Risks to Human Health from Mercury in Gold Mining in the Coastal Region of Ecuador.

Artisanal and small-scale gold mining (ASGM) plays a crucial role in global gold production. However, the adoption of poor mining practices or the use of mercury (Hg) in gold recovery processes has generated serious environmental contamination events. The focus of this study is assessing the concentration of Hg in surface waters within the coastal region of Ecuador. The results are used to conduct a human health risk assessment applying deterministic and probabilistic methods, specifically targeting groups vulnerable to exposure in affected mining environments. Between April and June 2022, 54 water samples were collected from rivers and streams adjacent to mining areas to determine Hg levels. In the health risk assessment, exposure routes through water ingestion and dermal contact were considered for both adults and children, following the model structures outlined by the U.S. Environmental Protection Agency. The results indicate elevated Hg concentrations in two of the five provinces studied, El Oro and Esmeraldas, where at least 88% and 75% of the samples, respectively, exceeded the maximum permissible limit (MPL) set by Ecuadorian regulations for the preservation of aquatic life. Furthermore, in El Oro province, 28% of the samples exceeded the MPL established for drinking water quality. The high concentrations of Hg could be related to illegal mining activity that uses Hg for gold recovery. Regarding the human health risk assessment, risk values above the safe exposure limit were estimated. Children were identified as the most vulnerable receptor. Therefore, there is an urgent need to establish effective regulations that guarantee the protection of river users in potentially contaminated areas. Finally, it is important to continue investigating the contamination caused by human practices in the coastal region.

Future climate-induced distribution shifts in a sexually dimorphic key predator of the Southern Ocean.

The response to climate change in highly dimorphic species can be hindered by differences between sexes in habitat preferences and movement patterns. The Antarctic fur seal, Arctocephalus gazella, is the most abundant pinniped in the Southern Hemisphere, and one of the main consumers of Antarctic krill, Euphausia superba, in the Southern Ocean. However, the populations breeding in the Atlantic Southern Ocean are decreasing, partly due to global warming. Male and female Antarctic fur seals differ greatly in body size and foraging ecology, and little is known about their sex-specific responses to climate change. We used satellite tracking data and Earth System Models to predict changes in habitat suitability for male and female Antarctic fur seals from the Western Antarctic Peninsula under different climate change scenarios. Under the most extreme scenario (SSP5-8.5; global average temperature +4.4°C projected by 2100), suitable habitat patches will shift southward during the non-breeding season, leading to a minor overall habitat loss. The impact will be more pronounced for females than for males. The reduction of winter foraging grounds might decrease the survival of post-weaned females, reducing recruitment and jeopardizing population viability. During the breeding season, when males fast on land, suitable foraging grounds for females off the South Shetland Islands will remain largely unmodified, and new ones will emerge in the Bellingshausen Sea. As Antarctic fur seals are income breeders, the foraging grounds of females should be reasonably close to the breeding colony. As a result, the new suitable foraging grounds will be useful for females only if nearby beaches currently covered by sea ice emerge by the end of the century. Furthermore, the colonization of these new, ice-free breeding locations might be limited by strong female philopatry. These results should be considered when managing the fisheries of Antarctic krill in the Southern Ocean.

