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ABSTRACT: Extremely low specific growth rates (below 0.01 h(-1) ) represent a largely unexplored area of microbial physiology. In this study, anaerobic, glucose-limited retentostats were used to analyse physiological and genome-wide transcriptional responses of Saccharomyces cerevisiae to cultivation at near-zero specific growth rates. While quiescence is typically investigated as a result of carbon starvation, cells in retentostat are fed by small, but continuous carbon and energy supply. Yeast cells cultivated near-zero specific growth rates, while metabolically active, exhibited characteristics previously associated with quiescence, including accumulation of storage polymers and an increased expression of genes involved in exit from the cell cycle into G(0) . Unexpectedly, analysis of transcriptome data from retentostat and chemostat cultures showed, as specific growth rate was decreased, that quiescence-related transcriptional responses were already set in at specific growth rates above 0.025 h(-1) . These observations stress the need for systematic dissection of physiological responses to slow growth, quiescence, ageing and starvation and indicate that controlled cultivation systems such as retentostats can contribute to this goal. Furthermore, cells in retentostat do not (or hardly) divide while remaining metabolically active, which emulates the physiological status of metazoan post-mitotic cells. We propose retentostat as a powerful cultivation tool to investigate chronological ageing-related processes.
FEMS Yeast Research 12/2011; 11(8):603-20. · 2.40 Impact Factor
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ABSTRACT: To establish more advanced models of molecular dynamics within cells, protein characteristics such as turnover rate and absolute instead of relative abundance have to be analyzed. We applied a proteomics strategy to analyze protein degradation and abundance in Saccharomyces cerevisiae. We used steady-state chemostat cultures to ascertain well-defined growth conditions and nitrogen limited media, which allowed us to rapidly switch from (14)N to (15)N-isotope containing media and to monitor the decay of the (14)N mono-isotope signals in time. We acquired both protein abundance information and degradation rates of 641 proteins. Half-lives of individual proteins were very diverse under nitrogen-limited steady-state conditions, ranging from less than 30 min to over 20 h. Proteins that act as single physical complexes do not always show alike half-lives. For example the chaperonin-containing TCP-1 complex showed similar intermediate half-lives ranging from 7 to 20 h. In contrast, the ribosome exhibited a wide diversity of half-lives ranging from 2.5 to over 20 h, although their cellular abundances were rather similar. The stabilities of proteins involved in the central sugar metabolism were found to be intermediary, except for the glycolytic enzymes Hxk1p and Fba1p and the TCA-cycle proteins Lsc2p and Kgd1p, which showed half-lives of over 20 h. These data stress the need for inclusion of quantitative data of protein turn-over rates in yeast systems biology.
Molecular BioSystems 12/2011; 7(12):3316-26. · 3.53 Impact Factor
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ABSTRACT: To investigate the effect of anaerobiosis on the Saccharomyces cerevisiae mitochondrial proteome and the formation of respiratory chain and other protein complexes, we analyzed mitochondrial protein extracts that were enriched from lysates of aerobic and anaerobic steady-state chemostat cultures. We chose an innovative approach in which native mitochondrial membrane protein complexes were separated by 1-D blue native PAGE, which was combined with quantitative analysis of each complex subunit using stable isotope labeling. LC-FT(ICR)-MS/MS analysis was applied to identify and quantify the mitochondrial proteins. In addition, to establish if changes in mitochondrial complex composition occurred under anaerobiosis, we investigated the 1-D blue native PAGE protein migration patterns by Pearson correlation analysis. Surprisingly, we discovered that under anaerobic conditions, where the yeast respiratory chain is not active, the respiratory chain supercomplexes, such as complex V dimer, complex (III)(2)(IV)(2) and complex (III)(2)(IV) were still present, although at reduced levels. Pearson correlation analysis showed that the composition of the mitochondrial complexes was unchanged under aerobic or anaerobic conditions, with the exception of complex II. In addition, this latter approach allowed screening for possible novel complex interaction partners, since for example protein Aim38p, with a yet unknown function, was identified as a possible component of respiratory chain complex IV.
