Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells.

The construction of energetically autonomous artificial protocells is one of the most ambitious goals in bottom-up synthetic biology. Here, we show an efficient manner to build adenosine 5'-triphosphate (ATP) synthesizing hybrid multicompartment protocells. Bacterial chromatophores from Rhodobacter sphaeroides accomplish the photophosphorylation of adenosine 5'-diphosphate (ADP) to ATP, functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles (ATP production rate up to ∼100 ATP∙s-1 per ATP synthase). The chromatophore morphology and the orientation of the photophosphorylation proteins were characterized by cryo-electron microscopy (cryo-EM) and time-resolved spectroscopy. The freshly synthesized ATP has been employed for sustaining the transcription of a DNA gene, following the RNA biosynthesis inside individual vesicles by confocal microscopy. The hybrid multicompartment approach here proposed is very promising for the construction of full-fledged artificial protocells because it relies on easy-to-obtain and ready-to-use chromatophores, paving the way for artificial simplified-autotroph protocells (ASAPs).

Supramolecular Biohybrid Construct for Photoconversion Based on a Bacterial Reaction Center Covalently Bound to Cytochrome c by an Organic Light Harvesting Bridge.

A supramolecular construct for solar energy conversion is developed by covalently bridging the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides and cytochrome c (Cyt c) proteins with a tailored organic light harvesting antenna (hCy2). The RC-hCy2-Cyt c biohybrid mimics the working mechanism of biological assemblies located in the bacterial cell membrane to convert sunlight into metabolic energy. hCy2 collects visible light and transfers energy to the RC, increasing the rate of photocycle between a RC and Cyt c that are linked in such a way that enhances proximity without preventing protein mobility. The biohybrid obtained with average 1 RC/10 hCy2/1.5 Cyt c molar ratio features an almost doubled photoactivity versus the pristine RC upon illumination at 660 nm, and ∼10 times higher photocurrent versus an equimolar mixture of the unbound proteins. Our results represent an interesting insight into photoenzyme chemical manipulation, opening the way to new eco-sustainable systems for biophotovoltaics.

The periodic table of photosynthetic purple non-sulfur bacteria: intact cell-metal ions interactions.

Photosynthetic purple non-sulfur bacteria (PNB) have been widely utilized as model organisms to study bacterial photosynthesis. More recently, the remarkable resistance of these microorganisms to several metals ions called particular interest. As a result, several research efforts were directed toward clarifying the interactions of metal ions with PNB. The mechanisms of metal ions active uptake and bioabsorption have been studied in detail, unveiling that PNB enable harvesting and removing various toxic ions, thus fostering applications in environmental remediation. Herein, we present the most important achievements in the understanding of intact cell-metal ions interactions and the approaches utilized to study such processes. Following, the application of PNB-metal ions interactions toward metal removal from contaminated environments is presented. Finally, the possible coupling of PNB with abiotic electrodes to obtain biohybrid electrochemical systems is proposed as a sustainable pathway to tune and enhance metal removal and monitoring.

Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocells.

Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP. Herein we show that synthetic protocells, based on giant lipid vesicles embedding an oriented population of reaction centers, are capable of generating a photoinduced proton gradient across the membrane. Under continuous illumination, the protocells generate a gradient of 0.061 pH units per min, equivalent to a proton motive force of 3.6 mV⋅min-1 Remarkably, the facile reconstitution of the photosynthetic reaction center in the artificial lipid membrane, obtained by the droplet transfer method, paves the way for the construction of novel and more functional protocells for synthetic biology.

About the origin of the large Stokes shift in aminoalkyl substituted heptamethine cyanine dyes.

Aminoalkyl-substituted heptamethine cyanine dyes are characterized by a large Stokes shift, an uncommon feature for cyanine molecules yet very promising for their application as fluorescent probes in bioimaging and as light harvesting antennas in biohybrid systems for solar energy conversion. The origin of this photophysical feature has not been adequately explored so far, and a combined experimental and theoretical work is herein provided to shed light on the role played by the central aminoalkyl substituent bound to the heptamethine cyanine backbone in defining the unusual properties of the dye. The spectra recorded in solvents of different polarities point to a marginal role of the medium in the definition of the Stokes shift, which conversely can be ascribed to the relaxation of the molecular geometry upon photoexcitation. This hypothesis is supported by an extensive theoretical investigation of the ground and excited states of the dye. TD-DFT results on the aminoalkyl-substituted dye and its unsubstituted precursor demonstrate a very similar cyanine-like structure for both molecules in the relaxed excited state. Conversely, in the ground state the amino substitution disrupts the conjugation in the polymethine chain, leading to a broken-symmetry, non-planar structure.