La resposta al canvi climàtic en espècies amb dimorfisme sexual pot veure's dificultada per les diferències entre sexes respecte a les seves preferències d'ús de l'hàbitat i els seus patrons de moviment. L'os marí antàrtic (Arctocephalus gazella), és el pinnípede més abundant a l'Hemisferi Sud i un dels principals consumidors de krill antàrtic, (Euphausia superba), a l'Oceà Antàrtic. No obstant això, les poblacions que es reprodueixen al sector Atlàntic de l'Oceà Antàrtic estan disminuint, en part a causa de l'escalfament global. Els mascles i les femelles de l'os marí antàrtic difereixen considerablement en la seva mida corporal i ecologia tròfica, i es té poc coneixement sobre les seves respostes específiques al canvi climàtic. En aquest estudi hem utilitzat dades de seguiment per satèl·lit i models del Sistema Terrestre per predir els canvis en la idoneïtat de l'hàbitat per als mascles i les femelles d'os marí antàrtic de la Península Antàrtica Occidental sota diferents escenaris de canvi climàtic. Sota l'escenari més extrem (SSP5-8.5; temperatura mitjana mundial +4.4°C prevista per a 2100), les zones d'hàbitat idoni es desplaçaran cap al sud durant l'època d'hivernada (no reproducció), provocant una lleugera pèrdua d'hàbitat idoni. Tot i això, l'impacte serà més pronunciat per a les femelles que per als mascles. Aquesta reducció dels territoris d'alimentació durant l'hivern podria disminuir la supervivència de les femelles postdeslletades, reduint-ne el reclutament i posant en perill la viabilitat de la població. Durant l'època de cria, quan els mascles es troben majoritàriament en dejú a terra, els territoris d'alimentació idonis per a les femelles al voltant de les Illes Shetland del Sud romandran en gran part sense modificar-se, i n'emergiran de nous al mar de Bellingshausen. Com que les femelles d'os marí antàrtic es continuen alimentant durant la cria, els territoris d'alimentació de les femelles han d'estar raonablement a prop de la colònia de cria. Com a resultat, aquestes noves zones d'alimentació seran útils només si les platges properes, actualment cobertes de gel marí, emergeixen al llarg del segle. A més, la colonització d'aquests nous llocs de reproducció lliures de gel podria veure's limitada per la forta filopatria de les femelles. Aquests resultats haurien de tenir-se en compte en la gestió de les pesqueries de krill a l'Oceà Antàrtic.

Expanding the Toolbox for Genetic Manipulation in Pseudogymnoascus: RNAi-Mediated Silencing and CRISPR/Cas9-Mediated Disruption of a Polyketide Synthase Gene Involved in Red Pigment Production in P. verrucosus.

Fungi belonging to the genus Pseudogymnoascus have garnered increasing attention in recent years. One of the members of the genus, P. destructans, has been identified as the causal agent of a severe bat disease. Simultaneously, the knowledge of Pseudogymnoascus species has expanded, in parallel with the increased availability of genome sequences. Moreover, Pseudogymnoascus exhibits great potential as a producer of specialized metabolites, displaying a diverse array of biological activities. Despite these significant advancements, the genetic landscape of Pseudogymnoascus remains largely unexplored due to the scarcity of suitable molecular tools for genetic manipulation. In this study, we successfully implemented RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption in Pseudogymnoascus, using an Antarctic strain of Pseudogymnoascus verrucosus as a model. Both methods were applied to target azpA, a gene involved in red pigment biosynthesis. Silencing of the azpA gene to levels of 90% or higher eliminated red pigment production, resulting in transformants exhibiting a white phenotype. On the other hand, the CRISPR/Cas9 system led to a high percentage (73%) of transformants with a one-nucleotide insertion, thereby inactivating azpA and abolishing red pigment production, resulting in a white phenotype. The successful application of RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption represents a significant advancement in Pseudogymnoascus research, opening avenues for comprehensive functional genetic investigations within this underexplored fungal genus.

PrlaeA Affects the Production of Roquefortine C, Mycophenolic Acid, and Andrastin A in Penicillium roqueforti, but It Has Little Impact on Asexual Development.

The regulation of fungal specialized metabolism is a complex process involving various regulators. Among these regulators, LaeA, a methyltransferase protein originally discovered in Aspergillus spp., plays a crucial role. Although the role of LaeA in specialized metabolism has been studied in different fungi, its function in Penicillium roqueforti remains unknown. In this study, we employed CRISPR-Cas9 technology to disrupt the laeA gene in P. roqueforti (PrlaeA) aiming to investigate its impact on the production of the specialized metabolites roquefortine C, mycophenolic acid, and andrastin A, as well as on asexual development, because they are processes that occur in the same temporal stages within the physiology of the fungus. Our results demonstrate a substantial reduction in the production of the three metabolites upon disruption of PrlaeA, suggesting a positive regulatory role of LaeA in their biosynthesis. These findings were further supported by qRT-PCR analysis, which revealed significant downregulation in the expression of genes associated with the biosynthetic gene clusters (BGCs) responsible for producing roquefortine C, mycophenolic acid, and andrastin A in the ΔPrlaeA strains compared with the wild-type P. roqueforti. Regarding asexual development, the disruption of PrlaeA led to a slight decrease in colony growth rate, while conidiation and conidial germination remained unaffected. Taken together, our results suggest that LaeA positively regulates the expression of the analyzed BGCs and the production of their corresponding metabolites in P. roqueforti, but it has little impact on asexual development.

