a probiotic bacterium, as an alternative supplement. The findings reveal that the R. sphaeroides extract surpasses FBS in enhancing cell growth, viability, and functionality. It also mitigates oxidative stress and stimulates lysosomal activity, critical for cellular health. The extract's abundance of glycine and arginine, amino acids with known growth-promoting effects, further highlights its potential. By providing a viable substitute for FBS, the R. sphaeroides extract addresses the need for consistent, ethical, and environmentally friendly cell culture supplements. This research paves the way for sustainable and reliable cell culture systems, revolutionizing biomedical research and applications in drug development and regenerative medicine.

Among the recombinant TSPOs, the BcTSPO.

Overexpression of recombinant Bacillus cereus TSPO (BcTSPO) in E. coli bacteria leads to its recovery with a bound hemin both in bacterial membrane (MB) and inclusion bodies (IB). Unlike mouse TSPO, BcTSPO purified in SDS detergent from IB is well structured and can bind various ligands such as high-affinity PK 11195, protoporphyrin IX (PPIX) and δ-aminolevulinic acid (ALA). For each of the three ligands, 1H-15N HSQC titration NMR experiments suggest that different amino acids of BcTSPO binding cavity are involved in the interaction. PPIX, an intermediate of heme biosynthesis, binds to the cavity of BcTSPO and its fluorescence can be significantly reduced in the presence of light and oxygen. The light irradiation leads to two products that have been isolated and characterized as photoporphyrins. They result from the addition of singlet oxygen to the two vinyl groups hence leading to the formation of hydroxyaldehydes. The involvement of water molecules, recently observed along with the binding of heme in Rhodobacter sphaeroides (RsTSPO) is highly probable. Altogether, these results raise the question of the role of TSPO in heme biosynthesis regulation as a possible scavenger of reactive intermediates.

Excitation energy transfer in proteoliposomes reconstituted with LH2 and RC-LH1 complexes from Rhodobacter sphaeroides.

Light-harvesting 2 (LH2) and reaction-centre light-harvesting 1 (RC-LH1) complexes purified from the photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were reconstituted into proteoliposomes either separately, or together at three different LH2:RC-LH1 ratios, for excitation energy transfer studies. Atomic force microscopy (AFM) was used to investigate the distribution and association of the complexes within the proteoliposome membranes. Absorption and fluorescence emission spectra were similar for LH2 complexes in detergent and liposomes, indicating that reconstitution retains the structural and optical properties of the LH2 complexes. Analysis of fluorescence emission shows that when LH2 forms an extensive series of contacts with other such complexes, fluorescence is quenched by 52.6 ± 1.4%. In mixed proteoliposomes, specific excitation of carotenoids in LH2 donor complexes resulted in emission of fluorescence from acceptor RC-LH1 complexes engineered to assemble with no carotenoids. Extents of energy transfer were measured by fluorescence lifetime microscopy; the 0.72 ± 0.08 ns lifetime in LH2-only membranes decreases to 0.43 ± 0.04 ns with a ratio of 2:1 LH2 to RC-LH1, and to 0.35 ± 0.05 ns for a 1:1 ratio, corresponding to energy transfer efficiencies of 40 ± 14% and 51 ± 18%, respectively. No further improvement is seen with a 0.5:1 LH2 to RC-LH1 ratio. Thus, LH2 and RC-LH1 complexes perform their light harvesting and energy transfer roles when reconstituted into proteoliposomes, providing a way to integrate native, non-native, engineered and de novo designed light-harvesting complexes into functional photosynthetic systems.

Construction of the Rhodobacter sphaeroides strain overproducing 5-aminolevulinic acid by insertion of endogenous promoter.

