Pheromone-inducible expression vectors for fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an attractive host for heterologous gene expression. However, expression systems for industrially viable large-scale fermentations are scarce. Several inducible expression vectors for S. pombe have been reported, with the strong thiamine-repressible nmt1+ promoter or derivatives thereof most commonly employed. Previously, the promoter regions of the genes sxa2+ and rep1+ were utilized to couple pheromone signaling to the expression of reporter genes for quantitative assessment of the cellular response to mating pheromones. Here, we exploit these promoters to serve as highly effective, plasmid-based inducible expression systems for S. pombe. Simply by adding synthetic P-factor pheromone, both promoters conferred 50-60% higher peak expression levels than the nmt1+ promoter. Full induction was significantly faster than observed for nmt1+-based expression platforms. Furthermore, the sxa2+ promoter showed very low basal activity and an overall 584-fold induction by synthetic P-factor pheromone. The dose-response curves of both promoters were assessed, providing the opportunity for facile tuning of the expression level by modulating P-factor concentration. Since the expression plasmids relying on the sxa2+ and rep1+ promoters require neither medium exchange nor glucose/thiamine starvation, they proved to be very convenient in handling. Hence, these expression vectors will improve the palette of valuable genetic tools for S. pombe, applicable to both basic research and biotechnology.

Tuning the Sensitivity of the PDR5 Promoter-Based Detection of Diclofenac in Yeast Biosensors.

The commonly used drug diclofenac is an important environmental anthropogenic pollutant. Currently, detection of diclofenac is mainly based on chemical and physical methods. Here we describe a yeast biosensor that drives the diclofenac-dependent expression of a recombinant fluorescent protein from the authentic promoter of the PDR5 gene. This key component of the pleiotropic drug response encodes a multidrug transporter that is involved in cellular detoxification. We analyse the effects on diclofenac sensitivity of artificial PDR5 promoter derivatives in wild-type and various yeast mutant strains. This approach enabled us to generate sensor strains with elevated drug sensitivity.

Hydrophobin-Based Surface Engineering for Sensitive and Robust Quantification of Yeast Pheromones.

Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.

A yeast pheromone-based inter-species communication system.

We report on a pheromone-based inter-species communication system, allowing for a controlled cell-cell communication between the two species Saccharomyces cerevisiae and Schizosaccharomyces pombe as a proof of principle. It exploits the mating response pathways of the two yeast species employing the pheromones, α- or P-factor, as signaling molecules. The authentic and chimeric pheromone-encoding genes were engineered to code for the P-factor in S. cerevisiae and the α-factor in S. pombe. Upon transformation of the respective constructs, cells were enabled to express the mating pheromone of the opposite species. The supernatant of cultures of S. pombe cells expressing α-factor were able to induce a G1 arrest in the cell cycle, a change in morphology to the typical shmoo effect and expression driven by the pheromone-responsive FIG1 promoter in S. cerevisiae. The supernatant of cultures of S. cerevisiae cells expressing P-factor similarly induced cell cycle arrest in G1, an alteration in morphology typical for mating as well as the activation of the pheromone-responsive promoters of the rep1 and sxa2 genes in a pheromone-hypersensitive reporter strain of S. pombe. Apparently, both heterologous pheromones were correctly processed and secreted in an active form by the cells of the other species. Our data clearly show that the species-specific pheromone systems of yeast species can be exploited for a controlled inter-species communication.

Genetic modules for α-factor pheromone controlled growth regulation of Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a commonly used microorganism in the biotechnological industry. For the industrial heterologous production of compounds, it is of great advantage to work with growth-controllable yeast strains. In our work, we utilized the natural pheromone system of S. cerevisiae and generated a set of different strains possessing an α-pheromone controllable growth behavior. Naturally, the α-factor pheromone is involved in communication between haploid S. cerevisiae cells. Perception of the pheromone initiates several cellular changes, enabling the cells to prepare for an upcoming mating event. We exploited this natural pheromone response system and developed two different plasmid-based modules, in which the target genes, MET15 and FAR1, are under control of the α-factor sensitive FIG1 promoter for a controlled expression in S. cerevisiae. Whereas expression of MET15 led to a growth induction, FAR1 expression inhibited growth. The utilization of low copy number or high copy number plasmids for target gene expression and different concentrations of α-factor allow a finely adjustable control of yeast growth rate.