CRISPR/Cas9-Mediated Disruption of the pcz1 Gene and Its Impact on Growth, Development, and Penicillin Production in Penicillium rubens.

Penicillium rubens is a filamentous fungus of great biotechnological importance due to its role as an industrial producer of the antibiotic penicillin. However, despite its significance, our understanding of the regulatory mechanisms governing biological processes in this fungus is still limited. In fungi, zinc finger proteins containing a Zn(II)2Cys6 domain are particularly interesting regulators. Although the P. rubens genome harbors many genes encoding proteins with this domain, only two of them have been investigated thus far. In this study, we employed CRISPR-Cas9 technology to disrupt the pcz1 gene, which encodes a Zn(II)2Cys6 protein in P. rubens. The disruption of pcz1 resulted in a decrease in the production of penicillin in P. rubens. This decrease in penicillin production was accompanied by the downregulation of the expression of pcbAB, pcbC and penDE genes, which form the biosynthetic gene cluster responsible for penicillin production. Moreover, the disruption of pcz1 also impacts on asexual development, leading to decreased growth and conidiation, as well as enhanced conidial germination. Collectively, our results indicate that pcz1 acts as a positive regulator of penicillin production, growth, and conidiation, while functioning as a negative regulator of conidial germination in P. rubens. To the best of our knowledge, this is the first report involving a gene encoding a Zn(II)2Cys6 protein in the regulation of penicillin biosynthesis in P. rubens.

Secondary Metabolites Produced by the Blue-Cheese Ripening Mold Penicillium roqueforti; Biosynthesis and Regulation Mechanisms.

Filamentous fungi are an important source of natural products. The mold Penicillium roqueforti, which is well-known for being responsible for the characteristic texture, blue-green spots, and aroma of the so-called blue-veined cheeses (French Bleu, Roquefort, Gorgonzola, Stilton, Cabrales, and Valdeón, among others), is able to synthesize different secondary metabolites, including andrastins and mycophenolic acid, as well as several mycotoxins, such as Roquefortines C and D, PR-toxin and eremofortins, Isofumigaclavines A and B, festuclavine, and Annullatins D and F. This review provides a detailed description of the biosynthetic gene clusters and pathways of the main secondary metabolites produced by P. roqueforti, as well as an overview of the regulatory mechanisms controlling secondary metabolism in this filamentous fungus.

Whiskers provide time-series of toxic and essential trace elements, Se:Hg molar ratios, and stable isotope values of an apex Antarctic predator, the leopard seal.

In an era of rapid environmental change and increasing human presence, researchers need efficient tools for tracking contaminants to monitor the health of Antarctic flora and fauna. Here, we examined the utility of leopard seal whiskers as a biomonitoring tool that reconstructs time-series of significant ecological and physiological biomarkers. Leopard seals (Hydrurga leptonyx) are a sentinel species in the Western Antarctic Peninsula due to their apex predator status and top-down effects on several Antarctic species. However, there are few data on their contaminant loads. We analyzed leopard seal whiskers (n = 18 individuals, n = 981 segments) collected during 2018-2019 field seasons to acquire longitudinal profiles of non-essential (Hg, Pb, and Cd) and essential (Se, Cu, and Zn) trace elements, stable isotope (ẟ15N and ẟ13C) values and to assess Hg risk with Se:Hg molar ratios. Whiskers provided between 46 and 286 cumulative days of growth with a mean ~ 125 days per whisker (n = 18). Adult whiskers showed variability in non-essential trace elements over time that could partly be explained by changes in diet. Whisker Hg levels were insufficient (<20 ppm) to consider most seals being at "high" risk for Hg toxicity. Nevertheless, maximum Hg concentrations observed in this study were greater than that of leopard seal hair measured two decades ago. However, variation in the Se:Hg molar ratios over time suggest that Se may detoxify Hg burden in leopard seals. Overall, we provide evidence that the analysis of leopard seal whiskers allows for the reconstruction of time-series ecological and physiological data and can be valuable for opportunistically monitoring the health of the leopard seal population and their Antarctic ecosystem during climate change.

Transcriptomic analysis of chloride tolerance in Leptospirillum ferriphilum DSM 14647 adapted to NaCl.

