Slr1670 from Synechocystis sp. PCC 6803 Is Required for the Re-assimilation of the Osmolyte Glucosylglycerol

When subjected to mild salt stress, the cyanobacterium Synechocystis sp. PCC 6803 produces small amounts of glycerol through an as of yet unidentified pathway. Here, we show that this glycerol is a degradation product of the main osmolyte of this organism, glucosylglycerol (GG). Inactivation of ggpS, encoding the first step of GG-synthesis, abolished de novo synthesis of glycerol, while the ability to hydrolyze exogenously supplied glucoslylglycerol was unimpaired. Inactivation of glpK, encoding glycerol kinase, had no effect on glycerol synthesis. Inactivation of slr1670, encoding a GHL5-type putative glycoside hydrolase, abolished de novo synthesis of glycerol, as well as hydrolysis of GG, and led to increased intracellular concentrations of this osmolyte. Slr1670 therefore presumably displays GG hydrolase activity. A gene homologous to the one encoded by slr1670 occurs in a wide range of cyanobacteria, proteobacteria, and archaea. In cyanobacteria, it co-occurs with genes involved in GG-synthesis

Using the AKAR3-EV biosensor to assess Sch9p- and PKA-signalling in budding yeast

Budding yeast uses the TORC1-Sch9p and cAMP-PKA signalling pathways to regulate adaptations to changing nutrient environments. Dynamic and single-cell measurements of the activity of these cascades will improve our understanding of the cellular adaptation of yeast. Here, we employed the AKAR3-EV biosensor developed for mammalian cells to measure the cellular phosphorylation status determined by Sch9p and PKA activity in budding yeast. Using various mutant strains and inhibitors, we show that AKAR3-EV measures the Sch9p- and PKA-dependent phosphorylation status in intact yeast cells. At the single-cell level, we found that the phosphorylation responses are homogenous for glucose, sucrose, and fructose, but heterogeneous for mannose. Cells that start to grow after a transition to mannose correspond to higher normalized Förster resonance energy transfer (FRET) levels, in line with the involvement of Sch9p and PKA pathways to stimulate growth-related processes. The Sch9p and PKA pathways have a relatively high affinity for glucose (K0.5 of 0.24 mM) under glucose-derepressed conditions. Lastly, steady-state FRET levels of AKAR3-EV seem to be independent of growth rates, suggesting that Sch9p- and PKA-dependent phosphorylation activities are transient responses to nutrient transitions. We believe that the AKAR3-EV sensor is an excellent addition to the biosensor arsenal for illuminating cellular adaptation in single yeast cells.</p

Candida albicans smFISH data processing pipeline

&lt;p&gt;Data processing pipeline connected to the publication: &quot;Single-molecule Fluorescent In Situ Hybridization (smFISH) for RNA detection in the fungal pathogen &lt;em&gt;Candida albicans&lt;/em&gt;&quot;&lt;/p&gt; &lt;p&gt;&lt;strong&gt;Abstract&lt;/strong&gt; &lt;em&gt;Candida albicans&lt;/em&gt; is the most prevalent human fungal pathogen. Its pathogenicity is linked to the ability of &lt;em&gt;C. albicans&lt;/em&gt; to reversibly change morphology and to grow as yeast, pseudohyphal or hyphal cells in response to environmental stimuli. Understanding the molecular regulation controlling those morphological switches remains a challenge that, if solved, could help fight &lt;em&gt;C. albicans&lt;/em&gt; infections. While numerous studies investigated gene expression changes occurring during &lt;em&gt;C. albicans&lt;/em&gt; morphological switches using bulk approaches (e.g., RNA sequencing), here we describe a single-cell and single-molecule RNA imaging and analysis protocol to measure absolute mRNA counts in morphologically intact cells. To detect endogenous mRNAs in single fixed cells, we optimized a single molecule fluorescent in situ hybridization (smFISH) protocol for &lt;em&gt;C. albicans&lt;/em&gt;, which allows one to quantify the differential expression of mRNAs in yeast, pseudohyphae or hyphal cells. We quantified the expression of two mRNAs, cell cycle-controlled mRNA (&lt;em&gt;CLB2&lt;/em&gt;) and a transcription regulator &lt;em&gt;(EFG1&lt;/em&gt;), which show differential expression in the different morphological cell types and in different nutrients conditions. In this protocol we described in detail the major steps of this approach: growth and fixation, hybridization, imaging, cell-segmentation and mRNA spot analysis. Raw data is provided with the protocol to favor reproducibility. This approach could benefit the molecular characterization of &lt;em&gt;C. albicans&lt;/em&gt; and other filamentous fungi, pathogenic or non-pathogenic.&lt;/p&gt;If you use this software, please cite it as below

