Soft-metal(loid)s induce protein aggregation in Escherichia coli.

Metal(loid) salts were used to treat infectious diseases in the past due to their exceptional biocidal properties at low concentrations. However, the mechanism of their toxicity has yet to be fully elucidated. The production of reactive oxygen species (ROS) has been linked to the toxicity of soft metal(loid)s such as Ag(I), Au(III), As(III), Cd(II), Hg(II), and Te(IV). Nevertheless, few reports have described the direct, or ROS-independent, effects of some of these soft-metal(loid)s on bacteria, including the dismantling of iron-sulfur clusters [4Fe-4S] and the accumulation of porphyrin IX. Here, we used genome-wide genetic, proteomic, and biochemical approaches under anaerobic conditions to evaluate the direct mechanisms of toxicity of these metal(loid)s in Escherichia coli. We found that certain soft-metal(loid)s promote protein aggregation in a ROS-independent manner. This aggregation occurs during translation in the presence of Ag(I), Au(III), Hg(II), or Te(IV) and post-translationally in cells exposed to Cd(II) or As(III). We determined that aggregated proteins were involved in several essential biological processes that could lead to cell death. For instance, several enzymes involved in amino acid biosynthesis were aggregated after soft-metal(loid) exposure, disrupting intracellular amino acid concentration. We also propose a possible mechanism to explain how soft-metal(loid)s act as proteotoxic agents.

Comparative genomics of the proteostasis network in extreme acidophiles.

Extreme acidophiles thrive in harsh environments characterized by acidic pH, high concentrations of dissolved metals and high osmolarity. Most of these microorganisms are chemolithoautotrophs that obtain energy from low redox potential sources, such as the oxidation of ferrous ions. Under these conditions, the mechanisms that maintain homeostasis of proteins (proteostasis), as the main organic components of the cells, are of utmost importance. Thus, the analysis of protein chaperones is critical for understanding how these organisms deal with proteostasis under such environmental conditions. In this work, using a bioinformatics approach, we performed a comparative genomic analysis of the genes encoding classical, periplasmic and stress chaperones, and the protease systems. The analysis included 35 genomes from iron- or sulfur-oxidizing autotrophic, heterotrophic, and mixotrophic acidophilic bacteria. The results showed that classical ATP-dependent chaperones, mostly folding chaperones, are widely distributed, although they are sub-represented in some groups. Acidophilic bacteria showed redundancy of genes coding for the ATP-independent holdase chaperones RidA and Hsp20. In addition, a systematically high redundancy of genes encoding periplasmic chaperones like HtrA and YidC was also detected. In the same way, the proteolytic ATPase complexes ClpPX and Lon presented redundancy and broad distribution. The presence of genes that encoded protein variants was noticeable. In addition, genes for chaperones and protease systems were clustered within the genomes, suggesting common regulation of these activities. Finally, some genes were differentially distributed between bacteria as a function of the autotrophic or heterotrophic character of their metabolism. These results suggest that acidophiles possess an abundant and flexible proteostasis network that protects proteins in organisms living in energy-limiting and extreme environmental conditions. Therefore, our results provide a means for understanding the diversity and significance of proteostasis mechanisms in extreme acidophilic bacteria.

Characterization and genomic analysis of two novel psychrotolerant Acidithiobacillus ferrooxidans strains from polar and subpolar environments.

The bioleaching process is carried out by aerobic acidophilic iron-oxidizing bacteria that are mainly mesophilic or moderately thermophilic. However, many mining sites are located in areas where the mean temperature is lower than the optimal growth temperature of these microorganisms. In this work, we report the obtaining and characterization of two psychrotolerant bioleaching bacterial strains from low-temperature sites that included an abandoned mine site in Chilean Patagonia (PG05) and an acid rock drainage in Marian Cove, King George Island in Antarctic (MC2.2). The PG05 and MC2.2 strains showed significant iron-oxidation activity and grew optimally at 20°C. Genome sequence analyses showed chromosomes of 2.76 and 2.84 Mbp for PG05 and MC2.2, respectively, and an average nucleotide identity estimation indicated that both strains clustered with the acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans. The Patagonian PG05 strain had a high content of genes coding for tolerance to metals such as lead, zinc, and copper. Concordantly, electron microscopy revealed the intracellular presence of polyphosphate-like granules, likely involved in tolerance to metals and other stress conditions. The Antarctic MC2.2 strain showed a high dosage of genes for mercury resistance and low temperature adaptation. This report of cold-adapted cultures of the At. ferrooxidans species opens novel perspectives to satisfy the current challenges of the metal bioleaching industry.

In vitro biosynthesis of Ag, Au and Te-containing nanostructures by Exiguobacterium cell-free extracts.