Chloride ions are toxic for most acidophilic microorganisms. In this study, the chloride tolerance mechanisms in the acidophilic iron-oxidizing bacterium Leptospirillum ferriphilum DSM 14647 adapted to 180 mM NaCl were investigated by a transcriptomic approach. Results showed that 99 genes were differentially expressed in the adapted versus the non-adapted cultures, of which 69 and 30 were significantly up-regulated or down-regulated, respectively. Genes that were up-regulated include carbonic anhydrase, cytochrome c oxidase (ccoN) and sulfide:quinone reductase (sqr), likely involved in intracellular pH regulation. Towards the same end, the cation/proton antiporter CzcA (czcA) was down-regulated. Adapted cells showed a higher oxygen consumption rate (2.2 x 10-9 ppm O2 s-1cell-1) than non-adapted cells (1.2 x 10-9 ppm O2 s-1cell-1). Genes coding for the antioxidants flavohemoprotein and cytochrome c peroxidase were also up-regulated. Measurements of the intracellular reactive oxygen species (ROS) level revealed that adapted cells had a lower level than non-adapted cells, suggesting that detoxification of ROS could be an important strategy to withstand NaCl. In addition, data analysis revealed the up-regulation of genes for Fe-S cluster biosynthesis (iscR), metal reduction (merA) and activation of a cellular response mediated by diffusible signal factors (DSFs) and the second messenger c-di-GMP. Several genes related to the synthesis of lipopolysaccharide and peptidoglycan were consistently down-regulated. Unexpectedly, the genes ectB, ectC and ectD involved in the biosynthesis of the compatible solutes (hydroxy)ectoine were also down-regulated. In line with these findings, although hydroxyectoine reached 20 nmol mg-1 of wet biomass in non-adapted cells, it was not detected in L. ferriphilum adapted to NaCl, suggesting that this canonical osmotic stress response was dispensable for salt adaptation. Differentially expressed transcripts and experimental validations suggest that adaptation to chloride in acidophilic microorganisms involves a multifactorial response that is different from the response in other bacteria studied.

Penicillium chrysogenum, a Vintage Model with a Cutting-Edge Profile in Biotechnology.

The discovery of penicillin entailed a decisive breakthrough in medicine. No other medical advance has ever had the same impact in the clinical practise. The fungus Penicillium chrysogenum (reclassified as P. rubens) has been used for industrial production of penicillin ever since the forties of the past century; industrial biotechnology developed hand in hand with it, and currently P. chrysogenum is a thoroughly studied model for secondary metabolite production and regulation. In addition to its role as penicillin producer, recent synthetic biology advances have put P. chrysogenum on the path to become a cell factory for the production of metabolites with biotechnological interest. In this review, we tell the history of P. chrysogenum, from the discovery of penicillin and the first isolation of strains with high production capacity to the most recent research advances with the fungus. We will describe how classical strain improvement programs achieved the goal of increasing production and how the development of different molecular tools allowed further improvements. The discovery of the penicillin gene cluster, the origin of the penicillin genes, the regulation of penicillin production, and a compilation of other P. chrysogenum secondary metabolites will also be covered and updated in this work.

The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region.

Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (ß/α)8-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end. Enzyme activity assays revealed that the elimination of the IDR resulted in a mutant enzyme (XynAΔ29) in which two new activities emerged: the ability to release xylose from xylan, and the ability to hydrolyze p-nitrophenyl-ß-d-xylopyranoside (pNPXyl), a substrate that wild-type enzyme cannot hydrolyze. Circular dichroism and tryptophan fluorescence quenching by acrylamide showed changes in secondary structure and increased flexibility of XynAΔ29. Molecular dynamics simulations revealed that the emergence of the pNPXyl-hydrolyzing activity correlated with a dynamic behavior not previously observed in GH10 endoxylanases: a hinge-bending motion of two symmetric regions within the (ß/α)8-barrel domain, whose hinge point is the active cleft. The hinge-bending motion is more intense in XynAΔ29 than in XynA and promotes the formation of a wider active site that allows the accommodation and hydrolysis of pNPXyl. Our results open new avenues for the study of the relationship between IDRs, dynamics and activity of endoxylanases, and other enzymes containing (ß/α)8-barrel domain.