pH dependencies of glycolytic enzymes of yeast under in vivo-like assay conditions

Under carbon source transitions, the intracellular pH of Saccharomyces cerevisiae is subject to change. Dynamics in pH modulate the activity of the glycolytic enzymes, resulting in a change in glycolytic flux and ultimately cell growth. To understand how pH affects the global behavior of glycolysis and ethanol fermentation, we measured the activity of the glycolytic and fermentative enzymes in S. cerevisiae under in vivo-like conditions at different pH. We demonstrate that glycolytic enzymes exhibit differential pH dependencies, and optima, in the pH range observed during carbon source transitions. The forward reaction of GAPDH shows the highest decrease in activity, 83%, during a simulated feast/famine regime upon glucose removal (cytosolic pH drop from 7.1 to 6.4). We complement our biochemical characterization of the glycolytic enzymes by fitting the V max to the progression curves of product formation or decay over time. The fitting analysis shows that the observed changes in enzyme activities require changes in V max , but changes in K m cannot be excluded. Our study highlights the relevance of pH as a key player in metabolic regulation and provides a large set of quantitative data that can be explored to improve our understanding of metabolism in dynamic environments

pH dependencies of glycolytic enzymes of yeast under in vivo-like assay conditions

Under carbon source transitions, the intracellular pH of Saccharomyces cerevisiae is subject to change. Dynamics in pH modulate the activity of the glycolytic enzymes, resulting in a change in glycolytic flux and ultimately cell growth. To understand how pH affects the global behavior of glycolysis and ethanol fermentation, we measured the activity of the glycolytic and fermentative enzymes in S. cerevisiae under in vivo-like conditions at different pH. We demonstrate that glycolytic enzymes exhibit differential pH dependencies, and optima, in the pH range observed during carbon source transitions. The forward reaction of GAPDH shows the highest decrease in activity, 83%, during a simulated feast/famine regime upon glucose removal (cytosolic pH drop from 7.1 to 6.4). We complement our biochemical characterization of the glycolytic enzymes by fitting the Vmax to the progression curves of product formation or decay over time. The fitting analysis shows that the observed changes in enzyme activities require changes in Vmax, but changes in Km cannot be excluded. Our study highlights the relevance of pH as a key player in metabolic regulation and provides a large set of quantitative data that can be explored to improve our understanding of metabolism in dynamic environments

Cyclin CLB2 mRNA localization determines efficient protein synthesis to orchestrate bud growth and cell cycle progression

mRNA localization to subcellular compartments has been reported across all kingdoms of life and it is generally believed to promote asymmetric protein synthesis and localization. In striking contrast to previous observations, we show that in S. cerevisiae the B-type cyclin CLB2 mRNA is localized and translated in the yeast bud, while the Clb2 protein, a key regulator of mitosis progression, is concentrated in the mother nucleus. Using single-molecule RNA imaging in fixed (smFISH) and living cells (MS2 system), we show that the CLB2 mRNA is transported to the yeast bud by the She2-She3 complex, via an mRNA ZIP-code situated in the coding sequence. In CLB2 mRNA localization mutants, Clb2 protein synthesis in the bud is decreased resulting in changes in cell cycle distribution and genetic instability. Altogether, we propose that CLB2 mRNA localization acts as a sensor for bud development to couple cell growth and cell cycle progression, revealing a novel function for mRNA localization