Zygosaccharomyces rouxii Combats Salt Stress by Maintaining Cell Membrane Structure and Functionality.

Zygosaccharomyces rouxii is an important yeast that is required in the food fermentation process due to its high salt tolerance. In this study, the responses and resistance strategies of Z. rouxii against salt stress were investigated by performing physiological analysis at membrane level. The results showed that under salt stress, cell integrity was destroyed, and the cell wall was ruptured, which was accompanied by intracellular substance spillover. With an increase of salt concentrations, intracellular Na+ content increased slightly, whereas intracellular K+ content decreased significantly, which caused the increase of the intracellular Na+/K+ ratio. In addition, in response to salt stress, the activity of Na+/K+-ATPase increased from 0.54 to 2.14 µmol/mg protein, and the ergosterol content increased to 2.42-fold to maintain membrane stability. Analysis of cell membrane fluidity and fatty acid composition showed that cell membrane fluidity decreased and unsaturated fatty acid proportions increased, leading to a 101.21% rise in the unsaturated/saturated fatty acid ratio. The results presented in this study offer guidance in understanding the salt tolerance mechanism of Z. rouxii, and in developing new strategies to increase the industrial utilization of this species under salt stress.

Zygosaccharomyces seidelii sp. nov. a new yeast species from the Maldives, and a revisit of the single-strain species debate.

Zygosaccharomyces seidelii, a new species in the genus Zygosaccharomyces is described. The description of the species is based on a single strain that was isolated from flowers collected on the Maldives. On this occasion, the description of yeast species from single strains was revisited. Sequence analysis of the D1/D2 domain of the nuclear large subunit rRNA gene revealed that Z. seidelii is closely related to Z. gambellarensis. Both species differ by 2.6% (one indel of 7 bp and 9 substitutions) in the D1/D2 domain, 71 substitutions and 23 indels in the ITS1-5.8S-ITS2 (ITS) region and by several physiological tests. Two divergent copies of the ITS region were detected in Z. seidelii. Asexual and sexual reproduction as well as the physiological properties of Z. seidelii fit well in the genus Zygosaccharomyces. (Holotype strain: CBS 16021, Isotype strain: CLIB 3343; MycoBank no.: MB830900).

Chimeric Sex-Determining Chromosomal Regions and Dysregulation of Cell-Type Identity in a Sterile Zygosaccharomyces Allodiploid Yeast.

Allodiploidization is a fundamental yet evolutionarily poorly characterized event, which impacts genome evolution and heredity, controlling organismal development and polyploid cell-types. In this study, we investigated the sex determination system in the allodiploid and sterile ATCC 42981 yeast, a member of the Zygosaccharomyces rouxii species complex, and used it to study how a chimeric mating-type gene repertoire contributes to hybrid reproductive isolation. We found that ATCC 42981 has 7 MAT-like (MTL) loci, 3 of which encode α-idiomorph and 4 encode a-idiomorph. Two phylogenetically divergent MAT expression loci were identified on different chromosomes, accounting for a hybrid a/α genotype. Furthermore, extra a-idimorph-encoding loci (termed MTLa copies 1 to 3) were recognized, which shared the same MATa1 ORFs but diverged for MATa2 genes. Each MAT expression locus was linked to a HML silent cassette, while the corresponding HMR loci were located on another chromosome. Two putative parental sex chromosome pairs contributed to this unusual genomic architecture: one came from an as-yet-undescribed taxon, which has the NCYC 3042 strain as a unique representative, while the other did not match any MAT-HML and HMR organizations previously described in Z. rouxii species. This chimeric rearrangement produces two copies of the HO gene, which encode for putatively functional endonucleases essential for mating-type switching. Although both a and α coding sequences, which are required to obtain a functional cell-type a1-α2 regulator, were present in the allodiploid ATCC 42981 genome, the transcriptional circuit, which regulates entry into meiosis in response to meiosis-inducing salt stress, appeared to be turned off. Furthermore, haploid and α-specific genes, such as MATα1 and HO, were observed to be actively transcribed and up-regulated under hypersaline stress. Overall, these evidences demonstrate that ATCC 42981 is unable to repress haploid α-specific genes and to activate meiosis in response to stress. We argue that sequence divergence within the chimeric a1-α2 heterodimer could be involved in the generation of negative epistasis, contributing to the allodiploid sterility and the dysregulation of cell identity.

