Structural and electrochemical characterization of lawsone-dependent production of tellurium-metal nanoprecipitates by photosynthetic cells of Rhodobacter capsulatus.

Cells of the facultative photosynthetic bacterium Rhodobacter capsulatus exploit the simultaneous presence in the cultural medium of the toxic oxyanion tellurite (TeO32-) and the redox mediator lawsone (2-hydroxy-1,4-naphthoquinone) by reducing tellurite to metal Te0 nanoprecipitates (TeNPs) outside the cells. Here we have studied the mechanism by which lawsone interacts with metabolically active cells and analysed both structure and composition of the TeNPs collected from the growth medium of phototrophycally grown R. capsulatus. High Resolution Transmission Electron Microscopy (HR-TEM) images and Energy-Dispersive X-ray (EDX) microanalysis of TeNPs showed a central core of polycrystalline tellurium interspersed in an organic matrix with a predominant protein-based composition. The main proteins from Te0 nanostructures were identified by Liquid Chromatography tandem-Mass Spectrometry and were all correlated with the cell outer membrane composition. The interaction of reduced lawsone with tellurite and with the bacterial cells was probed by Cyclic Voltammetry and Scanning ElectroChemical Microscopy (SECM). We concluded that lawsone is required for the reduction of tellurite to metal Te0 in a reaction mechanism dependent on reducing equivalents deriving from the cell photosynthetic metabolism. SECM experiments demonstrate that lawsone, by diffusing inside the bacterial cells, is effectively available at the membrane site of the photosynthetic electron transport chain.

Reprint of "Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus".

The toxic oxyanion tellurite (TeO32-) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te0 in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te0 nanoprecipitates (TeNPs) outside the cells upon addition of the redox mediator lawsone (2-hydroxy-1,4-naphtoquinone). TeNPs generation kinetics were monitored to define the optimal conditions to produce TeNPs as a function of various carbon sources and lawsone concentration. We report that growing cultures over a 10 days period with daily additions of 1mM tellurite led to the accumulation in the growth medium of TeNPs with dimensions from 200 up to 600-700nm in length as determined by atomic force microscopy (AFM). This result suggests that nucleation of TeNPs takes place over the entire cell growth period although the addition of new tellurium Te0 to pre-formed TeNPs is the main strategy used by R. capsulatus to generate TeNPs outside the cells. Finally, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) analysis of TeNPs indicate they are coated with an organic material which keeps the particles in solution in aqueous solvents.

On the role of a specific insert in acetate permeases (ActP) for tellurite uptake in bacteria: Functional and structural studies.

The oxyanion tellurite (TeO32-) is extremely toxic to bacterial cells. In Rhodobacter capsulatus, tellurite enters the cytosol by means of the high uptake-rate acetate permease RcActP2, encoded by one of the three actP genes present in this species (actP1, actP2 and actP3). Conversely, in Escherichia coli a low rate influx of the oxyanion is measured, which depends mainly on the phosphate transporter EcPitA, even though E. coli contains its own EcActP acetate permease. Here we report that when the actP2 gene from R. capsulatus is expressed in wild-type E. coli HB101 and in E. coli JW3460 ΔpitA mutant, the cellular intake of tellurite increases up to four times, suggesting intrinsic structural differences between EcActP and RcActP2. Indeed, a sequence analysis indicated the presence in RcActP2 of an insert of 15-16 residues, located between trans-membrane (TM) helices 6 and 7, which is absent in both EcActP and RcActP1. Based on this observation, the molecular models of homodimeric RcActP1 and RcActP2 were calculated and analyzed. In the RcActP2 model, the insert induces a perturbation in the conformation of the loop between TM helices 6 and 7, located at the RcActP2 dimerization interface. This perturbation opens a cavity on the periplasmic side that is closed, instead, in the RcActP1 model. This cavity also features an increase of the positive electric potential on the protein surface, an effect ascribed to specific residues Lys261, Lys281 and Arg560. We propose that this positively charged patch in RcActP2 is involved in recognition and translocation of the TeO32- anion, attributing to RcActP2 a greater ability as compared to RcActP1 to transport this inorganic poison inside the cells.

Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus.

The toxic oxyanion tellurite (TeO3(2-)) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te(0) in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te(0) nanoprecipitates (TeNPs) outside the cells upon addition of the redox mediator lawsone (2-hydroxy-1,4-naphtoquinone). TeNPs generation kinetics were monitored to define the optimal conditions to produce TeNPs as a function of various carbon sources and lawsone concentration. We report that growing cultures over a 10 days period with daily additions of 1mM tellurite led to the accumulation in the growth medium of TeNPs with dimensions from 200 up to 600-700 nm in length as determined by atomic force microscopy (AFM). This result suggests that nucleation of TeNPs takes place over the entire cell growth period although the addition of new tellurium Te(0) to pre-formed TeNPs is the main strategy used by R. capsulatus to generate TeNPs outside the cells. Finally, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) analysis of TeNPs indicate they are coated with an organic material which keeps the particles in solution in aqueous solvents.

Reduction of chalcogen oxyanions and generation of nanoprecipitates by the photosynthetic bacterium Rhodobacter capsulatus.

The facultative photosynthetic bacterium Rhodobacter capsulatus is characterized in its interaction with the toxic oxyanions tellurite (Te(IV)) and selenite (Se(IV)) by a highly variable level of resistance that is dependent on the growth mode making this bacterium an ideal organism for the study of the microbial interaction with chalcogens. As we have reported in the past, while the oxyanion tellurite is taken up by R. capsulatus cells via acetate permease and it is reduced to Te(0) in the cytoplasm in the form of splinter-like black intracellular deposits no clear mechanism was described for Se(0) precipitation. Here, we present the first report on the biotransformation of tellurium and selenium oxyanions into extracellular Te(0) and Se(0)nanoprecipitates (NPs) by anaerobic photosynthetically growing cultures of R. capsulatus as a function of exogenously added redox-mediator lawsone, i.e. 2-hydroxy-1,4-naphthoquinone. The NPs formation was dependent on the carbon source used for the bacterial growth and the rate of chalcogen reduction was constant at different lawsone concentrations, in line with a catalytic role for the redox mediator. X-ray diffraction (XRD) analysis demonstrated the Te(0) and Se(0) nature of the nanoparticles.

Microbial processing of tellurium as a tool in biotechnology.

Here, we overview the most recent advances in understanding the bacterial mechanisms that stay behind the reduction of tellurium oxyanions in both planktonic cells and biofilms. This is a topic of interest for basic and applied research because microorganisms are deeply involved in the transformation of metals and metalloids in the environment. In particular, the recent observation that toxic tellurite can be precipitated either inside or outside the cells being used as electron sink to support bacterial growth, opens new perspectives for both microbial physiologists and biotechnologists. As promising nanomaterials, tellurium based nanoparticles show unique electronic and optical properties due to quantum confinement effects to be used in the area of chemistry, electronics, medicine and environmental biotechnologies.

Fructose increases the resistance of Rhodobacter capsulatus to the toxic oxyanion tellurite through repression of acetate permease (ActP).

The highly toxic oxyanion tellurite (TeO(3) (2-)) enters the cells of the facultative photosynthetic bacterium Rhodobacter capsulatus through an acetate permease. Here we show that actP gene expression is down-regulated by fructose and this in turn determines a strong decrease of tellurite uptake and a parallel increase in the cells resistance to the toxic metalloid (from a minimal inhibitory concentration of 8 µM up to 400 µM tellurite under aerobic growth conditions). This demonstrates that there exists a direct connection between the level of tellurite uptake and the sensitivity of the cells to the oxyanion.

Acetate permease (ActP) Is responsible for tellurite (TeO32-) uptake and resistance in cells of the facultative phototroph Rhodobacter capsulatus.

