Synthesis of salt-stable fluorescent nanoparticles (quantum dots) by polyextremophile halophilic bacteria.

Here we report the biological synthesis of CdS fluorescent nanoparticles (Quantum Dots, QDs) by polyextremophile halophilic bacteria isolated from Atacama Salt Flat (Chile), Uyuni Salt Flat (Bolivia) and the Dead Sea (Israel). In particular, a Halobacillus sp. DS2, a strain presenting high resistance to NaCl (3-22%), acidic pH (1-4) and cadmium (CdCl2 MIC: 1,375 mM) was used for QDs biosynthesis studies. Halobacillus sp. synthesize CdS QDs in presence of high NaCl concentrations in a process related with their capacity to generate S2- in these conditions. Biosynthesized QDs were purified, characterized and their stability at different NaCl concentrations determined. Hexagonal nanoparticles with highly defined structures (hexagonal phase), monodisperse size distribution (2-5 nm) and composed by CdS, NaCl and cysteine were determined by TEM, EDX, HRXPS and FTIR. In addition, QDs biosynthesized by Halobacillus sp. DS2 displayed increased tolerance to NaCl when compared to QDs produced chemically or biosynthesized by non-halophilic bacteria. This is the first report of biological synthesis of salt-stable QDs and confirms the potential of using extremophile microorganisms to produce novel nanoparticles. Obtained results constitute a new alternative to improve QDs properties, and as consequence, to increase their industrial and biomedical applications.

"Use of acidophilic bacteria of the genus Acidithiobacillus to biosynthesize CdS fluorescent nanoparticles (quantum dots) with high tolerance to acidic pH".

The use of bacterial cells to produce fluorescent semiconductor nanoparticles (quantum dots, QDs) represents a green alternative with promising economic potential. In the present work, we report for the first time the biosynthesis of CdS QDs by acidophilic bacteria of the Acidithiobacillus genus. CdS QDs were obtained by exposing A. ferrooxidans, A. thiooxidans and A. caldus cells to sublethal Cd2+ concentrations in the presence of cysteine and glutathione. The fluorescence of cadmium-exposed cells moves from green to red with incubation time, a characteristic property of QDs associated with nanocrystals growth. Biosynthesized nanoparticles (NPs) display an absorption peak at 360nm and a broad emission spectra between 450 and 650nm when excited at 370nm, both characteristic of CdS QDs. Average sizes of 6 and 10nm were determined for green and red NPs, respectively. The importance of cysteine and glutathione on QDs biosynthesis in Acidithiobacillus was related with the generation of H2S. Interestingly, QDs produced by acidophilic bacteria display high tolerance to acidic pH. Absorbance and fluorescence properties of QDs was not affected at pH 2.0, a condition that totally inhibits the fluorescence of QDs produced chemically or biosynthesized by mesophilic bacteria (stable until pH 4.5-5.0). Results presented here constitute the first report of the generation of QDs with improved properties by using extremophile microorganisms.

Low-temperature biosynthesis of fluorescent semiconductor nanoparticles (CdS) by oxidative stress resistant Antarctic bacteria.

Bacterial biosynthesis of nanoparticles represents a green alternative for the production of nanostructures with novel properties. Recently, the importance of antioxidant molecules on the biosynthesis of semiconductor fluorescent nanoparticles (quantum dots, QDs) by mesophilic bacteria was reported. The objective of this work was the isolation of psychrotolerant, oxidative stress-resistant bacteria from Antarctica to determine their ability for biosynthesizing CdS QDs at low temperatures. QDs biosynthesis at 15 °C was evaluated by determining their spectroscopic properties after exposing oxidative-stress resistant isolates identified as Pseudomonas spp. to Cd(2+) salts. To characterize the QDs biosynthetic process, the effect of metal exposure on bacterial fluorescence was determined at different times. Time-dependent changes in fluorescence color (green to red), characteristic of QDs, were observed. Electron microscopy analysis of fluorescent cells revealed that biosynthesized nanometric structures localize at the cell periphery. QDs were purified from the bacterial isolates and their fluorescence properties were characterized. Emission spectra displayed classical CdS peaks when excited with UV light. Thiol content, peroxidase activity, lipopolysaccharide synthesis, metabolic profiles and sulfide generation were determined in QDs-producing isolates. No relationship between QDs production and cellular thiol content or peroxidase activity was found. However, sulfide production enhanced CdS QDs biosynthesis. In this work, the use of Antarctic psychrotolerant Pseudomonas spp. for QDs biosynthesis at low temperature is reported for the first time.

Salmonella enterica serovar Typhimurium BaeSR two-component system positively regulates sodA in response to ciprofloxacin.

In response to antibiotics, bacteria activate regulatory systems that control the expression of genes that participate in detoxifying these compounds, like multidrug efflux systems. We previously demonstrated that the BaeSR two-component system from Salmonella enterica serovar Typhimurium (S. Typhimurium) participates in the detection of ciprofloxacin, a bactericidal antibiotic, and in the positive regulation of mdtA, an efflux pump implicated in antibiotic resistance. In the present work, we provide further evidence for a role of the S. Typhimurium BaeSR two-component system in response to ciprofloxacin treatment and show that it regulates sodA expression. We demonstrate that, in the absence of BaeSR, the transcript levels of sodA and the activity of its gene product are lower. Using electrophoretic mobility shift assays and transcriptional fusions, we demonstrate that BaeR regulates sodA by a direct interaction with the promoter region.

ompW is cooperatively upregulated by MarA and SoxS in response to menadione.

