Scientific Report 2010–2014 LOEWE Center for Synthetic 3

Preface

Research on microorganisms has been a long-stand- The collaboration between groups from the Philipps- ing focus of the Philipps-Universität and has made Universität with departments and research groups Marburg one of the best places in Germany to study from the Max Planck Institute for Terrestrial Microbi- microbiology. The fruitful collaborations between ology laid the foundation for further projects like the groups working at the university and the local Max international Max Planck Research School and DFG- Planck Institute for Terrestrial Microbiology have funded coordinated programs, including the micro- heightened the national and international visibility -oriented Collaborative Research Center 987. of Marburg and have fortified its reputation for excel- Regionally, SYNMIKRO interacts with groups at the lence. These concerted activities provided the appro- Justus-Liebig-Universität Giessen and the Technische priate framework for the foundation of the Center for Hochschule Mittelhessen and thereby strengthens the Synthetic Microbiology (SYNMIKRO) within the LOEWE research alliance among the universities. SYNMIKRO excellence program of the state of Hessen in a joint is thus also a prime example of fruitful collaborations effort betweenP hilipps-Universität and Max Planck between institutions. Society in the year 2010. We are grateful for the con- tinued support of the state of Hessen without which The manifold research activities and the broad range the center could not have been established. On the oc- of microorganisms studied under the umbrella of casion of the fifth anniversary since its inception, this SYNMIKRO extend to dedicated teaching activities brochure surveys the many activities of SYNMIKRO. that provide superb training for the next generations As the president of the Philipps-Universität Marburg, of microbiologists. Excellence in teaching in the field I am very proud of what has been achieved in this time. of microbiology is a nationally and internationally recognized hallmark of the Philipps-Universität. Scientific progress requires creative minds, excellent working conditions and a stimulating environment. Along with the many scientists working in SYNMIKRO, All these elements come together in an ideal manner I feel that it is both necessary and worthwhile to sum- in SYNMIKRO. Research at the center is carried out by marize for a broader audience what has been accom- internationally highly regarded and recognized scien- plished so far and to look forward to future activities tists. The center provides access to a state-of-the-art of the center. I thank the group leaders, scientists, infrastructure for modern molecular biology. The large and the large number of students for their hard work number of cooperations across the traditional disci- and their concentrated efforts to make SYNMIKRO a plines from life sciences and natural sciences all the success. They have all contributed to making this bro- way to the humanities shows that Marburg provides chure an interesting and stimulating read that I hope an atmosphere that is conducive for interdisciplinary you will enjoy. and trans-disciplinary research. In the near future, key groups in synthetic microbiology from both the Prof. Katharina Krause Philipps-Universität and the Max Planck Institute President will be housed in a large, well-equipped new research Philipps-Universität Marburg building in the center of the science campus Lahn- berge. This new building will foster even closer sci- entific interactions and the cost-effective sharing of equipment. 4 Welcome 5

Freisleben

Mösch Hüllermeier

Klebe Kolb Thauer Bölker Li

Kahmann

Essen Lill Bremer

Becker Structural Biology

Thanbichler Maier Lenz Super Buckel Resolution Microscopy

Voigt Albers Mass Spectrometry Synthetic Microbiology in Marburg Laboratory Heider Automation

Electron Microorganisms are omnipresent in the biosphere. and interaction of cellular components generates Microscopy Conrad They occupy even the most exotic ecological niches emergent properties is required before we can use Liesack Bange Marahiel and have developed an impressive variety of physi- natural systems as prototypes for the construction Sourjik Waldming- ological and sensory functions that allow them to of predictable, streamlined and robust systems with haus S gaard- respond to and cope with environmental challenges. novel properties. Vice versa, the process of building Andersen Some of these traits already provide the basis for synthetic systems or of modifying natural systems Randau Fritz large-scale production of chemicals and pharmaceu- facilitates understanding, as the performance of Eckhardt ticals, for biofuels, -processing, biosensing or bio- these constructs helps to understand their function.