Overexpression of ctr1Δ300, a high-affinity copper transporter with deletion of the cytosolic C-terminus in Saccharomyces cerevisiae under excess copper, leads to disruption of transition metal homeostasis and transcriptional remodelling of cellular processes.

In an approach to generating Saccharomyces cerevisiae strains with increased intracellular copper amounts for technical applications, we overexpressed the copper transporter CTR1 and a variant of CTR1 with a truncation in the C-terminus after the 300th amino acid (ctr1Δ300). We determined the copper sensitivity of the generated strains and used inductively coupled plasma spectrometry analysis (ICP-OES and ICP-MS) to investigate the effects of overexpression of both constructs under excess copper on the cellular content of different elements in S. cerevisiae. In addition, we performed DNA microarray analysis to obtain the gene expression profile under the changed element contents. Overexpression of CTR1 increased the copper content in the cells to 160% and 78 genes were differentially regulated. Overexpression of the truncated ctr1Δ300 resulted in an increased copper, iron and zinc content of > 200% and 980 genes showed differential expression. We found that transition metal ion homeostasis was disrupted in ctr1Δ300-overexpressing strains under excess copper and that this was combined with a transcriptional remodelling of cellular processes.

Temporal and spatial properties of a yeast multi-cellular amplification system based on signal molecule diffusion.

We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α-factor as key signaling molecule between two cell types. One cell type represents the α-factor secreting sensor part and the other the reporter part emitting fluorescence upon activation. Although multi-cellular signaling systems promise higher specificity and modularity, the complex interaction of the cells makes prediction of sensor performance difficult. To test the maximum distance and response time between sensor and reporter cells, the two cell types were spatially separated in defined compartments of agarose hydrogel (5 x 5 mm) and reconnected by diffusion of the yeast pheromone. Different ratios of sensor to reporter cells were tested to evaluate the minimum amount of sensor cells required for signal transduction. Even the smallest ratio, one α-factor-secreting cell to twenty reporter cells, generated a distinct fluorescence signal. When using a 1:1 ratio, the secreted pheromone induced fluorescence in a distance of up to four millimeters after six hours. We conclude from both our experimental results and a mathematical diffusion model that in our approach: (1) the maximum dimension of separated compartments should not exceed five millimeters in gradient direction; and (2) the time-limiting step is not diffusion of the signaling molecule but production of the reporter protein.

Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium.

Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

Layer thickness of hydrophobin films leads to oscillation in wettability.

In nanobiotechnology, the properties of surfaces are often key to sensor applications. If analytes possess a low tolerance or affinity regarding the sensory substrate (surface), then the setup of mediators may be indicated. Hydrophobins enable biocompatible surface functionalization without significant restrictions of the physicochemical substrate properties. Because of the imperfect formation of hydrophobin films, a high variation in surface properties is observed. In this study, we report on the relation between the film thickness of hydrophobin-coated solid surfaces and their wettability. We found that the wettability of protein-coated surfaces strictly depends on the amount of adsorbed protein, as reflected in an oscillation of the contact angles of hydrophobin-coated silicon wafers. Fusion proteins of Ccg2 and HFBI, representatives of class I and II hydrophobins, document the influence of fused peptide tags on the wettability. The orientation of the first crystal nuclei plays a decisive role in the formation of the growing hydrophobin layers. Here, a simple method of deducing the film thickness of hydrophobin assemblies on solid surfaces is presented. The determination of the static contact angle allows the prediction of which part of the protein is exposed to possible analytes.

Nested Formation of Calcium Carbonate Polymorphs in a Bacterial Surface Membrane with a Graded Nanoconfinement: An Evolutionary Strategy to Ensure Bacterial Survival.

