Intracellular Proteomic Analysis of Streptomyces sp. MC1 When Exposed to Cr(VI) by Gel-Based and Gel-Free Methods.

The actinobacterium Streptomyces sp. MC1 has previously shown the capacity to resist and remove Cr(VI) from liquid culture media. The aim of this work is to analyze the differential expression pattern of intracellular proteins when Streptomyces sp. MC1 is exposed to Cr(VI) in order to explain the molecular mechanisms of resistance that this microorganism possesses. For this purpose, 2D-PAGE and shotgun proteomic analyses (2D-nanoUPLC-ESI-MS/MS) were applied. The presence of Cr(VI) induced the expression of proteins involved in molecular biosynthesis and energy generation, chaperones with a key role in the repair of misfolded proteins and stress response, transcription proteins, proteins of importance in the DNA supercoiling, repair and replication, and dehydrogenases involved in oxidation-reduction processes. These dehydrogenases can be associated with the reduction of Cr(VI) to Cr(III). The results of this study show that proteins from the groups mentioned before are important to face the stress caused by the Cr(VI) presence and help the microorganism to counteract the toxicity of the metal. The use of two proteomic approaches resulted in a larger number of peptides identified, which is also transduced in a significant number of protein ID. This decreased the potential complexity of the sample because of the protein dynamic range, as well as increased the recovery of peptides from the gel after digestion.

Biogenic nanoparticles: Synthesis, stability and biocompatibility mediated by proteins of Pseudomonas aeruginosa.

The development of environmental friendly new procedures for the synthesis of metallic nanoparticles is one of the main objectives of nanotechnology. Plants, algae, fungi and bacteria for the production of nanomaterials are viable alternatives due to their low cost, the absence of toxic waste production and their highly energy efficiency. It is also known that biosynthesized silver nanoparticles (AgNPs) show higher biocompatibility compared to the chemically-synthesized ones. In previous results, biosynthesized AgNPs were obtained from the supernatant of Pseudomonas aeruginosa, and they showed a bigger antimicrobial activity against different bacterial species compared to the chemically-synthesized ones. The aim of this work was to analyze the capping of biosynthesized AgNPs using techniques such as transmission electron microscopy (TEM), infrared spectroscopy (IR), and protein identification through mass spectrometry (MS) in order to identify the compounds responsible for their formation, stability and biocompatibility. The TEM images showed that AgNPs were surrounded by an irregular coverage. The IR spectrum showed that this coverage was composed of carbohydrates and/or proteins. Different proteins were identified in the capping associated to biosynthesized AgNPs. Some proteins seem to be important for their formation (Alkyl hydroperoxide reductase and Azurin) and stabilization (Outer membrane protein OprG and Glycine zipper 2 T M domain-containing protein). The proteins identified with the capability to interact with some biomolecules can be responsible for the biocompatibility and may be responsible for the bigger antimicrobial activity than AgNPs have previously shown. These results are pioneers in the identification of proteins in the capping of biosynthesized AgNPs.

Prokaryotic and eukaryotic community structure affected by the presence of an acid mine drainage from an abandoned gold mine.

The acid mine drainage that originates in the abandoned gold mine in San Luis, Argentina, is released into La Carolina stream. The aim of this study was to determine the influence of this mine drainage on the physicochemical parameters of the area studied and on both prokaryotic and eukaryotic community structure. In addition, specific relationships between microbial taxonomic groups and physicochemical parameters were established. The drainage that flows into La Carolina stream acidifies the stream and increases its sulfate, Zn, Cd and Te concentrations. Microbial analysis showed that prokaryotic community structure is mainly affected by pH values. Actinobacteria and Gammaproteobacteria were abundant in samples characterized by low pH values, while Nitrospirae, Chloroflexi, Deltaproteobacteria, Thaumarchaeota and Euryarchaeota were associated with high concentrations of heavy metals. Otherwise, Alphaproteobacteria was present in samples taken in sunlit areas. Regarding eukaryotic community structure, the sunlight had the greatest impact. Inside the mine, in the absence of light, fungi and protists members were the most abundant microorganisms, while those samples taken in the presence of light displayed algae (green algae and diatoms) as the most abundant ones. After receiving the mine drainage, the stream showed a decrease in the diatom abundance and green algae predominated.

Raw sugarcane bagasse as carbon source for xylanase production by Paenibacillus species: a potential degrader of agricultural wastes.

Paenibacillus species isolated from a variety of natural sources have shown to be important glycoside hydrolases producers. These enzymes play a key role in bio-refining applications, as they are central biocatalysts for the processing of different types of polymers from vegetal biomass. Xylanase production by three native isolates belonging to the genus Paenibacillus was approached by utilizing mineral-based medium and agricultural by-products as a convenient source to produce biocatalysts suitable for their degradation. While varieties of alkali pretreated sugarcane bagasse were useful substrates for the strains from Paenibacillus genus evaluated, raw sugarcane bagasse was the most effective substrate for endoxylanase production by Paenibacillus sp. AR247. This strain was then selected to further improvement of its enzyme production by means of a two-step statistical approach. It was determined that the carbon source, provided as an inexpensive agro-waste, as well as phosphate and magnesium were the culture media components that most influenced the enzyme production, which was improved three times compared to the screening results.

Cu(II) removal by Rhodotorula mucilaginosa RCL-11 in sequential batch cultures.

