Rubrica Bimestrale sulle proteine

The photosynthetic reaction center (RC) from the purple Rhodobacter (R.) sphaeroides bacterium is a protein with unique photoconversion capability that can be exploited in hybrid systems for energy conversion. We have developed a tailored aryleneethynylene organic fluorophore (AE750) acting as efficient light harvesting antenna and successfully bioconjugated it to the photosynthetic RC. We have also demonstrated that, under suitable conditions, the biohybrid AE750-RC system can outperform the energy photoconversion ability of the native protein.

Humankind is in great need of new energy sources. The use of solar radiation for powering the planet would fulfil the energy requirements of Earth's inhabitants as well as greatly mitigate tension flares arising from the uneven distribution of fossil fuels and environmental problems associated to their extraction procedures. How to proceed than? Easy to say! Mother Nature is inspiring: all life on earth is based on the conversion of the solar radiation into high energy molecules, including gas and oil human beings are consuming these days, by mean of the so-called primary photoconverters, i.e. the photosynthetic organisms, plants, algae and some kind of bacteria. So, let's learn from Nature and assemble in our laboratories artificial systems capable of exploiting solar energy for photocatalysis and electrical energy production, i.e. mimic photosynthesis. Not an easy task of course, but a large number of laboratory are heavily involved since the last 25 years in the field of artificial photosynthesis and are obtaining encouraging results. The photosynthetic apparatus used by photosynthetic organisms to convert solar energy and drive their metabolism is the photochemical core where photoconversion takes place, and is constituted by a protein portion allocating several pigments directly involved in the harvesting of solar light and in the subsequent sequence of electron transfer reactions which eventually lead to the formation of an electron-hole couple to be used for any energy requiring process. In artificial photosynthesis the role of the protein scaffold in often ignored and attention is devoted to assembly molecular system for optimising light harvest and electron-transfer reactions, focussing to the "less-complex" portion of the photosynthetic apparatus. What would be a different paradigm in artificial photosynthesis? Assemble artificial photoconverters using genuine natural components formed by hybrid organic-biologic systems. The hybrids have a central protein, the so-called photosynthetic reaction center (RC) that converts sunlight into a charge-separated state having a lifetime sufficient to allow ancillary chemistry to take place. The RCs can be eventually garnished with opportune organic moieties to be used for different applications.The state of the art of these hybrid organic-biologic photosynthetic assemblies will be reviewed.

The reconstitution of the integral membrane protein photosynthetic reaction center (RC) in polymersomes, i.e. artificial closed vesicles, was achieved by the micelle-to-vesicle transition technique, a very mild protocol based on size exclusion chromatography often used to drive the incorporation of proteins contemporarily to liposome formation. An optimized protocol was used to successfully reconstitute the protein in a fully active state in polymersomes formed by the tri-block copolymers PMOXA<inf>22</inf>-PDMS<inf>61</inf>-PMOXA<inf>22</inf>. The RC is very sensitive to its solubilizing environment and was used to probe the positioning of the protein in the vesicles. According to charge-recombination experiments and to the enzymatic activity assay, the RC is found to accommodate in the PMOXA<inf>22</inf> region of the polymersome, facing the water bulk solution, rather than in the PDMS<inf>61</inf> transmembrane-like region. Furthermore, polymersomes were found to preserve protein integrity efficiently as the biomimetic lipid bilayers but show a much longer temporal stability than lipid based vesicles.

Aim of this study is the identification of an appropriate internal reference gene to quantifygene transcripts isolated from Rhodobacter (R.) sphaeroides cells grown in presence of highconcentrations of cobalt ions. RNA was isolated using a commercial kit protocol ad-hocmodified. Several primer pairs were used to perform reverse transcription PCR and real-timePCR to assess the suitable internal reference gene whose expression is not affected by cobaltions, identified with the gene rsp0154.This finding can be of definite help in the investigation of the response to heavy metals ofthe chosen strain, a potential candidate for environmental applications.

This study shows the direct effect of atmospheric particulate matter on plant growth. Tomato (Solanum lycopersicum L.) plants were grown for 18d directly on PM10 collected on quartz fiber filters. Organic and elemental carbon and polycyclic aromatic hydrocarbons (PAHs) contents were analyzed on all the tested filters. The toxicity indicators (i.e., seed germination, root elongation, shoot and/or fresh root weight, chlorophyll and carotenoids content) were quantified to study the negative and/or positive effects in the plants via root uptake. Substantial differences were found in the growth of the root apparatus with respect to that of the control plants. A 17-58% decrease of primary root elongation, a large amount of secondary roots and a decrease in shoot (32%) and root (53-70%) weights were found. Quantitative analysis of the reactive oxygen species (ROS) indicated that an oxidative burst in response to abiotic stress occurred in roots directly grown on PM10, and this detrimental effect was also confirmed by the findings on the chlorophyll content and chlorophyll-to-carotenoid ratio.

Hexavalent chromium represents an outstanding risk for the environment and the health of human beings, as it is considerably involved in the genesis of cancer and other fatal diseases. Biological reduction of Cr(VI) to Cr(III) is a potentially useful mechanism to remediate chromium (VI) pollution and to detoxify contaminated wastes. The photosynthetic purple bacterium Rhodobacter sphaeroides is known for its ability to tolerate high concentrations of several heavy metal ions, to bioaccumulate nickel and cobalt, and to reduce oxyanions as tellurite, selenite and chromate. The response of the carotenoidless mutant R26 to chromate stress under phototrophic conditions has been recently investigated by biochemical and spectroscopic measurements, proteomic analysis and cell imaging, revealing good Cr(VI) reduction ability associated with morphological and compositional changes of the cell envelope, while no specific stress-induced chromate-reductase activity was found in the soluble proteome. Phototrophic biomass of Rhodobacter sphaeroides strain R26, harvested, washed, and stored at -20°C, was just thawed and used as Cr(VI) reduction catalyst. Chromate solutions, buffered at neutral pH and supplemented with a mixture of succinate, malonate and glucose as electron donors, have been employed for simulating the waste-water environment. The decrease of Cr(VI) concentration triggered by cells addition was evaluated by the diphenylcarbazide (DPC) assay. The analysis of reaction kinetics revealed that Rhodobacter sphaeroides resting biomass acts as an excellent bio-catalyst promoting chromate reduction by oxidizable carbon compounds. The role of abiotic variables such as pH, light, temperature and oxygen concentration was also assessed. Our data extend the information available about this phototrophic microorganism and elucidate its potential in Cr(VI) bioremediation applications.

