Francesca Italiano

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.

Oligomeric characterization of the photosynthetic apparatus of Rhodobacter sphaeroides R26.1 by nondenaturing electrophoresis methods.

Blue and colorless native gel electrophoresis in combination with LC-ESI-MS/MS are powerful tools in the analysis of protein networks in biological membranes. We used these techniques in the present study to generate a comprehensive overview on a proteome-wide scale of intracytoplasmic membrane (ICM) associated proteins in order to investigate the native supramolecular organization of Rhodobacter sphaeroides R26.1 photosynthetic apparatus. The results obtained were compared with past proteomic data, as well as with models for the topology of photosynthetic membranes as derived from previously published atomic force microscopy studies. We identified 52 proteins organized in 10 different multiprotein complexes. We were able to demonstrate the existence of different oligomeric states for the integral membrane pigment-protein complexes dedicated to bacterial photosynthesis. Specifically, we found dimers and trimers, as well as supercomplexes of light-harvesting (LH) 2 at very high molecular weights (around 10,000 kDa). We recovered the monomeric form of the photochemical reaction center (RC), as well as the monomer and dimer of the reaction center-light harvesting 1-PufX (RC-LH1-PufX) complex. Curiously, no type of LH1 complex was detected. Lastly, ATP synthase and cytochrome bc(1) complexes were only recovered in their monomeric states. Purified ICM vesicles were shown to be rich in newly discovered gene products, including three proteins with unknown functions (RSP_2125, RSP_3238, RSP_6207), a possible alkane hydroxylase and a spheroidene monooxygenase. Other multiprotein complexes were found to be localized in the ICM, including succinate dehydrogenase in trimeric form and sarcosine oxidase in two different aggregation states. These findings contribute to the growing body of evidence that the bacterial ICM is a specialized bioenergetic membrane hosting, not only photosynthesis, but many other critical activities.

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.

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.

Effect of Cobalt Ions on the Soluble Proteome of a Rhodobacter sphaeroides Carotenoidless Mutant

Rhodobacter sphaeroides strain R26.1 showed tolerance to Co2+ ions, up to 10 mM concentration. Interestingly the bacteriochlorophyll biosynthesis was found to decrease upon addition of such metal in the growth medium. Analysis of R. sphaeroides proteome from cells grown in control and in Co2+ enriched media was performed by two dimensional electrophoresis (2DE) followed by mass spectrometry. Proteome and functional analyses of differentially expressed proteins in cobalt response clearly highlighted the involvement of several metabolic pathways, and in particular of some enzymes involved in tetrapyrrole biosynthesis pathway.

Rhodobacter sphaeroides adaptation to high concentrations of cobalt ions requires energetic metabolism changes

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.

Assessment of an internal reference gene in Rhodobacter sphaeroides grown under cobalt exposure

Aim of this study is the identification of an appropriate internal reference gene to quantify gene transcripts isolated from Rhodobacter (R.) sphaeroides cells grown in presence of high concentrations of cobalt ions. RNA was isolated using a commercial kit protocol ad‐hoc modified. Several primer pairs were used to perform reverse transcription PCR and real‐time PCR to assess the suitable internal reference gene whose expression is not affected by cobalt ions, identified with the gene rsp0154.

Magnesium chemical rescue to cobalt-poisoned cells from Rhodobacter sphaeroides

Rhodobacter sphaeroides is able to tolerate high cobaltous ion concentrations, notwithstanding the detrimental effects on growth parameters and bacteriochlorophyll content (Giotta et al. 2006). In order to study the influence of magnesium concentration on cobalt toxiCity, growth experiments were performed with variable magnesium and cobalt concentrations. At high cobalt concentration the increase of Mg2+ in the growth medium results in a significant increase in growth rate and population size reached at the stationary phase, contrasting cobalt toxic effect. Moreover cobalt-exposed bacteria showed a reduced Mg content with respect to control cells. These results demonstrate the existence of an interrelationship in the metabolism of magnesium and cobalt.

Transcriptomic analysis of nickel exposure in Sphingobium sp. ba1 cells using RNA-seq

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.