BACKGROUND: The bacterial genus Exiguobacterium includes several species that inhabit environments with a wide range of temperature, salinity, and pH. This is why the microorganisms from this genus are known generically as polyextremophiles. Several environmental isolates have been explored and characterized for enzyme production as well as for bioremediation purposes. In this line, toxic metal(loid) reduction by these microorganisms represents an approach to decontaminate soluble metal ions via their transformation into less toxic, insoluble derivatives. Microbial-mediated metal(loid) reduction frequently results in the synthesis of nanoscale structures-nanostructures (NS) -. Thus, microorganisms could be used as an ecofriendly way to get NS. RESULTS: We analyzed the tolerance of Exiguobacterium acetylicum MF03, E. aurantiacum MF06, and E. profundum MF08 to Silver (I), gold (III), and tellurium (IV) compounds. Specifically, we explored the ability of cell-free extracts from these bacteria to reduce these toxicants and synthesize NS in vitro, both in the presence or absence of oxygen. All isolates exhibited higher tolerance to these toxicants in anaerobiosis. While in the absence of oxygen they showed high tellurite- and silver-reducing activity at pH 9.0, whereas AuCl4- which was reduced at pH 7.0 in both conditions. Given these results, cell-free extracts were used to synthesize NS containing silver, gold or tellurium, characterizing their size, morphology and chemical composition. Silver and tellurium NS exhibited smaller size under anaerobiosis and their morphology was circular (silver NS), starred (tellurium NS) or amorphous (gold NS). CONCLUSIONS: This nanostructure-synthesizing ability makes these isolates interesting candidates to get NS with biotechnological potential.

Synthesis and Antibacterial Activity of Metal(loid) Nanostructures by Environmental Multi-Metal(loid) Resistant Bacteria and Metal(loid)-Reducing Flavoproteins.

Microbes are suitable candidates to recover and decontaminate different environments from soluble metal ions, either via reduction or precipitation to generate insoluble, non-toxic derivatives. In general, microorganisms reduce toxic metal ions generating nanostructures (NS), which display great applicability in biotechnological processes. Since the molecular bases of bacterial reduction are still unknown, the search for new -environmentally safe and less expensive- methods to synthesize NS have made biological systems attractive candidates. Here, 47 microorganisms isolated from a number of environmental samples were analyzed for their tolerance or sensitivity to 19 metal(loid)s. Ten of them were highly tolerant to some of them and were assessed for their ability to reduce these toxicants in vitro. All isolates were analyzed by 16S rRNA gene sequencing, fatty acids composition, biochemical tests and electron microscopy. Results showed that they belong to the Enterobacter, Staphylococcus, Acinetobacter, and Exiguobacterium genera. Most strains displayed metal(loid)-reducing activity using either NADH or NADPH as cofactor. While Acinetobacter schindleri showed the highest tellurite ( TeO32- ) and tetrachloro aurate ( AuCl4- ) reducing activity, Staphylococcus sciuri and Exiguobacterium acetylicum exhibited selenite ( SeO32- ) and silver (Ag+) reducing activity, respectively. Based on these results, we used these bacteria to synthetize, in vivo and in vitro Te, Se, Au, and Ag-containing nanostructures. On the other hand, we also used purified E. cloacae glutathione reductase to synthesize in vitro Te-, Ag-, and Se-containing NS, whose morphology, size, composition, and chemical composition were evaluated. Finally, we assessed the putative anti-bacterial activity exhibited by the in vitro synthesized NS: Te-containing NS were more effective than Au-NS in inhibiting Escherichia coli and Listeria monocytogenes growth. Aerobically synthesized TeNS using MF09 crude extracts showed MICs of 45- and 66- µg/ml for E. coli and L. monocytogenes, respectively. Similar MIC values (40 and 82 µg/ml, respectively) were observed for TeNS generated using crude extracts from gorA-overexpressing E. coli. In turn, AuNS MICs for E. coli and L. monocytogenes were 64- and 68- µg/ml, respectively.

Comparative genomic analysis of a new tellurite-resistant Psychrobacter strain isolated from the Antarctic Peninsula.