The intensification of industrial technology increased heavy metal contamination in aquatic systems. Since inorganic pollutants cannot be degraded, an efficient removal system must be designed in order to detoxify heavy metal-contaminated wastewaters. Metal ion biosorption by microorganisms is an interesting mechanism which can be exploited for this purpose. The purple bacterium Rhodobacter sphaeroides is known for its ability to tolerate under phototrophic conditions high concentrations of several heavy metal ions and to bioaccumulate Ni2+ and Co2+ ions. In this work Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy and X-Ray Photoelectron Spectroscopy (XPS) have been employed for getting information about Ni2+ binding onto R. sphaeroides cell surface. The ability to bind nickel ions was evaluated both in free cells and in calcium alginate-immobilized biomass. Before Ni2+ exposure the bacterial biomass was washed thoroughly with KCl 0.1 M in order to fully saturate with K+ ions the negatively charged cell envelopes. XPS measurements revealed that treatment with Ni2+ resulted in full displacement of K+ ions from free R. sphaeroides cells, indicating high affinity between nickel ions and surface functional groups. Moreover ATR-FTIR measurements showed that Ni2+-treatment induce the shift of absorption bands arising from symmetric and asymmetric stretching modes of cell surface carboxylate groups, in agreement with their involvement in metal complexation. Calcium alginate beads entrapping bacterial biomass were prepared dropping a cell suspension supplemented with sodium alginate into 2% CaCl . XPS analysis of Ni2+-treated beads revealed that the exposure of cells to Ca2+ strongly inhibited Ni2+ uptake suggesting that displacement of Ca2+ by nickel ions does not occur. These data are of interest in order to identify optimal conditions for the efficient removal of Ni2+ by means of phototrophic bacterial biomass.

Photosynthesis represents one of the most important biological reactions in the biosphere, since all life on Earth, directly or indirectly, depends on it as a source of energy. Nature performs the photosynthetic process using specialized protein - pigments complexes organized to ensure up to 98% conversion of the absorbed photons in stable, long - living charge separated states. A proper combination of the photosynthetic core , the reaction center (RC), with engineered materials, i.e. metals or inorganic semiconductor electrodes, has attracted great attention for the building of new versatile hybrid devices for solar energy conversion 1 . Here we propose a covalent approach able to stably anchor RCs onto evaporated thin films of the hydrogen - bonded pigments epindolidione (EPI) and quinacridone (QNC) 2 , a well - known class of organic colorants which have recently emerged as promising semiconductors, demonstrating hole mobility in the range of 0.1 - 1 cm 2 /Vs and outstanding operatio nal stability in both air and aqueous media with pH 3 - 10. Due to low - toxicity, biocom patibility and potential low - cost, EPI and QNC substrates are envisioned to go where other traditional semicond uctors simply cannot be applied, resulting to be amenable to direct surface bioconjuga tion. T he NH functional group of these molecules in thin film reacts spontaneously with N - hydroxysuccinimide functionalized linke rs as disuccinimidyl sub erate. The protruding linker s are then used to covalently bind the lysines residues of the Rhodobacter sphaeroides reaction center, by forming an amide linkage. Our pro tocol is shown to preserve the semiconducting properties of the pigment s while maintaini ng the protein's photoactivity. Multiple - ref lection infrared spectroscopy and atomic force microscopy demonstrated the effective covalent binding and the robustness of the protein anchoring even after buffer washing procedures compared to the weakness of the physisorbed RC interactions. Furthermore, RC charge recombination kinetic measurements confirmed the fully functionality of bioconjugated proteins and ruled out any possible hindering effect from the organic films. As key results of our work, we have shown that semiconductors preserve their favo rable electrical properties: the proposed photoconductor devices operate under water, before and after RCs anchoring . These are enabling steps for usin g hydrogen - bonded pigments as a platform for multifunct ional bioelectronics devices, paving the way in designing and realizing new photosynthetic protein - based hybrid systems.

Gold nanoparticles exhibit unique electronic, optical, and catalytic properties that are different from those of bulk metal and have several applications in optoelectronics, imaging technology, catalysis, and drug delivery. Currently, there is a growing need to develop eco-friendly nanoparticle synthesis processes using living organisms, such as bacteria, fungi and algae. In particular, microorganisms are well known to protect themselves from metal ion stress either by intracellular-segregation mechanism or by secreting them into the external medium. This defensive behaviour can be exploited to obtain a more efficient fabrication of advanced functional nanomaterials than chemical synthesis routes: biological syntheses do not require hazardous organic solvents and surfactants , and can work at environmental temperature and pressure, preserving high selectivity and reproducibility.Rhodobacter sphaeroides is a facultative phototrophic anoxygenic proteobacterium known for its capacity to grow under a wide range of environmental conditions, with promising applications in bioremediation [1, 2].The response of the photosynthetic bacterium Rhodobacter sphaeroides to gold exposure and its reducing capability of Au(III) to produce stable Au(0) nanoparticles is reported in this study. The properties of prepared nanoparticles were characterized by UV-Visible (UV-Vis) spectroscopy, Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy, Transmission Electron Microscopy (TEM), X-ray Photoelectron Spectroscopy (XPS), X-ray Fluorescence Spectrometry (XRF) and X-ray Absorption Spectroscopy (XAS) measurements. Gold nanoparticles (AuNPs) were spherical in shape with an average size of 10±3 nm. Based on our experiments, the particles were likely fabricated by the aid of reducing sugars present in the bacterial cell membrane and were capped by a protein/peptide coat. The nanoparticles were hydrophilic and resisted to aggregation for several months. Gold nanoparticles were also positively tested for their catalytic activity in nitroaromatic compounds degradation.

The covalent functionalization of photosynthetic proteins with properly tailored organic molecular antennas represents a powerful approach to build a new generation of hybrid systems capable of exploiting solar energy. In this paper the strategy for the synthesis of the tailored aryleneethynylene organic fluorophore (AE) properly designed to act as light harvesting antenna is presented along with its successful bioconjugation to the photosynthetic reaction center RC from the bacterium Rhodobacter sphaeroides

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.

Chromate is a highly soluble and toxic non-essential oxyanion for most organisms. A number of chromate resistant bacteria have been investigated and diverse resistance mechanisms were found [1].We are investigating the potentialities of the photosynthetic facultative bacterium Rhodobacter sphaeroides, known for its ability to tolerate high concentrations of several heavy metal ions [2] and bioaccumulate some of them, such as nickel and cobalt [3, 4], in the bioremediation of chromate polluted sites. Employing an interdisciplinary approach, the response to chromate stress was investigated by combining biochemical and spectroscopic measurements, proteomic characterization and cell imaging.An efficient resistance mechanism to chromate is suggested both by the high EC50 value and the lag-phase lengthening induced at concentrations above 0.05 mM. R. sphaeroides is also able to reduce chromate to the less toxic and soluble form Cr(III) with reductase activity preferentially associated with the protein soluble fraction. Chromate effect on soluble enzymes was investigated by a proteomic approach: soluble protein expression profiles of cells exposed to chromate were compared with those of untreated control cells through two-dimensional gel electrophoresis analysis. Upon exposure to chromate at least 30 soluble proteins were differentially expressed. The wide variety of differentially expressed proteins suggests that different metabolic pathways are involved as response to chromate exposure. The accompanying physiological response to Cr(VI) exposure included marked changes in cellular morphology as revealed by atomic force microscopy.

