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The gray mold (Botrytis cinerea; Botrytis) is the main disease affecting grapevines production in Chile. Succinate Dehydrogenase Inhibitors (SDHI) belonging to the carboxamides fungicide family are a key tool for the control of Botrytis in grapevines from Chilean Central Valley. This study aimed to determine the sensitivity of Chilean Botrytis population to the new generation carboxamide pydiflumetofen. Conidial germination (CG) and germ-tube elongation (GTE) sensitivity assays were conducted on 200 single-spore isolates collected during the 2016-2017 season. The mean effective concentration that inhibited 50% (EC50) of CG in the Botrytis population was 0.0545 µg/mL, with mean values of 0.066 µg/mL and 0.042 µg/mL, for table and wine grapes, respectively. The mean EC50 value of GTE was 0.000245 µg/mL, 0.0003 µg/mL, and 0.0019 µg/mL for the total, table grape, and wine grape populations, respectively. The comparison between pydiflumetofen and fludioxonil, a highly-efficient fungicide carrying a different mode of action, showed the 87.5% and 97.5% of Botrytis control with an EC50 threshold of 0.1 µg/mL, in table grape, and wine grape populations, respectively. No cross-resistance between pydiflumetofen and fludioxonil was detected. For nine isolates with reduced pydiflumetofen sensitivity, we evaluated SdhB mutations using a qPCR-HRM diagnostic system. Two isolates carried the sdhBP225/H272R genotype and two the sdhBP225/H272Y. Additional analysis of SdhB mutant isolates determined that pydiflumetofen controls wild-type as well as sdhBP225/H272R and sdhBP225H/H272 mutants. Pydiflumetofen does not control CG in the sdhBP225/H272Y mutant but is effective in the GTE control. Pydiflumetofen significantly controls Botrytis independently of the SdhB genotype in wounded berry assays. This condition resembles the berry cracking due to heavy rainfall right before harvest, as seen in recent years in the Chilean Central Valley. The findings demonstrate that pydiflumetofen effectively controls the grapevine Botrytis population, suggest a moderate risk of pydiflumetofen resistance, and highlight the significance of incorporating genetic data into the design of control programs.

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Fludioxonil is a highly effective phenylpyrrole fungicide for controlling Botrytis cinerea. Although the field efficacy of fludioxonil remains high, Botrytis cinerea isolates with reduced sensitivity have been reported globally. The molecular target of fludioxonil still remains unknown; however, a mechanism of reduced sensitivity to fludioxonil underlies the overexpression of the ATP binding cassette (ABC) transporter AtrB in a dependent pathway of the Mrr1 transcription factor. Fludioxonil is a key player in controlling B. cinerea infection in table grapes in Chile. However, some isolates with a reduced sensitivity to fludioxonil were detected. This study observed endogenous atrB overexpression in Chilean isolates with reduced sensitivity to fludioxonil (n = 22) compared to the sensitive isolates (n = 10). All isolates increased the expression of atrB in a growth medium supplemented with fludioxonil (0.05 µg/mL). However, sensitive isolates showed lower atrB expression than those with reduced fludioxonil sensitivity. Remarkably, a mutant version of the transcription factor Mrr1 carrying 21 amino acid modifications was identified in all isolates with reduced sensitivity to fludioxonil. These changes alter the protein's transcription factor domain and the C-terminal portion of the protein but not the Zn (2)-C6 fungal-type DNA-binding domain. These results suggest a direct relationship between the conserved and divergent mutant version of mrr1 and sensitivity to fludioxonil. This study provides a new target for developing molecular diagnostic strategies to monitor B. cinerea's sensitivity to fludioxonil in the field.