The Psychrobacter genus is a cosmopolitan and diverse group of aerobic, cold-adapted, Gram-negative bacteria exhibiting biotechnological potential for low-temperature applications including bioremediation. Here, we present the draft genome sequence of a bacterium from the Psychrobacter genus isolated from a sediment sample from King George Island, Antarctica (3,490,622 bp; 18 scaffolds; G + C = 42.76%). Using phylogenetic analysis, biochemical properties and scanning electron microscopy the bacterium was identified as Psychrobacter glacincola BNF20, making it the first genome sequence reported for this species. P. glacincola BNF20 showed high tellurite (MIC 2.3 mM) and chromate (MIC 6.0 mM) resistance, respectively. Genome-wide nucleotide identity comparisons revealed that P. glacincola BNF20 is highly similar (>90%) to other uncharacterized Psychrobacter spp. such as JCM18903, JCM18902, and P11F6. Bayesian multi-locus phylogenetic analysis showed that P. glacincola BNF20 belongs to a polyphyletic clade with other bacteria isolated from polar regions. A high number of genes related to metal(loid) resistance were found, including tellurite resistance genetic determinants located in two contigs: Contig LIQB01000002.1 exhibited five ter genes, each showing putative promoter sequences (terACDEZ), whereas contig LIQB1000003.2 showed a variant of the terZ gene. Finally, investigating the presence and taxonomic distribution of ter genes in the NCBI's RefSeq bacterial database (5,398 genomes, as January 2017), revealed that 2,623 (48.59%) genomes showed at least one ter gene. At the family level, most (68.7%) genomes harbored one ter gene and 15.6% exhibited five (including P. glacincola BNF20). Overall, our results highlight the diverse nature (genetic and geographic diversity) of the Psychrobacter genus, provide insights into potential mechanisms of metal resistance, and exemplify the benefits of sampling remote locations for prospecting new molecular determinants.

Reduction of Gold (III) and Tellurium (IV) by Enterobacter cloacae MF01 Results in Nanostructure Formation Both in Aerobic and Anaerobic Conditions.

Microorganism survival in the presence of toxic substances such as metal(loid)s lies chiefly on their ability to resist (or tolerate) such elements through specific resistance mechanisms. Among them, toxicant reduction has attracted the attention of researchers because metal(loid)-reducing bacteria are being used to recover and/or decontaminate polluted sites. Particularly, our interest is to analyze the toxicity of gold and tellurium compounds for the environmental microorganism Enterobacter cloacae MF01 and also to explore the generation of nanostructures to be used in future biotechnological processes. Resistance of E. cloacae MF01 to gold and tellurium salts as well as the putative mechanisms involved -both in aerobic and anaerobic growth conditions- was evaluated. These metal(loid)s were selected because of their potential application in biotechnology. Resistance to auric tetrachloride acid (HAuCl4) and potassium tellurite (K2TeO3) was assessed by determining areas of growth inhibition, minimum inhibitory concentrations, and growth curves as well as by viability tests. E. cloacae MF01 exhibited higher resistance to HAuCl4 and K2TeO3 under aerobic and anaerobic conditions, respectively. In general, their toxicity is mediated by the generation of reactive oxygen species and by a decrease of intracellular reduced thiols (RSH). To assess if resistance implies toxicant reduction, intra- and extra-cellular toxicant-reducing activities were evaluated. While E. cloacae MF01 exhibited intra- and extra-cellular HAuCl4-reducing activity, tellurite reduction was observed only intracellularly. Then, Au- and Te-containing nanostructures (AuNS and TeNS, respectively) were synthesized using crude extracts from E. cloacae MF01 and their size, morphology, and chemical composition was evaluated.

Accumulation of heme biosynthetic intermediates contributes to the antibacterial action of the metalloid tellurite.

The metalloid tellurite is highly toxic to microorganisms. Several mechanisms of action have been proposed, including thiol depletion and generation of hydrogen peroxide and superoxide, but none of them can fully explain its toxicity. Here we use a combination of directed evolution and chemical and biochemical approaches to demonstrate that tellurite inhibits heme biosynthesis, leading to the accumulation of intermediates of this pathway and hydroxyl radical. Unexpectedly, the development of tellurite resistance is accompanied by increased susceptibility to hydrogen peroxide. Furthermore, we show that the heme precursor 5-aminolevulinic acid, which is used as an antimicrobial agent in photodynamic therapy, potentiates tellurite toxicity. Our results define a mechanism of tellurite toxicity and warrant further research on the potential use of the combination of tellurite and 5-aminolevulinic acid in antimicrobial therapy.

Flavoprotein-Mediated Tellurite Reduction: Structural Basis and Applications to the Synthesis of Tellurium-Containing Nanostructures.