Unveiling the compositional variety of cardiolipins in Rhodobacter sphaeroides by liquid chromatography with electrospray ionization and multistage collision-induced dissociation mass spectrometry.

Cardiolipins (CL) contained in the lipid extracts of the photosynthetic bacterium Rhodobacter sphaeroides (strain R26) were systematically characterized by reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry, performed in single (MS), tandem (MS/MS) and sequential (MS3) modes using a linear ion trap mass spectrometer. The total number of carbon atoms and C=C bonds of each CL and, subsequently, those related to each of the constituting phosphatidic acid (PA) units, along with the location of the latter on the central glycerol backbone, were inferred from MS and MS/MS data, respectively. Moreover, the composition and location of both acyl chains on the glycerol backbone of each PA unit was obtained by MS3 measurements, an approach used for the first time for the structural elucidation of CL in R. sphaeroides. As a result, an unprecedented profile of CL in this bacterium was obtained, with 27 main species characterized, many of which are represented by compositional or regiochemical isomers. Interestingly, such a variability is generated from a limited number of different acyl chains, either saturated (i.e. 12:0, 16:0, 17:0, 18:0, 19:0) or mono-unsaturated (16:1, 18:1). The absence of polyunsaturated chains, more susceptible to oxidation damage, appeared to be indirectly related to the lack of carotenoids potentially acting as antioxidant agents, a specific feature of R. sphaeroides R26. The occurrence of odd-numbered acyl chains was ascribed to the need to guarantee membrane fluidity, through a less compact packing of CL, thus compensating for the lack of CL bearing polyunsaturated side chains. Graphical abstract Representation of MS signals due to carboxylate anions that would be detected, as separate couples, in the fragmentation spectra of the anions resulting from the two phosphatidic acid units included in a cardiolipin molecule bearing four different acyl chains.

Synthetic Antenna Functioning As Light Harvester in the Whole Visible Region for Enhanced Hybrid Photosynthetic Reaction Centers.

The photosynthetic reaction center (RC) from the Rhodobacter sphaeroides bacterium has been covalently bioconjugated with a NIR-emitting fluorophore (AE800) whose synthesis was specifically tailored to act as artificial antenna harvesting light in the entire visible region. AE800 has a broad absorption spectrum with peaks centered in the absorption gaps of the RC and its emission overlaps the most intense RC absorption bands, ensuring a consistent increase of the protein optical cross section. The covalent hybrid AE800-RC is stable and fully functional. The energy collected by the artificial antenna is transferred to the protein via FRET mechanism, and the hybrid system outperforms by a noteworthy 30% the overall photochemical activity of the native protein under the entire range of visible light. This improvement in the optical characteristic of the photoenzyme demonstrates the effectiveness of the bioconjugation approach as a suitable route to new biohybrid materials for energy conversion, photocatalysis, and biosensing.

Semiquinone oscillations as a tool for investigating the ubiquinone binding to photosynthetic reaction centers.

Semiquinone oscillations induced by light pulses in the presence of exogenous electron donors are a valuable source of information on the kinetics and thermodynamics of ubiquinone chemistry relevant to the QB site of the photosynthetic reaction center (RC). In previous attempts to achieve the quantitative interpretation of data, the ubiquinone concentration was considered constant during the experiment since it was much bigger than that of RC. In this work, we extended existing models to low ubiquinone concentrations revealing several hidden processes taking place during the ubiquinone photoreduction and enabling the evaluation of the ubiquinone binding constant K Q at the QB site. The proposed approach provides for the first time the evaluation of K Q without any preliminary treatment of ubiquinone extraction from the binding site, thereby better preserving its native structure.

The binding of quinone to the photosynthetic reaction centers: kinetics and thermodynamics of reactions occurring at the QB-site in zwitterionic and anionic liposomes.

