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.

The mitochondrial copper chaperone COX11 has an additional role in cellular redox homeostasis.

Mitochondria are sites of cellular respiration, which is accompanied by the generation of dangerous reactive oxygen species (ROS). Cells have multiple mechanisms to mitigate the dangers of ROS. Here we investigate the involvement of the COX complex assembly chaperone COX11 (cytochrome c oxidase 11) in cellular redox homeostasis, using homologs from the flowering plant Arabidopsis thaliana (AtCOX11) and yeast Saccharomyces cerevisiae (ScCOX11). We found that AtCOX11 is upregulated in Arabidopsis seedlings in response to various oxidative stresses, suggesting a defensive role. In line with this, the overexpression of either AtCOX11 or ScCOX11 reduced ROS levels in yeast cells exposed to the oxidative stressor paraquat. Under normal growth conditions, both Arabidopsis and yeast COX11 overexpressing cells had the same ROS levels as the corresponding WT. In contrast, the COX11 knock-down and knock-out in Arabidopsis and yeast, respectively, significantly reduced ROS levels. In yeast cells, the ScCOX11 appears to be functionally redundant with superoxide dismutase 1 (ScSOD1), a superoxide detoxifying enzyme. The ΔSccox11ΔScsod1 mutants had dramatically reduced growth on paraquat, compared with the WT or single mutants. This growth retardation does not seem to be linked to the status of the COX complex and cellular respiration. Overexpression of putatively soluble COX11 variants substantially improved the resistance of yeast cells to the ROS inducer menadione. This shows that COX11 proteins can provide antioxidative protection likely independently from their COX assembly function. The conserved Cys219 (in AtCOX11) and Cys208 (in ScCOX11) are important for this function. Altogether, these results suggest that COX11 homologs, in addition to participating in COX complex assembly, have a distinct and evolutionary conserved role in protecting cells during heightened oxidative stress.

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.

The antioxidant function of Sco proteins depends on a critical surface-exposed residue.

BACKGROUND: Besides their role in copper metabolism, Sco proteins from different organisms have been shown to play a defensive role against oxidative stress. In the present study, we set out to identify crucial amino acid residues for the antioxidant activity. METHODS: Native and mutated Sco proteins from human, Arabidopsis thaliana and the yeast Kluyveromyces lactis were expressed in the model organism Saccharomyces cerevisiae. The oxidative stress resistance of the respective transformants was determined by growth and lipid peroxidation assays. RESULTS: A functionally important site, located 15 amino acids downstream of the well-conserved copper binding CxxxC motif, was identified. Mutational analysis revealed that a positive charge at this position has a detrimental effect on the antioxidant capacity. Bioinformatic analysis predicts that this site is surface-exposed, and according to Co-IP data it is required for binding of proteins that are connected to known antioxidant pathways. CONCLUSION: This study shows that the antioxidant capacity of eukaryotic Sco proteins is conserved and depends on the presence of functional site(s) rather than the extent of overall sequence homology. GENERAL SIGNIFICANCE: These findings provide an insight into the conserved functional sites of eukaryotic Sco proteins that are crucial for combating oxidative stress. This capacity is probably not due to an enzymatic activity but rather is indirectly mediated by interaction with other proteins.

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.

Mitochondrial Sco proteins are involved in oxidative stress defense.

Members of the evolutionary conserved Sco protein family have been intensively studied regarding their role in the assembly of the mitochondrial cytochrome c oxidase. However, experimental and structural data, specifically the presence of a thioredoxin-like fold, suggest that Sco proteins may also play a role in redox homeostasis. In our study, we addressed this putative function of Sco proteins using Saccharomyces cerevisiae as a model system. Like many eukaryotes, this yeast possesses two SCO homologs (SCO1 and SCO2). Mutants bearing a deletion of either of the two genes are not affected in their growth under oxidative stress. However, the concomitant deletion of the SOD1 gene encoding the superoxide dismutase 1 resulted in a distinct phenotype: double deletion strains lacking SCO1 or SCO2 and SOD1 are highly sensitive to oxidative stress and show dramatically increased ROS levels. The respiratory competent double deletion strain Δsco2Δsod1 paved the way to investigate the putative antioxidant function of SCO homologs apart from their role in respiration by complementation analysis. Sco homologs from Drosophila, Arabidopsis, human and two other yeast species were integrated into the genome of the double deletion mutant and the transformants were analyzed for their growth under oxidative stress. Interestingly, all homologs except for Kluyveromyces lactis K07152 and Arabidopsis thaliana HCC1 were able to complement the phenotype, indicating their role in oxidative stress defense. We further applied this complementation-based system to investigate whether pathogenic point mutations affect the putative antioxidant role of hSco2. Surprisingly, all of the mutant alleles failed to restore the ROS-sensitivity of the Δsco2Δsod1 strain. In conclusion, our data not only provide clear evidence for the function of Sco proteins in oxidative stress defense but also offer a valuable tool to investigate this role for other homologous proteins.