5-Aminolevulinic acid (ALA) is a precursor of heme and a natural amino acid synthesized in the cells of most living organisms. Currently, ALA is used as an ingredient in pharmaceuticals, supplements, cosmetics, feed, fertilizers, and other products. ALA is mainly produced by industrial fermentation by the photosynthetic bacterium Rhodobacter sphaeroides. In this study, we tried to improve the ALA productivity by R. sphaeroides using a genetic strategy to highly express ALA synthase (ALAS) genes. We inserted a constitutive promoter (PrrnB or Prsp_7571) upstream of genes encoding ALAS (hemA and/or hemT) to construct strains that constitutively express ALAS. The highest transcript levels of hemA were observed in the strain where PrrnB was inserted into the hemA promoter region and were 3.5-fold higher than those in the wild-type. The highest transcript levels of hemT were observed in the strain where PrrnB was inserted into the hemT promoter region and were 46-fold higher than those in the wild-type. The maximum ALAS activity was observed in crude cell extracts of the strain where PrrnB was inserted into the hemT promoter region under optimized growth conditions that was 2.7-fold higher than that in the wild type. This strain showed 12-fold accumulation of ALA compared to the wild-type. Thus, we improved ALA productivity without using exogenous DNA sequences. In the future, further improvement in ALA productivity may be expected by applying this approach to current industrial ALA-producing bacteria.

Roadmap of electrons from donor side to the reaction center of photosynthetic purple bacteria with mutated cytochromes.

In photosynthetic bacteria, the absorbed light drives the canonical cyclic electron transfer between the reaction center and the cytochrome bc1 complexes via the pools of mobile electron carriers. If kinetic or structural barriers hinder the participation of the bc1 complex in the cyclic flow of electrons, then the pools of mobile redox agents must supply the electrons for the multiple turnovers of the reaction center. These conditions were achieved by continuous high light excitation of intact cells of bacterial strains Rba. sphaeroides and Rvx. gelatinosus with depleted donor side cytochromes c2 (cycA) and tetraheme cytochrome subunit (pufC), respectively. The gradual oxidation by ferricyanide further reduced the availability of electron donors to pufC. Electron transfer through the reaction center was tracked by absorption change and by induction and relaxation of the fluorescence of the bacteriochlorophyll dimer. The rate constants of the electron transfer (~ 3 × 103 s‒1) from the mobile donors of Rvx. gelatinosus bound either to the RC (pufC) or to the tetraheme subunit (wild type) were similar. The electrons transferred through the reaction center dimer were supplied entirely by the donor pool; their number amounted to about 5 in wild type Rvx. gelatinosus and decreased to 1 in pufC oxidized by ferricyanide. Fluorescence yield was measured as a function of the oxidized fraction of the dimer and its complex shape reveals the contribution of two competing processes: the migration of the excitation energy among the photosynthetic units and the availability of electron donors to the oxidized dimer. The experimental results were simulated and rationalized by a simple kinetic model of the two-electron cycling of the acceptor side combined with aperiodic one-electron redox function of the donor side.

Generation of Electric Potential Difference by Chromatophores from Photosynthetic Bacteria in the Presence of Trehalose under Continuous Illumination.

Measurement of electrical potential difference (Δψ) in membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides associated with the surface of a nitrocellulose membrane filter (MF) impregnated with a phospholipid solution in decane or immersed into it in the presence of exogenous mediators and disaccharide trehalose demonstrated an increase in the amplitude and stabilization of the signal under continuous illumination. The mediators were the ascorbate/N,N,N'N'-tetramethyl-p-phenylenediamine pair and ubiquinone-0 (electron donor and acceptor, respectively). Although stabilization of photoelectric responses upon long-term continuous illumination was observed for both variants of chromatophore immobilization, only the samples immersed into the MF retained the functional activity of reaction centers (RCs) for a month when stored in the dark at room temperature, which might be due to the preservation of integrity of chromatophore proteins inside the MF pores. The stabilizing effect of the bioprotector trehalose could be related to its effect on both the RC proteins and the phospholipid bilayer membrane. The results obtained will expand current ideas on the use of semi-synthetic structures based on various intact photosynthetic systems capable of converting solar energy into its electrochemical form.

Functional Coupling of Biohybrid Photosynthetic Antennae and Reaction Center Complexes: Quantitative Comparison with Native Antennae.

