Analysis of the sensitivity of different fungal species to uracil

Abstract

The use of auxotrophies as a tool for genetic modification of microbial strains is widely employed both in fundamental (e.g., molecular characterization of genes based on reverse genetics approaches) and applied research (e.g., maintenance of plasmids used for the expression of genes of interest). Moreover, the manipulation of auxotrophic strains is a common practice in microbiology laboratories. Among them, one of the most prominent auxotrophies used is that for uracil. In this case, the gene encoding the essential enzyme oritidine-5-phosphate decarboxylase of the de novo biosynthesis of pyrimidines pathway is deleted (e.g., URA3 gene in Saccharomyces cerevisiae and Ashbya gossypii, pyrG in Aspergillus nidulans) conferring to the mutants the incapacity of growing in medium lacking uracil. Consequently, these auxotrophic strains require the supply of exogenous uracil to compensate their nutritional deficiency. In some some studies exogenous uridine supplementation is used instead of uracil. Recently, the presence of high levels of uracil showed to affect the growth of wild and uracil auxotrophic strains of A. gossypii and A. nidulans [1,2]. However, this sensitivity was more pronounced in the uracil auxotrophic strains than in their respective parent strains. Moreover, the A. gossypii Agura3 strain was unable to grow at the same level of the parent strain when the medium was supplemented with uracil alone, being necessary the addition of uridine to overcome this effect [3]. Given the importance and the prominence of these uracil auxotrophic strains, it is of extreme pertinence to elucidate this sensitivity to uracil in A. gossypii and analyze if this effect is widespread among different fungal species. It is believed that the cellular levels of uracil must be firmly controlled in order to avoid its incorporation into DNA [2]. Therefore, in this work we systematically characterized the physiological sensitivity of A. gossypii and other fungal species (auxotrophic and prototrophic) to uracil and verified that uridine is more adequate for growth supplementation of uracil auxotrophic strains.