Center for Synthetic Microbiology

Marburg an der Lahn, Germany

Center for Synthetic Microbiology

Marburg an der Lahn, Germany
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Peschke M.,University of Marburg | Peschke M.,Max Planck Institute for Medical Research | Moog D.,Center for Synthetic Microbiology | Klingl A.,Center for Synthetic Microbiology | And 3 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2013

Diatoms are microalgae that possess so-called "complex plastids," which evolved by secondary endosymbiosis and are surrounded by four membranes. Thus, in contrast to primary plastids, which are surrounded by only two membranes, nucleus-encoded proteins of complex plastids face additional barriers, i.e., during evolution, mechanisms had to evolve to transport preproteins across all four membranes. This study reveals that there exist glycoproteins not only in primary but also in complex plastids, making transport issues even more complicated, as most translocation machineries are not believed to be able to transport bulky proteins. We show that plastidal reporter proteins with artificial N-glycosylation sites are indeed glycosylated during transport into the complex plastid of the diatom Phaeodactylum tricornutum. Additionally, we identified five endogenous glycoproteins, which are transported into different compartments of the complex plastid. These proteins get N-glycosylated during transport across the outermost plastid membrane and thereafter are transported across the second, third, and fourth plastid membranes in the case of stromal proteins. The results of this study provide insights into the evolutionary pressure on translocation mechanisms and pose unique questions on the operating mode of well-known transport machineries like the translocons of the outer/inner chloroplast membranes (Toc/Tic).

Stiebler A.C.,University of Marburg | Freitag J.,University of Marburg | Freitag J.,LOEWE Excellence Cluster for Integrative Fungal Research IPF | Schink K.O.,University of Oslo | And 6 more authors.
PLoS Genetics | Year: 2014

Translation of mRNA into a polypeptide chain is a highly accurate process. Many prokaryotic and eukaryotic viruses, however, use leaky termination of translation to optimize their coding capacity. Although growing evidence indicates the occurrence of ribosomal readthrough also in higher organisms, a biological function for the resulting extended proteins has been elucidated only in very few cases. Here, we report that in human cells programmed stop codon readthrough is used to generate peroxisomal isoforms of cytosolic enzymes. We could show for NAD-dependent lactate dehydrogenase B (LDHB) and NAD-dependent malate dehydrogenase 1 (MDH1) that translational readthrough results in C-terminally extended protein variants containing a peroxisomal targeting signal 1 (PTS1). Efficient readthrough occurs at a short sequence motif consisting of a UGA termination codon followed by the dinucleotide CU. Leaky termination at this stop codon context was observed in fungi and mammals. Comparative genome analysis allowed us to identify further readthrough-derived peroxisomal isoforms of metabolic enzymes in diverse model organisms. Overall, our study highlights that a defined stop codon context can trigger efficient ribosomal readthrough to generate dually targeted protein isoforms. We speculate that beyond peroxisomal targeting stop codon readthrough may have also other important biological functions, which remain to be elucidated. © 2014 Stiebler et al.

Kiekebusch D.,Max Planck Institute for Terrestrial Microbiology | Kiekebusch D.,University of Marburg | Kiekebusch D.,Center for Synthetic Microbiology | Michie K.,University of Cambridge | And 6 more authors.
Molecular Cell | Year: 2012

Protein gradients play a central role in the spatial organization of cells, but the mechanisms of their formation are incompletely understood. This study analyzes the determinants responsible for establishing bipolar gradients of the ATPase MipZ, a key regulator of division site placement in . Caulobacter crescentus. We have solved the crystal structure of MipZ in different nucleotide states, dissected its ATPase cycle, and investigated its interaction with FtsZ, ParB, and the nucleoid. Our results suggest that the polar ParB complexes locally stimulate the formation of ATP-bound MipZ dimers, which are then retained near the cell poles through association with chromosomal DNA. Due to their intrinsic ATPase activity, dimers eventually dissociate into freely diffusible monomers that undergo spontaneous nucleotide exchange and are recaptured by ParB. These findings clarify the molecular function of a conserved gradient-forming system and reveal mechanistic principles that might be commonly used to sustain protein gradients within cells. © 2012 Elsevier Inc.

