Seda-Miró, Jasmine M.

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    Phenotypic and genetic characterization of Debaryomyces hansenii strains exposed to cobalt and saline stress
    (2012) Seda-Miró, Jasmine M.; Nadathur, Govind S.; College of Arts and Sciences - Sciences; Corredor, Jorge E.; Cotta, Michael; Sen, Arup; Kubaryk, John; Department of Marine Sciences; López Garriga, Juan
    Debaryomyces (Torulaspora) hansenii, an osmotolerant yeast of mostly marine origin, has become of keen interest for physiological, metabolic, genetic and molecular studies due to its versatility to survive and thrive under conditions that may be unbearable for other microorganisms. Having been isolated from several habitats that exhibit low water activity, high sugar content, limited carbon sources, high salt concentrations, D. hansenii is a model organism with appealing characteristics for studies ranging from environmental stresses, industrial metabolic capabilities, ecological implications and biotechnological applications. However, the analyses of various D. hansenii strains upon exposure to metal (oxidative) and saline (osmotic) stress, and whether a direct correlation with genotype can be postulated, has not yet been addressed. Therefore, the objectives of this study consisted of: (i) determining the potential of D. hansenii as a biosensor of metal contaminated environments using riboflavin (RF) as an indicator and the description of a RF-impaired mutant, (ii) evalulating the phenotypic diversity of over 30 D. hansenii strains was upon exposure to cobalt (oxidative) and saline (osmotic) stress over a range of concentrations by growth pattern and RF production, and (iii) profiling the genotype of the functional and inducible genes FET3 and ENA1, which have been associated with cobalt and salt stress, using restriction fragment length polymorphism (RFLP) and subsequent analysis on the possible effect of base substitutions on the predicted translated products. Growth and RF production were compared between wild-type D. hansenii and a RF production-impaired metal-tolerant ura3 mutant in the presence of sublethal cobalt(II) concentrations. Debaryomyces hansenii J26 (wild type) exhibited an extended lag phase with an increase in RF synthesis. Supplementation of exogenous uracil shortened the lag phase at the highest concentration of cobalt(II) used, suggesting that uracil has a possible role in metal acclimation. The D. hansenii ura3- mutant exhibited a higher level of metal tolerance, no extended lag phase, and no marked increase in RF synthesis. Restoration of uracil phenotype by transformation with URA3 from Saccharyomyces cerevisiae or D. hansenii did not restore wild-type characteristics, suggesting a second mutation that impairs RF oversynthesis. These results demonstrate that growth, metal sensitivity, and RF biosynthesis are linked. Thirty four D. hansenii strains isolated from various sources were submitted to cobalt and saline stress to evaluate phenotypic variation by colony growth on agar nutrient media plates supplemented with different concentrations of cobalt(II) and NaCl, respectively. Visible RF production was also monitored for strains exposed to cobalt(II). The results demonstrated that no direct pattern of phenotypic variation among the strains could be correlated to the source of isolation. Upon cobalt stress, most strains (~65%) were described as either highly tolerant (dense growth at 1.5 mM) or tolerant (slightly reduced growth at 1.5 mM Co(II)). Very few (three) strains were characterized as being sensitive. Four different strains produced substantial extracellular RF. When exposed to salt stress, no strains were considered highly tolerant but were mostly described as tolerant (~67%; dense growth up to 2 M NaCl). Overall, the results demonstrate mounting evidence that individual strains may express distinct phenotypes, which may depend on different genetic and molecular mechanisms involved in surviving under harsh conditions. To profile for genotypic heterogeneity among the 34 D. hansenii strains analyzed for phenotypic variability, the DNA sequences of the multicopper oxidase gene FET3 and the P-type ATPase sodium efflux gene ENA1 were analyzed using RFLP. Amino acid sequence comparisons of the predicted translated products of each gene were also analyzed to identify conserved regions or domains that may have suffered from base substitutions as detected by RFLP. The results obtained from the FET3 RFLP profiling revealed six distinct genotype patterns, excluding the type sequence pattern of D. hansenii CBS 767T. On the other hand, the ENA1 RFLP analysis discriminated two patterns that varied from the pattern exhibited by the type strain. Amino acid sequence comparison of the FET3 predicted translated product of CBS 767T and possible residue changes based on the RFLP analysis revealed that most regions important for ligands were conserved although changes in residues were found close to these areas and in putative transmembrane helices, which may affect its catalytic activity. Results of the comparison of the putative transmembrane helices identified in the ENA1 predicted translated product showed that at least 3 out of 10 helices may be affected by changes in residues and, thus in conformational structure. To summarize, the observations in this dissertation provide substantial evidence to support D. hansenii as a model organism for understanding the complexity involved in physiological and metabolic processes of higher level organisms and requires an integrated approach that involves further genomic, transcriptomic, and proteomic analyses.