It is the intention of this study to elucidate the nested formation of calcium carbonate polymorphs or polyamorphs in the different nanosized compartments. With these observations, it can be concluded how the bacteria can survive in a harsh environment with high calcium carbonate supersaturation. The mechanisms of calcium carbonate precipitation at the surface membrane and at the underlying cell wall membrane of the thermophilic soil bacterium Geobacillus stearothermophilus DSM 13240 have been revealed by high-resolution transmission electron microscopy and atomic force microscopy. In this Gram-positive bacterium, nanopores in the surface layer (S-layer) and in the supporting cell wall polymers are nucleation sites for metastable calcium carbonate polymorphs and polyamorphs. In order to observe the different metastable forms, various reaction times and a low reaction temperature (4 °C) have been chosen. Calcium carbonate polymorphs nucleate in the confinement of nanosized pores (⌀ 3-5 nm) of the S-layer. The hydrous crystalline calcium carbonate (ikaite) is formed initially with [110] as the favored growth direction. It transforms into the anhydrous metastable vaterite by a solid-state transition. In a following reaction step, calcite is precipitated, caused by dissolution of vaterite in the aqueous solution. In the larger pores of the cell wall (⌀ 20-50 nm), hydrated amorphous calcium carbonate is grown, which transforms into metastable monohydrocalcite, aragonite, or calcite. Due to the sequence of reaction steps via various metastable phases, the bacteria gain time for chipping the partially mineralized S-layer, and forming a fresh S-layer (characteristic growth time about 20 min). Thus, the bacteria can survive in solutions with high calcium carbonate supersaturation under the conditions of forced biomineralization.

Formation of tubes during self-assembly of bacterial surface layers.

Based on experimental studies on tube formation during self-assembly of bacterial surface (S)-layers, a mechanistic model for describing the underlying basic mechanisms is proposed and the effect of process parameters on growth velocity and tube radius is investigated. The S-layer is modeled as a curved sheet with discrete binding sites for the association of monomers distributed along the S-layer edges. Reported changes of the tube radius owing to genetic protein modifications are explained within the framework of continuum mechanics. S-layer growth velocity and shape development are analyzed by Monte Carlo simulation in their dependence on the attachment and detachment frequencies of monomers at the S-layer. For curved S-layer patches, a criterion for the formation of S-layer tubes is derived. Accordingly, tubes can form only within a certain range of the initial monomer concentration. Furthermore, the effect of calcium ion concentration on tube formation is discussed, including recent experimental findings on the calcium effect.

Calcium dependent formation of tubular assemblies by recombinant S-layer proteins in vivo and in vitro.

Surface layer proteins have the appealing property to self-assemble in nanosized arrays in solution and on solid substrates. In this work, we characterize the formation of assembly structures of the recombinant surface layer protein SbsC of Geobacillus stearothermophilus ATTC 12980, which was tagged with enhanced green fluorescent protein and expressed in the yeast Saccharomyces cerevisiae. The tubular structures formed by the protein in vivo are retained upon bursting the cells by osmotic shock; however, their average length is decreased. During dialysis, monomers obtained by treatment with chaotropic chemicals recrystallize again to form tube-like structures. This process is strictly dependent on calcium (Ca(2+)) ions, with an optimal concentration of 10 mM. Further increase of the Ca(2+) concentration results in multiple non-productive nucleation points. We further show that the lengths of the S-layer assemblies increase with time and can be controlled by pH. After 48 h, the average length at pH 9.0 is 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications.

Hydrophobin signal sequence mediates efficient secretion of recombinant proteins in Pichia pastoris.

Pichia pastoris is an important eukaryotic organism for the expression, processing, and secretion of recombinant proteins. Here, the secretion of enhanced green fluorescent protein (EGFP) in P. pastoris by using three novel secretion signals originating from the HFBI and HFBII class 2 hydrophobins of Trichoderma reesei was investigated. EGFP was fused to the carboxyl terminus of hydrophobin secretion signals and expressed under the control of the constitutive GAP promoter. In every case, recombinant EGFP entered the secretory pathway of P. pastoris. SDS-polyacrylamide gel electrophoresis, Western blot analysis of the cells' supernatant, and fluorescence measurements on single-cell level via flow cytometry confirmed the efficient secretion of EGFP mediated by the novel secretion sequences. In conclusion, the data clearly show that the secretion sequences derived from HFBI and HFBII of T. reesei have the potential to achieve an efficient secretion of heterologous proteins in P. pastoris. Due to the small size of the hydrophobin-derived secretion signals, their coding sequence can be easily introduced to the gene of interest by PCR.

