Highlights d Rtg1/3 are key determinants of sphingolipid synthesis in the fungus Candida albicans d Rtg1/3 regulate enzymes involved in the synthesis of sphingolipids' building blocks d The stability of Rtg1/3 is dependent upon TOR and the ubiquitin ligase Ubr1 d The Rtg1/3 system is activated upon engulfment of C. albicans by human neutrophils
A yeast deletion mutation in the nuclear-encoded gene, AFO1, which codes for a mitochondrial ribosomal protein, led to slow growth on glucose, the inability to grow on glycerol or ethanol, and loss of mitochondrial DNA and respiration. We noticed that afo1-yeast readily obtains secondary mutations that suppress aspects of this phenotype, including its growth defect. We characterized and identified a dominant missense suppressor mutation in the ATP3 gene. Comparing isogenic slowly growing rho-zero and rapidly growing suppressed afo1- strains under carefully controlled fermentation conditions showed that energy charge was not significantly different between strains and was not causal for the observed growth properties. Surprisingly, in a wild-type background, the dominant suppressor allele of ATP3 still allowed respiratory growth but increased the petite frequency. Similarly, a slow-growing respiratory deficient afo1-strain displayed an about twofold increase in spontaneous frequency of point mutations (comparable to the rho-zero strain) while the suppressed strain showed mutation frequency comparable to the repiratory-competent WT strain. We conclude, that phenotypes that result from afo1- are mostly explained by rapidly emerging mutations that compensate for the slow growth that typically follows respiratory deficiency.
Abstract:The multi-fixed-bed bioreactor "Multiferm" provides a downscaled system for the development of fixed-bed processes with immobilized microorganisms or cells. It consists of twelve single fixed-bed units that can be operated independently. Different bacterial strains, carriers used for immobilization, media or flow rates can be studied in one compact experimental set-up. Experiments can be run in parallel to allow for determination of experimental errors. In these studies several continuous cultivations were carried out with Lactobacillus delbrueckii ssp. bulgaricus using different dilution rates and culture durations as proof-ofconcept for the Multiferm. Parameters relevant for process design such as reproducibility of simultaneously performed cultures, optimal dilution rate and long-term performance were determined by means of volume specific lactate and biomass productivity. First indications on a reduced growth rate of the cells due to immobilization were found.
Many yeasts differentiate into multicellular phenotypes in adverse environmental conditions. Here we investigate pseudohyphal growth in Komagataella phaffii and the involvement of the flocculin (FLO) gene family in its regulation. The K. phaffii FLO family consists of 13 members, and the conditions inducing pseudohyphal growth are different from Saccharomyces cerevisiae. So far, this phenotype was only observed when K. phaffii was cultivated at slow growth rates in glucose-limited chemostats, but not upon nitrogen starvation or the presence of fusel alcohols. Transcriptional analysis identified that FLO11, FLO400 and FLO5–1 are involved in the phenotype, all being controlled by the transcriptional regulator Flo8. The three genes exhibit a complex mechanism of expression and repression during transition from yeast to pseudohyphal form. Unlike in S. cerevisiae, deletion of FLO11 does not completely prevent the phenotype. In contrast, deletion of FLO400 or FLO5–1 prevents pseudohyphae formation, and hampers FLO11 expression. FAIRE-Seq data shows that the expression and repression of FLO400 and FLO5–1 are correlated to open or closed chromatin regions upstream of these genes, respectively. Our findings indicate that K. phaffii Flo400 and/or Flo5–1 act as upstream signals that lead to the induction of FLO11 upon glucose limitation in chemostats at slow growth and chromatin modulation is involved in the regulation of their expression.
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