Light-harvesting (LH) complexes in photosynthetic organisms absorb photons within limited wavelength ranges over a broad solar spectrum. Extension of the LH wavelength has been realized by attaching artificial fluorophores to LH complexes (biohybrid LH complexes) for complementing the limited-wavelength regions. However, how efficiently such fluorophores in biohybrid LH complexes function to drive the photocatalytic reaction center (RC) has not been quantitatively evaluated, specifically in comparison with native LH antenna complexes. In this study, we prepared various biohybrid LH1-RC complexes (from Rhodopseudomonas palustris), to quantitatively evaluate the LH activity of the attached external chromophores through a photocurrent generation reaction by LH1-RC on an electrode. For a direct comparison of the LH activity among the LH chromophores that were examined, we introduced the k1 term, which represents the extent of the functional coupling of LH and the photochemical reactions in the RC. We determined that the hydrophobic fluorophore ATTO647N attached to LH1 possesses the highest LH activity among the examined hydrophilic fluorophores such as Alexa647, and its activity is comparable to that of native LH1(-RC). The LH activity of LH2 (from Rhodoblastus acidophilus strain 10050) and its biohybrid LH2s were examined for the comprehensive assessment of their LH activity.

RNase III participates in control of quorum sensing, pigmentation and oxidative stress resistance in Rhodobacter sphaeroides.

RNase III is a dsRNA-specific endoribonuclease, highly conserved in bacteria and eukarya. In this study, we analysed the effects of inactivation of RNase III on the transcriptome and the phenotype of the facultative phototrophic α-proteobacterium Rhodobacter sphaeroides. RNA-seq revealed an unexpectedly high amount of genes with increased expression located directly downstream to the rRNA operons. Chromosomal insertion of additional transcription terminators restored wild type-like expression of the downstream genes, indicating that RNase III may modulate the rRNA transcription termination in R. sphaeroides. Furthermore, we identified RNase III as a major regulator of quorum-sensing autoinducer synthesis in R. sphaeroides. It negatively controls the expression of the autoinducer synthase CerI by reducing cerI mRNA stability. In addition, RNase III inactivation caused altered resistance against oxidative stress and impaired formation of photosynthetically active pigment-protein complexes. We also observed an increase in the CcsR small RNAs that were previously shown to promote resistance to oxidative stress. Taken together, our data present interesting insights into RNase III-mediated regulation and expand the knowledge on the function of this important enzyme in bacteria.

Unravelling the Roles of Integral Polypeptides in Excitation Energy Transfer of Photosynthetic RC-LH1 Supercomplexes.

Elucidating the photosynthetic processes that occur within the reaction center-light-harvesting 1 (RC-LH1) supercomplexes from purple bacteria is crucial for uncovering the assembly and functional mechanisms of natural photosynthetic systems and underpinning the development of artificial photosynthesis. Here, we examined excitation energy transfer of various RC-LH1 supercomplexes of Rhodobacter sphaeroides using transient absorption spectroscopy, coupled with lifetime density analysis, and studied the roles of the integral transmembrane polypeptides, PufX and PufY, in energy transfer within the RC-LH1 core complex. Our results show that the absence of PufX increases both the LH1 → RC excitation energy transfer lifetime and distribution due to the role of PufX in defining the interaction and orientation of the RC within the LH1 ring. While the absence of PufY leads to the conformational shift of several LH1 subunits toward the RC, it does not result in a marked change in the excitation energy transfer lifetime.

Selective expression of light-harvesting complexes alters phospholipid composition in the intracytoplasmic membrane and core complex of purple phototrophic bacteria.