Liposomes represent a versatile biomimetic environment for studying the interaction between integral membrane proteins and hydrophobic ligands. In this paper, the quinone binding to the QB-site of the photosynthetic reaction centers (RC) from Rhodobacter sphaeroides has been investigated in liposomes prepared with either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG) to highlight the role of the different phospholipid polar heads. Quinone binding (K Q) and interquinone electron transfer (L AB) equilibrium constants in the two type of liposomes were obtained by charge recombination reaction of QB-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 °C. The kinetic of the charge recombination reactions has been fitted by numerically solving the ordinary differential equations set associated with a detailed kinetic scheme involving electron transfer reactions coupled with quinone release and uptake. The entire set of traces at each temperature was accurately fitted using the sole quinone release constants (both in a neutral and a charge separated state) as adjustable parameters. The temperature dependence of the quinone exchange rate at the QB-site was, hence, obtained. It was found that the quinone exchange regime was always fast for PC while it switched from slow to fast in PG as the temperature rose above 20 °C. A new method was introduced in this paper for the evaluation of constant K Q using the area underneath the charge recombination traces as the indicator of the amount of quinone bound to the QB-site.

The lipidome of the photosynthetic bacterium Rhodobacter sphaeroides R26 is affected by cobalt and chromate ions stress.

A detailed characterization of membrane lipids of the photosynthetic bacterium Rhodobacter (R.) sphaeroides was accomplished by thin-layer chromatography coupled with matrix-assisted laser desorption ionization mass spectrometry. Such an approach allowed the identification of the main membrane lipids belonging to different classes, namely cardiolipins (CLs), phosphatidylethanolamines, phosphatidylglycerols (PGs), phosphatidylcholines, and sulfoquinovosyldiacylglycerols (SQDGs). Thus, the lipidomic profile of R. sphaeroides R26 grown in abiotic stressed conditions by exposure to bivalent cobalt cation and chromate oxyanion, was investigated. Compared to bacteria grown under control conditions, significant lipid alterations take place under both stress conditions; cobalt exposure stress results in the relative content increase of CLs and SQDGs, most likely compensating the decrease in PGs content, whereas chromate stress conditions result in the relative content decrease of both PGs and SQDGs, leaving CLs unaltered. For the first time, the response of R. sphaeroides to heavy metals as Co(2+) and CrO4 (2-) is reported and changes in membrane lipid profiles were rationalised.

Polydopamine-immobilized yeast cells for portable electrochemical biosensors applied in environmental copper sensing.

The coupling of biological organisms with electrodes enables the development of sustainable, low cost, and potentially self-sustained biosensors. A critical aspect is to obtain portable bioelectrodes where the biological material is immobilized on the electrode surface to be utilized on demand. Herein, we developed an approach for the rapid entrapment and immobilization of metabolically active yeast cells in a biocompatible polydopamine layer, which does not require a separate and time-consuming synthesis. The reported approach allows obtaining the "electrical wire" of intact and active yeast cells with resulting current generation from glucose oxidation. Additionally, the electrochemical performance of the biohybrid yeast-based system has been characterized in the presence of CuSO4, a widely used pesticide, in the environmentally relevant concentration range of 20-100 µM. The system enabled the rapid preliminary monitoring of the contaminant based on variations in current generation, with a limit of detection of 12.5 µM CuSO4. The present approach for the facile preparation of portable yeast-based electrochemical biosensors paves the way for the future development of sustainable systems for environmental monitoring.

Cobalt binding in the photosynthetic bacterium R. sphaeroides by X-ray absorption spectroscopy.

Cobalt is an important oligoelement required for bacteria; if present in high concentration, exhibits toxic effects that, depending on the microorganism under investigation, may even result in growth inhibition. The photosynthetic bacterium Rhodobacter (R.) sphaeroides tolerates high cobalt concentration and bioaccumulates Co(2+) ion, mostly on the cellular surface. Very little is known on the chemical fate of the bioaccumulated cobalt, thus an X-ray absorption spectroscopy investigation was conducted on R. sphaeroides cells to gain structural insights into the Co(2+) binding to cellular components. X-ray absorption near-edge spectroscopy and extended X-ray absorption fine structure measurements were performed on R. sphaeroides samples containing whole cells and cell-free fractions obtained from cultures exposed to 5 mM Co(2+). An octahedral coordination geometry was found for the cobalt ion, with six oxygen-ligand atoms in the first shell. In the soluble portion of the cell, cobalt was found bound to carboxylate groups, while a mixed pattern containing equivalent amount of two sulfur and two carbon atoms was found in the cell envelope fraction, suggesting the presence of carboxylate and sulfonate metal-binding functional groups, the latter arising from sulfolipids of the cell envelope.

