Publicação
Heavy metal resistance in extremophilic yeasts: a molecular and physiological approach
| Resumo: | Heavy metal resistant microorganisms are often associated with acidic environments, since metals are easily solubilized in acidic milieus. A unique yeast species from the Cryptococcus genus was isolated from two sulfur-rich acidic environments: acid mine drainage in the south of Portugal and a volcanic river in Argentina. The uniqueness of this species lies on the fact that it is the first acidophilic basidiomycetous yeast known to date. Additionally, the two strains chosen for this work (one from each environment) are resistant to high levels of heavy metals (arsenic, cadmium, copper and zinc). Metal resistance mechanisms are only described for neutrophilic yeasts, and mainly involve thiolated peptides and efflux transporters. To unveil the mechanisms that allow this Cryptococcus species to resist high levels of heavy metals, physiological, cytological and molecular approaches were conducted. Since both isolation sites are sulfur-rich, the possibility that this element influences metal resistance was assessed with microscale growth assays by determining minimal inhibitory concentrations of the four metals in differential sulfate availability conditions. Assessment of thiolmediated resistance mechanisms was achieved by incubating cells (grown with and without metal) with the thiol-specific fluorescent probe 5-chloromethylfluorescein-diacetate. Dot blot hybridization was applied to detect gene homologues involved in arsenic extrusion, and vacuolar metal-thiol accumulation in response to arsenic and cadmium. Also, suppression subtractive hybridization was conducted to investigate resistance to cadmium by analyzing transcripts induced upon exposure to this metal. Our results indicate that thiolated peptides are involved in resistance to arsenic and zinc in the Portuguese strain, cadmium in the Argentinean strain, and copper in both strains. Also, both strains presented evidence of an arsenic extrusion mechanism, and of a Cd-induced demand in protein synthesis and folding. Thus, the present work allowed unveiling of heavy metal resistance mechanisms in two strains of this unique novel yeast species, Cryptococcus sp. |
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| Autores principais: | Fidalgo, Cátia Isabel Assis, 1986- |
| Assunto: | Microbiologia Leveduras Metais pesados Cryptococcus Expressão génica Biologia molecular Teses de mestrado - 2011 |
| Ano: | 2011 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Heavy metal resistant microorganisms are often associated with acidic environments, since metals are easily solubilized in acidic milieus. A unique yeast species from the Cryptococcus genus was isolated from two sulfur-rich acidic environments: acid mine drainage in the south of Portugal and a volcanic river in Argentina. The uniqueness of this species lies on the fact that it is the first acidophilic basidiomycetous yeast known to date. Additionally, the two strains chosen for this work (one from each environment) are resistant to high levels of heavy metals (arsenic, cadmium, copper and zinc). Metal resistance mechanisms are only described for neutrophilic yeasts, and mainly involve thiolated peptides and efflux transporters. To unveil the mechanisms that allow this Cryptococcus species to resist high levels of heavy metals, physiological, cytological and molecular approaches were conducted. Since both isolation sites are sulfur-rich, the possibility that this element influences metal resistance was assessed with microscale growth assays by determining minimal inhibitory concentrations of the four metals in differential sulfate availability conditions. Assessment of thiolmediated resistance mechanisms was achieved by incubating cells (grown with and without metal) with the thiol-specific fluorescent probe 5-chloromethylfluorescein-diacetate. Dot blot hybridization was applied to detect gene homologues involved in arsenic extrusion, and vacuolar metal-thiol accumulation in response to arsenic and cadmium. Also, suppression subtractive hybridization was conducted to investigate resistance to cadmium by analyzing transcripts induced upon exposure to this metal. Our results indicate that thiolated peptides are involved in resistance to arsenic and zinc in the Portuguese strain, cadmium in the Argentinean strain, and copper in both strains. Also, both strains presented evidence of an arsenic extrusion mechanism, and of a Cd-induced demand in protein synthesis and folding. Thus, the present work allowed unveiling of heavy metal resistance mechanisms in two strains of this unique novel yeast species, Cryptococcus sp. |
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