A novel gene from the acidophilic bacterium Leptospirillum sp. CF-1 and its role in oxidative stress and chromate tolerance

BACKGROUND: Acidophilic microorganisms like Leptospirillum sp. CF 1 thrive in environments with extremely low pH and high concentrations of dissolved heavy metals that can induce the generation of reactive oxygen species (ROS). Several hypothetical genes and proteins from Leptospirillum sp. CF 1 are known to be up regulated under oxidative stress conditions. RESULTS: In the present work, the function of hypothetical gene ABH19_09590 from Leptospirillum sp. CF 1 was studied. Heterologous expression of this gene in Escherichia coli led to an increase in the ability to grow under oxidant conditions with 5 mM K2CrO4 or 5 mM H2O2. Similarly, a significant reduction in ROS production in E. coli transformed with a plasmid carrying ABH19_09590 was observed after exposure to these oxidative stress elicitors for 30 min, compared to a strain complemented with the empty vector. A co transcriptional study using RT PCR showed that ABH19_09590 is contained in an operon, here named the "och" operon, that also contains ABH19_09585, ABH19_09595 and ABH19_09600 genes. The expression of the och operon was significantly up regulated in Leptospirillum sp. CF 1 exposed to 5 mM K2CrO4 for 15 and 30 min. Genes of this operon potentially encode a NADH:ubiquinone oxidoreductase, a CXXC motif containing protein likely involved in thiol/disulfide exchange, a hypothetical protein, and a di hydroxy acid dehydratase. A comparative genomic analysis revealed that the och operon is a characteristic genetic determinant of the Leptospirillum genus that is not present in other acidophiles. CONCLUSIONS: Altogether, these results suggest that the och operon plays a protective role against chromate and hydrogen peroxide and is an important mechanism required to face polyextremophilic conditions in acid environments.

Description of the First Four Species of the Genus Pseudogymnoascus From Antarctica.

The genus Pseudogymnoascus represents a diverse group of fungi widely distributed in different cold regions on Earth. Our current knowledge of the species of Pseudogymnoascus is still very limited. Currently, there are only 15 accepted species of Pseudogymnoascus that have been isolated from different environments in the Northern Hemisphere. In contrast, species of Pseudogymnoascus from the Southern Hemisphere have not yet been described. In this work, we characterized four fungal strains obtained from Antarctic marine sponges. Based on multilocus phylogenetic analyses and morphological characterizations we determined that these strains are new species, for which the names Pseudogymnoascus antarcticus sp. nov., Pseudogymnoascus australis sp. nov., Pseudogymnoascus griseus sp. nov., and Pseudogymnoascus lanuginosus sp. nov. are proposed. Phylogenetic analyses indicate that the new species form distinct lineages separated from other species of Pseudogymnoascus with strong support. The new species do not form sexual structures and differ from the currently known species mainly in the shape and size of their conidia, the presence of chains of arthroconidia, and the appearance of their colonies. This is the first report of new species of Pseudogymnoascus not only from Antarctica but also from the Southern Hemisphere.

Nonionizing Electromagnetic Field: A Promising Alternative for Growing Control Yeast.

In the food industry, some fungi are considered to be common spoilage microorganisms which reduce the shelf life of products. To avoid this outcome, different technologies are being developed to control their growth. Electromagnetic fields (EMF) have been used to combat bacterial growth, but there are few studies on yeasts and their possible action mechanisms. For this reason, we studied the effect of EMF between 1 to 5.9 GHz bands on the growth of Saccharomyces cerevisiae yeast and observed that all the frequencies of the band used cause the reduction of the viability of this yeast. In addition, we observed that the distance between the antenna and the sample is an important factor to consider to control the growing yeast. By using transmission electron microscopy, we found that the EMF caused a loss of continuity of the yeast cell membrane. Therefore, EMF may be used as a control method for yeast growth.

Redox stress response and UV tolerance in the acidophilic iron-oxidizing bacteria Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans.

The oxidative stress response represents a sum of antioxidative mechanisms that are essential for determining the adaptation and abundance of microorganisms in the environment. Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans are chemolithotrophic bacteria that obtain their energy from the oxidation of ferrous ion. Both microorganisms are important for bioleaching of sulfidic ores and both are tolerant to high levels of heavy metals and other factors that can induce oxidative stress. In this work, we compared the tolerance and response of L. ferriphilum and At. ferrooxidans to Fe3+, H2O2, K2CrO4, and UV-C radiation. We evaluated growth, generation of reactive oxygen species (ROS), oxidative damage to lipid membranes and DNA, and the activity of antioxidative proteins in cells exposed to these stressors. L. ferriphilum had higher cell density, lower ROS content and less lipid and DNA damage than At. ferrooxidans. Consistent with this, the activity levels of thioredoxin and superoxide dismutase in L. ferriphilum were upregulated and higher than in At. ferrooxidans. This indicated that L. ferriphilum has a higher capacity to respond to oxidative stress and to manage redox homeostasis. This capacity could largely contribute to the high abundance of this species in natural and anthropogenic sites.