Production and characterization of a novel yeast extracellular invertase activity towards improved dibenzothiophene biodesulfurization.

The main goal of this work was the production and characterization of a novel invertase activity from Zygosaccharomyces bailii strain Talf1 for further application to biodesulfurization (BDS) in order to expand the exploitable alternative carbon sources to renewable sucrose-rich feedstock. The maximum invertase activity (163 U ml(-1)) was achieved after 7 days of Z. bailii strain Talf1 cultivation at pH 5.5-6.0, 25 °C, and 150 rpm in Yeast Malt Broth with 25 % Jerusalem artichoke pulp as inducer substrate. The optimum pH and temperature for the crude enzyme activity were 5.5 and 50 °C, respectively, and moreover, high stability was observed at 30 °C for pH 5.5-6.5. The application of Talf1 crude invertase extract (1 %) to a BDS process by Gordonia alkanivorans strain 1B at 30 °C and pH 7.5 was carried out through a simultaneous saccharification and fermentation (SSF) approach in which 10 g l(-1) sucrose and 250 µM dibenzothiophene were used as sole carbon and sulfur sources, respectively. Growth and desulfurization profiles were evaluated and compared with those of BDS without invertase addition. Despite its lower stability at pH 7.5 (loss of activity within 24 h), Talf1 invertase was able to catalyze the full hydrolysis of 10 g l(-1) sucrose in culture medium into invert sugar, contributing to a faster uptake of the monosaccharides by strain 1B during BDS. In SSF approach, the desulfurizing bacterium increased its µmax from 0.035 to 0.070 h(-1) and attained a 2-hydroxybiphenyl productivity of 5.80 µM/h in about 3 days instead of 7 days, corresponding to an improvement of 2.6-fold in relation to the productivity obtained in BDS process without invertase addition.

Effect of inorganic salts on the growth and Cd2+ bioaccumulation of Zygosaccharomyces rouxii cultured under Cd2+ stress.

Using living cells to remove heavy metals was limited by the sensitivity of living cells to heavy metal ions. Effect of inorganic salts on the growth and Cd(2+) bioaccumulation of Zygosaccharomyces rouxii (Z. rouxii) was studied. Results showed that NaCl, KCl, CaCl(2) and MgCl(2) could markedly reduce the growth inhibition ratio while NaNO(3), KNO(3), Na(2)SO(4) and K(2)SO(4) significantly increased it. Effect of inorganic salts on the Cd(2+) bioaccumulation of Z. rouxii indicated that different inorganic salts could promote or restrain the growth of Z. rouxii under Cd(2+) stress by weakening or enhancing Cd(2+) bioaccumulation. Although NaCl and KCl lowered the Cd(2+) accumulation per unit weight of Z. rouxii biomass, the total Cd(2+) bioaccumulation and Cd(2+) percentage removal both markedly increased. It suggested that NaCl and KCl could be introduced to enhance the growth and Cd(2+) bioaccumulation of Z. rouxii under Cd(2+) stress.

Improved transformation of the halo-tolerant yeast Zygosaccharomyces rouxii by electroporation.

To improve the transformation efficiency of Zygosaccharomyces rouxii by electroporation, glycerol was added to the electroporation buffer and the cells were frozen at -80 degrees C. These alterations drastically increased transformation efficiency, and we found that competent cells can be preserved at -80 degrees C without decreasing their transformation efficiency for at least 30 d.

Caffeine induces macroautophagy and confers a cytocidal effect on food spoilage yeast in combination with benzoic acid.