The present study explored the ability of the yeast Rhodotorula mucilaginosa RCL-11 to adapt to increasing Cu(II) concentrations, measuring oxidative stress through superoxide dismutase and catalase activity in two parallel sequential batch assays. One assay was performed in Erlenmeyer flasks without aeration and a second in a fermentor in which the dissolved oxygen was maintained at 30% saturation. Both assays were carried out by increasing Cu(II) concentrations in five sequential steps: 0; 0.1; 0.2; 0.5 and 1 mM. Each assay was incubated at 30 degrees C, 250 rpm and pH 5.5. While growth parameters of R. mucilaginosa RCL-11 decreased 90-95% with increasing Cu(II) concentration in the culture medium, the oxidative stress level increased from 30 to 55% in both assays. Cells grown under controlled oxygen conditions showed 30% more copper bioaccumulation and 10% glucose consumption when compared with cells grown without aeration. SOD activity was higher under controlled than without aeration, whereas CAT activity was similar under both test conditions. Cu(II) bioaccumulation by R. mucilaginosa RCL-11 and a possible increase in this capacity by adaptation of the strain under controlled aeration represent a potential valuable tool for treatment of effluents or water bioremediation with high copper contents.

Responses of Candida fukuyamaensis RCL-3 and Rhodotorula mucilaginosa RCL-11 to copper stress.

The effect of high Cu(II) concentrations on superoxide dismutase (SOD) and catalase (CAT) activity in Candida fukuyamaensis RCL-3 and Rhodotorula mucilaginosa RCL-11, previously isolated from a copper filter at a mine plant in Argentina, was studied. Addition of 0.1, 0.2 and 0.5 mM Cu(II) to the culture medium increased total SOD and CAT activity in both strains. Native polyacrylamide gel electrophoresis revealed two bands with SOD activity for C. fukuyamaensis RCL-3 and only one for R. mucilaginosa RCL-11; the three bands corresponded to MnSOD.Intracellular accumulation of copper and morphological changes was observed using electron microscopy. Dark bodies examined with transmission electron microscopy (TEM) after 48 h of incubation probably corresponded to copper deposits. The number of dark bodies in R. mucilaginosa RCL-11 grew with increasing incubation time, whereas in C. fukuyamaensis RCL-3 the amount decreased. Scanning electron micrographs (SEM) of C. fukuyamaensis RCL-3 did not reveal any differences compared with the control, but R. mucilaginosa RCL-11 cells were bigger than control ones. TEM confirmed absence of compartmentalization mechanisms in Cu(II) detoxification since electron-dense bodies were mainly found in the cytoplasm.

Chromate removal by yeasts isolated from sediments of a tanning factory and a mine site in Argentina.

Twenty-one yeast-like microorganisms were isolated from tannery effluents and from a nickel-copper mine in Argentina. They were tested for their Cu(II), Ni(II), Cd(II) and Cr(VI) tolerance in qualitative assays on solid medium. Three isolates were selected for their multiple tolerance to the different heavy metals and highest tolerance to Cr(VI). According to morphological and physiological analysis and 26S rDNA D1/D2 domain sequences the isolates were characterized as: Lecythophora sp. NGV-1, Candida sp. NGV-9 and Aureobasidium pullulans VR-8. Resistance of the three strains to high Cr(VI) concentrations and their ability to remove Cr(VI) were assessed using YNB-glucose medium supplemented with 0.5 and 1 mM Cr(VI). Chromate removal activity was estimated by measuring remaining Cr(VI) concentration in the supernatant using the colorimetric 1,5-diphenylcarbazide method and total chromium was determined by flame atomic absorption spectroscopy. The results indicate that the initial Cr(VI) concentration negatively influenced growth and the specific growth rate but stimulated the metabolic activity of the three strains; resistance to Cr(VI) by these strains was mainly due to reduction of Cr(VI) rather than chromium bioaccumulation. This study showed the potential ability of these strains as tools for bioremediation of Cr(VI) from contaminated sites.

Copper tolerant yeasts isolated from polluted area of Argentina.

Eleven yeasts were isolated from wastewater sediment samples collected from a copper filter mine plant, located in the province of Tucumán, Argentina, and tested for their heavy metal tolerance. Two isolates were selected based on their multiple tolerance to different heavy metals and their copper biosorption capacity was studied. Analysis of the 26S rDNA D1/D2 domain sequences indicates that isolate RCL-3 showed similarity with Candida sp. and RCL-11 with Rhodotorula mucilaginosa . Growth performance and copper toxicity of both yeasts were evaluated using YNB-glucose medium supplemented with 0.1, 0.2, 0.5 and 1 mM of Cu2+ solutions. Candida sp. RCL-3 was able to grow up to 7 mg ml(-1) biomass in the presence of either 0.1 or 0.2 mM Cu2+, and at 0.5 mM Cu2+ growth reached 5.5 mg ml(-1). R. mucilaginosa RCL-11 reached 8 mg ml(-1) in the presence of 0.1 mM Cu2+, and values of 6.5 and 5.5 mg ml(-1) biomass were obtained at 0.2 and 0.5 mM Cu2+, respectively. Copper accumulation profiles were different: the metal was librated from the intact cells by Candida sp. whereas R. mucilaginosa did not show release from the cells indicating intracellular storage. Specific biosorption of copper by both isolated yeasts showed increase with the initial copper supplied with the medium (up to 11.5 and 8.0 mg Cu g(-1) biomass for Candida sp. and R. mucilaginosa , respectively). However, specific biosorption decreased with time.