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BACKGROUND: Quantum Dots (QDs) are fluorescent nanoparticles with exceptional optical and optoelectronic properties, finding widespread utility in diverse industrial applications. Presently, chemically synthesized QDs are employed in solar cells, bioimaging, and various technological domains. However, many applications demand QDs with prolonged lifespans under conditions of high-energy radiation. Over the past decade, microbial biosynthesis of nanomaterials has emerged as a sustainable and cost-effective process. In this context, the utilization of extremophile microorganisms for synthesizing QDs with unique properties has recently been reported. RESULTS: In this study, UV-resistant bacteria were isolated from one of the most extreme environments in Antarctica, Union Glacier at the Ellsworth Mountains. Bacterial isolates, identified through 16 S sequencing, belong to the genera Rhodococcus, Pseudarthrobacter, and Arthrobacter. Notably, Rhodococcus sp. (EXRC-4 A-4), Pseudarthrobacter sp. (RC-2-3), and Arthrobacter sp. (EH-1B-1) tolerate UV-C radiation doses ≥ 120 J/m². Isolated UV-resistant bacteria biosynthesized CdS QDs with fluorescence intensities 4 to 8 times higher than those biosynthesized by E. coli, a mesophilic organism tolerating low doses of UV radiation. Transmission electron microscopy (TEM) analysis determined QD sizes ranging from 6 to 23 nm, and Fourier-transform infrared (FTIR) analysis demonstrated the presence of biomolecules. QDs produced by UV-resistant Antarctic bacteria exhibit high photostability after exposure to UV-B radiation, particularly in comparison to those biosynthesized by E. coli. Interestingly, red fluorescence-emitting QDs biosynthesized by Rhodococcus sp. (EXRC-4 A-4) and Arthrobacter sp. (EH-1B-1) increased their fluorescence emission after irradiation. Analysis of methylene blue degradation after exposure to irradiated QDs biosynthesized by UV-resistant bacteria, indicates that the QDs transfer their electrons to O2 for the formation of reactive oxygen species (ROS) at different levels. CONCLUSIONS: UV-resistant Antarctic bacteria represent a novel alternative for the sustainable generation of nanostructures with increased radiation tolerance-two characteristics favoring their potential application in technologies requiring continuous exposure to high-energy radiation.

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Conserved Histidine Alpha-helical Domain (CHAD) proteins attached to the surface of polyphosphate (PolyP) have been studied in some bacteria and one archaeon. However, the activity of CHAD proteins is unknown beyond their interaction with PolyP granules. By using bioinformatic analysis, we report that several species of the biomining acidophilic bacteria contain orthologs of CHAD proteins with high sequence identity. Furthermore, the gene coding for the CHAD protein is in the same genetic context of the enzyme polyphosphate kinase (PPK), which is in charge of PolyP synthesis. Particularly, the group of ppk and CHAD genes is highly conserved. Metallosphaera sedula and other acidophilic archaea used in biomining also contain CHAD proteins. These archaea show high levels of identity in genes coding for a cluster having the same organization. Amongst these genes are chad and ppx. In general, both biomining bacteria and archaea contain high PolyP levels and are highly resistant to heavy metals. Therefore, the presence of this conserved genetic organization suggests a high relevance for their metabolism. It has been formerly reported that a crystallized CHAD protein contains a copper-binding site. Based on this previous knowledge, in the present report, it was determined that all analyzed CHAD proteins are very conserved at their structural level. In addition, it was found that the lack of YgiF, an Escherichia coli CHAD-containing protein, decreases copper resistance in this bacterium. This phenotype was not only complemented by transforming E. coli with YgiF but also by expressing CHAD from Acidithiobacillus ferrooxidans in it. Interestingly, the strains in which the possible copper-binding sites were mutated were also more metal sensitive. Based on these results, we propose that CHAD proteins are involved in copper resistance in microorganisms. These findings are very interesting and may eventually improve biomining operations in the future.