Expression and assembly of recombinant surface layer proteins in Saccharomyces cerevisiae.

Most bacterial surface layers (SLs) are formed by self-assembly of a single type of protein. Native and recombinant surface layer monomers are capable to self-assemble on solid substrates and in solution to highly regular nanosized arrays which make them attractive for nanobiotechnological applications. In this study, we expressed the surface layer protein SbsC of Bacillus stearothermophilus ATTC 12980, tagged with Enhanced Green Fluorescent Protein, in the yeast Saccharomyces cerevisiae. We observed a network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis, SL structures were formed de novo in the buds. SL assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point.

Applying dimorphic yeasts as model organisms to study mycelial growth: part 2. Use of mathematical simulations to identify different construction principles in yeast colonies.

The dimorphic yeasts Candida boidinii and Yarrowia lipolytica were applied as model organisms to study mycelial growth. A mathematical model of hybrid cellular automaton type was developed to analyze the impact of different biological assumptions on the predicted development of filamentous yeast colonies. The one-dimensional model described discrete cells and continuous distribution of nutrients. The simulation algorithm accounted for proliferation of cells, diffusion of nutrient, as well as biomass decay and recycling inside the mycelium. Simulations reproduced the spatio-temporal development of C. boidinii colonies when a diffusion-limited growth algorithm based on the growth of pseudohyphal cells was applied. Development of Y. lipolytica colonies could only be reproduced when proliferation was restricted to the colony boundary, and cell decay and biomass recycling were incorporated into the model. The results suggested that cytoplasm, which served as the secondary nutrient resource, had to be translocated inside the hyphal network.

Applying dimorphic yeasts as model organisms to study mycelial growth: part 1. Experimental investigation of the spatio-temporal development of filamentous yeast colonies.

Colony development of the dimorphic yeasts Yarrowia lipolytica and Candida boidinii on solid agar substrates under glucose limitation served as a model system for mycelial development of higher filamentous fungi. Strong differences were observed in the behaviour of both yeasts: C. boidinii colonies reached a final colony extension which was small compared to the size of the growth field. They formed cell-density profiles which steeply declined along the colony radius and no biomass decay processes could be detected. The stop of colony extension coincided with the depletion of glucose from the growth substrate. These findings supported the hypothesis that glucose-limited C. boidinii colonies can be regarded as populations of single cells which grow according to a diffusion-limited growth mechanism. Y. lipolytica colonies continued to extend after the depletion of the primary nutrient resource, glucose, until the populations covered the entire growth field which was accomplished by utilization of mycelial biomass.

A fluorescence-based yeast sensor for monitoring acetic acid.

Accumulation of acetic acid indicates an imbalance of the process due to a disturbed composition of the microorganisms. Hence, monitoring the acetic acid concentration is an important parameter to control the biogas process. Here, we describe the generation and validation of a fluorescence-based whole cell sensor for the detection of acetic acid based on the yeast Saccharomyces cerevisiae. Acetic acid induces the transcription of a subset of genes. The 5´-regulatory sequences (5´ URS) of these genes were cloned into a multicopy plasmid to drive the expression of a red fluorescent reporter gene. The 5´ URS of YGP1, encoding a cell wall-related glycoprotein, led to a 20-fold increase of fluorescence upon addition of 30 mM acetic acid to the media. We show that the system allows estimating the approximate concentration of acetic acid in condensation samples from a biogas plant. To avoid plasmid loss and increase the long-term stability of the sensor, we integrated the reporter construct into the yeast genome and tested the suitability of spores for long-term storage of sensor cells. Lowering the reporter gene's copy number resulted in a significant drop of the fluorescence, which can be compensated by applying a yeast pheromone-based signal amplification system.