Cobalt is an important oligoelement required for bacteria; if present in high concentration, exhibits toxic effects that, depending on the microor-ganism 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 ofthe cell envelope.

Hydrogen-bonded pigments are a class of organic colorants, which features many natural-origin molecules that have been used for centuries, as well as numerous mass-produced industrial synthetic compounds used in applications as various as out- door paints, cosmetics, and printing inks. Their widespread use in the dye and pigment industry is motivated by three favorable properties: low-cost production, excellent stability, and low toxicity, with some of them considered less hazardous than even water-soluble food dyes. Recently, H-bonded pigments have emerged also as promising organic semiconductors, with epindolidione (EPI) and quinacridone (QNC) demonstrating hole mobility in the range of 0.1-1 cm2 V-1 s-1 and outstanding operational stability in both air and in aqueous environments with pH 3-10 [1]. This latter finding is motivating for deploying these materials in applications requiring direct interfacing with biological ''wet'' environments. Their N-H and C=O functional groups are the "chemical handles" that are in principle amenable for direct bioconjugation. A proper combination of the photosynthetic reaction center (RC), the pivotal protein in photosynthesis, with engineered materials such as metals or inorganic semiconductor electrodes, has attracted great attention for the building of new versatile hybrid devices for solar energy conversion. Here we propose a covalent approach able to stably anchor RCs onto evaporated thin films of EPI and QNC. The N-H functional group of these molecules in thin film reacts spontaneously with N-hydroxysuccinimide functionalized linkers as disuccinimidyl suberate. The protruding linkers are then used to covalently bind the lysines residues of the Rb. sphaeroides RC, by forming an amide linkage (right panel in the figure). Our protocol is shown to preserve the semiconducting properties of the pigments while maintaining the protein's photoactivity. Multiple reflection IR spectroscopy and AFM demonstrated the effective covalent binding and the robustness of the protein anchoring even after buffer washing. Furthermore, RC charge recombination kinetic measurements confirmed the full functionality of bioconjugated proteins ruling out any possible hindering effect from the organic films. As key results of our work, we show that semiconductors preserve their favorable electrical properties and the proposed photoconductor device operates under water, before and after RCs anchoring. These are enabling steps for using H-bonded pigments as a platform for multifunctional bioelectronics devices.

A high-throughput crystallographic investigation on several crystals of photosynthetic reaction center covalently bound to an ad-hoc synthesized artificial antenna (AE600) is presented. The investigation did not show a preferential binding site of the antenna molecule AE600 to the reaction center in the solid phase. An accurate crystallographic study allowed identifying a lysine residue sitting on periplasmic side of the protein as one of the bioconjugation sites. The residue sits on subunit M of the protein, in close proximity to the bacteriochlorophylls of the reaction center involved in the light absorption and conversion processes. Distances obtained from the crystallographic structure confirm that energy transfer between the antenna and the protein proceed with the Förster resonance mechanism.

The bacterial photosynthetic reaction centre (RC) is a membrane spanning protein that, upon illumination, promotes the reduction of a ubiquinone molecule withdrawing electrons from cytochrome c2. This photo-activated reaction has been often exploited, in suitably designed photoelectrochemical cells, to generate photocurrents sustained by the reduction at the working electrode of the photo-oxidized electron donor or by the oxidation of the electron acceptor. In this work we have explored in more detail the factors affecting the photocurrent generation in commercially available screen-printed electrochemical cells containing an electrolyte solution where RC proteins and suitable mediators are solubilized. In particular, the role of the applied potential and the influence of concentration and structure of acceptor and donor molecules have been assessed. We show that efficient generation of cathodic photocurrents in a three electrode configuration occurs at an applied potential of 0.0 V versus quasi-ref Ag (the open circuit potential of the system measured in the dark) in presence of ferrocenemethanol and decylubiquinone, which proved to guarantee high performances as electron donor and acceptor respectively. Moreover, we employed a set of differential equations, describing reaction and diffusion processes, for modelling with high accuracy the chronoamperometry profiles recorded at variable RC concentrations. This model allowed us to estimate the kinetic parameters relevant to the chemical and electrochemical reactions triggered by light and to get a snapshot of the electrolyte composition in the bulk and electrode surroundings at different times from the light exposure. The characteristic time course of the photocurrent, showing a fast rise to a peak value followed by a slower decay, has been therefore explained as the result of the strict interconnection between the dynamical processes involved.

The genome sequence of a Sphingobium strain capable of tolerating high concentrations of Ni ions, and exhibiting natural kanamycin resistance, is presented. The presence of a transposon derived kanamycin resistance gene and several genes for efflux-mediated metal resistance may explain the observed characteristics of the new Sphingobium isolate.

The genome sequences of three new strains of Staphylococcus arlettae named Bari1, Bari2, and Bari3 are presented. The strains exhibited tolerance to hexavalent chromium ions. An sprC gene encoding a putative chromium transporter was present in each of the three draft genome sequences.

The induction (sudden dark-to-light transition) of fluorescence of photosynthetic bacteria has proved to be sensitive tool for early detection of mercury (Hg2+) contamination of the culture medium. The major characteristics of the induction (dark, variable and maximum fluorescence levels together with rise time) offer an easier, faster and more informative assay of indication of the contamination than the conventional techniques. The inhibition of Hg2+ is stronger in the light than in the dark and follows complex kinetics. The fast component (in minutes) reflects the damage of the quinone acceptor pool of the RC and the slow component (in hours) is sensitive to the disintegration of the light harvesting system including the loss of the structural organization and of the pigments. By use of fluorescence induction, the dependence of the diverse pathways and kinetics of the mercury-induced effects on the age and the metabolic state of the bacteria were revealed.