Unveiling the Influence of Hydrated Deep Eutectic Solvents on the Dynamics of Water-Soluble Proteins.

Deep eutectic solvents (DESs) are mixtures of two or more pure compounds (e.g., Lewis or Brønsted acids and bases, anionic and/or cationic species) in a well-defined stoichiometric proportion, with a melting point lower to that of an ideal liquid mixture. These neoteric solvents are highly tunable through varying the structure or relative ratio of parent components and have been evaluated as solvents able to improve biomolecules' performance, specifically their stability and biocatalytic properties. Inspired by a recent crystallographic study, we have explored through molecular dynamics (MD) simulations the dynamic properties of two different proteins (hen egg-white lysozyme and the human VH antibody fragment HEL4) in a (20% w/w) hydrated solution of choline chloride-glycerol (1:2). We have developed proper force fields to account for DES, protein, and DES-protein interactions, which have been calibrated using pair distribution function measurements of pure DES solutions. MD results show that the presence of DES quenches the protein motion, increasing the rigidity of the overall protein structure. Specific interactions among DES components and protein residues, such as those between choline ions and two Tryptophan residues of lysozyme, may amplify the protein-DES interactions and lead to protein crystallization in the presence of hydrated DES. These findings open new horizons to improve or achieve control on protein properties by a proper choice of hydrated DESs used as solvents.

Light induced transmembrane proton gradient in artificial lipid vesicles reconstituted with photosynthetic reaction centers.

Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c2 and K4[Fe(CN)6]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.

The reaction center is the sensitive target of the mercury(II) ion in intact cells of photosynthetic bacteria.

The sensitivity of intact cells of purple photosynthetic bacterium Rhodobacter sphaeroides wild type to low level (<100 µM) of mercury (Hg²âº) contamination was evaluated by absorption and fluorescence spectroscopies of the bacteriochlorophyll-protein complexes. All assays related to the function of the reaction center (RC) protein (induction of the bacteriochlorophyll fluorescence, delayed fluorescence and light-induced oxidation and reduction of the bacteriochlorophyll dimer and energization of the photosynthetic membrane) showed prompt and later effects of the mercury ions. The damage expressed by decrease of the magnitude and changes of rates of the electron transfer kinetics followed complex (spatial and temporal) pattern according to the different Hg²âº sensitivities of the electron transport (donor/acceptor) sites including the reduced bound and free cytochrome c2 and the primary reduced quinone. In contrast to the RC, the light harvesting system and the bc1 complex demonstrated much higher resistance against the mercury pollution. The 850 and 875 nm components of the peripheral and core complexes were particularly insensitive to the mercury(II) ions. The concentration of the photoactive RCs and the connectivity of the photosynthetic units decreased upon mercury treatment. The degree of inhibition of the photosynthetic apparatus was always higher when the cells were kept in the light than in the dark indicating the importance of metabolism in active transport of the mercury ions from outside to the intracytoplasmic membrane. Any of the tests applied in this study can be used for detection of changes in photosynthetic bacteria at the early stages of the action of toxicants.

Changes in morphology, cell wall composition and soluble proteome in Rhodobacter sphaeroides cells exposed to chromate.

The response of the carotenoidless Rhodobacter sphaeroides mutant R26 to chromate stress under photosynthetic conditions is investigated by biochemical and spectroscopic measurements, proteomic analysis and cell imaging. Cell cultures were found able to reduce chromate within 3-4 days. Chromate induces marked changes in the cellular dimension and morphology, as revealed by atomic force microscopy, along with compositional changes in the cell wall revealed by infrared spectroscopy. These effects are accompanied by significant changes in the level of several proteins: 15 proteins were found up-regulated and 15 down-regulated. The protein content found in chromate exposed cells is in good agreement with the biochemical, spectroscopic and microscopic results. Moreover at the present stage no specific chromate-reductase could be found in the soluble proteome, indicating that detoxification of the pollutant proceeds via aspecific reductants.

