Evaluation and comparison of colorimetric outputs for yeast-based biosensors in laboratory and point-of-use settings.

Recent research has shown the potential of yeast-based biosensors (YBBs) for point-of-use detection of pathogens and target molecules in saliva, blood, and urine samples. The choice of output can greatly affect the sensitivity, dynamic range, detection time, and ease-of-use of a sensor. For visual detection without the need for additional reagents or machinery, colorimetric outputs have shown great potential. Here, we evaluated the inducible generation of prodeoxyviolacein and proviolacein as colorimetric YBB outputs and benchmarked these against lycopene. The outputs were induced via the yeast mating pathway and were compared on agar plates, in liquid culture, and on paper slips. We found that all three outputs produced comparable pigment intensity on agar plates, making them applicable for bioengineering settings. In liquid media and on paper slips, lycopene resulted in a higher intensity pigment and a decreased time-of-detection.

Comparison of Experimental Approaches Used to Determine the Structure and Function of the Class D G Protein-Coupled Yeast α-Factor Receptor.

The Saccharomyces cerevisiae α-factor mating pheromone receptor (Ste2p) has been studied as a model for the large medically important family of G protein-coupled receptors. Diverse yeast genetic screens and high-throughput mutagenesis of STE2 identified a large number of loss-of-function, constitutively-active, dominant-negative, and intragenic second-site suppressor mutants as well as mutations that specifically affect pheromone binding. Facile genetic manipulation of Ste2p also aided in targeted biochemical approaches, such as probing the aqueous accessibility of substituted cysteine residues in order to identify the boundaries of the seven transmembrane segments, and the use of cysteine disulfide crosslinking to identify sites of intramolecular contacts in the transmembrane helix bundle of Ste2p and sites of contacts between the monomers in a Ste2p dimer. Recent publication of a series of high-resolution cryo-EM structures of Ste2p in ligand-free, agonist-bound and antagonist-bound states now makes it possible to evaluate the results of these genetic and biochemical strategies, in comparison to three-dimensional structures showing activation-related conformational changes. The results indicate that the genetic and biochemical strategies were generally effective, and provide guidance as to how best to apply these experimental strategies to other proteins. These strategies continue to be useful in defining mechanisms of signal transduction in the context of the available structures and suggest aspects of receptor function beyond what can be discerned from the available structures.

Multiplexed mRNA assembly into ribonucleoprotein particles plays an operon-like role in the control of yeast cell physiology.

Prokaryotes utilize polycistronic messages (operons) to co-translate proteins involved in the same biological processes. Whether eukaryotes achieve similar regulation by selectively assembling and translating monocistronic messages derived from different chromosomes is unknown. We employed transcript-specific RNA pulldowns and RNA-seq/RT-PCR to identify yeast mRNAs that co-precipitate as ribonucleoprotein (RNP) complexes. Consistent with the hypothesis of eukaryotic RNA operons, mRNAs encoding components of the mating pathway, heat shock proteins, and mitochondrial outer membrane proteins multiplex in trans, forming discrete messenger ribonucleoprotein (mRNP) complexes (called transperons). Chromatin capture and allele tagging experiments reveal that genes encoding multiplexed mRNAs physically interact; thus, RNA assembly may result from co-regulated gene expression. Transperon assembly and function depends upon histone H4, and its depletion leads to defects in RNA multiplexing, decreased pheromone responsiveness and mating, and increased heat shock sensitivity. We propose that intergenic associations and non-canonical histone H4 functions contribute to transperon formation in eukaryotic cells and regulate cell physiology.

Engineering G protein-coupled receptor signalling in yeast for biotechnological and medical purposes.

G protein-coupled receptors (GPCRs) comprise the largest class of membrane proteins in the human genome, with a common denominator of seven-transmembrane domains largely conserved among eukaryotes. Yeast is naturally armoured with three different GPCRs for pheromone and sugar sensing, with the pheromone pathway being extensively hijacked for characterising heterologous GPCR signalling in a model eukaryote. This review focusses on functional GPCR studies performed in yeast and on the elucidated hotspots for engineering, and discusses both endogenous and heterologous GPCR signalling. Key emphasis will be devoted to studies describing important engineering parameters to consider for successful coupling of GPCRs to the yeast mating pathway. We also review the various means of applying yeast for studying GPCRs, including the use of yeast armed with heterologous GPCRs as a platform for (i) deorphanisation of orphan receptors, (ii) metabolic engineering of yeast for production of bioactive products and (iii) medical applications related to pathogen detection and drug discovery. Finally, this review summarises the current challenges related to expression of functional membrane-bound GPCRs in yeast and discusses the opportunities to continue capitalising on yeast as a model chassis for functional GPCR signalling studies.

A second HD mating type sublocus of Flammulina velutipes is at least di-allelic and active: new primers for identification of HD-a and HD-b subloci.

