Genetic evidence for independence between fermentative metabolism (ethanol accumulation) and yeast-cell development in the dimorphic fungus Mucor rouxii

Abstract

Three allyl-alcohol-resistant mutants were isolated in the dimorphic fungus Mucor rouxii and characterized with regard to their alcohol dehydrogenase (ADH) activity in vitro and in vivo as well as their ability to execute the morphological alternatives of dimorphism under different environmental stimuli, either in the absence or in the presence of oxygen. These studies indicated that fermentation and yeast-cell development are independent events and that ADH activity is essential for growth of the fungus in the absence of oxygen. Heterokaryon construction and analysis indicated that in the three mutant strains the corresponding genetic alterations are recessive nuclear mutations which behave as allelic in complementation tests.

Developmental and environmental influences in the production of a single NAD-dependent fermentative alcohol dehydrogenase by the zygomycete Mucor rouxii

“…In M. rouxii, genetic studies have indicated the existence of a nuclear gene (ADH1) involved in the synthesis of the NAD + -dependent ADH (Torres-Guzmán et al 1994). The results of the present work indicate that this enzyme is constitutive since it is produced under different growth conditions that include the two morphogenetic alternatives of the yeast/mold dimorphism (both with and without oxygen) and the absence or the presence of glucose or ethanol in the growth medium.…”

“…Our observation that the optimum pH for the reduction of acetaldehyde to ethanol is approximately pH 7.5 (whereas that for ethanol oxidation is approximately pH 8.5) may also be indicative of a role of ADH in acetaldehyde reduction under physiological conditions since the intracellular (cytoplasmic) pH in M. rouxii is most likely close to pH 7.0. Additional support for this interpretation comes from the observations that adh mutations impair growth of M. rouxii in the absence of oxygen and reduce ethanol production in aerobically growing mycelium (Torres-Guzmán et al 1994).…”

“…Strain G3, an allyl-alcohol-resistant derivative of G1, was used as the ADH-deficient (Adh -) strain (Torres Guzmán et al 1994). Yeast-peptone-glucose (YPG) complete medium (Bartnicki-García and Nickerson 1962a) and minimal medium (Bartnicki-García and Nickerson 1962b) were used for cultivation of the fungus.…”

“…Mycelium or yeast cells were processed and broken as described by Torres-Guzmán et al (1994) with some modifications. Briefly, mycelial or yeast cells were washed and suspended in TP6.5 buffer [20 mM Tris-HCl (pH 6.5) containing 1 mM phenylmethanesulfonyl fluoride].…”

A multicopy vector system for genetic studies in Mucor circinelloides and other zygomycetes

“…However, this vector is not useful for transformation of zygomycetes (Mackenzie et al 2000), probably because the gpdA promoter does not function properly in this group of fungi. We therefore investigated whether pEUKA7-kan might be useful for transformation of other zygomycetes, and tested the M. rouxii strain IM-80 (Torres-Guzman et al 1994) and the R. pusillus strain B49-7 (Wada et al 1996). In both species, transformants were selected for geneticin resistance using the procedure developed for M. circinelloides.…”