Carlos Andrés Martínez-Garay

Aquatic environments could be reservoirs of pathogenic yeasts with acquired antifungal resistance. The susceptibility to antifungal agents of yeasts present in the wastewater and natural waters of the city of Cali was evaluated. Samples were taken from two types of water: drinking water (Meléndez River, drinking water treatment plant “Puerto Mallarino” in the Cauca River) and wastewater (South Channel of the Cauca River, “Cañaveralejo-PTAR” wastewater treatment plant). Physico-chemical parameters, heavy metal concentration, and yeast levels were determined using standard procedures. Yeasts were identified using API 20 C AUX (BioMérieux) and sequence analysis of the ITS1-5.8S-ITS2 and D1/D2 regions of the large subunit of the ribosome. Susceptibility assays against fluconazole and amphotericin B using the minimum inhibitory concentration (MIC) test were determined using the microdilution method. The influence of physico-chemical parameters and heavy metals was established using principal component analysis (PCA). Yeast counts were higher at WWTP “PTAR” and lower at Melendez River, as expected. A total of 14 genera and 21 yeast species was identified, and the genus Candida was present at all locations. Susceptibility tests showed a 32.7% resistance profile to fluconazole in the order DWTP “Puerto Mallarino = WWTP “PTAR” > South Channel “Navarro”. There were significant differences (p < 0.05) in the physico-chemical parameters/concentration of heavy metals and yeast levels between the aquatic systems under study. A positive association was observed between yeast levels and total dissolved solids, nitrate levels, and Cr at the “PTAR” WWTP; conductivity, Zn, and Cu in the South Channel; and the presence of Pb in the “Puerto Mallarino” DWTP. Rhodotorula mucilaginosa, Candida albicans, and Candida sp. 1 were influenced by Cr and Cd, and Diutina catelunata was influenced by Fe (p < 0.05). The water systems explored in this study showed different yeast levels and susceptibility profiles, and, therefore, possible genetic differences among populations of the same species, and different physico-chemical and heavy metals concentrations, which were probably modulating the antifungal-resistant yeasts. All these aquatic systems discharge their content into the Cauca River. We highlight the importance to further investigate if these resistant communities continue to other locations in the second largest river of Colombia and to determine the risk posed to humans and animals.

Introducción: Los carotenoides son fuente importante de actividades biológicas funcionales, tales como antioxidantes o antimicrobianas, además de tener gran impacto a nivel industrial, ya sea en cosmética o suplementación animal en acuacultura. Se han reportado varias moléculas novedosas a partir de aislamientos en Latinoamérica, principalmente en la Patagonia, Argentina. Sin embargo, no hay reportes en Colombia que evalúen la producción de carotenoides en levaduras nativas pigmentadas. Objetivo: Se evaluó la capacidad de producción de carotenoides en levaduras nativas aisladas de lagos, ríos y aguas residuales de la ciudad de Cali, Colombia. Materiales y métodos: Se caracterizaron 30 levaduras provenientes de dos colecciones. De estas se obtuvo su biomasa, rendimiento de carotenoides totales y producción de β-caroteno. Las cepas promisorias fueron identificadas secuenciando la región ITS1-5.8S-ITS2. Resultados: El mayor rendimiento en la extracción de pigmentos se obtuvo para las cepas P11A (84,36 ± 5,24 µg/g) y Rhodotorula paludigena CS13 (56,26 ± 7,08 µg/g), mientras que las concentraciones más altas de β-caroteno fueron 10,2 µg/mL (R. paludigena CS13) y 9,7 µg/mL (R. mucilaginosa/alborubescens P10A). La cinética de crecimiento y producción de pigmentos durante cinco días fue óptima para la cepa P11A, ya que hubo un aumento en el rendimiento de carotenoides totales 10 veces mayor (48 h: 109,62 µg/g, 120 h: 1403,10 µg/g). Conclusiones: En este estudio se encontró que levaduras aisladas de sistemas acuáticos son promisorias para la producción de pigmentos carotenoides (incluyendo β-caroteno), siendo su extracción y caracterización viable para futuros estudios biotecnológicos.