BACKGROUND: Sexual development in Flammulina velutipes is controlled by two different mating type loci (HD and PR). The HD locus contains homeodomain (Hd) genes on two separate HD subloci: HD-a and HD-b. While the functionality of the HD-b sublocus has been largely confirmed, the status and content of the HD-a sublocus has remained unclear. METHODS: To examine the function of the HD-a sublocus, genome sequences of a series of F. velutipes strains were analyzed and tested through series of amplification by specific primer sets. Furthermore, activity of di-allelic HD-a locus was confirmed by crossing strains with different combinations of HD-a and HD-b subloci. RESULTS: Sublocus HD-b contained a large variety of fixed Hd1/Hd2 gene pairs, while the HD-a sublocus either contained a conserved Hd2 gene or, a newly discovered Hd1 gene that was also conserved. Identification of whole HD loci, that is, the contents of HD-a and HD-b subloci in a strain, revealed that strains with similar HD-b subloci could still form normal dikaryons if the two genes at the HD-a sublocus differed. At least di-allelic HD-a sublocus, is thus indicated to be actively involved in mating type compatibility. CONCLUSIONS: HD-a sublocus is active and di-allelic. Using the new information on the HD subloci, primers sets were developed that specifically amplify HD-a or HD-b subloci in the majority of F. velutipes strains. In this way, unknown HD mating types of F. velutipes can now be quickly identified, and HD mating type compatibility conferred by HD-a or HD-b can be confirmed by PCR.

A System for Gene Expression Noise Control in Yeast.

Gene expression noise arises from stochastic variation in the synthesis and degradation of mRNA and protein molecules and creates differences in protein numbers across populations of genetically identical cells. Such variability can lead to imprecision and reduced performance of both native and synthetic networks. In principle, gene expression noise can be controlled through the rates of transcription, translation and degradation, such that different combinations of those rates lead to the same protein concentrations but at different noise levels. Here, we present a "noise tuner" which allows orthogonal control over the transcription and the mRNA degradation rates by two different inducer molecules. Combining experiments with theoretical analysis, we show that in this system the noise is largely determined by the transcription rate, whereas the mean expression is determined by both the transcription rate and mRNA stability and can thus be decoupled from the noise. This noise tuner enables 2-fold changes in gene expression noise over a 5-fold range of mean protein levels. We demonstrated the efficacy of the noise tuner in a complex regulatory network by varying gene expression noise in the mating pathway of Saccharomyces cerevisiae, which allowed us to control the output noise and the mutual information transduced through the pathway. The noise tuner thus represents an effective tool of gene expression noise control, both to interrogate noise sensitivity of natural networks and enhance performance of synthetic circuits.

Real-Time Genetic Compensation Defines the Dynamic Demands of Feedback Control.

Biological signaling networks use feedback control to dynamically adjust their operation in real time. Traditional static genetic methods such as gene knockouts or rescue experiments can often identify the existence of feedback interactions but are unable to determine what feedback dynamics are required. Here, we implement a new strategy, closed-loop optogenetic compensation (CLOC), to address this problem. Using a custom-built hardware and software infrastructure, CLOC monitors, in real time, the output of a pathway deleted for a feedback regulator. A minimal model uses these measurements to calculate and deliver-on the fly-an optogenetically enabled transcriptional input designed to compensate for the effects of the feedback deletion. Application of CLOC to the yeast pheromone response pathway revealed surprisingly distinct dynamic requirements for three well-studied feedback regulators. CLOC, a marriage of control theory and traditional genetics, presents a broadly applicable methodology for defining the dynamic function of biological feedback regulators.

Robust Synthetic Circuits for Two-Dimensional Control of Gene Expression in Yeast.

Cellular phenotypes are the result of complex interactions between many components. Understanding and predicting the system level properties of the resulting networks requires the development of perturbation tools that can simultaneously and independently modulate multiple cellular variables. Here, we develop synthetic modules that use different arrangements of two transcriptional regulators to achieve either concurrent and independent control of the expression of two genes, or decoupled control of the mean and variance of a single gene. These modules constitute powerful tools to probe the quantitative attributes of network wiring and function.

Single-cell dynamics and variability of MAPK activity in a yeast differentiation pathway.

In response to pheromones, yeast cells activate a MAPK pathway to direct processes important for mating, including gene induction, cell-cycle arrest, and polarized cell growth. Although a variety of assays have been able to elucidate signaling activities at multiple steps in the pathway, measurements of MAPK activity during the pheromone response have remained elusive, and our understanding of single-cell signaling behavior is incomplete. Using a yeast-optimized FRET-based mammalian Erk-activity reporter to monitor Fus3 and Kss1 activity in live yeast cells, we demonstrate that overall mating MAPK activity exhibits distinct temporal dynamics, rapid reversibility, and a graded dose dependence around the KD of the receptor, where phenotypic transitions occur. The complex dose response was found to be largely a consequence of two feedbacks involving cyclin-mediated scaffold phosphorylation and Fus3 autoregulation. Distinct cell cycle-dependent response patterns comprised a large portion of the cell-to-cell variability at each dose, constituting the major source of extrinsic noise in coupling activity to downstream gene-expression responses. Additionally, we found diverse spatial MAPK activity patterns to emerge over time in cells undergoing default, gradient, and true mating responses. Furthermore, ramping up and rapid loss of activity were closely associated with zygote formation in mating-cell pairs, supporting a role for elevated MAPK activity in successful cell fusion and morphogenic reorganization. Altogether, these findings present a detailed view of spatiotemporal MAPK activity during the pheromone response, elucidating its role in mediating complex long-term developmental fates in a unicellular differentiation system.