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The main phytosanitary problem for table grape production in Chile is gray mold caused by the fungus Botrytis cinerea. To manage this issue, the primary method utilized is chemical control. Fludioxonil, a phenylpyrrole, is highly effective in controlling B. cinerea and other plant pathogens. Consistently, there have been no field reports of reduced efficacy of fludioxonil; however, subpopulations with reduced sensitivity to fludioxonil are on the rise globally, as per increasing reports. Our study involved a large-scale evaluation of B. cinerea's sensitivity to fludioxonil in the Central Valley of Chile's primary table grape production area during the growing seasons from 2015 to 2018. Out of 2,207 isolates, only 1.04% of the isolates (n = 23) exceeded the sensitivity threshold value of 1 µg/ml. Remarkably, 95.7% are concentrated in a geographic region (Valparaíso Region). Isolates with reduced sensitivity to fludioxonil showed growth comparable with sensitive isolates and even more robust growth under nutritional deficit, temperature, or osmotic stress, suggesting greater environmental adaptation. When table grape detached berries were stored at 0°C, isolates less sensitive to fludioxonil caused larger lesions than sensitive isolates (2.82 mm compared with 1.48 mm). However, the lesions generated by both types of isolates were equivalent at room temperature. This study found no cross-resistance between fludioxonil and fenhexamid, an essential fungicide integrated with fludioxonil in Chilean B. cinerea control programs. All the Chilean isolates with reduced sensitivity to fludioxonil were controlled by the fludioxonil/cyprodinil mixture, a commonly employed form of fludioxonil. The cyprodinil sensitivity in the isolates with reduced sensitivity to fludioxonil explains their low field frequency despite their null fitness penalties. However, the emergence of fludioxonil-resistant isolates inside the Chilean B. cinerea population demands a comprehensive analysis of their genetic bases, accompanied by monitoring tools that allow the permanence of field fludioxonil efficacy.

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BACKGROUND: Bacterial biosynthesis of fluorescent nanoparticles or quantum dots (QDs) has emerged as a unique mechanism for heavy metal tolerance. However, the physiological pathways governing the removal of QDs from bacterial cells remains elusive. This study investigates the role of minicells, previously identified as a means of eliminating damaged proteins and enhancing bacterial resistance to stress. Building on our prior work, which unveiled the formation of minicells during cadmium QDs biosynthesis in Escherichia coli, we hypothesize that minicells serve as a mechanism for the accumulation and detoxification of QDs in bacterial cells. RESULTS: Intracellular biosynthesis of CdS QDs was performed in E. coli mutants ΔminC and ΔminCDE, known for their minicell-producing capabilities. Fluorescence microscopy analysis demonstrated that the generated minicells exhibited fluorescence emission, indicative of QD loading. Transmission electron microscopy (TEM) confirmed the presence of nanoparticles in minicells, while energy dispersive spectroscopy (EDS) revealed the coexistence of cadmium and sulfur. Cadmium quantification through flame atomic absorption spectrometry (FAAS) demonstrated that minicells accumulated a higher cadmium content compared to rod cells. Moreover, fluorescence intensity analysis suggested that minicells accumulated a greater quantity of fluorescent nanoparticles, underscoring their efficacy in QD removal. Biosynthesis dynamics in minicell-producing strains indicated that biosynthesized QDs maintained high fluorescence intensity even during prolonged biosynthesis times, suggesting continuous QD clearance in minicells. CONCLUSIONS: These findings support a model wherein E. coli utilizes minicells for the accumulation and removal of nanoparticles, highlighting their physiological role in eliminating harmful elements and maintaining cellular fitness. Additionally, this biosynthesis system presents an opportunity for generating minicell-coated nanoparticles with enhanced biocompatibility for diverse applications.

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A Gram-stain-positive, catalase-positive, non-motile bacteria, with a rod-coccus cycle (designated as EH-1B-1T) was isolated from a soil sample from Union Glacier in Ellsworth Mountains, Antarctica. Strain EH-1B-1T had an optimal growth temperature of 28 °C and grew at pH 7-10. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0, C16 : 0 and anteiso-C17 : 0. The G+C content based on the whole genome sequence was 63.1 mol%. Strain EH-1B-1T was most closely related to members of the genus Arthrobacter, namely Arthrobacter subterraneus and Arthrobacter tumbae. The strain grew on tryptic soy agar, Reasoner's 2A agar, lysogeny broth agar and nutrient agar. The average nucleotide identity and digital DNA-DNA hybridization values between strain EH-1B-1T and its closest reference type strains ranged from 78 to 88 % and from 20.9 to 36.3 %, respectively. Based on phenotypic, chemotypic and genotypic evidence, it is proposed that strain EH-1B-1T represents a novel species of Arthrobacter, for which the name Arthrobacter vasquezii sp. nov. is proposed, with strain EH-1B-1T (RGM 3386T=LMG 32961T) as the type strain.