Weak organic acids are an important class of food preservatives that are particularly efficacious towards yeast and fungal spoilage. While acids with small aliphatic chains appear to function by acidification of the cytosol and are required at high concentrations to inhibit growth, more hydrophobic organic acids such as sorbic and benzoic acid have been suggested to function by perturbing membrane dynamics and are growth-inhibitory at much lower concentrations. We previously demonstrated that benzoic acid has selective effects on membrane trafficking in Saccharomyces cerevisiae. Benzoic acid selectively blocks macroautophagy in S. cerevisiae while acetic acid does not, and sorbic acid does so to a lesser extent. Indeed, while both benzoic acid and nitrogen starvation are cytostatic when assayed separately, the combination of these treatments is cytocidal, because macroautophagy is essential for survival during nitrogen starvation. In this report, we demonstrate that Zygosaccharomyces bailii, a food spoilage yeast with relatively high resistance to weak acid stress, also exhibits a cytocidal response to the combination of benzoic acid and nitrogen starvation. In addition, we show that nitrogen starvation can be replaced by caffeine supplementation. Caffeine induces a starvation response that includes the induction of macroautophagy, and the combination of caffeine and benzoic acid is cytocidal, as predicted from the nitrogen starvation data.

Differences in osmotolerant and cell-wall properties of two Zygosaccharomyces rouxii strains.

The osmotolerant and cell wall properties of the two most studied wild-type Zygosaccharomyces rouxii strains (CBS 732 and ATCC 42981) were examined. Differences in their (1) tolerance to high salt content in the medium, (2) resistance to the lysing enzymes Lyticase and Zymolyase, (3) cell-wall polymer content and (4) cell wall micromorphology suggested that the less osmotolerant CBS 732 strain possesses a more rigid cell wall than the more osmotolerant ATCC 42981, whose cell wall seems to be more flexible and elastic.

Osmoresistant yeast Zygosaccharomyces rouxii: the two most studied wild-type strains (ATCC 2623 and ATCC 42981) differ in osmotolerance and glycerol metabolism.

The yeast Zygosaccharomyces rouxii is known for its high tolerance to osmotic stress, which is thought to be caused by sets of specific genes. Relatively few Z. rouxii genes have been identified so far, all of them having homologues in Saccharomyces cerevisiae; none of them was Z. rouxii-specific. Most of the known Z. rouxii genes were isolated from two wild-type strains, ATCC 2623 and ATCC 42981. In this study, we compared these two strains with regard to some of their morphological, physiological and genomic properties. Important differences were found in their salt tolerance and assimilation of glycerol and karyotype; slight differences were also present in their cell morphology. The ATCC 42981 strain showed a higher resistance to salts, higher glycerol production and, unlike ATCC 2623, was able to assimilate glycerol. Under conditions of osmotic stress, the glycerol production in both Z. rouxii strains was much lower than in a S. cerevisiae S288c culture, which suggested the presence of a system that efficiently retains glycerol inside Z. rouxii cells. The karyotype analysis revealed that ATCC 42981 cells contain more chromosomes and have a bigger genome size than those of ATCC 2623.

Acetic acid induces a programmed cell death process in the food spoilage yeast Zygosaccharomyces bailii.

Here we show that 320-800 mM acetic acid induces in Zygosaccharomyces bailii a programmed cell death (PCD) process that is inhibited by cycloheximide, is accompanied by structural and biochemical alterations typical of apoptosis, and occurs in cells with preserved mitochondrial and plasma membrane integrity (as revealed by rhodamine 123 (Rh123) and propidium iodide (PI) staining, respectively). Mitochondrial ultrastructural changes, namely decrease of the cristae number, formation of myelinic bodies and swelling were also seen. Exposure to acetic acid above 800 mM resulted in killing by necrosis. The occurrence of an acetic acid-induced active cell death process in Z. bailii reinforces the concept of a physiological role of the PCD in the normal yeast life cycle.