Ultrasounds are used in many industrial, medical and research applications. Properties and function of proteins are strongly influenced by the interaction with the ultrasonic waves and their bioactivity can be lost because of alteration of protein structure. Surprisingly, to the best of our knowledge no study was carried out on Integral Membrane Proteins (IMPs), which are responsible for a variety of fundamental biological functions. In this work, the photosynthetic Reaction Center (RC) of the bacterium Rhodobacter sphaeroides has been used as a model for the study of the ultrasound-induced IMP denaturation. Purified RCs were suspended in i) detergent micelles, in ii) detergent-free buffer and iii) reconstituted in liposomes, and then treated with ultrasound at 30 W and 20 kHz at increasing times. The optical absorption spectra showed a progressive and irreversible denaturation in all cases, resulting from the perturbation of the protein scaffold structure, as confirmed by circular dichroism spectra that showed progressive alterations of the RC secondary structure. Charge recombination kinetics were studied to assess the protein photoactivity. The lifetime for the loss of RC photoactivity was 32 min in detergent micelles, ranged from 3.8 to 6.5 min in the different proteoliposomes formulations, and 5.5 min in detergent-free buffer. Atomic force microscopy revealed the formation of large RC aggregates related to the sonication-induced denaturation, in agreement with the scattering increase observed in solution.

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

Following a bottom-up synthetic biology approach it is shown that vesicle-based cell-like systems (shortly "synthetic cells") can be designed and assembled to perform specific function (for biotechnological applications) and for studies in the origin-of-life field. We recently focused on the construction of synthetic cells capable to converting light into chemical energy. Here we first present our approach, which has been realized so far by the reconstitution of photosynthetic reaction centre in the membrane of giant lipid vesicles. Next, the details of our ongoing research program are presented. It involves the use of the reaction centre, the coenzyme Q-cytochrome c oxidoreductase, and the ATP synthase for creating an autonomous synthetic cell. We show experimental results on the chemistry of the first two proteins showing that they can efficiently sustain light-driven chemical oscillations. Moreover, the cyclic pattern has been reproduced in silico by a minimal kinetic model.

Deep eutectic solvents (DESs) are emerging as a new class of green solvents with the potential to replace organic solvents in several fundamental and applied processes. In this work, we offer an unprecedented characterization of the behavior of the bacterial photosynthetic reaction center (RC) from Rhodobacter sphaeroides in a series of choline chloride based DESs. RC is a membrane-spanning three-subunit pigment protein complex that, upon illumination, is capable of producing a stable charge-separated state. Thus, it represents the ideal model for carrying out basic studies of protein solvent interactions. Herein, we first report that, in many DES mixtures investigated, RC (a) is stable, (b) is capable of generating the charge-separated state, and (c) is even able to perform its natural photocycle. It proved, indeed, to be effective in reducing quinone molecules to quinol by withdrawing electrons from cytochrome c. As an example of biotechnological application, a photoelectrochemical cell based on DES-dissolved RC has also been designed and successfully employed to generate photocurrents arising from the reduction of the electron-donor ferrocenemethanol.

The development of an amperometric biosensor for herbicide detection, using bacterial reaction centers (RC) as biorecognition element, is presented. RC immobilization on gold screen printed electrodes was achieved by LIFT, a powerful physisorption-based immobilization technique that enhances the intimate contact between the protein and the electrode surface. As a result, stable photocurrents driven by direct electron transfer at the donor side were observed, both in the presence and in the absence of a quinone substrate in solution. The addition of quinone UQ(0) increased the photocurrents, while the UQ(0)-free system showed higher sensitivity to the herbicide terbutryn, a model inhibitor, acting as photocurrent attenuator. In spite of its simple design, the performances achieved by our mediatorless device are comparable or superior to those reported for analogous RC-based photoelectrochemical cells, in terms of both terbutryn sensing and photocurrent generation. (C) 2016 Elsevier B.V. All rights reserved.

The photosynthetic reaction center is an extraordinarily efficient natural photoconverter, which can be ideally used in combination with conducting or semiconducting interfaces to produce electrical signals in response to absorption of photons. The actual applicability of this protein in bioelectronic devices critically depends on the finding of (a) suitable deposition methods enabling controlled addressing and precise orientation of the protein on electrode interfaces and (b) chemical manipulation protocols able to tune and enhance protein light absorption in specific or broader spectral regions. Literature reports several examples of approaches to fulfill these requirements, which have faced in different ways the fundamental issues of assembling the biological component and non-natural systems, such as electrode surfaces and artificial light harvesting components. Here we present a short overview of the main methods reported to accomplish both the objectives by properly "garnishing" the photosynthetic reaction center (RC) via chemical modifications.

The non-sulfur, purple, facultatively phototrophic bacterium Rhodobacter (R.) sphaeroides represents a unique model for the investigation of the structure, function and biosynthesis of the energy-transducing system in photosynthetic machineries. Photosynthetic units share a basic architecture, composed by an efficient light collecting system, which funnels light to the reaction center, where photons are converted into chemical potential energy with a quantum yield close to unity. In the R. sphaeroides wild type (strain 2.4.1), two distinct antenna complexes are present: the LH-II, absorbing at 800 and 850 nm, and the LH-I, with a maximum at 870 nm, appearing as a shoulder of the 850 nm LH-II band. Strong dependence of the absorption spectrum on changing growing conditions, i.e light intensity or heavy metal ions concentration, requires to find out a robust, non-destructive instrumental method of investigation which could help to quickly solve the complex structure of the absorption signals in the presence of different stress factors. In this work, we present second-order derivative spectroscopy as the ideal technique to tackle this issue. Peaks that originate from derivation correspond to a maximum in the original spectrum, but with the advantage of a sharpening effect, which enables the precise assessment of relevant wavelengths. Such effect was successfully tested on potassium permanganate solutions, whose composite band, presenting seven overlapped peaks located in the visible region of the electromagnetic spectrum, was separated in well-distinct signals, even at very low concentrations. Interestingly, Lambert-Beer Law is maintained, allowing an accurate calculation of peak ratios directly on the sharpened second derivative spectrum. This great advantage was exploited to investigate the effect of the presence of heavy metal ions on the LH complexes within cultures of R. sphaeroides 2.4.1 cells. This was possible through the precise individuation of absorption maxima and the evaluation of relative ratios between the three LH-I and LH-II peaks. Preliminary results clearly show a direct influence of tested metals on the biosynthesis of the LH-I complex, thus confirming the potentialities of the proposed technique as a promising tool for the evaluation of the chronic effect of exposure to pollutants during bacterial growth.

Nature solved the problem of converting solar light into chemical energy in a very elegant way through the photosynthesis. The natural photosynthetic process takes place thanks to a very precise protein scaffolding architecture and pigments arrangement that lead to convert 98% of the absorbed photons into a stable charge separated state with a lifetime in the second timescale. An intelligent way towards the design of artificial devices for solar conversion is to try to learn the lesson from Nature by mimicking the organization of the natural photosynthetic process. The interest of the scientific and industrial world towards a system capable of storing sunlight and convert it into a suitable form of energy has been increasing markedly in recent years. The idea is to focus on the natural candidates capable of performing these tasks, the photosynthetic reaction center proteins (RC), whose photochemical properties, selected by nature in the evolution course, however, are complicated to reproduce in artificial device. Hybrids have as central protein the photosynthetic reaction center from the purple no sulphur bacterium Rhodobacter sphaeroides strain R26, that converts solar light into a electron-hole couple state having an adequate lifetime to allow the ancillary chemistry to happen.

