Single-Molecule Fluorescent In Situ Hybridization (smFISH) for RNA Detection in the Fungal Pathogen Candida albicans.

Candida albicans is the most prevalent human fungal pathogen. Its pathogenicity is linked to the ability of C. albicans to reversibly change morphology and to grow as yeast, pseudohyphae, or hyphal cells in response to environmental stimuli. Understanding the molecular regulation controlling those morphological switches remains a challenge that, if solved, could help eradicate C. albicans infections.While numerous studies investigated gene expression changes occurring during C. albicans 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 C. albicans, which allows one to quantify the differential expression of mRNAs in yeast, pseudohyphae, or hyphal cells. We quantified the expression of two mRNAs, a cell cycle-controlled mRNA (CLB2) and a transcription factor (EFG1), which show expression changes in the different morphological cell types and nutrient 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 C. albicans and other filamentous fungi, pathogenic or nonpathogenic.

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.

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.

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.

Photosynthetic production of glycerol by a recombinant cyanobacterium.

Cyanobacteria are prokaryotic organisms capable of oxygenic photosynthesis. Glycerol is an important commodity chemical. Introduction of phosphoglycerol phosphatase 2 from Saccharomyces cerevisiae into the model cyanobacterium Synechocystis sp. PCC6803 resulted in a mutant strain that produced a considerable amount of glycerol from light, water and COPhotosynthetic production . Mild salt stress (200 mM NaCl) on the cells led to an increase of the extracellular glycerol concentration of more than 20%. Under these conditions the mutant accumulated glycerol to an extracellular concentration of 14.3 mM after 17 days of culturing.

Engineering cyanobacteria for direct biofuel production from CO2.

For a sustainable future of our society it is essential to close the global carbon cycle. Oxidised forms of carbon, in particular CO2, can be used to synthesise energy-rich organic molecules. Engineered cyanobacteria have attracted attention as catalysts for the direct conversion of CO2 into reduced fuel compounds. Proof of principle for this approach has been provided for a vast range of commodity chemicals, mostly energy carriers, such as short chain and medium chain alcohols. More recently, research has focused on the photosynthetic production of compounds with higher added value, most notably terpenoids. Below we review the recent developments that have improved the state-of-the-art of this approach and speculate on future developments.

Synthesis of 2,3-butanediol by Synechocystis sp. PCC6803 via heterologous expression of a catabolic pathway from lactic acid- and enterobacteria.

The direct and efficient conversion of CO2 into liquid energy carriers and/or bulk chemicals is crucial for a sustainable future of modern society. Here we describe the production of 2,3-butanediol in Synechocystis sp. PCC6803 expressing a heterologous catabolic pathway derived from enteric- and lactic acid bacteria. This pathway is composed of an acetolactate synthase, an acetolactate decarboxylase and an acetoin reductase. Levels of up to 0.72 g/l (corresponding to 8 mmol/L) of C(4) products, including a level of 0.43 g/l (corresponding to 4.7 mmol/L) 2,3-butanediol production are observed with the genes encoding these three enzymes integrated into the cyanobacterial genome, as well as when they are plasmid encoded. Further optimization studies revealed that Synechocystis expresses significant levels of acetolactate synthase endogenously, particularly under conditions of restricted CO2 supply to the cells. Co-expression of a soluble transhydrogenase or of an NADPH-dependent acetoin reductase allows one to drive the last step of the engineered pathway to near completion, resulting in pure meso-2,3-butanediol being produced.

Light-induced alteration of c-di-GMP level controls motility of Synechocystis sp. PCC 6803.

Cph2 from the cyanobacterium Synechocystis sp. PCC 6803 is a hybrid photoreceptor that comprises an N-terminal module for red/far-red light reception and a C-terminal module switching between a blue- and a green-receptive state. This unusual photoreceptor exerts complex, light quality-dependent control of the motility of Synechocystis sp. PCC 6803 cells by inhibiting phototaxis towards blue light. Cph2 perceives blue light by its third GAF domain that bears all characteristics of a cyanobacteriochrome (CBCR) including photoconversion between green- and blue-absorbing states as well as formation of a bilin species simultaneously tethered to two cysteines, C994 and C1022. Upon blue light illumination the CBCR domain activates the subsequent C-terminal GGDEF domain, which catalyses formation of the second messenger c-di-GMP. Accordingly, expression of the CBCR-GGDEF module in Δcph2 mutant cells restores the blue light-dependent inhibition of motility. Additional expression of the N-terminal Cph2 fragment harbouring a red/far-red interconverting phytochrome fused to a c-di-GMP degrading EAL domain restores the complex behaviour of the intact Cph2 photosensor. c-di-GMP was shown to regulate flagellar and pili-based motility in several bacteria. Here we provide the first evidence that this universal bacterial second messenger is directly involved in the light-dependent regulation of cyanobacterial phototaxis.