IMA genome - F14 : Draft genome sequences of Penicillium roqueforti, Fusarium sororula, Chrysoporthe puriensis, and Chalaropsis populi.

Draft genomes of Penicillium roqueforti, Fusarium sororula, Chalaropsis populi, and Chrysoporthe puriensis are presented. Penicillium roqueforti is a model fungus for genetics, physiological and metabolic studies, as well as for biotechnological applications. Fusarium sororula and Chrysoporthe puriensis are important tree pathogens, and Chalaropsis populi is a soil-borne root-pathogen. The genome sequences presented here thus contribute towards a better understanding of both the pathogenicity and biotechnological potential of these species.

Deciphering the Role of Multiple Thioredoxin Fold Proteins of Leptospirillum sp. in Oxidative Stress Tolerance.

Thioredoxin fold proteins (TFPs) form a family of diverse proteins involved in thiol/disulfide exchange in cells from all domains of life. Leptospirillum spp. are bioleaching bacteria naturally exposed to extreme conditions like acidic pH and high concentrations of metals that can contribute to the generation of reactive oxygen species (ROS) and consequently the induction of thiol oxidative damage. Bioinformatic studies have predicted 13 genes that encode for TFP proteins in Leptospirillum spp. We analyzed the participation of individual tfp genes from Leptospirillum sp. CF-1 in the response to oxidative conditions. Genomic context analysis predicted the involvement of these genes in the general thiol-reducing system, cofactor biosynthesis, carbon fixation, cytochrome c biogenesis, signal transduction, and pilus and fimbria assembly. All tfp genes identified were transcriptionally active, although they responded differentially to ferric sulfate and diamide stress. Some of these genes confer oxidative protection to a thioredoxin-deficient Escherichia coli strain by restoring the wild-type phenotype under oxidative stress conditions. These findings contribute to our understanding of the diversity and complexity of thiol/disulfide systems, and of adaptations that emerge in acidophilic microorganisms that allow them to thrive in highly oxidative environments. These findings also give new insights into the physiology of these microorganisms during industrial bioleaching operations.

Genetic Transformation of the Filamentous Fungus Pseudogymnoascus verrucosus of Antarctic Origin.

Cold-adapted fungi isolated from Antarctica, in particular those belonging to the genus Pseudogymnoascus, are producers of secondary metabolites with interesting bioactive properties as well as enzymes with potential biotechnological applications. However, at genetic level, the study of these fungi has been hindered by the lack of suitable genetic tools such as transformation systems. In fungi, the availability of transformation systems is a key to address the functional analysis of genes related with the production of a particular metabolite or enzyme. To the best of our knowledge, the transformation of Pseudogymnoascus strains of Antarctic origin has not been achieved yet. In this work, we describe for the first time the successful transformation of a Pseudogymnoascus verrucosus strain of Antarctic origin, using two methodologies: the polyethylene glycol (PEG)-mediated transformation, and the electroporation of germinated conidia. We achieved transformation efficiencies of 15.87 ± 5.16 transformants per µg of DNA and 2.67 ± 1.15 transformants per µg of DNA for PEG-mediated transformation and electroporation of germinated conidia, respectively. These results indicate that PEG-mediated transformation is a very efficient method for the transformation of this Antarctic fungus. The genetic transformation of Pseudogymnoascus verrucosus described in this work represents the first example of transformation of a filamentous fungus of Antarctic origin.

Osmotic Imbalance, Cytoplasm Acidification and Oxidative Stress Induction Support the High Toxicity of Chloride in Acidophilic Bacteria.