The bioconjugation of photosynthetic proteins with efficient organic light harvesting antennas is a very intriguing approach to build novel hybrid organic-biological machineries that, mimicking nature, employ solar energy to generate photocurrents or to drive thermodynamically unfavoured reactions, reaching efficiencies higher than those obtainable by their natural conterparts. Such hybrid systems are potentially useful as active materials in new generation devices for photovoltaics and biosensing. In the frame of our studies on organic-biological hybrids for solar energy conversion,[1] here we present the design, synthesis and preliminary characterization of a series of heptamethine cyanine dyes (such as Cian-1 in Figure 1a) particularly suitable as light harvesting antennas for the photosynthetic Reaction Center (RC) of the purple bacterium Rhodobacter sphaeroides strain R26. These molecules have been properly tailored to have efficient light absorption in the visible spectral range, where the RC absorbance is very low, and efficient emission in the near infrared region, in correspondence of the highest RC absorption peaks.Moreover, the charged sites within their molecular structure make these molecules highly soluble in detergent aqueous environment where the RC is stable, this allowing them to approach the bioconjugation sites of the protein. Finally, the synthesized cyanines are endowed with a carboxylic moiety useful for their covalent binding to the amino groups of the RC lysine residues. Our preliminary results show that the bioconjugation of these organic antennas to the RC is expected to be a very profitable strategy to afford highly efficient organic-biological hybrids for solar energy conversion.

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.

Rubrica Bimestrale sulle Proteine

Rubrica Bimestrale sulle Proteine

Rubrica bimestrale "Proteine Operaie

Rubrica bimestrale "Proteine Operaie

Rubrica bimestrale "Proteine Operaie

Rubrica bimestrale "Proteine Operaie

Biological processes using microorganisms for nanoparticle synthesis are appealing as eco-friendly nanofac-tories. The response of the photosynthetic bacterium Rhodobacter sphaeroides to gold exposure and its reducingcapability of Au(III) to produce stable gold nanoparticles (AuNPs), using metabolically active bacteria andquiescent biomass, is reported in this study.In the former case, bacterial cells were grown in presence of gold chloride at physiological pH. Gold exposurewas found to cause a significant increase of the lag-phase duration at concentrations higher than 10 ?M, sug-gesting the involvement of a resistance mechanism activated by Au(III). Transmission Electron Microscopy(TEM) and Scanning Electron Microscopy/Energy Dispersive X-ray Spectrometry (SEM/EDS) analysis of bac-terial cells confirmed the extracellular formation of AuNPs.Further studies were carried out on metabolically quiescent biomass incubated with gold chloride solution.The biosynthesized AuNPs were spherical in shape with an average size of 10 ± 3 nm, as analysed byTransmission Electron Microscopy (TEM). The nanoparticles were hydrophilic and stable against aggregation forseveral months.In order to identify the functional groups responsible for the reduction and stabilization of nanoparticles,AuNPs were analysed by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy, X-ray Photoelectron Spectroscopy (XPS), X-ray Fluorescence Spectrometry (XRF) and X-ray AbsorptionSpectroscopy (XAS) measurements. The obtained results indicate that gold ions bind to functional groups of cellmembrane and are subsequently reduced by reducing sugars to gold nanoparticles and capped by a protein/peptide coat.Gold nanoparticles demonstrated to be efficient homogeneous catalysts in the degradation of nitroaromaticcompounds.

Il particolato atmosferico (PM) è costituito da una miscela di particelle solide e liquide aventiorigine primaria e secondaria. La composizione chimica del PM varia notevolmente e dipende dafattori quali le fonti di combustione, il clima, la stagione e il tipo di inquinamento. Il PM è costituitoda particelle di materiale carbonioso, da composti organici volatili o semi-volatili adsorbiti sulleparticelle carboniose, da ioni, metalli di transizione, materiali di origine biologica e minerali. Seclassificato in base alla sua granulometria, il PM è distinto in "coarse" e "fine" e le due frazionicomprendono le particelle aventi rispettivamente diametro aerodinamico superiore e inferiore ai 2.5µm (PM2.5). Il particolato fine ed ultrafine è quello maggiormente associato agli effetti negativisulla salute umana perché può raggiungere le vie respiratorie più profonde fino ad arrivare aglialveoli polmonari, tuttavia non si può escludere la pericolosità delle particelle, caratterizzate dadiametro aerodinamico inferiore ai 10 µm (PM10). In generale gli effetti del PM sui diversiorganismi variano a seconda della concentrazione in atmosfera, delle loro caratteristiche fisicochimichee dal tempo di esposizione degli organismi a tale inquinante.In questo lavoro è stata studiata l'interazione del PM10 con tre differenti organismi. Estratti organicidi PM10 (EOM) e soluzioni standard di Idrocarburi Policiclici Aromatici (IPA) sono stati testatisugli organismi modello Rhodobacter (R.) sphaeroides 2.4.1 e Caenorhabditis elegans. Campionidi PM10 raccolti su filtri in fibra di quarzo sono stati utilizzati come supporto per la crescita dipiantine di pomodoro (Solanum lycopersicon). I risultati ottenuti indicano che gli effetti degliestratti organici di particolato atmosferico sono fortemente dipendenti dal tipo di organismo che adessi viene esposto. Mentre il C. elegans subisce un effetto negativo, con una mortalità fino al 50% apartire dal secondo stadio larvale (Liuzzi et al, 2012), l'esposizione di R. sphaeroides agli EOM nonmostra effetti dannosi, eccettuata una contenuta diminuzione della velocità di crescita. Le pianteesposte al particolato mostrano un evidente cambiamento nella morfologia dell'apparato radicale estress ossidativo rappresentato da un aumento del contenuto di radicali dell'ossigeno (ROS).

The life cycle of the bacterium Rhodobacter sphaeroides was investigated by isothermal microcalorimetry using two different procedures based on the use of a static ampoule and a flow cell, respectively. In the static ampoule method it is possible to follow the growth phase and also the death phase which cannot be revealed by total biomass based techniques like turbidimetry. However, different cellular metabolisms, possibly due do the oxygen limitation occurring during the bacterial life cycle, produce complex behavior in the experimental curves. In the stop- flow cell mode this limitation is overcome as the bacteria are grown outside the calorimeter under well-defined aerobic conditions and aliquots of cell suspension are transferred in the calorimeter at different time intervals. The complex behavior shown in the static ampoule mode was successfully analyzed by a population evolution model based on a Fujikawa modified logistic equation which provides a quantitative description of the process.