Mitochondrial ROS direct the differentiation of murine pluripotent P19 cells.

ROS are frequently associated with deleterious effects caused by oxidative stress. Despite the harmful effects of non-specific oxidation, ROS also function as signal transduction molecules that regulate various biological processes, including stem cell proliferation and differentiation. Here we show that mitochondrial ROS level determines cell fate during differentiation of the pluripotent stem cell line P19. As stem cells in general, P19 cells are characterized by a low respiration activity, accompanied by a low level of ROS formation. Nevertheless, we found that P19 cells contain fully assembled mitochondrial electron transport chain supercomplexes (respirasomes), suggesting that low respiration activity may serve as a protective mechanism against ROS. Upon elevated mitochondrial ROS formation, the proliferative potential of P19 cells is decreased due to longer S phase of the cell cycle. Our data show that besides being harmful, mitochondrial ROS production regulates the differentiation potential of P19 cells: elevated mitochondrial ROS level favours trophoblast differentiation, whereas preventing neuron differentiation. Therefore, our results suggest that mitochondrial ROS level serves as an important factor that directs differentiation towards certain cell types while preventing others.

New approaches in bioprocess-control: Consortium guidance by synthetic cell-cell communication based on fungal pheromones.

Bioconversions in industrial processes are currently dominated by single-strain approaches. With the growing complexity of tasks to be carried out, microbial consortia become increasingly advantageous and eventually may outperform single-strain fermentations. Consortium approaches benefit from the combined metabolic capabilities of highly specialized strains and species, and the inherent division of labor reduces the metabolic burden for each strain while increasing product yields and reaction specificities. However, consortium-based designs still suffer from a lack of available tools to control the behavior and performance of the individual subpopulations and of the entire consortium. Here, we propose to implement novel control elements for microbial consortia based on artificial cell-cell communication via fungal mating pheromones. Coupling to the desired output is mediated by pheromone-responsive gene expression, thereby creating pheromone-dependent communication channels between different subpopulations of the consortia. We highlight the benefits of artificial communication to specifically target individual subpopulations of microbial consortia and to control e.g. their metabolic profile or proliferation rate in a predefined and customized manner. Due to the steadily increasing knowledge of sexual cycles of industrially relevant fungi, a growing number of strains and species can be integrated into pheromone-controlled sensor-actor systems, exploiting their unique metabolic properties for microbial consortia approaches.

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.

Artificial cell-cell communication as an emerging tool in synthetic biology applications.

Cell-cell communication is a widespread phenomenon in nature, ranging from bacterial quorum sensing and fungal pheromone communication to cellular crosstalk in multicellular eukaryotes. These communication modes offer the possibility to control the behavior of an entire community by modifying the performance of individual cells in specific ways. Synthetic biology, i.e., the implementation of artificial functions within biological systems, is a promising approach towards the engineering of sophisticated, autonomous devices based on specifically functionalized cells. With the growing complexity of the functions performed by such systems, both the risk of circuit crosstalk and the metabolic burden resulting from the expression of numerous foreign genes are increasing. Therefore, systems based on a single type of cells are no longer feasible. Synthetic biology approaches with multiple subpopulations of specifically functionalized cells, wired by artificial cell-cell communication systems, provide an attractive and powerful alternative. Here we review recent applications of synthetic cell-cell communication systems with a specific focus on recent advances with fungal hosts.

Protein kinase Ymr291w/Tda1 is essential for glucose signaling in saccharomyces cerevisiae on the level of hexokinase isoenzyme ScHxk2 phosphorylation*.

