Massimo Trotta

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.

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.

Early detection of mercury contamination by fluorescence induction of photosynthetic bacteria

The induction (sudden dark-to-light transition) of fluorescence of photosynthetic bacteria has proved to be sensitive tool for early detection of mercury (Hg(2+)) 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 Hg(2+) 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.

Response of membrane protein to the environment: the case of photosynthetic Reaction Centre

The role of the environment on the function and structure of enzymes is discussed in the case of the photosynthetic Reaction Centre (RC) reconstituted in proteoliposomes. The reconstitution procedure is illustrated in detail and the effect of the bilayer upon the activity of the enzyme is discussed. The combined use of ENDOR spectroscopy and flash photolysis clearly demonstrate that the enzyme isolation procedure induces some structural changes, relative to the native state, that are completely reversible once the protein is reconstituted in the phospholipid bilayer.

Trapping of a long-living charge separated state of photosynthetic reaction centers in proteoliposomes of negatively charged phospholipids

AbstractReaction centers from the purple bacterium Rhodobacter sphaeroides strain R-26.1 were purified and reconstituted in proteoliposomes formed by the anionic phospholipids phosphatidylglycerol, phosphatidylserine and phosphatidylinositol and by the zwitterionic phospholipid phosphatidylcholine by size-exclusion chromatography in the dark and under illumination. We report the large stabilizing effect induced by anionic phospholipids on the protein charge separated state which results trapped in a long-living (up to tens of minutes) state with a yield up to 80%. This fully reversible state is formed in oxygenic conditions regardless the presence of the secondary quinone QB and its lifetime and relative yield increase at low pH. In proteoliposomes formed with QA-depleted reaction centers (RCs) the resulting protein is very light-sensitive and the long living charge separated state is not observed. The data collected in negatively charged proteoliposomes are discussed in terms of the electrostatic effect on the primary quinone acceptor and compared with similar long living species reported in literature and obtained in anionic, zwitterionic, and non-ionic detergents.

ROLE OF PALMITIC ACID ON THE ISOLATION AND PROPERTIES OF HALORHODOPSIN

Purified halorhodopsin was isolated from Halobacterium halobium as previously described (Duschl, A. et al. (1988) J. Biol. Chem. 263, 17016-17022). Two purple bands were eluted from phenyl-Sepharose column, indicating the presence of differently retained halorhodopsin forms; the absorption spectra of the two halorhodopsin bands in the dark were not different. By gas chromatography/mass spectrometry we could identify palmitate (which is only a minor lipid component of archaeal cells) among lipids associated with purple fractions. Typically the palmitate content of the first eluted band was higher than that of the second, indicating a correlation between the palmitate content and the retention time; from one to two fatty acid molecules per halorhodopsin molecule were present depending on the fraction analysed. Very little or no palmitate was released from denatured halorhodopsin. By adding palmitate to buffers used in the phenyl-Sepharose chromatography, only one sharp purple band was collected, corresponding to the less retained halorhodopsin fraction. Pentadecanoic fatty acid could also affect the halorhodopsin chromatography. Chromatography of halorhodopsin in the presence of beta-mercaptoethanol showed only one band, corresponding to the more retrained halorhodopsin form. The two halorhodopsin fractions had different photoreactivity; the less retained halorhodopsin fraction (at higher palmitate content) showed an higher rate of decay of the absorbance at 570 nm upon illumination. By following the decay of the absorbance at 570 nm upon addition of alkali in the dark, we found that the two halorhodopsin fractions had different pKa values of deprotonation.

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

Significance The photosynthetic reaction center (RC), an integral membrane protein at the core of bioenergetics of all autotrophic organisms, has been reconstituted in the membrane of giant unilamellar vesicles (RC@GUV) by retaining the physiological orientation at a very high percentage (90 ± 1%). Owing to this uniform orientation, it has been possible to demonstrate that, under red-light illumination, photosynthetic RCs operate as nanoscopic machines that convert light energy into chemical energy, in the form of a proton gradient across the vesicle membrane. This result is of great relevance in the field of synthetic cell construction, proving that such systems can easily transduce light energy into chemical energy eventually exploitable for the synthesis of ATP. 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.

''Garnishing'' the photosynthetic bacterial reaction center for bioelectronics

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.

Characterisation of RC-proteoliposomes at different RC/lipid ratios

Reconstitution of membrane proteins in phospholipid vesicles allows the investigation of such macromolecules in a biomimetic simplified environment. The often employed micelle-to-vesicle-transition method for proteoliposome preparation is a fast and reproducible technique. In this, communication is shown that the lipid/protein ratio influences the size of the proteoliposomes and the actual protein reconstitution. The results indicate that for photosynthetic reaction centres, the best conditions for ligand-interaction experiments are achieved with a lipid/protein value of 1000:1, while for complete protein incorporation, the 2000:1 ratio should be chosen.

Atmospheric particulate matter (PM) effect on the growth of Solanum lycopersicum cv. Roma plants.

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.