Phospholipid-protein interactions play important roles in regulating the function and morphology of photosynthetic membranes in purple phototrophic bacteria. Here, we characterize the phospholipid composition of intracytoplasmic membrane (ICM) from Rhodobacter (Rba.) sphaeroides that has been genetically altered to selectively express light-harvesting (LH) complexes. In the mutant strain (DP2) that lacks a peripheral light-harvesting (LH2) complex, the phospholipid composition was significantly different from that of the wild-type strain; strain DP2 showed a marked decrease in phosphatidylglycerol (PG) and large increases in cardiolipin (CL) and phosphatidylcholine (PC) indicating preferential interactions between the complexes and specific phospholipids. Substitution of the core light-harvesting (LH1) complex of Rba. sphaeroides strain DP2 with that from the purple sulfur bacterium Thermochromatium tepidum further altered the phospholipid composition, with substantial increases in PG and PE and decreases in CL and PC, indicating that the phospholipids incorporated into the ICM depend on the nature of the LH1 complex expressed. Purified LH1-reaction center core complexes (LH1-RC) from the selectively expressing strains also contained different phospholipid compositions than did core complexes from their corresponding wild-type strains, suggesting different patterns of phospholipid association between the selectively expressed LH1-RC complexes and those purified from native strains. Effects of carotenoids on the phospholipid composition were also investigated using carotenoid-suppressed cells and carotenoid-deficient species. The findings are discussed in relation to ICM morphology and specific LH complex-phospholipid interactions.

Protonation-State Dependence of Hydration and Interactions in the Two Proton-Conducting Channels of Cytochrome c Oxidase.

Cytochrome c Oxidase (CcO), a membrane protein of the respiratory chain, pumps protons against an electrochemical gradient by using the energy of oxygen reduction to water. The ("chemical") protons required for this reaction and those pumped are taken up via two distinct channels, named D-channel and K-channel, in a step-wise and highly regulated fashion. In the reductive phase of the catalytic cycle, both channels transport protons so that the pumped proton passes the D-channel before the "chemical" proton has crossed the K-channel. By performing molecular dynamics simulations of CcO in the O→E redox state (after the arrival of the first reducing electron) with various combinations of protonation states of the D- and K-channels, we analysed the effect of protonation on the two channels. In agreement with previous work, the amount of water observed in the D-channel was significantly higher when the terminal residue E286 was not (yet) protonated than when the proton arrived at this end of the D-channel and E286 was neutral. Since a sufficient number of water molecules in the channel is necessary for proton transport, this can be understood as E286 facilitating its own protonation. K-channel hydration shows an even higher dependence on the location of the excess proton in the K-channel. Also in agreement with previous work, the K-channel exhibits a very low hydration level that likely hinders proton transfer when the excess proton is located in the lower part of the K-channel, that is, on the N-side of S365. Once the proton has passed S365 (towards the reaction site, the bi-nuclear centre (BNC)), the amount of water in the K-channel provides hydrogen-bond connectivity that renders proton transfer up to Y288 at the BNC feasible. No significant direct effect of the protonation state of one channel on the hydration level, hydrogen-bond connectivity, or interactions between protein residues in the other channel could be observed, rendering proton conductivity in the two channels independent of each other. Regulation of the order of proton uptake and proton passage in the two channels such that the "chemical" proton leaves its channel last must, therefore, be achieved by other means of communication, such as the location of the reducing electron.

Single-photon absorption and emission from a natural photosynthetic complex.

Photosynthesis is generally assumed to be initiated by a single photon1-3 from the Sun, which, as a weak light source, delivers at most a few tens of photons per nanometre squared per second within a chlorophyll absorption band1. Yet much experimental and theoretical work over the past 40 years has explored the events during photosynthesis subsequent to absorption of light from intense, ultrashort laser pulses2-15. Here, we use single photons to excite under ambient conditions the light-harvesting 2 (LH2) complex of the purple bacterium Rhodobacter sphaeroides, comprising B800 and B850 rings that contain 9 and 18 bacteriochlorophyll molecules, respectively. Excitation of the B800 ring leads to electronic energy transfer to the B850 ring in approximately 0.7 ps, followed by rapid B850-to-B850 energy transfer on an approximately 100-fs timescale and light emission at 850-875 nm (refs. 16-19). Using a heralded single-photon source20,21 along with coincidence counting, we establish time correlation functions for B800 excitation and B850 fluorescence emission and demonstrate that both events involve single photons. We also find that the probability distribution of the number of heralds per detected fluorescence photon supports the view that a single photon can upon absorption drive the subsequent energy transfer and fluorescence emission and hence, by extension, the primary charge separation of photosynthesis. An analytical stochastic model and a Monte Carlo numerical model capture the data, further confirming that absorption of single photons is correlated with emission of single photons in a natural light-harvesting complex.