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Sucrose is a central regulator of plant growth and development, coordinating cell division and cell elongation according to the energy status of plants. Sucrose is known to stimulate bulk endocytosis in cultured cells; however, its physiological role has not been described to date. Our work shows that sucrose supplementation induces root cell elongation and endocytosis. Sucrose targets clathrin-mediated endocytosis (CME) in epidermal cells. Its presence decreases the abundance of both the clathrin coating complex and phosphatidylinositol 4,5-biphosphate at the plasma membrane, while increasing clathrin complex abundance in intracellular spaces. Sucrose decreases the plasma membrane residence time of the clathrin complex, indicating that it controls the kinetics of endocytic vesicle formation and internalization. CME regulation by sucrose is inducible and reversible; this on/off mechanism reveals an endocytosis-mediated mechanism for sensing plant energy status and signaling root elongation. The sucrose monosaccharide fructose also induces CME, while glucose and mannitol have no effect, demonstrating the specificity of the process. Overall, our data show that sucrose can mediate CME, which demonstrates that sucrose signaling for plant growth and development is dependent on endomembrane trafficking.

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Anthracnose caused by Colletotrichum species is one of the most frequent and damaging fungal diseases affecting avocado fruits (Persea americana Mill.) worldwide. In Chile, the disease incidence has increased over the last decades due to the establishment of commercial groves in more humid areas. Since 2018, unusual symptoms of anthracnose have been observed on Hass avocado fruits, with lesions developing a white to gray sporulation. Morphological features and multi-locus phylogenetic analyses using six DNA barcodes (act, chs-1, gapdh, his3, ITS, and tub2) allowed the identification of the causal agent as Colletotrichum anthrisci, a member of the dematium species complex. Pathogenicity was confirmed by inoculating healthy Hass avocado fruits with representative isolates, reproducing the same symptoms initially observed, and successfully reisolating the same isolates from the margin of the necrotic pulp. Previously, several Colletotrichum species belonging to other species complexes have been associated with avocado anthracnose in other countries. To our knowledge, this is the first record of C. anthrisci and of a species of the dematium species complex causing anthracnose on avocado fruits in Chile and worldwide.

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Due to the current and future scenario in which phenomena such as global warming, massive industrial waste, excessive pollution of the ecosystem, water scarcity, among other negative variables, our planet and society, faces the urgent need to advance in the generation of more sustainable and environmentally friendly mining methods. The decline in the quality of the geological resources, specifically the increase of low-grade minerals, has created a scenario under which mining companies must make great efforts to maintain their current production levels.

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Polyphosphates (polyP) are polymers of orthophosphate residues linked by high-energy phosphoanhydride bonds that are important in all domains of life and function in many different processes, including biofilm development. To study the effect of polyP in archaeal biofilm formation, our previously described Sa. solfataricus polyP (-) strain and a new polyP (-) S. acidocaldarius strain generated in this report were used. These two strains lack the polymer due to the overexpression of their respective exopolyphosphatase gene (ppx). Both strains showed a reduction in biofilm formation, decreased motility on semi-solid plates and a diminished adherence to glass surfaces as seen by DAPI (4',6-diamidino-2-phenylindole) staining using fluorescence microscopy. Even though arlB (encoding the archaellum subunit) was highly upregulated in S. acidocardarius polyP (-), no archaellated cells were observed. These results suggest that polyP might be involved in the regulation of the expression of archaellum components and their assembly, possibly by affecting energy availability, phosphorylation or other phenomena. This is the first evidence indicating polyP affects biofilm formation and other related processes in archaea.