The tellurium oxyanion tellurite (TeO3 (2-)) is extremely harmful for most organisms. It has been suggested that a potential bacterial tellurite resistance mechanism would consist of an enzymatic, NAD(P)H-dependent, reduction to the less toxic form elemental tellurium (Te(0)). To date, a number of enzymes such as catalase, type II NADH dehydrogenase and terminal oxidases from the electron transport chain, nitrate reductases, and dihydrolipoamide dehydrogenase (E3), among others, have been shown to display tellurite-reducing activity. This activity is generically referred to as tellurite reductase (TR). Bioinformatic data resting on some of the abovementioned enzymes enabled the identification of common structures involved in tellurite reduction including vicinal catalytic cysteine residues and the FAD/NAD(P)(+)-binding domain, which is characteristic of some flavoproteins. Along this line, thioredoxin reductase (TrxB), alkyl hydroperoxide reductase (AhpF), glutathione reductase (GorA), mercuric reductase (MerA), NADH: flavorubredoxin reductase (NorW), dihydrolipoamide dehydrogenase, and the putative oxidoreductase YkgC from Escherichia coli or environmental bacteria were purified and assessed for TR activity. All of them displayed in vitro TR activity at the expense of NADH or NADPH oxidation. In general, optimal reducing conditions occurred around pH 9-10 and 37°C. Enzymes exhibiting strong TR activity produced Te-containing nanostructures (TeNS). While GorA and AhpF generated TeNS of 75 nm average diameter, E3 and YkgC produced larger structures (>100 nm). Electron-dense structures were observed in cells over-expressing genes encoding TrxB, GorA, and YkgC.

Glutathione reductase-mediated synthesis of tellurium-containing nanostructures exhibiting antibacterial properties.

Tellurium, a metalloid belonging to group 16 of the periodic table, displays very interesting physical and chemical properties and lately has attracted significant attention for its use in nanotechnology. In this context, the use of microorganisms for synthesizing nanostructures emerges as an eco-friendly and exciting approach compared to their chemical synthesis. To generate Te-containing nanostructures, bacteria enzymatically reduce tellurite to elemental tellurium. In this work, using a classic biochemical approach, we looked for a novel tellurite reductase from the Antarctic bacterium Pseudomonas sp. strain BNF22 and used it to generate tellurium-containing nanostructures. A new tellurite reductase was identified as glutathione reductase, which was subsequently overproduced in Escherichia coli. The characterization of this enzyme showed that it is an NADPH-dependent tellurite reductase, with optimum reducing activity at 30°C and pH 9.0. Finally, the enzyme was able to generate Te-containing nanostructures, about 68 nm in size, which exhibit interesting antibacterial properties against E. coli, with no apparent cytotoxicity against eukaryotic cells.

DNA, cell wall and general oxidative damage underlie the tellurite/cefotaxime synergistic effect in Escherichia coli.

The constant emergence of antibiotic multi-resistant pathogens is a concern worldwide. An alternative for bacterial treatment using nM concentrations of tellurite was recently proposed to boost antibiotic-toxicity and a synergistic effect of tellurite/cefotaxime (CTX) was described. In this work, the molecular mechanism underlying this phenomenon is proposed. Global changes of the transcriptional profile of Escherichia coli exposed to tellurite/CTX were determined by DNA microarrays. Induction of a number of stress regulators (as SoxS), genes related to oxidative damage and membrane transporters was observed. Accordingly, increased tellurite adsorption/uptake and oxidative injuries to proteins and DNA were determined in cells exposed to the mixture of toxicants, suggesting that the tellurite-mediated CTX-potentiating effect is dependent, at least in part, on oxidative stress. Thus, the synergistic tellurite-mediated CTX-potentiating effect depends on increased tellurite uptake/adsorption which results in damage to proteins, DNA and probably other macromolecules. Our findings represent a contribution to the current knowledge of bacterial physiology under antibiotic stress and can be of great interest in the development of new antibiotic-potentiating strategies.

Enhancing the antibiotic antibacterial effect by sub lethal tellurite concentrations: tellurite and cefotaxime act synergistically in Escherichia coli.

The emergence of antibiotic-resistant pathogenic bacteria during the last decades has become a public health concern worldwide. Aiming to explore new alternatives to treat antibiotic-resistant bacteria and given that the tellurium oxyanion tellurite is highly toxic for most microorganisms, we evaluated the ability of sub lethal tellurite concentrations to strengthen the effect of several antibiotics. Tellurite, at nM or µM concentrations, increased importantly the toxicity of defined antibacterials. This was observed with both gram negative and gram positive bacteria, irrespective of the antibiotic or tellurite tolerance of the particular microorganism. The tellurite-mediated antibiotic-potentiating effect occurs in laboratory and clinical, uropathogenic Escherichia coli, especially with antibiotics disturbing the cell wall (ampicillin, cefotaxime) or protein synthesis (tetracycline, chloramphenicol, gentamicin). In particular, the effect of tellurite on the activity of the clinically-relevant, third-generation cephalosporin (cefotaxime), was evaluated. Cell viability assays showed that tellurite and cefotaxime act synergistically against E. coli. In conclusion, using tellurite like an adjuvant could be of great help to cope with several multi-resistant pathogens.