In acidophilic microorganisms, anions like chloride have higher toxicity than their neutrophilic counterparts. In addition to the osmotic imbalance, chloride can also induce acidification of the cytoplasm. We predicted that intracellular acidification produces an increase in respiratory rate and generation of reactive oxygen species, and so oxidative stress can also be induced. In this study, the multifactorial effect as inducing osmotic imbalance, cytoplasm acidification and oxidative stress in the iron-oxidizing bacterium Leptospirillum ferriphilum DSM 14647 exposed to up to 150 mM NaCl was investigated. Results showed that chloride stress up-regulated genes for synthesis of potassium transporters (kdpC and kdpD), and biosynthesis of the compatible solutes (hydroxy)ectoine (ectC and ectD) and trehalose (otsB). As a consequence, the intracellular levels of both hydroxyectoine and trehalose increased significantly, suggesting a strong response to keep osmotic homeostasis. On the other hand, the intracellular pH significantly decreased from 6.7 to pH 5.5 and oxygen consumption increased significantly when the cells were exposed to NaCl stress. Furthermore, this stress condition led to a significant increase of the intracellular content of reactive oxygen species, and to a rise of the antioxidative cytochrome c peroxidase (CcP) and thioredoxin (Trx) activities. In agreement, ccp and trx genes were up-regulated under this condition, suggesting that this bacterium displayed a transcriptionally regulated response against oxidative stress induced by chloride. Altogether, these data reveal that chloride has a dramatic multifaceted effect on acidophile physiology that involves osmotic, acidic and oxidative stresses. Exploration of the adaptive mechanisms to anion stress in iron-oxidizing acidophilic microorganisms may result in new strategies that facilitate the bioleaching of ores for recovery of precious metals in presence of chloride.

Novel penguin Avian avulaviruses 17, 18 and 19 are widely distributed in the Antarctic Peninsula.

Three novel Avian avulavirus species were discovered and isolated during 2017 from Gentoo penguins (Pygoscelis papua) at Kopaitic island in the Northwestern region of the Antarctic Peninsula. The viruses were officially named as Avian avulavirus 17 (AAV17), Avian avulavirus 18 (AAV18) and Avian avulavirus 19 (AAV19), collectively referred to as penguin avulaviruses (PAVs). To determine whether these viruses are capable of infecting the three species of Pygoscelis spp. penguins (Gentoo, Adelie and Chinstrap) and assess its geographical distribution, serum samples were collected from seven locations across the Antarctic Peninsula and Southern Shetland Islands. The samples were tested by Hemagglutination inhibition assay using reference viruses for AAV17, AAV18 and AAV19. A total of 498 sera were tested, and 40 were positive for antibodies against AAV17, 20 for AAV18 and 45 for AAV19. Positive sera were obtained for the penguin's species for each virus; however, antibodies against AAV18 were not identified in Adelie penguins. Positive penguins were identified in all regions studied. Positive locations include Ardley Island and Cape Shirreff at Livingston Island (Southern Shetland Region); Anvers Island, Doumer Island and Paradise Bay in the Central Western region; and Avian Island at Southwestern region of the Antarctic Peninsula. The lowest occurrence was observed at the Southwestern region at Lagotellerie Island, where all samples were negative. On the other hand, Cape Shirreff and Paradise Bay showed the highest antibody titres. Field samples did not evidence cross-reactivity between viruses, and detection was significantly higher for AAV19 and lower for AAV18. This is the first serologic study on the prevalence of the novel Avian avulaviruses including different locations in the white continent. The results indicate that these novel viruses can infect the three Pygoscelis spp. penguins, which extend across large distances of the Antarctic Peninsula.

Cold-active pectinolytic activity produced by filamentous fungi associated with Antarctic marine sponges.

BACKGROUND: Pectinase enzymes catalyze the breakdown of pectin, a key component of the plant cell wall. At industrial level, pectinases are used in diverse applications, especially in food-processing industry. Currently, most of the industrial pectinases have optimal activity at mesophilic temperatures. On the contrary, very little is known about the pectinolytic activities from organisms from cold climates such as Antarctica. In this work, 27 filamentous fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new source of cold-active pectinases. RESULTS: In semi-quantitative plate assays, 8 out 27 of these isolates showed pectinolytic activities at 15 °C and one of them, Geomyces sp. strain F09-T3-2, showed the highest production of pectinases in liquid medium containing pectin as sole carbon source. More interesting, Geomyces sp. F09-T3-2 showed optimal pectinolytic activity at 30 °C, 10 °C under the temperature of currently available commercial mesophilic pectinases. CONCLUSION: Filamentous fungi associated with Antarctic marine sponges are a promising source of pectinolytic activity. In particular, pectinases from Geomyces sp. F09-T3-2 may be potentially suitable for biotechnological applications needing cold-active pectinases. To the best of our knowledge, this is the first report describing the production of pectinolytic activity from filamentous fungi from any environment in Antarctica.