Hempel F.,Center for Synthetic Microbiology | Maier U.G.,Center for Synthetic Microbiology | Maier U.G.,University of Marburg
Microbial Cell Factories | Year: 2012

Background: Although there are many different expression systems for recombinant production of pharmaceutical proteins, many of these suffer from drawbacks such as yield, cost, complexity of purification, and possible contamination with human pathogens. Microalgae have enormous potential for diverse biotechnological applications and currently attract much attention in the biofuel sector. Still underestimated, though, is the idea of using microalgae as solar-fueled expression system for the production of recombinant proteins.Results: In this study, we show for the first time that completely assembled and functional human IgG antibodies can not only be expressed to high levels in algal systems, but also secreted very efficiently into the culture medium. We engineered the diatom Phaeodactylum tricornutum to synthesize and secrete a human IgG antibody against the Hepatitis B Virus surface protein. As the diatom P. tricornutum is not known to naturally secrete many endogenous proteins, the secreted antibodies are already very pure making extensive purification steps redundant and production extremely cost efficient.Conclusions: Microalgae combine rapid growth rates with all the advantages of eukaryotic expression systems, and offer great potential for solar-powered, low cost production of pharmaceutical proteins. © 2012 Hempel and Maier; licensee BioMed Central Ltd.

Schlimpert S.,Max Planck Institute for Terrestrial Microbiology | Schlimpert S.,University of Marburg | Klein E.A.,Princeton University | Briegel A.,California Institute of Technology | And 12 more authors.
Cell | Year: 2012

In eukaryotes, the differentiation of cellular extensions such as cilia or neuronal axons depends on the partitioning of proteins to distinct plasma membrane domains by specialized diffusion barriers. However, examples of this compartmentalization strategy are still missing for prokaryotes, although complex cellular architectures are also widespread among this group of organisms. This study reveals the existence of a protein-mediated membrane diffusion barrier in the stalked bacterium Caulobacter crescentus. We show that the Caulobacter cell envelope is compartmentalized by macromolecular complexes that prevent the exchange of both membrane and soluble proteins between the polar stalk extension and the cell body. The barrier structures span the cross-sectional area of the stalk and comprise at least four proteins that assemble in a cell-cycle-dependent manner. Their presence is critical for cellular fitness because they minimize the effective cell volume, allowing faster adaptation to environmental changes that require de novo synthesis of envelope proteins. © 2012 Elsevier Inc.

Specht M.,Albert Ludwigs University of Freiburg | Dempwolff F.,Albert Ludwigs University of Freiburg | Schatzle S.,University Hospital Freiburg | Schatzle S.,Albert Ludwigs University of Freiburg | And 4 more authors.
Journal of Bacteriology | Year: 2013

Of the various kinds of cell division, the most common mode is binary fission, the division of a cell into two morphologically identical daughter cells. However, in the case of asymmetric cell division, Caulobacter crescentus produces two morphologically and functionally distinct cell types. Here, we have studied cell cycle progression of the human pathogen Helicobacter pylori using a functional green fluorescent protein (GFP) fusion of FtsZ protein and membrane staining. In small cells, representing newly divided cells, FtsZ localizes to a single cell pole. During the cell cycle, spiral intermediates are formed until an FtsZ ring is positioned with very little precision, such that central as well as acentral rings can be observed. Daughter cells showed considerably different sizes, suggesting that H. pylori divides asymmetrically. Fluorescence recovery after photobleaching (FRAP) analyses demonstrate that the H. pylori FtsZ ring is about as dynamic as that of Escherichia coli but that polar assemblies show less turnover. Strikingly, our results demonstrate that H. pylori cell division follows a different route from that in E. coli and Bacillus subtilis. It is also different from that in C. crescentus, where cytokinesis regulation proteins like MipZ play a role. Therefore, this report provides the first cell-biological analysis of FtsZ dynamics in the human pathogen H. pylori and even in epsilonproteobacteria to our knowledge. In addition, analysis of the filament architecture of H. pylori and E. coli FtsZ filaments in the heterologous system of Drosophila melanogaster S2 Schneider cells revealed that both have different filamentation properties in vivo, suggesting a unique intrinsic characteristic of each protein. © 2013, American Society for Microbiology.