OmpW is a minor porin whose biological function has not been clearly defined. Evidence obtained in our laboratory indicates that in Salmonella enterica serovar Typhimurium the expression of OmpW is activated by SoxS upon exposure to paraquat and it is required for resistance. SoxS belongs to the AraC family of transcriptional regulators, like MarA and Rob. Due to their high structural similarity, the genes under their control have been grouped in the mar/sox/rob regulon, which presents a DNA-binding consensus sequence denominated the marsox box. In this work, we evaluated the role of the transcription factors MarA, SoxS and Rob of S. enterica serovar Typhimurium in regulating ompW expression in response to menadione. We determined the transcript and protein levels of OmpW in different genetic backgrounds; in the wild-type and Δrob strains ompW was upregulated in response to menadione, while in the ΔmarA and ΔsoxS strains the induction was abolished. In a double marA soxS mutant, ompW transcript levels were lowered after exposure to menadione, and only complementation in trans with both genes restored the positive regulation. Using transcriptional fusions and electrophoretic mobility shift assays with mutant versions of the promoter region we demonstrated that two of the predicted sites were functional. Additionally, we demonstrated that MarA increases the affinity of SoxS for the ompW promoter region. In conclusion, our study shows that ompW is upregulated in response to menadione in a cooperative manner by MarA and SoxS through a direct interaction with the promoter region.

Differential expression of the transcription factors MarA, Rob, and SoxS of Salmonella Typhimurium in response to sodium hypochlorite: down-regulation of rob by MarA and SoxS.

To survive, Salmonella enterica serovar Typhimurium (S. Typhimurium) must sense signals found in phagocytic cells and modulate gene expression. In the present work, we evaluated the expression and cross-regulation of the transcription factors MarA, Rob, and SoxS in response to NaOCl. We generated strains ΔsoxS and ΔmarA, which were 20 times more sensitive to NaOCl as compared to the wild-type strain; while Δrob only 5 times. Subsequently, we determined that marA and soxS transcript and protein levels were increased while those of rob decreased in a wild-type strain treated with NaOCl. To assess if changes in S. Typhimurium after exposure to NaOCl were due to a cross-regulation, as in Escherichia coli, we evaluated the expression of marA, soxS, and rob in the different genetic backgrounds. The positive regulation observed in the wild-type strain of marA and soxS was retained in the Δrob strain. As in the wild-type strain, rob was down-regulated in the ΔmarA and ΔsoxS treated with NaOCl; however, this effect was decreased. Since rob was down-regulated by both factors, we generated a ΔmarA ΔsoxS strain finding that the negative regulation was abolished, confirming our hypothesis. Electrophoretic mobility shift assays using MarA and SoxS confirmed an interaction with the promoter of rob.

Characterization of the BaeSR two-component system from Salmonella Typhimurium and its role in ciprofloxacin-induced mdtA expression.

Two-component systems are one of the most prevalent mechanisms by which bacteria sense, respond and adapt to changes in their environment. The activation of a sensor histidine kinase leads to autophosphorylation of a conserved histidine residue followed by transfer of the phosphoryl group to a cognate response regulator in an aspartate residue. The search for antibiotics that inhibit molecular targets has led to study prokaryotic two-component systems. In this study, we characterized in vitro and in vivo the BaeSR two-component system from Salmonella Typhimurium and evaluated its role in mdtA regulation in response to ciprofloxacin treatment. We demonstrated in vitro that residue histidine 250 is essential for BaeS autophosphorylation and aspartic acid 61 for BaeR transphosphorylation. By real-time PCR, we showed that mdtA activation in the presence of ciprofloxacin depends on both members of this system and that histidine 250 of BaeS and aspartic acid 61 of BaeR are needed for this. Moreover, the mdtA expression is directly regulated by binding of BaeR at the promoter region, and this interaction is enhanced when the protein is phosphorylated. In agreement, a BaeR mutant unable to phosphorylate at aspartic acid 61 presents a lower affinity with the mdtA promoter.

SoxS regulates the expression of the Salmonella enterica serovar Typhimurium ompW gene.

OmpW of Salmonella enterica serovar Typhimurium has been described as a minor porin involved in osmoregulation, and is also affected by environmental conditions. Biochemical and genetic evidence from our laboratory indicates that OmpW is involved in efflux of and resistance towards paraquat (PQ), and its expression has been shown to be activated in response to oxidative stress. In this study we have explored ompW expression in response to PQ. Primer extension and transcriptional fusions showed that its expression was induced in the presence of PQ. In silico analyses suggested a putative binding site for the SoxS transcriptional factor at the ompW regulatory region. Electrophoretic mobility shift assays (EMSAs) and footprinting experiments showed that SoxS binds at a region that starts close to -54 and ends at about -197 upstream of the transcription start site. Transcriptional fusions support the relevance of this region in ompW activation. The SoxS site is in the forward orientation and its location suggests that the ompW gene has a class I SoxS-dependent promoter.