Proteomics 09/2009; 9(20):4787-98. · 4.43 Impact Factor
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ABSTRACT: Growth at near-zero specific growth rates is a largely unexplored area of yeast physiology. To investigate the physiology of Saccharomyces cerevisiae under these conditions, the effluent removal pipe of anaerobic, glucose-limited chemostat culture (dilution rate, 0.025 h(-1)) was fitted with a 0.22-microm-pore-size polypropylene filter unit. This setup enabled prolonged cultivation with complete cell retention. After 22 days of cultivation, specific growth rates had decreased below 0.001 h(-1) (doubling time of >700 h). Over this period, viability of the retentostat cultures decreased to ca. 80%. The viable biomass concentration in the retentostats could be accurately predicted by a maintenance coefficient of 0.50 mmol of glucose g(-1) of biomass h(-1) calculated from anaerobic, glucose-limited chemostat cultures grown at dilution rates of 0.025 to 0.20 h(-1). This indicated that, in contrast to the situation in several prokaryotes, maintenance energy requirements in S. cerevisiae do not substantially change at near-zero specific growth rates. After 22 days of retentostat cultivation, glucose metabolism was predominantly geared toward alcoholic fermentation to meet maintenance energy requirements. The strict correlation between glycerol production and biomass formation observed at higher specific growth rates was not maintained at the near-zero growth rates reached in the retentostat cultures. In addition to glycerol, the organic acids acetate, d-lactate, and succinate were produced at low rates during prolonged retentostat cultivation. This study identifies robustness and by-product formation as key issues in attempts to uncouple growth and product formation in S. cerevisiae.
Applied and environmental microbiology 07/2009; 75(17):5607-14. · 3.69 Impact Factor
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ABSTRACT: Transcriptional responses of the yeast Saccharomyces cerevisiae to Zn availability were investigated at a fixed specific growth rate under limiting and abundant Zn concentrations in chemostat culture. To investigate the context dependency of this transcriptional response and eliminate growth rate-dependent variations in transcription, yeast was grown under several chemostat regimens, resulting in various carbon (glucose), nitrogen (ammonium), zinc, and oxygen supplies. A robust set of genes that responded consistently to Zn limitation was identified, and the set enabled the definition of the Zn-specific Zap1p regulon, comprised of 26 genes and characterized by a broader zinc-responsive element consensus (MHHAACCBYNMRGGT) than so far described. Most surprising was the Zn-dependent regulation of genes involved in storage carbohydrate metabolism. Their concerted down-regulation was physiologically relevant as revealed by a substantial decrease in glycogen and trehalose cellular content under Zn limitation. An unexpectedly large number of genes were synergistically or antagonistically regulated by oxygen and Zn availability. This combinatorial regulation suggested a more prominent involvement of Zn in mitochondrial biogenesis and function than hitherto identified.
Applied and environmental microbiology 01/2008; 73(23):7680-92. · 3.69 Impact Factor
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ABSTRACT: Saccharomyces cerevisiae is unique among yeasts in its ability to grow rapidly in the complete absence of oxygen. S. cerevisiae is therefore an ideal eukaryotic model to study physiological adaptation to anaerobiosis. Recent transcriptome analyses have identified hundreds of genes that are transcriptionally regulated by oxygen availability but the relevance of this cellular response has not been systematically investigated at the key control level of the proteome. Therefore, the proteomic response of S. cerevisiae to anaerobiosis was investigated using metabolic stable-isotope labelling in aerobic and anaerobic glucose-limited chemostat cultures, followed by relative quantification of protein expression. Using independent replicate cultures and stringent statistical filtering, a robust dataset of 474 quantified proteins was generated, of which 249 showed differential expression levels. While some of these changes were consistent with previous transcriptome studies, many of the responses of S. cerevisiae to oxygen availability were, to our knowledge, previously unreported. Comparison of transcriptomes and proteomes from identical cultivations yielded strong evidence for post-transcriptional regulation of key cellular processes, including glycolysis, amino-acyl-tRNA synthesis, purine nucleotide synthesis and amino acid biosynthesis. The use of chemostat cultures provided well-controlled and reproducible culture conditions, which are essential for generating robust datasets at different cellular information levels. Integration of transcriptome and proteome data led to new insights into the physiology of anaerobically growing yeast that would not have been apparent from differential analyses at either the mRNA or protein level alone, thus illustrating the power of multi-level studies in yeast systems biology.
Microbiology 12/2007; 153(Pt 11):3864-78. · 3.06 Impact Factor