Enhancing the light harvesting capability of a photosynthetic reaction center by a tailored molecular fluorophore.

Light machine: The simplest photosynthetic protein able to convert sunlight into other energy forms is covalently functionalized with a tailored organic dye to obtain a fully functional hybrid complex that outperforms the natural system in light harvesting and conversion ability.

Electroactivity of weak electricigen Bacillus subtilis biofilms in solution containing deep eutectic solvent components.

Bacillus subtilis is a Gram-positive, spore-forming bacterium with a versatile and adaptable metabolism, which makes it a viable cell factory for microbial production. Electroactivity has recently been identified as a cellular characteristic linked with the metabolic activity of B. subtilis. The enhancement of B. subtilis electroactivity can positively enhance bioproduction of high-added value metabolites under electrofermentative conditions. Here, we explored the use of deep eutectic solvents (DESs) and DES components as biocompatible nutrient additives for enhancing electroactivity of B. subtilis. The strongest electroactivity was obtained in an aqueous choline chloride: glycerol (1:2 mol mol-1) eutectic mixture. At low concentration (50-500 mM), this mixture induced a pseudo-diauxic increase in planktonic growth and increased biofilm formation, likely due to a nutritional and osmoprotectant effect. Similarities in electroactivity enhancements of choline chloride-based eutectic mixtures and quinone redox metabolism in B. subtilis were detected using high performance liquid chromatography and differential pulse voltammetry. Results show that choline chloride-based aqueous eutectic mixtures can enhance biomass and productivity in biofilm-based electrofermentation. However, the specific mechanism needs to be fully elucidated.

Bio-Inspired Redox-Adhesive Polydopamine Matrix for Intact Bacteria Biohybrid Photoanodes.

Interfacing intact and metabolically active photosynthetic bacteria with abiotic electrodes requires both establishing extracellular electron transfer and immobilizing the biocatalyst on electrode surfaces. Artificial approaches for photoinduced electron harvesting through redox polymers reported in literature require the separate synthesis of artificial polymeric matrices and their subsequent combination with bacterial cells, making the development of biophotoanodes complex and less sustainable. Herein, we report a one-pot biocompatible and sustainable approach, inspired by the byssus of mussels, that provides bacterial cells adhesion on multiple surfaces under wet conditions to obtain biohybrid photoanodes with facilitated photoinduced electron harvesting. Purple bacteria were utilized as a model organism, as they are of great interest for the development of photobioelectrochemical systems for H2 and NH3 synthesis, biosensing, and bioremediation purposes. The polydopamine matrix preparation strategy allowed the entrapment of active purple bacteria cells by initial oxygenic polymerization followed by electrochemical polymerization. Our results unveil that the deposition of bacterial cells with simultaneous polymerization of polydopamine on the electrode surface enables a 5-fold enhancement in extracellular electron transfer at the biotic/abiotic interface while maintaining the viability of the cells. The presented approach paves the way for a more sustainable development of biohybrid photoelectrodes.

Reversible binding of metal ions onto bacterial layers revealed by protonation-induced ATR-FTIR difference spectroscopy.

The ability of microorganisms to adhere to abiotic surfaces and the potentialities of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy have been exploited to study protonation and heavy metal binding events onto bacterial surfaces. This work represents the first attempt to apply on bacteria the recently developed method known as perfusion-induced ATR-FTIR difference spectroscopy. Such a technique allows measurement of even slight changes in the infrared spectrum of the sample, deposited as a thin layer on an ATR crystal, while an aqueous solution is perfused over its surface. Solutions at different pH have been used for inducing protonation/deprotonation of functional groups lying on the surface of Rhodobacter sphaeroides cells, chosen as a model system. The interaction of Ni(2+) with surface protonable groups of this microorganism has been investigated with a double-difference approach exploiting competition between nickel cations and protons. Protonation-induced difference spectra of simple model compounds have been acquired to guide band assignment in bacterial spectra, thus allowing identification of major components involved in proton uptake and metal binding. The data collected reveal that carboxylate moieties on the bacterial surface of R. sphaeroides play a role in extracellular biosorption of Ni(2+), establishing with this ion relatively weak coordinative bonds.