Rubrica bimestrale "Proteine Operaie

Rubrica

Rubrica Bimestrale sulle Proteine

Rubrica Bimestrale sulle Proteine

The fabrication of low-cost, stable, sensitive and selective biosensors for herbicides has gained considerable attention in recent years as modern agriculture makes massive use of pesticides that are known to be harmful for human health. The use of photosynthetic proteins for herbicide detection is the most straightforward strategy for this aim, as these are the natural target of this class of chemicals. Among the possible biosensor devices, the most successful that have led to commercial products are based on amperometric detection. In particular, in this type of biosensors, the monitored photocurrent is progressively attenuated in the presence of increasing amount of analyte, minimizing the mathematical treatment of the data. In the present work, we show the functionalization of screen-printed electrodes with an ordered film of photoactive biological material, by means of laser induced forward transfer (LIFT) technique, in order to fabricate a biohybrid device for energy conversion and/or biosensing. LIFT is an advanced tool for achieving the direct immobilization of biosystems [1] with high spatial resolution, due to the high impact pressure of the transferred droplets, at the receiver substrate. As a result, it enhances physical adsorption onto the electrode surface and high photocurrents can be attained by using extremely low quantities of deposited samples. The biomaterial used is the photosynthetic reaction center (RC) from the bacterium Rhodobacter (Rb.) sphaeroides. Cathodic or anodic photocurrents are detected depending on the potential applied to the working electrode: in particular, cathodic photocurrents are detected at the donor side under reducing potentials and anodic ones at the acceptor side under oxidizing potentials. Rb. sphaeroides RC is classified as Q-type and resembles the more evolved photosystem II (PSII) found in plants, algae and cyanobacteria, sharing the sensitivity to the same class of herbicides. However, bacterial RC has a simpler architecture, is more stable and is more selective to a particular class of herbicides, the triazinic ones, while PSII is sensitive to different classes of pesticides [2]. In the present case, thanks to the intimate contact between the RC and the electrode, we could detect cathodic photocurrents in the absence of any mediator at both acceptor and donor side, bringing about several advantages: simpler biosensor architecture, ideal square-wave shape of the photocurrents, and independence on the mediator concentration. Most importantly, the absence of quinone, often used as mediator at the acceptor side makes the biosensor particularly sensitive to the herbicide tested, due to the competitive nature of the binding reaction. We tested the herbicide terbutryn which, although is no longer permitted in the EU, has the strongest binding affinity to the RC and is more suitable to show the potentiality of the final device. A limit of detection for the terbutryn was found in the range 8-30 nM w

Rubrica bimestrale "Proteine Operaie

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 c(2) and K-4[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.

Articolo privo abstract

Photosynthetic organisms are able to convert photons in chemical energy thanks to unique light-harvesting antenna systems occurring inside the cytoplasmic membrane. These antenna complexes are composed of a wide variety of proteins and different chlorophyll pigments acting as binders [1]. Their structures can be divided into chlorophylls (Chls) class found in cyanobacteria and algae up through plants, and into bacteriochlorophylls (BChls), found in phototrophic bacteria [1]. Both pigments consist of a macrocyclic tetrapyrrolic ring system named porphyrin, coordinating a Mg2+ ion, and several different side chains, usually including phytol [2]. The main difference between these two types of pigments is related to the saturation state of the porphyrin macrocycle, with bacteriochlorophylls having a much more unsaturated structure than chlorophylls. Fast identification of BChls and related compounds may be carried out by matrix assisted laser desorption ionization (MALDI) time-of-flight (ToF) MS because of some characteristic advantages as rapid and easy sample preparation, tolerance to salts, and high sensitivity. However, previous analysis of BChls showed that demetalation of magnesium porphyrins occurs by using conventional acidic matrices. Indeed pheophitinization (i.e., release of the metal ion) was observed by Persson et al., using a-cyano-4-hydroxycinnamic acid (CHCA) as MALDI matrix, for BChls extracted from Chlorobium tepidum green sulfur bacterium that were detected as bacteriopheophytins [3]. Very recently, 1,5-diaminonaphthalene (DAN) was introduced as an electron-transfer secondary reaction matrix for the analysis of chlorophylls [4]. DAN was proved to outperform conventional matrices such as CHCA, dithranol, antracene and even terthiophene, since loss of the metal ion and fragmentation of the phytol-ester linkage are negligible. Here, we report the identification of intact bacteriochlorophylls by MALDI MS in the purple non-sulfur bacterium Rhodobacter sphaeroides, a model system for studying both bacteriochlorophyll biosynthesis and assembly of bacterial photosynthetic complexes [5]. These results show the great capability of MALDI MS to follow bacteriochlorophylls biotransformation occurring in different growth conditions of bacteria.

Le recenti politiche europee sono rivolte alla valutazione dei possibili effetti nocivi di miscele complesse di sostanze chimiche (REACH) utilizzando metodi che escludano l'impiego di vertebrati. In questa relazione vengono illustrati gli effetti di tossicità di particolato atmosferico su due organismi modello: Caenorhabditis elegans e Rhodobacter sphaeroides. I campioni di particolato sono stati raccolti in due siti della città di Bari e da questi sono stati estratti i composti organici solubili in acetone ed esano. I risultati ottenuti da tali test hanno mostrato un effetto tossico sul nematode C. elegans, mentre nel caso del batterio fotosintetico gli effetti sono limitati ad una leggera diminuzione della velocità di crescita. Gli sviluppi futuri di questo studio saranno orientati verso frazioni granulometriche quali PM1 e nano particolato e verso altri sistemi modello.

The functionalization of screen-printed electrodes (SPEs) with a thin film of reaction centre (RC) proteins from the phototrophic bacterium Rhodobacter (R.) sphaeroides, by means of laser induced forward transfer (LIFT) technique, allowed the fabrication of robust and sensitive bio-hybrid devices for terbutryn detection and analysis. The optimal wiring between RCs and the gold electrode surface, achieved by LIFT, led to the generation of cathodic photocurrents sustained by a direct electron transfer (DET) mechanism, which were attenuated by addition of the herbicide inhibitor. (C) 2016 The Authors. Published by Elsevier Ltd.

The photosynthetic reaction center (RC) is an integral membrane protein that, upon absorption of photons, generates a hole-electron couple with a yield close to one. This energetic state has numerous possible applications in several biotechnological fields given that its lifetime is long enough to allow non-metabolic ancillary redox chemistry to take place. Here we focus on RCs reconstituted in liposomes, formed with sole phospholipids or in blends with other lipids, and show that the electrical charge sitting on the polar head of such hydrophobic molecules does play an important role on the stability of the hole-electron couple. More specifically this study shows that the presence of negative charges in the surrounding of the protein stabilizes the charge-separated state while positive charges have a strong opposite effect.