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In Chile, the 2019-2020 sweet cherry season yielded 228,548 t, produced on 38,392 hectares and an average annual crop value about US$1.6 billion (http://www.iqonsulting.com/yb/). Between autumn 2019 and summer of 2020, branch and limbs dieback symptoms were observed in two 12-year-old sweet cherry (Prunus avium L.) orchards located in the O'Higgins region (Chile Central Valley). Furthermore, other symptoms such as wilting leaves, cankers, bark cracking, emission of gum exudates and internal wood necrosis were detected on trees of "Bing", "Santina" and "Sweetheart" cultivars (Cainelli et al. 2017). Wood fragments from symptomatic branches were surface sterilized with 95% ethanol, flaming and placed onto potato dextrose agar (PDA) amended with 0.5 g liter-1 of streptomycin sulfate (Berbegal et al. 2014). After 7 days of incubation at 25°C, pink to red colonies with white margins were isolated. Each isolate was characterized by having hyaline and oblong-ellipsoidal conidia of 5.76 ± 0.88 × 1.76 ± 0.36 µm (n=100) (Trouillas et al. 2012). According to these morphological features, the fungus was identified as Calosphaeria pulchella (Pers.: Fr.) J. Schröt (anamorph Calosphaeriosphora pulchella Réblová,L. Mostert, W. Gams & Crous) (Réblová et al. 2004). ITS (Internal Transcribed Spacer region of the rDNA) sequence comparison using BLAST analysis revealed a 99.48% identity and 100% query coverage between C. pulchella sequence HM237297 and the Chilean isolates. Moreover, the Chilean isolates were confirmed by means of phylogenetic analysis using ITS sequences of C. pulchella available in GenBank database. The maximum-parsimony phylogenetic tree supported the cluster analysis of the Chilean C. pulchella isolates with those obtained in other regions of the world with a bootstrap value of 95% (Berbegal et al. 2014; Trouillas et al. 2012). The Chilean ITS sequences were deposited into GenBank (MT378444 to MT378447). Two-year-old sweet cherry trees cv. Bing were inoculated with the Chilean isolates. Six trees were used as replicates. To accomplish this goal, two punctures of 5mm diameter were made in two branches per tree with a cork borer and a plug of mycelium from 7-day-old colonies was laid on the wound mycelium side down. Six trees were inoculated with sterile agar plugs. Every puncture was sealed with petroleum jelly and wrapped with parafilm. Four months after inoculation, the vascular streaking developing from the inoculated wounds was measured. The average lesion lengths on inoculated and non-inoculated shoots were 43.79 and 21.79 mm, respectively, which were significantly different according LSD Fisher test (p<0.05). C. pulchella was recovered from all the inoculated branches. No fungus was isolated from the controls, confirming Koch's postulates (Trouillas et al. 2012). To our knowledge this is the first report of C. pulchella causing canker and branch dieback in sweet cherry trees in Chile. This new disease represents a serious threat to the Chilean cherry industry, and further research on disease control is needed.

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Table grapes are highly susceptible to Botrytis cinerea infections during the bloom period. After reaching the flower development stage, B. cinerea remains quiescent until berry ripening or gives rise to blossom blight under specific climate conditions. A research study was conducted on the Chilean Central Valley during the 2018-2019 growing season. Flowers of Vitis vinifera cv. Thompson Seedless were collected and B. cinerea was isolated together to a second and morphologically different species, characterized by white mycelium and low to no sporulation (11.4% of total isolates). Three randomly selected isolates within this population were genetically examined and identified as Botrytis prunorum based on a phylogenetic multilocus approach using partial regions of genes RPB2, HSP60, and G3PDH or NEP1 and NEP2. Pathogenicity tests showed that B. prunorum infects and causes wilting in healthy table grape flowers. B. prunorum isolates were able to infect Thompson Seedless berries, inducing lesions between 13.11 and 41.53% with respect to the lesion diameter generated by B. cinerea B05.10. The fungicide sensitivity was evaluated. The three genetically characterized isolates were sensitive to boscalid and to cyprodinil/fludioxonil mixture with a mean EC50 value of 5.5 µg/ml and 0.065 µg/ml, respectively. However, loss of sensitivity to fenhexamid was determined, with a mean EC50 value of 5.13 µg/ml. Our understanding about blossom blight in V. vinifera has been limited to B. cinerea. Here we associated B. prunorum as a second causal agent of this disease in Chile. This data represents a first approach to the epidemiological characteristics of B. prunorum associated with blossom blight in table grapes.