The highly toxic oxyanion tellurite has to enter the cytoplasm of microbial cells in order to fully express its toxicity. Here we show that in the phototroph Rhodobacter capsulatus, tellurite exploits acetate permease (ActP) to get into the cytoplasm and that the levels of resistance and uptake are linked.

The highly toxic oxyanion tellurite (TeO (3) (2-) ) enters the phototrophic bacterium Rhodobacter capsulatus via an as yet uncharacterized monocarboxylate transport system.

The facultative phototroph Rhodobacter capsulatus takes up the highly toxic oxyanion tellurite when grown under both photosynthetic and respiratory growth conditions. Previous works on Escherichia coli and R. capsulatus suggested that tellurite uptake occurred through a phosphate transporter. Here we present evidences indicating that tellurite enters R. capsulatus cells via a monocarboxylate transport system. Indeed, intracellular accumulation of tellurite was inhibited by the addition of monocarboxylates such as pyruvate, lactate and acetate, but not by dicarboxylates like malate or succinate. Acetate was the strongest tellurite uptake antagonist and this effect was concentration dependent, being already evident at 1 microM acetate. Conversely, tellurite at 100 microM was able to restrict the acetate entry into the cells. Both tellurite and acetate uptakes were energy dependent processes, since they were abolished by the protonophore FCCP and by the respiratory electron transport inhibitor KCN. Interestingly, cells grown on acetate, lactate or pyruvate showed a high level resistance to tellurite, whereas cells grown on malate or succinate proved to be very sensitive to the oxyanion. Taking these data together, we propose that: (a) tellurite enters R. capsulatus cells via an as yet uncharacterized monocarboxylate(s) transporter, (b) competition between acetate and tellurite results in a much higher level of tolerance against the oxyanion and (c) the toxic action of tellurite at the cytosolic level is significantly restricted by preventing tellurite uptake.

Tellurite effects on Rhodobacter capsulatus cell viability and superoxide dismutase activity under oxidative stress conditions.

Cells of the facultative photosynthetic bacterium Rhodobacter capsulatus (MT1131 strain) incubated with 10 microg ml-1 of the toxic oxyanion tellurite (TeO2-(3)) exhibited an increase in superoxide dismutase activity. The latter effect was also seen upon incubation with sublethal amounts of paraquat, a cytosolic generator of superoxide anions (O2-), in parallel with a strong increase in tellurite resistance (TeR). A mutant strain (CW10) deficient in SenC, a protein with similarities to peroxiredoxin/thiol:disulfide oxidoreductases and a homologue of mitochondrial Sco proteins, was constructed by interposon mutagenesis via the gene transfer agent system. Notably, the absence of SenC affected R. capsulatus resistance to periplasmic O2- generated by xanthine/xanthine oxidase but not to cytosolic O2- produced by paraquat. Further, the absence of SenC did not affect R. capsulatus tellurite resistance. We conclude that: (1) cytosolic-generated O2- enhances TeR of this bacterial species; (2) small amounts of tellurite increase SOD activity so as to mimic the early cell response to oxidative stress; (3) SenC protein is required in protection of R. capsulatus against periplasmic oxidative stress; and finally, (4) SenC protein is not involved in TeR, possibly because tellurite does not generate O-2 at the periplasmic space level.

Effects of the metalloid oxyanion tellurite (TeO32-) on growth characteristics of the phototrophic bacterium Rhodobacter capsulatus.

This work examines the effects of potassium tellurite (K2TeO3) on the cell viability of the facultative phototroph Rhodobacter capsulatus. There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 microg of tellurite (TeO3(2-)) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 microg/ml. The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium. Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (< or =50% viable cells); however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te0 crystallites, recovery of cytoplasmic membrane integrity was apparent (> or =90% viable cells), which was supported by the development of a significant membrane potential (Deltapsi = 120 mV). These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph R. capsulatus might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.