The enzyme ScHxk2 of Saccharomyces cerevisiae is a dual-function hexokinase that besides its catalytic role in glycolysis is involved in the transcriptional regulation of glucose-repressible genes. Relief from glucose repression is accompanied by the phosphorylation of the nuclear fraction of ScHxk2 at serine 15 and the translocation of the phosphoenzyme into the cytosol. Different studies suggest different serine/threonine protein kinases, Ymr291w/Tda1 or Snf1, to accomplish ScHxk2-S15 phosphorylation. The current paper provides evidence that Ymr291w/Tda1 is essential for that modification, whereas protein kinases Ydr477w/Snf1, Ynl307c/Mck1, Yfr014c/Cmk1, and Ykl126w/Ypk1, which are co-purified during Ymr291w/Tda1 tandem affinity purification, as well as protein kinase PKA and PKB homolog Sch9 are dispensable. Taking into account the detection of a significantly higher amount of the Ymr291w/Tda1 protein in cells grown in low-glucose media as compared with a high-glucose environment, Ymr291w/Tda1 is likely to contribute to glucose signaling in S. cerevisiae on the level of ScHxk2-S15 phosphorylation in a situation of limited external glucose availability. The evolutionary conservation of amino acid residue serine 15 in yeast hexokinases and its phosphorylation is illustrated by the finding that YMR291W/TDA1 of S. cerevisiae and the homologous KLLA0A09713 gene of Kluyveromyces lactis allow for cross-complementation of the respective protein kinase single-gene deletion strains.

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.

The Arabidopsis COX11 Homolog is Essential for Cytochrome c Oxidase Activity.

Members of the ubiquitous COX11 (cytochrome c oxidase 11) protein family are involved in copper delivery to the COX complex. In this work, we characterize the Arabidopsis thaliana COX11 homolog (encoded by locus At1g02410). Western blot analyses and confocal microscopy identified Arabidopsis COX11 as an integral mitochondrial protein. Despite sharing high sequence and structural similarities, the Arabidopsis COX11 is not able to functionally replace the Saccharomyces cerevisiae COX11 homolog. Nevertheless, further analysis confirmed the hypothesis that Arabidopsis COX11 is essential for COX activity. Disturbance of COX11 expression through knockdown (KD) or overexpression (OE) affected COX activity. In KD lines, the activity was reduced by ~50%, resulting in root growth inhibition, smaller rosettes and leaf curling. In OE lines, the reduction was less pronounced (~80% of the wild type), still resulting in root growth inhibition. Additionally, pollen germination was impaired in COX11 KD and OE plants. This effect on pollen germination can only partially be attributed to COX deficiency and may indicate a possible auxiliary role of COX11 in ROS metabolism. In agreement with its role in energy production, the COX11 promoter is highly active in cells and tissues with high-energy demand for example shoot and root meristems, or vascular tissues of source and sink organs. In COX11 KD lines, the expression of the plasma-membrane copper transporter COPT2 and of several copper chaperones was altered, indicative of a retrograde signaling pathway pertinent to copper homeostasis. Based on our data, we postulate that COX11 is a mitochondrial chaperone, which plays an important role for plant growth and pollen germination as an essential COX complex assembly factor.

Proteomic analysis reveals a novel function of the kinase Sat4p in Saccharomyces cerevisiae mitochondria.

The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt) localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE) approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p) and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p). The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p), Lys114 (Kgd2p) and Lys102 (Gcv3p), respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.

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.

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.

OXPHOS supercomplexes as a hallmark of the mitochondrial phenotype of adipogenic differentiated human MSCs.

Mitochondria are essential organelles with multiple functions, especially in energy metabolism. Recently, an increasing number of data has highlighted the role of mitochondria for cellular differentiation processes. Metabolic differences between stem cells and mature derivatives require an adaptation of mitochondrial function during differentiation. In this study we investigated alterations of the mitochondrial phenotype of human mesenchymal stem cells undergoing adipogenic differentiation. Maturation of adipocytes is accompanied by mitochondrial biogenesis and an increase of oxidative metabolism. Adaptation of the mt phenotype during differentiation is reflected by changes in the distribution of the mitochondrial network as well as marked alterations of gene expression and organization of the oxidative phosphorylation system (OXPHOS). Distinct differences in the supramolecular organization forms of cytochrome c oxidase (COX) were detected using 2D blue native (BN)-PAGE analysis. Most remarkably we observed a significant increase in the abundance of OXPHOS supercomplexes in mitochondria, emphasizing the change of the mitochondrial phenotype during adipogenic differentiation.