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Lateral roots (LRs) increase the contact area of the root with the rhizosphere and thereby improve water and nutrient uptake from the soil. LRs are generated either via a developmentally controlled mechanism or through induction by external stimuli, such as water and nutrient availability. Auxin regulates LR organogenesis via transcriptional activation by an auxin complex receptor. Endocytic trafficking to the vacuole positively regulates LR organogenesis independently of the auxin complex receptor in Arabidopsis (Arabidopsis thaliana). Here, we demonstrate that phosphatidylinositol 4-phosphate (PI4P) biosynthesis regulated by the phosphatidylinositol 4-kinases PI4KIIIß1 and PI4KIIIß2 is essential for the LR organogenesis driven by endocytic trafficking to the vacuole. Stimulation with Sortin2, a biomodulator that promotes protein targeting to the vacuole, altered PI4P abundance at both the plasma membrane and endosomal compartments, a process dependent on PI4K activity. These findings suggest that endocytic trafficking to the vacuole regulated by the enzymatic activities of PI4KIIIß1 and PI4KIIIß2 participates in a mechanism independent of the auxin complex receptor that regulates LR organogenesis in Arabidopsis. Surprisingly, loss-of-function of PI4KIIIß1 and PI4KIIIß2 induced both LR primordium formation and endocytic trafficking toward the vacuole. This LR primordium induction was alleviated by exogenous PI4P, suggesting that PI4KIIIß1 and PI4KIIIß2 activity constitutively negatively regulates LR primordium formation. Overall, this research demonstrates a dual role of PI4KIIIß1 and PI4KIIIß2 in LR primordium formation in Arabidopsis.

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Cadmium is a heavy metal present in contaminated soils. It has no biological role but when entering cells generates DNA damage, overexpression of stress response proteins and misfolded proteins, amongst other deleterious effects. Acidithiobacillus ferrooxidans is an acidophilic bacterium resisting high concentrations of heavy metals such as cadmium. This is important for industrial bioleaching processes where Cd+2 concentrations can be 5-100 mM. Cadmium resistance mechanisms in these microorganisms have not been fully characterized. A. ferrooxidans ATCC 53993 contains genes coding for possible metal resistance determinants such as efflux systems: P-type ATPases, RND transporters and cation diffusion facilitators. In addition, it has extra copies of these genes in its exclusive genomic island (GI). Several of these putative genes were characterized in the present report by determining their transcriptional expression profiles and functionality. Moreover, an iTRAQ proteomic analysis was carried out to explore new cadmium resistance determinants in this bacterium. Changes in iron oxidation components, upregulation of transport proteins and variations in ribosomal protein levels were seen. Finally, increased concentrations of exclusive putative cadmium ATPases present in strain ATCC 53993 GI and other non-identified proteins such as Lferr_0210, forming part of a possible operon, could explain its extreme cadmium resistance. SIGNIFICANCE: Cadmium is a very toxic heavy metal present in mining operations and contaminated environments, it can affect all living organisms, including humans. Therefore, it is important to know the resistance mechanisms of bacteria highly resistant to this metal. These microorganisms in turn, can be used to bioremediate more efficiently environments highly polluted with metals. The results obtained suggest A. ferrooxidans strain ATCC 53993 can be an efficient bacterium to remove cadmium, copper and other metals from contaminated sites.

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Acidithiobacillus ferrooxidans resists extremely high concentrations of copper. Strain ATCC 53993 is much more resistant to the metal compared with strain ATCC 23270, possibly due to the presence of a genomic island in the former one. The global response of strain ATCC 53993 to copper was analyzed using iTRAQ (isobaric tag for relative and absolute quantitation) quantitative proteomics. Sixty-seven proteins changed their levels of synthesis in the presence of the metal. On addition of CusCBA efflux system proteins, increased levels of other envelope proteins, such as a putative periplasmic glucan biosynthesis protein (MdoG) involved in the osmoregulated synthesis of glucans and a putative antigen O polymerase (Wzy), were seen in the presence of copper. The expression of A. ferrooxidansmdoG or wzy genes in a copper sensitive Escherichia coli conferred it a higher metal resistance, suggesting the possible role of these components in copper resistance of A. ferrooxidans. Transcriptional levels of genes wzy, rfaE and wzz also increased in strain ATCC 23270 grown in the presence of copper, but not in strain ATCC 53993. Additionally, in the absence of this metal, lipopolysaccharide (LPS) amounts were 3-fold higher in A. ferrooxidans ATCC 53993 compared with strain 23270. Nevertheless, both strains grown in the presence of copper contained similar LPS quantities, suggesting that strain 23270 synthesizes higher amounts of LPS to resist the metal. On the other hand, several porins diminished their levels in the presence of copper. The data presented here point to an essential role for several envelope components in the extreme copper resistance by this industrially important acidophilic bacterium.