The role of CenKR in the coordination of Rhodobacter sphaeroides cell elongation and division.

Cell elongation and division are essential aspects of the bacterial life cycle that must be coordinated for viability and replication. The impact of misregulation of these processes is not well understood as these systems are often not amenable to traditional genetic manipulation. Recently, we reported on the CenKR two-component system (TCS) in the Gram-negative bacterium Rhodobacter sphaeroides that is genetically tractable, widely conserved in α-proteobacteria, and directly regulates the expression of components crucial for cell elongation and division, including genes encoding subunit of the Tol-Pal complex. In this work, we show that overexpression of cenK results in cell filamentation and chaining. Using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET), we generated high-resolution two-dimensional (2D) images and three-dimensional (3D) volumes of the cell envelope and division septum of wild-type cells and a cenK overexpression strain finding that these morphological changes stem from defects in outer membrane (OM) and peptidoglycan (PG) constriction. By monitoring the localization of Pal, PG biosynthesis, and the bacterial cytoskeletal proteins MreB and FtsZ, we developed a model for how increased CenKR activity leads to changes in cell elongation and division. This model predicts that increased CenKR activity decreases the mobility of Pal, delaying OM constriction, and ultimately disrupting the midcell positioning of MreB and FtsZ and interfering with the spatial regulation of PG synthesis and remodeling. IMPORTANCE By coordinating cell elongation and division, bacteria maintain their shape, support critical envelope functions, and orchestrate division. Regulatory and assembly systems have been implicated in these processes in some well-studied Gram-negative bacteria. However, we lack information on these processes and their conservation across the bacterial phylogeny. In R. sphaeroides and other α-proteobacteria, CenKR is an essential two-component system (TCS) that regulates the expression of genes known or predicted to function in cell envelope biosynthesis, elongation, and/or division. Here, we leverage unique features of CenKR to understand how increasing its activity impacts cell elongation/division and use antibiotics to identify how modulating the activity of this TCS leads to changes in cell morphology. Our results provide new insight into how CenKR activity controls the structure and function of the bacterial envelope, the localization of cell elongation and division machinery, and cellular processes in organisms with importance in health, host-microbe interactions, and biotechnology.

Grafting Rhodobacter sphaeroides with red algae Rubisco to accelerate catalysis and plant growth.

Improving the carboxylation properties of Rubisco has primarily arisen from unforeseen amino acid substitutions remote from the catalytic site. The unpredictability has frustrated rational design efforts to enhance plant Rubisco towards the prized growth-enhancing carboxylation properties of red algae Griffithsia monilis GmRubisco. To address this, we determined the crystal structure of GmRubisco to 1.7 Å. Three structurally divergent domains were identified relative to the red-type bacterial Rhodobacter sphaeroides RsRubisco that, unlike GmRubisco, are expressed in Escherichia coli and plants. Kinetic comparison of 11 RsRubisco chimaeras revealed that incorporating C329A and A332V substitutions from GmRubisco Loop 6 (corresponding to plant residues 328 and 331) into RsRubisco increased the carboxylation rate (kcatc) by 60%, the carboxylation efficiency in air by 22% and the CO2/O2 specificity (Sc/o) by 7%. Plastome transformation of this RsRubisco Loop 6 mutant into tobacco enhanced photosynthesis and growth up to twofold over tobacco producing wild-type RsRubisco. Our findings demonstrate the utility of RsRubisco for the identification and in planta testing of amino acid grafts from algal Rubisco that can enhance the enzyme's carboxylase potential.