Roberto Borghese

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.

Effects of the Metalloid Oxyanion Tellurite (TeO32−) on Growth Characteristics of the Phototrophic Bacterium Rhodobacter capsulatus

ABSTRACT 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 μg of tellurite (TeO32−) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 μg/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 (≤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 (≥90% viable cells), which was supported by the development of a significant membrane potential (Δψ = 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.

Reduction of potassium tellurite to elemental tellurium and its effect on the plasma membrane redox components of the facultative phototroph Rhodobacter capsulatus.

Summary. Anaerobically light-grown cells of Rhodobacter capsulatus B100 are highly resistant to the toxic oxyanion tellurite (TeO32−; minimal inhibitory concentration, 250 μg/ml). This study examines, for the first time, some structural and biochemical features of cells and plasma membrane fragments of this facultative phototroph grown in the presence of 50μg of K2TeO3 per ml. Through the use of transmission microscopy and X-ray microanalysis we show that several “needlelike” shaped granules of elemental tellurium are accumulated into the cytosol near the intracytoplasmic membrane system. Flash-spectroscopy, oxygen consumption measurements, and difference spectra analysis indicated that membrane vesicles (chromatophores) isolated from tellurite-grown cells are able to catalyze both photosynthetic and respiratory electron transport activities, although they are characterized by a low c-type cytochrome content (mostly soluble cytochrome c2). This feature is paralleled by a low cytochrome c oxidase activity and with an NADH-dependent respiration which is catalyzed by a pathway leading to a quinol oxidase (Qox) inhibited by high (millimolar) concentrations of cyanide (CN−). Conversely, membranes from R. capsulatus B100 cells grown in the absence of tellurite are characterized by a branched respiratory chain in which the cytochrome c oxidase pathway (blocked by CN− in the micromolar range) accounts for 35–40% of the total NADH-dependent oxygen consumption, while the remaining activity is catalyzed by the quinol oxidase pathway. These data have been interpreted to show that tellurite resistance of R. capsulatus B100 is characterized by the presence of a modified plasma-membrane-associated electron transport system.

Acetate Permease (ActP) Is Responsible for Tellurite (TeO32−) Uptake and Resistance in Cells of the Facultative Phototroph Rhodobacter capsulatus

ABSTRACT 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.

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.

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.

The atpIBEXF operon coding for the F0 sector of the ATP synthase from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus

Abstract The atpIBEXF operon coding for the F0 sector of the ATP synthase from Rhodobacter capsulatus was cloned and sequenced. The genes for the five subunits were present in the order: atpI (subunit I), atpB (subunit a), atpE (subunit c), atpX (subunit b′), and atpF (subunit b). The transcription initiation site was defined by primer-extension analysis. A duplicated and divergent copy of the b subunit gene (subunit b′) was present. This duplication is found only in photosynthetic prokaryotes and in plant chloroplasts. F0 deletion mutants formed tiny colonies during anaerobic growth in the dark but could not sustain continuous growth. Based on the results of the present work, we conclude that a functioning ATP synthase is essential for normal growth under all conditions tested.

The respiratory chain of the halophilic anoxygenic purple bacterium Rhodospirillum sodomense.

The halophilic purple nonsulfur bacterium Rhodospirillum sodomense has been previously described as an obligate phototroph that requires yeast extract and a limited number of organic compounds for photoheterotrophic growth. In this work, we report on chemoheterotrophic growth of R. sodomense in media containing either acetate or succinate supplemented with 0.3–0.5% yeast extract. Plasma membranes isolated from cells grown aerobically in the dark contained three b-type and three c-type membrane-bound cytochromes with Em,7 of +171 ± 10, +62 ± 10 and –45 ± 13 mV (561–575 nm), and +268 ± 6, +137 ± 10 and –43 ± 12 mV (551–540 nm). A small amount of a soluble c-type cytochrome with a mol. mass of 15 kDa (Em,7≥ +150 mV) was identified. Spectroscopic and immunological methods excluded the presence of cytochrome of the c2 class and high-potential iron-sulfur proteins. Inhibitory studies indicated that only 60–70% of the respiratory activity was blocked by low concentrations of cyanide, antimycin A, and myxothiazol (10, 0.1, and 0.2 μM, respectively). These results were interpreted to show that the oxidative electron transport chain of R. sodomense is branched, leads to a quinol oxidase that is fully blocked by 1 mM cyanide and that is involved in light-dependent oxygen reduction, and leads to a cytochrome c oxidase that is inhibited by 10 μM cyanide. These features taken together suggest that R. sodomense differs from the closely related species Rhodospirillum salinarum and from other species of the genus Rhodospirillum in that it contains multiple membrane-bound cytochromes c.

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.

Respiration in Phototrophic Microorganisms

Phototrophic microorganisms include anoxygenic phototrophs, which are bacteria capable of growing photosynthetically with no oxygen generation, and Cyanobacteria which are oxygenic phototrophs because their photosynthetic apparatus generates oxygen. Several genera of anoxygenic phototrophs are capable of obtaining energy also from aerobic and anaerobic respiration in darkness; conversely, only a few filamentous cyanobacteria can grow in the dark on glucose or other sugars using the organic material as both carbon and energy source. The latter observation suggests that besides the bioenergetic aspect, respiration in cyanobacteria plays other roles such as to control the redox balance or to act as a scavenger for O 2 during nitrogen fixation. Facultative phototrophs (capable of both respiration and photosynthesis) contain a photosynthetic apparatus whose synthesis is repressed by oxygen; an exception to this rule is the group of aerobic-anoxygenic phototrophs, mainly marine microorganisms, requiring the presence of oxygen to synthesize their photosynthetic apparatus.