The yeast β-karyopherin Msn5 controls the SBF cell-cycle transcription factor, responsible for the periodic expression of CLN2 cyclin gene at G1/S, and the nuclear export of Cln2 protein. Here we show that Msn5 regulates Cln2 by an additional mechanism. Inactivation of Msn5 causes a severe reduction in the cellular content of Cln2. This occurs by a post-transcriptional mechanism, since CLN2 mRNA level is not importantly affected in asynchronous cultures. Cln2 stability is not significantly altered in msn5 cells and inactivation of Msn5 causes a reduction in protein level even when Cln2 is stabilized. Therefore, the reduced amount of Cln2 in msn5 cells is mainly due not to a higher rate of protein degradation but to a defect in Cln2 synthesis. In fact, analysis of polysome profiles indicated that Msn5 inactivation causes a shift of CLN2 and SWI5 mRNAs from heavy-polysomal to light-polysomal and non-polysomal fractions, supporting a defect in Cln2 and Swi5 protein synthesis in the msn5 mutant. The analysis of truncated versions of Cln2 and of chimeric cyclins combining distinct domains from Cln2 and the related Cln1 cyclin identified an internal region in Cln2 from 181 to 225 residues that when fused to GFP is able to confer Msn5-dependent regulation of protein cellular content. Finally, we showed that a high level of Cln2 is toxic in the absence of Msn5. In summary, we described that Msn5 is required for the proper protein synthesis of specific proteins, introducing a new level of control of cell cycle regulators. Graphical Abstract

All eukaryotic organisms rely on iron as an essential micronutrient for life because it participates as a redox-active cofactor in multiple biological processes. However, excess iron can generate reactive oxygen species that damage cellular macromolecules. The low solubility of ferric iron under physiological conditions increases the prevalence of iron deficiency anemia. A common strategy to treat iron deficiency consists of dietary iron supplementation. The baker's yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, but also as a feed supplement. In response to iron deficiency, the yeast Aft1 transcription factor activates cellular iron acquisition. However, when constitutively active, Aft1 inhibits growth probably due to iron toxicity. In this report, we have studied the consequences of using hyperactive AFT1 alleles, including AFT1-1UP, to increase yeast iron accumulation. We first characterized the iron sensitivity of cells expressing different constitutively active AFT1 alleles. We rescued the high iron sensitivity conferred by the AFT1 alleles by deleting the sphingolipid signaling kinase YPK1. We observed that the deletion of YPK1 exerts different effects on iron accumulation depending on the AFT1 allele and the environmental iron. Moreover, we determined that the impairment of the high-affinity iron transport system partially rescues the high iron toxicity of AFT1-1UP-expressing cells. Finally, we observed that AFT1-1UP inhibits oxygen consumption through activation of the RNA-binding protein Cth2. Deletion of CTH2 partially rescues the AFT1-1UP negative respiratory effect. Collectively, these results contribute to understand how the Aft1 transcription factor functions and the multiple consequences derived from its constitutive activation.

Fungi, including the yeast Saccharomyces cerevisiae, lack ferritin and use vacuoles as iron storage organelles. This work explored how plant ferritin expression influenced baker's yeast iron metabolism. Soybean seed ferritin H1 (SFerH1) and SFerH2 genes were cloned and expressed in yeast cells. Both soybean ferritins assembled as multimeric complexes, which bound yeast intracellular iron in vivo and, consequently, induced the activation of the genes expressed during iron scarcity. Soybean ferritin protected yeast cells that lacked the Ccc1 vacuolar iron detoxification transporter from toxic iron levels by reducing cellular oxidation, thus allowing growth at high iron concentrations. Interestingly, when simultaneously expressed in ccc1Δ cells, SFerH1 and SFerH2 assembled as heteropolymers, which further increased iron resistance and reduced the oxidative stress produced by excess iron compared to ferritin homopolymer complexes. Finally, soybean ferritin expression led to increased iron accumulation in both wild-type and ccc1Δ yeast cells at certain environmental iron concentrations.

Iron is an essential micronutrient for all eukaryotic organisms. However, the low solubility of ferric iron has tremendously increased the prevalence of iron deficiency anemia, especially in women and children, with dramatic consequences. Baker's yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, a fermentative microorganism, and a feed supplement. In this report, we explore the genetic diversity of 123 wild and domestic strains of S. cerevisiae isolated from different geographical origins and sources to characterize how yeast cells respond to elevated iron concentrations in the environment. By using two different forms of iron, we selected and characterized both iron-sensitive and iron-resistant yeast strains. We observed that when the iron concentration in the medium increases, iron-sensitive strains accumulate iron more rapidly than iron-resistant isolates. We observed that, consistent with excess iron leading to oxidative stress, the redox state of iron-sensitive strains was more oxidized than that of iron-resistant strains. Growth assays in the presence of different oxidative reagents ruled out that this phenotype was due to alterations in the general oxidative stress protection machinery. It was noteworthy that iron-resistant strains were more sensitive to iron deficiency conditions than iron-sensitive strains, which suggests that adaptation to either high or low iron is detrimental for the opposite condition. An initial gene expression analysis suggested that alterations in iron homeostasis genes could contribute to the different responses of distant iron-sensitive and iron-resistant yeast strains to elevated environmental iron levels.

Rot1 is an essential yeast protein that has been related to cell wall biosynthesis, actin cytoskeleton dynamics and protein folding. Rot1 is an N-gly