The reflectivity of transparent polymers can be reduced with a proper nanotextured layer on the surface according to the moth eye effect. Plasma etching has been proved to be a reliable method to generate self-organized nanostructures on the surface of various polymers. In the present work this method, directly carried out in one step, has been tested on polycarbonate for application as low reflective transparent material. CF4 and O2 fed plasma processes have been compared at different treatment time. Chemical (X-ray photoelectron spectroscopy), morphological (scanning electron microscopy), and optical (diffuse and specular reflectance, normal and grazing incidence) features have been evaluated. Results indicate that the CF4-to-O2 feed ratio significantly affects shape and distribution of the generated structures. O2 plasma, in particular, leads to taller structures with wire-like aspect and more homogeneous distribution, which are more effective in reducing reflectance with a broadband character (visible and near-infrared). Treatment duration, which instead affect mainly the dimension scale of the structures, must be tailored in order to control diffuse component of reflectance.

Photosynthetic Reaction Center (RC) is a transmembrane photoenzyme capable of converting absorbed photons into electron-hole pairs with almost unitary efficiency. The unique properties of this natural photoconverter attract considerable interest for its use as functional component in nanomaterials and bioelectronics devices. Implementation of RC into nanostructures or anchoring on devices' electrode surfaces require the development of suitable chemical manipulation. Here we report our methods to embed this protein in soft nanostructures or to covalently attach it on surfaces without denaturating it or altering its chemical properties. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

A critical selection of the recent literature reports on the use of photosynthetic and photoresponsive bacteria as a source of materials for optoelectronics and photonic devices is discussed, together with the applications foreseen in solar energy conversion and storage and light information technologies.The use of both photoactive cellular components and entire living cells is reviewed, aiming to highlight the great conceptual impact of these studies.These studies point out possible deep changes in the paradigm of design, and synthesis of materials and devices for optoelectronics. Although the possible technological impact of this technology is still hard to be predicted, these studies advance the understanding of photonics of living organisms and develop new intriguing concepts in biomaterials research.

Heavy metal pollution is a sensitive environmental topic with enormous impact on public opinion. Traditional chemical and physical techniques are employed in tackling this problem, often originating from anthropogenic activities. Recently the possibility of using the microbial word in heavy metal polluted sites has been proposed, and scientific literature is now witnessing an increase of publication in this field. The NIH on-line resource founds almost 1000 hits upon searching for bioremediation in the article title starting from 1988 when such definition appears for the first time. Bioremediation has shown promising results that have even spurred entrepreneurial activity in USA and northern Europe. A recent review [1] gives an up-to-date description of the state-of-the-art of the field including example of photosynthetic organisms, plants and algae. In this broad framework, anoxygenic photosynthetic bacteria and in particular the purple bacteria Rhodobacter sphaeroides, are being tested since 2005 in our laboratory [2-5] as tool in heavy metal removal from polluted sites. The effects of several metal ions and oxyanions on the metabolism of R. sphaeroides have been scrutinised using a multidisciplinary approach. The interaction with several metal species will be discussed and the state-of-the-art of our research will be presented along with future directions.References1. Gadd, G.M., Metals, minerals and microbes: geomicrobiology and bioremediation. Microbiology, 2010. 156(Pt 3): p. 609-43.2. Buccolieri, A., et al., Testing the photosynthetic bacterium Rhodobacter sphaeroides as heavy metal removal tool. Ann Chim -Rome, 2006. 96: p. 195-204.3. Giotta, L., et al., Reversible Binding of Metal Ions onto Bacterial Layers Revealed by Protonation-Induced ATR-FTIR Difference Spectroscopy. Langmuir, 2011. 27(7): p. 3762-3773.4. Italiano, F., et al., The photosynthetic membrane proteome of Rhodobacter sphaeroides R-26.1 exposed to cobalt. Res Microbiol, 2011. 162(5): p. 520-527.5. Pisani, F., et al., Soluble proteome investigation of cobalt effect on the carotenoidless mutant of Rhodobacter sphaeroides. J Appl Microbiol, 2009. 106(1): p. 338-49.

Ornithine lipids (OLs), a sub-group of the large (and of emerging interest) family of lipoamino acids of bacterial origin, contain a 3-hydroxy fatty acyl chain linked via an amide bond to the a-amino group of ornithine and via an ester bond to a second fatty acyl chain. OLs in extracts of Rhodobacter sphaeroides (R. sphaeroides) were investigated by high-performance reversed phase liquid chromatography (RPLC) with electrospray ionization mass spectrometry (ESI-MS) in negative ion mode using a linear ion trap (LIT). The presence of OLs bearing both saturated (i.e, 16:0, 17:0, 18:0, 19:0 and 20:0) and unsaturated chains (i.e., 18:1, 19:1, 19:2 and 20:1) was ascertained and their identification, even for isomeric, low abundance and partially co-eluting species, was achieved by low-energy collision induced dissociation(CID) multistage mass spectrometry (MSn n¼ 2e4). OLs signatures found in two R. sphaeroides strains, i.e., wild type 2.4.1 and mutant R26, were examined and up to 16 and 17 different OL species were successfully identified, respectively. OLs in both bacterial strains were characterized by several combinationsof fatty chains on ester-linked and amide-linked 3-OH fatty acids. Multistage MS spectra ofmonoenoic amide-linked 3-OH acyl chains, allowed the identification of positional isomer of OL containing18:1 (i.e. 9-octadecenoic) and 20:1 (i.e. 11-eicosenoic) fatty acids. The most abundant OL ([MH]at m/z 717.5) in R. sphaeroides R26 was identified as OL 3-OH 20:1/19:1 (i.e., 3-OH-eicosenoic acid amidelinkedto ornithine and esterified to a nonadecenoic chain containing a cyclopropane ring). An unusual OL (m/z 689.5 for the [MH] ion), most likely containing a cyclopropene ester-linked acyl chain (i.e., OL 3-OH 18:0/19:2), was retrieved only in the carotenoidless mutant strain R26. Based on the biosynthetic pathways already known for cyclopropa(e)ne ring-including acyl chains, a plausible explanation was invoked for the enzymatic generation of this ester-linked chain in R. sphaeroides.

Rubrica bimestrale "Proteine Operaie

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.(1, 2) 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 Ni2+ 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 Ni2+, establishing with this ion relatively weak coordinative bonds.