Biomimetic estrogen sensor based on soft colloidal probes.

An increasing number of reports substantiate the link between emerging estrogenic pollutants and a variety of adverse effects including developmental disorders, infertility, cancer and neurological disorders, threatening public health as well as environment. The detection of the diverse classes of estrogenic and antiestrogenic substances is still challenging due to analytics which needs to cover the whole range of compounds acting on estrogen receptors and the complex estrogen pathways. In this proof-of-concept study, we report a novel biomimetic detection scheme based on the specific recognition of estrogenic ligands by estrogen sulfotransferase 1E1 (SULT1E1), which acts as one of the key enzymes in estrogen homeostasis. SULT1E1 was site-specifically immobilized on transparent glass slides via a hexahistidine-tag in a multi-step procedure. Soft colloidal probes (SCPs) covalently functionalized with ligands of SULT1E1, namely estrone and estradiol 17-(ß-D-glucuronide), served as adhesion probes. The various functionalization steps were analyzed and optimized using epifluorescence, confocal laser scanning as well as reflection interference contrast microscopy (RICM). A competitive SCP binding assay probing the elastic SCP deformation driven by the specific interaction between SCPs and the SULT1E1 decorated glass slides was employed in conjunction with an optical readout by RICM and automated image analysis to detect estrogenic compounds by their inhibition of SCP adhesion. This sensing concept has demonstrated exceptional specificity for estrogenic steroid compounds compared to structurally related substance classes and provides promising options for multiplexed assays and incorporation of other proteins of the endocrine system to fully capture the whole ensemble of hormonally active substances.

Picomolar glyphosate sensitivity of an optical particle-based sensor utilizing biomimetic interaction principles.

The continually growing use of glyphosate and its critically discussed health and biodiversity risks ask for fast, low cost, on-site sensing technologies for food and water. To address this problem, we designed a highly sensitive sensor built on the remarkably specific recognition of glyphosate by its physiological target enzyme 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPs). This principle is implemented in an interferometric sensor by using the recently established soft colloidal probe (SCP) technique. EPSPs was site-specifically immobilized on a transparent surface utilizing the self-assembling properties of circadian clock gene 2 hydrophobin chimera and homogeneity of the layer was evidenced by atomic force microscopy. Exposure of the enzyme decorated biochip to glyphosate containing samples causes formation of enzyme-analyte complexes and a competitive loss of available binding sites for glyphosate-functionalized poly(ethylene glycol) SCPs. Functionalization of the SCPs with different types of linker molecules and glyphosate was assessed employing confocal laser scanning microscopy as well as confocal Raman microspectroscopy. Overall, reflection interference contrast microscopy analysis of SCP-biochip interactions revealed a strong influence of linker length and glyphosate coupling position on the sensitivity of the sensor. In employing a combination of pentaglycine linker and tethering glyphosate via its secondary amino group, concentrations in aqueous solutions down to 100 pM could be measured by the differential adhesion between SCP and biochip surface, supported by automated image analysis algorithms. This sensing concept could even prove its exceptional pM sensitivity in combination with a superior discrimination against structurally related compounds.

Surface Functionalization by Hydrophobin-EPSPS Fusion Protein Allows for the Fast and Simple Detection of Glyphosate.

Glyphosate, the most widely used pesticide worldwide, is under debate due to its potentially cancerogenic effects and harmful influence on biodiversity and environment. Therefore, the detection of glyphosate in water, food or environmental probes is of high interest. Currently detection of glyphosate usually requires specialized, costly instruments, is labor intensive and time consuming. Here we present a fast and simple method to detect glyphosate in the nanomolar range based on the surface immobilization of glyphosate's target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) via fusion to the hydrophobin Ccg2 and determination of enzyme activity with a malachite green assay, which is a common photometric technique to measure inorganic phosphate (Pi). The assay demonstrates a new approach for a fast and simple detection of pesticides.