RESUMO
An ecological study on Saccharomyces cerevisiae populations in spontaneous fermentation has been conducted in three vats of a cachaça distillery in Minas Gerais, Brazil. Ninety-seven yeast isolates were collected at the beginning, the middle and at the end of the production period, and were identified by standard methods. Differentiation between the indigenous S. cerevisiae strains isolated was performed by mitochondrial DNA (mtDNA) restriction analysis, RAPD-PCR, and PCR fingerprint using an intron splice primer. Analysis of the mtDNA restriction profiles revealed 12 different patterns, 11 corresponding to indigenous yeasts (I to XI) and one (XII) to a commercial strain of the bakery yeast. Pattern II (53.6 percent of the population) and pattern IV strains were present in all the vats. Pattern IV strain raised from the middle to the end of the period reaching proportions near those of pattern II strain. PCR methods allowed the differentiation of 41 molecular profiles. Both methods showed population fluctuation of S. cerevisiae strains along the period of cachaça production and among different vats of the distillery.
Um estudo ecológico das populações de Saccharomyces cerevisiae em fermentações espontâneas foi conduzido em três dornas de uma destilaria de cachaça em Minas Gerais, Brasil. Noventa e sete isolados foram coletados no início, meio e final do período de produção, e identificados por métodos padrões. A diferenciação entre as linhagens isoladas de S. cerevisiae indígenas foi feita pela analise de restrição do DNA mitocondrial (mtDNA), RAPD-PCR, e PCR por impressão digital do DNA utilizando um iniciador complementar a sítios de processamento de íntron. As análises dos perfis de restrição do mtDNA mostraram a ocorrência de 12 perfis diferentes, sendo 11 correspondentes as leveduras indígenas (I ao XI) e um (XII) a uma linhagem comercial de levedura de panificação. Linhagens com o perfil II (53,6 por cento da população) e o perfil IV estiveram presentes em todas as dornas. A linhagem com perfil IV aumentou do meio para o final do período de fermentação, alcançando proporções próximas a aquelas encontradas para a linhagem com o perfil II. Os métodos baseados em PCR permitiram a diferenciação de 41 perfis moleculares. Ambos os métodos mostraram flutuações populacionais nas linhagens de S. cerevisiae durante o período de produção da cachaça e entre as diferentes dornas da destilaria.
RESUMO
Drug resistance is a complex phenomenon in Leishmania and commonly involves gene amplification. Active efflux and metal sequestration through a P-glycoprotein have been pointed to as the major mechanisms used by drug-resistant Leishmania. A gene amplification from a glucantime-resistant Leishmania (Viannia) guyanensis cell line was characterised in an attempt to understand the mechanism of metal resistance in this pathogenic species. We show that the amplification is present as an extrachromosomal amplicon of 30 kb and contains a PGPA gene ( LgPGPA), which is overexpressed in the resistant line as shown by Northern and Western blot analyses. In addition, we gathered evidence from transfection experiments for the role of the LgPGPA gene in oxyanion resistance in L. (V.) guyanensis. Our work indicates that, in this pathogenic New World Leishmania species, amplification of the PGPA gene is the major determinant in oxyanion resistance.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Resistência a Medicamentos , Amplificação de Genes , Genes de Protozoários , Leishmania guyanensis/genética , Meglumina/farmacologia , Glicoproteínas de Membrana/genética , Compostos Organometálicos/farmacologia , Proteínas de Protozoários , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Eletroforese em Gel de Campo Pulsado/métodos , Regulação da Expressão Gênica , Leishmania guyanensis/efeitos dos fármacos , Antimoniato de Meglumina , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mapeamento por Restrição , Alinhamento de Sequência , TransfecçãoRESUMO
Twenty-seven Schizosaccharomyces pombe isolates from seven cachaça distilleries were tested for maximum temperature of growth and fermentation, osmotolerance, ethanol resistance, invertase production, and trehalose accumulation. Two isolates were selected for studies of trehalose accumulation under heat shock and ethanol stress. The S. pombe isolates were also characterized by RAPD-PCR. The isolates were able to grow and ferment at 41 degrees C, resisted concentrations of 10% ethanol, and grew on 50% glucose medium. Four isolates yielded invertase activity of more than 100 micromol of reducing sugar x mg(-1) x min(-1). The S. pombe isolates were able to accumulate trehalose during stationary phase. Two isolates, strains UFMG-A533 and UFMG-A1000, submitted to a 15 min heat shock, were able to accumulate high trehalose levels. Strain UFMG-A533 had a marked reduction in viability during heat shock, but strain UFMG-A1000 preserved a viability rate of almost 20% after 15 min at 48 degrees C. No clear correlation was observed between trehalose accumulation and cell survival during ethanol stress. Strain UFMG-A1000 had higher trehalose accumulation levels than strain UFMG-A533 under conditions of combined heat treatment and ethanol stress. Molecular analysis showed that some strains are maintained during the whole cachaça production period; using the RAPD-PCR profiles, it was possible to group the isolates according to their isolation sites.