Rhodobacter sphaeroides has for a long time been investigated for its adaptive capacities to different environmental and nutritional conditions, including presence of heavy metals, which make it a valuable model organism for understanding bacterial adaptation to metal stress conditions and future environmental applications, such as bioremediation of polluted sites. To further characterize the capability of R. sphaeroides to cope with high cobalt ion concentrations, we combined the selection of adaptive defective mutants, carried out by negative selection of transposon insertional libraries on 5 mM Co(2+) -enriched solid medium, with the analysis of growing capacities and transcriptome profiling of a selected mutant (R95). A comparative analysis of results from the mutant and wild-type strains clearly indicated that the adaptive ability of R. sphaeroides strongly relies on its ability to exploit any available energy-supplying metabolisms, being able to behave as photo- or chemotrophic microorganism. The selected R95 mutant, indeed, exhibits a severe down-expression of an ABC sugar transporter, which results nonpermissive for its growth in cobalt-enriched media under aerobic conditions. Interestingly, the defective expression of the transporter does not have dramatic effects on the growth ability of the mutant when cultivated under photosynthetic conditions.

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. © 2016 American Chemical Society.

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.

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 Co2+ and CrO4 (2-) is reported and changes in membrane lipid profiles were rationalised.

We have isolated and characterized the light-driven proton pump Bop I from the ultrathin square archaeon Haloquadratum walsbyi, the most abundant component of the dense microbial community inhabiting hypersaline environments. The disruption of cells by hypo-osmotic shock yielded Bop I retinal protein highly enriched membranes, which contain one main 27 kDa protein band together with a high content of the carotenoid bacterioruberin. Light-induced pH changes were observed in suspensions of Bop I retinal protein-enriched membranes under sustained illumination. Solubilization of H. walsbyi cells with Triton X-100, followed by phenyl-Sepharose chromatography, resulted in isolation of two purified Bop I retinal protein bands; mass spectrometry analysis revealed that the Bop I was present as only protein in both the bands. The study of light/dark adaptations, M-decay kinetics, responses to titration with alkali in the dark and endogenous lipid compositions of the two Bop I retinal protein bands showed functional differences that could be attributed to different protein aggregation states. Proton-pumping activity of Bop I during the photocycle was observed in liposomes constituted of archaeal lipids. Similarities and differences of Bop I with other archaeal proton-pumping retinal proteins will be discussed. Haloquadratum walsbyi is a peculiar organism which often dominates the microbial communities of the hypersaline ecosystems, such as salt lakes and solar saltern crystallizer ponds. It is extremely thin and possesses a unique square-like shape, with sharp edges and acute straight corners. The flat cells form large sheets similar to solar panels, able to efficiently collect light as an energy source for metabolism. Like other archaeal extremely halophilic microorganisms, square cells encode light-activated retinal-proteins to survive in hypersaline environments. In this study we describe the biochemical properties and the photochemistry of the light-activated proton pump Bop I of H. walsbyi, grown in laboratory.

Cells of the carotenoidless strain R-26.1 of Rhodobacter sphaeroides were grown in the presence of a high concentration (5 mM) of cobalt ions. The photosynthetic intracytoplasmic membranes were isolated and investigated by proteomic analysis using non-denaturating blue native electrophoresis in combination with LC-ESI-MS/MS. Comparison with intracytoplasmic membranes of cells grown under control conditions showed a change in the relative amount of proteins belonging to the photosynthetic apparatus, with net downregulation of light-harvesting complexes and increased concentration of the nude reaction center (RC), as well as upregulation of enzymes related to chemoorganotrophy. These effects represent possible bacterial adaptation so as to retrieve energy for metabolic processes from sources alternative to less efficient photosynthesis. The influence of cobalt on the photochemistry of the RC in cell extracts was also investigated by charge recombination. The kinetics of the charge recombination reaction was found to be slower in extracts from cells exposed to Co(2+), indicating that the reorganization of the photosynthetic apparatus also involves its photochemical core.

The sensitivity of intact cells of purple photosynthetic bacterium Rhodobacter sphaeroides wild type to low level (< 100 mu M) of mercury (Hg2+) 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 Hg2+ sensitivities of the electron transport (donor/acceptor) sites including the reduced bound and free cytochrome c (2) and the primary reduced quinone. In contrast to the RC, the light harvesting system and the bc(1) 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.

Urban particulate matter (PM) can affect green plants either via deposition on the above-ground biomass, where the contaminants can penetrate the leaf surface, or indirectly via soil-root interaction. In our investigation, a model experiment was carried out to demonstrate the direct effect of PM on tomato (Solanum lycopersicum L.) plant growth. A monitoring campaign of PM10 was conducted at an urban background site of Canosa (Apulia, Southern Italy) in four different days (1, 2, 3, 4). PM10 samples were collected for 24 hours on quartz fiber filter. The filters were then cut into two parts, one of which was used for the chemical characterization of the PM10 and one for the growth of tomato. Organic and elemental carbon and polycyclic aromatic hydrocarbons (PAHs) content were analysed for all the tested filters. Tomato plants were grown for 18 days directly on filters absorbed with PM10. The germination rate of tomato seeds and some parameters of seedlings primary growth of this plant species (length of root and shoot, their fresh weight and content of photosynthetic pigments in shoot) were used as laboratory indicators of phytotoxicity. Substantial differences were found in the growth of root apparatus respect to that of control plants. A significant decrease of primary root elongation, a large amount of secondary roots and a decrease in plant and root weights were found. To assess if the direct exposition of roots to PM10 induced an oxidative stress, reactive oxygen species (ROS) concentration was evaluated by measuring the fluorescence arising from oxidation of DCFH-DA in both control and treated roots. Quantitative analysis of ROS indicated that an oxidative burst in response to abiotic stress occurred in roots directly grown on PM10, whose detrimental effect was also confirmed by the findings on chlorophyll content and chlorophyll-to-carotenoid ratio.

Nickel acts as cofactor for a number of enzymes of many bacteria species. Its homeostasis is ensured by proteins working as ion efflux or accumulation systems. These mechanisms are also generally adopted to counteract life-threatening high extra-cellular Ni2+ concentrations. Little is known regarding nickel tolerance in the genus Sphingobium. We studied the response of the novel Sphingobium sp. ba1 strain, able to adapt to high Ni2+ concentrations. Differential gene expression in cells cultured in 10 mM Ni2+, investigated by RNA-seq analysis, identified 118 differentially expressed genes. Among the 90 up-regulated genes, a cluster including genes coding for nickel and other metal ion efflux systems (similar to either cnrCBA, nccCBA or cznABC) and for a NreB-like permease was found. Comparative analyses among thirty genomes of Sphingobium species show that this cluster is conserved only in two cases, while in the other genomes it is partially present or even absent. The differential expression of genes encoding proteins which could also work as Ni2+-accumulators (HupE/UreJ-like protein, NreA and components of TonB-associated transport and copper-homeostasis systems) was also detected. The identification of Sphingobium sp. ba1 strain adaptive mechanisms to nickel ions, can foster its possible use for biodegradation of poly-aromatic compounds in metal-rich environments.

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.