RESUMO
The euryhaline marine yeast Debaromyces hansenii is a model system for the study of processes related to osmoadaptation. In this study, microarray-based gene expression analyses of the entire genome of D. hansenii was used to study its response to osmotic stress. Differential gene expression, compared to control, was examined at three time points (0.5, 3 and 6 h) after exposure of D. hansenii cultures to high salt concentration. Among the 1.72% of genes showing statistically significant differences in expression, only 65 genes displayed at least three-fold increases in mRNA levels after treatment with 2 M NaCl. On the other hand, 44 genes showed three-fold repression. Upregulated as well as the downregulated genes were grouped into functional categories to identify biochemical processes possibly affected by osmotic stress and involved in osmoadaptation. The observation that only a limited number of genes are upregulated in D. hansenii in response to osmotic stress supports the notion that D. hansenii is pre-adapted to survive in extreme saline environments. In addition, since more than one-half of the upregulated genes encode for ribosomal proteins, it is possible that a translational gene regulatory mechanism plays a key role in D. hansenii's osmoregulatory response. Validation studies for ENA1 and for hyphal wall/cell elongation protein genes, using real-time PCR, confirmed patterns of gene expression observed in our microarray experiments. To our knowledge, this study is the first of its kind in this organism and provides the foundation for future molecular studies assessing the significance of the genes identified here in D. hansenii's osmoadaptation.
Assuntos
Debaryomyces/fisiologia , Perfilação da Expressão Gênica , Genoma Fúngico , Resposta ao Choque Térmico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Pressão Osmótica , Adaptação Fisiológica , Debaryomyces/efeitos dos fármacos , Debaryomyces/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Cloreto de Sódio/farmacologiaRESUMO
The small subunit ribosomal RNA gene (SSU rDNA) from the euryhaline yeast Debaryomyces hansenii has been isolated and sequenced. After appropriate alignment of this sequence with SSU rDNA sequences from 30 other taxa, phylogenetic reconstruction using distance matrix and maximum parsimony methods indicates that D. hansenii is most closely affiliated with Candida albicans, and occurs in the cluster of the yeasts Saccharomyces cerevisiae, Torulaspora delbruekii, Candida glabrata, and Kluyveromyces lactis. It appears that the capacity to tolerate high salt is independent of phylogenetic affiliations based on SSU rDNA analyses.
Assuntos
DNA Fúngico/genética , DNA Ribossômico/genética , RNA Ribossômico/genética , Leveduras/genética , Sequência de Bases , DNA Fúngico/química , DNA Ribossômico/química , Genes Fúngicos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA Fúngico/química , RNA Fúngico/genética , RNA Ribossômico/química , Alinhamento de Sequência , Leveduras/classificaçãoRESUMO
The marine yeast Debaryomyces hansenii is known to tolerate salinities ranging from 0 to 24%. As a first step toward the molecular analysis of halotolerance in this organism, we report the isolation of an autonomously replicating sequence (ARS) and its use in the construction of a shuttle vector. The ARS from D. hansenii (ARSD) is 0.4 kbp long, and the function rests in 0.13 kbp of the sequence. Sequence analysis of ARSD shows strong homology to ARS from other organisms, including a 12-bp consensus sequence common to all ARS functional in Saccharomyces cerevisiae.