Heimel K.,University of Gottingen | Heimel K.,Karlsruhe Institute of Technology | Freitag J.,University of Marburg | Freitag J.,Center for Synthetic Microbiology | And 5 more authors.
Plant Cell | Year: 2013

The unfolded protein response (UPR) is a conserved eukaryotic signaling pathway regulating endoplasmic reticulum (ER) homeostasis during ER stress, which results, for example, from an increased demand for protein secretion. Here, we characterize the homologs of the central UPR regulatory proteins Hac1 (for Homologous to ATF/CREB1) and Inositol Requiring Enzyme1 in the plant pathogenic fungus Ustilago maydis and demonstrate that the UPR is tightly interlinked with the b mating-type-dependent signaling pathway that regulates pathogenic development. Exact timing of UPR is required for virulence, since premature activation interferes with the b-dependent switch from budding to filamentous growth. In addition, we found crosstalk between UPR and the b target Clampless1 (Clp1), which is essential for cell cycle release and proliferation in planta. The unusual C-terminal extension of the U. maydis Hac1 homolog, Cib1 (for Clp1 interacting bZIP1), mediates direct interaction with Clp1. The interaction between Clp1 and Cib1 promotes stabilization of Clp1, resulting in enhanced ER stress tolerance that prevents deleterious UPR hyperactivation. Thus, the interaction between Cib1 and Clp1 constitutes a checkpoint to time developmental progression and increased secretion of effector proteins at the onset of biotrophic development. Crosstalk between UPR and the b mating-type regulated developmental program adapts ER homeostasis to the changing demands during biotrophy. © 2013 American Society of Plant Biologists.

Kiekebusch D.,Max Planck Institute for Terrestrial Microbiology | Kiekebusch D.,University of Marburg | Kiekebusch D.,Center for Synthetic Microbiology | Thanbichler M.,Max Planck Institute for Terrestrial Microbiology | And 2 more authors.
Trends in Microbiology | Year: 2014

The formation of protein concentration gradients is an effective means to restrict the activity of regulatory factors in space, thereby critically contributing to the spatiotemporal organization of biological systems. Although widely observed for extracellular proteins involved in tissue patterning, the implementation of this regulatory strategy was thought to be impossible in single, micron-sized cells. Recently, however, several intracellular proteins were shown to establish gradient-like distribution patterns, thereby relaying positional information to their downstream targets. In this review, we discuss gradient-forming systems from different microbial species, with an emphasis on their mode of action and the common principles that underlie their function. © 2013 Elsevier Ltd.

Stehlik T.,University of Marburg | Stehlik T.,Center for Synthetic Microbiology | Sandrock B.,University of Marburg | Ast J.,University of Marburg | And 2 more authors.
Current Opinion in Microbiology | Year: 2014

Peroxisomes are nearly ubiquitous single-membrane organelles harboring multiple metabolic pathways beside their prominent role in the β-oxidation of fatty acids. Here we review the diverse metabolic functions of peroxisomes in fungi. A variety of fungal metabolites are at least partially synthesized inside peroxisomes. These include the essential co-factor biotin but also different types of secondary metabolites. Peroxisomal metabolites are often derived from acyl-CoA esters in for example β-oxidation intermediates. In several ascomycetes a subtype of peroxisomes has been identified that is metabolically inactive but is required to plug the septal pores of wounded hyphae. Thus, peroxisomes are versatile organelles that can adapt their function to the life style of an organism. This remarkable variability suggests that the full extent of the biosynthetic capacity of peroxisomes is still elusive. Moreover, in fungi peroxisomes are non-essential under laboratory conditions making them attractive organelles for biotechnological approaches and the design of novel metabolic pathways in customized peroxisomes. © 2014 Elsevier Ltd. All rights reserved.

Dempwolff F.,Center for Synthetic Microbiology | Graumann P.L.,Center for Synthetic Microbiology
Communicative and Integrative Biology | Year: 2014

Dynamin is a membrane-associated GTPase that confers motor-like functions in membrane dynamics, such as endocytosis, in eukaryotic cells. Flotillin (reggie) proteins are also a widely conserved class of membrane proteins, associated with the formation of protein assemblies within the membrane, and with endocytotic processes. Bacterial dynamin has been shown to bind to membranes in vitro and to mediate membrane fusion. Bacillus subtilis DynA localizes to the cell division septum, and it was recently shown that it indeed plays a role in cell division. Interestingly, dynamin shows a genetic interaction with flotillin proteins in this prokaryotic model organism and the absence of both proteins results in a cell division and cell shape defect. Here, we show that in addition to the morphological phenotypes, a dynamin/flotillin double deletion strain shows a synthetic defect in cell motility, much stronger than that of flotillin single mutant cells. While the contribution of altered cell shape and slower growth of the double deletion strain on motility cannot be clearly assessed, our data emphasize the fact that dynamin and flotillin proteins play tightly connected functions in a wide range of aspects in membrane processes in bacteria.

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