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Chemical compounds are useful to perturb biological functions in the same way as classical genetic approaches take advantage of mutations at the DNA level to perturb gene function. The use of bioactive chemicals currently called chemical genetic is especially valuable for cell biology. Chemical genetic approaches allow perturbations of cellular processes post-germination in a given time window controlling the severity of the effect by modifying or modulating the dose and/or the period of the treatment. Additionally, compounds can be applied directly to different mutants and translational fluorescent reporters/marker lines, expanding the repertoire of experimental setups addressing cell biology research. In this chapter, we describe standard protocols to visualize vacuole morphology and trafficking to the vacuole and the use of bioactive compounds as a proxy to study these biological processes.

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Polyphosphates (PolyP) are linear polymers of orthophosphate residues that have been proposed to participate in metal resistance in bacteria and archaea. In addition of having a CopA/CopB copper efflux system, the thermoacidophilic archaeon Metallosphaera sedula contains electron-dense PolyP-like granules and a putative exopolyphosphatase (PPX Msed , Msed_0891) and four presumed pho84-like phosphate transporters (Msed_0846, Msed_0866, Msed_1094, and Msed_1512) encoded in its genome. In the present report, the existence of a possible PolyP-based copper-resistance mechanism in M. sedula DSM 5348T was evaluated. M. sedula DSM 5348T accumulated high levels of phosphorous in the form of granules, and its growth was affected in the presence of 16 mM copper. PolyP levels were highly reduced after the archaeon was subjected to an 8 mM CuSO4 shift. PPX Msed was purified, and the enzyme was found to hydrolyze PolyP in vitro. Essential residues for catalysis of PPX Msed were E111 and E113 as shown by a site-directed mutagenesis of the implied residues. Furthermore, M. sedula ppx, pho84-like, and copTMA genes were upregulated upon copper exposure, as determined by qRT-PCR analysis. The results obtained support the existence of a PolyP-dependent copper-resistance system that may be of great importance in the adaptation of this thermoacidophilic archaeon to its harsh environment.

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Industrial biomining has been extensively used for many years to recover valuable metals such as copper, gold, uranium and others. Furthermore, microorganisms involved in these processes can also be used to bioremediate places contaminated with acid and metals. These uses are possible due to the great metal resistance that these extreme acidophilic microorganisms possess. In this review, the most recent findings related to copper resistance mechanisms of bacteria and archaea related to biohydrometallurgy are described. The recent search for novel metal resistance determinants is not only of scientific interest but also of industrial importance, as reflected by the genomic sequencing of microorganisms present in mining operations and the search of those bacteria with extreme metal resistance to improve the extraction processes used by the biomining companies.

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UNLABELLED: Acidithiobacillus ferrooxidans is used in industrial bioleaching of minerals to extract valuable metals. A. ferrooxidans strain ATCC 53993 is much more resistant to copper than other strains of this microorganism and it has been proposed that genes present in an exclusive genomic island (GI) of this strain would contribute to its extreme copper tolerance. ICPL (isotope-coded protein labeling) quantitative proteomics was used to study in detail the response of this bacterium to copper. A high overexpression of RND efflux systems and CusF copper chaperones, both present in the genome and the GI of strain ATCC 53993 was found. Also, changes in the levels of the respiratory system proteins such as AcoP and Rus copper binding proteins and several proteins with other predicted functions suggest that numerous metabolic changes are apparently involved in controlling the effects of the toxic metal on this acidophile. SIGNIFICANCE: Using quantitative proteomics we overview the adaptation mechanisms that biomining acidophiles use to stand their harsh environment. The overexpression of several genes present in an exclusive genomic island strongly suggests the importance of the proteins coded in this DNA region in the high tolerance of A. ferrooxidans ATCC 53993 to metals.

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