remediation. Nevertheless, the vast natural toolbox All of this is made possible by advances and new tech- Jonas Graumann developed by microorganisms over billions of years of nologies in several disciplines, in particular in cell Dahlke evolution undoubtedly contains many undiscovered biology, , molecular biology, structural Brune Kostina Flow processes that wait to be explored and harnessed. The biology, , and computational biology. Interactome map of SYNMIKRO. Cytometry field of synthetic microbiology is at the center of this enterprise. Much of the fascination with this emer- In 2010, the Philipps-Universität Marburg and the Max gent field derives from the ability to freely combine Planck Institute for Terrestrial Microbiology joined myriads of genes, cellular processes, and biochemical forces to bring synthetic microbiology to Marburg. develop synthetic microbiology, from the identification discourse on pioneering developments. The public is functions in very much the same way as engineers They established SYNMIKRO, the Center for Synthetic and characterization of modules in natural systems regularly invited to learn about synthetic microbiol- combine different parts to form larger functional Microbiology, within the LOEWE excellence program of to the development of synthetic modules and their ogy in open lectures and plenary discussions, and a units and products. the state of Hessen. Research at the center follows the integration into natural host organisms or synthetic fruitful dialogue between basic natural sciences and two-pronged approach – building to understand and chassis-cells. the humanities was initiated. understanding to build – to gain insights into the basic Modeling & Bioethics principles of microbial life and to provide the funda- For optimal support of the scientific projects, With this brochure, we introduce the members of mental knowledge and tools needed to tap in novel SYNMIKRO has invested in state-of-the-art infra- SYNMIKRO and their projects, explain the work of the Microbial Communities ways the potential of microorganisms as cell factories structural units for laboratory automation, flow infrastructural units and describe key activities of Chassis and Genomes or sensor/reporter systems. Since its foundation, the cytometry, spectroscopy, super resolution the center in the years 2010-2014. We hope that you center has grown to become a major, internationally microscopy, electron microscopy, structural biology can see from this report that SYNMIKRO provides a Cellular visible research institution that today represents one and mass spectrometry. These infrastructural units vibrant environment for top-level education and first- Organization of the hot spots of research in quantitative and syn- provide critical support for the projects pursued by Cellular Signal class science, and that it’s worth to keep an eye on Processing and thetic microbiology in Europe. SYNMIKRO researchers and their collaboration part- SYNMIKRO and its diverse activities. Regulation ners, and are engaged in the development of enabling SYNMIKRO is an interdisciplinary center that consists technologies in key areas in synthetic microbiology. Bruno Eckhardt Anke Becker of members from seven faculties of the university Director Vice Director and the four departments of the Max Planck Insti- The impact of SYNMIKRO goes well beyond the dedi- tute. Drawing on Marburg’s highly-rated expertise in cated research projects. With the thematic focus in Topics integrated within SYNMIKRO. microbiology, it integrates almost 30 laboratory synthetic microbiology within the M.Sc. program groups at the university and the Max Planck Insti- Molecular and Cellular Biology and with our success- However, the challenges encountered by synthetic tute to cover all aspects of microbial life from the ful iGEM team, we recruit graduate and undergradu- biology in its early years revealed that many processes molecular level to whole cells and communities. In ate students to this fascinating new field early in their within cells are still poorly understood and that not addition, theory- and ethics-focused groups provide career. Furthermore, a structured doctoral program, all synthetic pathways sketched on the drawing board testable mathematical models, systematic overviews summer schools, workshops and seminars aim at actually work in cells. A better quantitative, dynamic, and guidance. With its inter- and trans-disciplinary scientists in academia and industry. In addition, we and theoretical understanding of how organization expertise, SYNMIKRO offers a unique opportunity to are committed to promoting the public and scientific 6 Welcome 7

Scientific Advisory Board SYNMIKRO seeks feedback from independent ex- advises the center regarding research planning and perts: Its Scientific Advisory Board of internationally organization. We thank the members of the Scientific renowned scientists convenes every two years and Advisory Board for this valuable contribution.

Dr. Jürgen Eck Prof. Dr. BRAIN AG, Zwingenberg Regine Hengge Humboldt University Berlin

Content Prof. Dr. Prof. Dr. Vitor Oscar Kuipers Martins dos Santos 1 rESEArch , Wageningen University, Netherlands 1.1 cellular Signal Processing and Regulation 8 1.2 Metabolism 24 1.3 Cellular Organization 44 1.4 Chassis and Genomes 62 1.5 Microbial Communities 72 1.6 Bioethics 82 2 Infrastructure 86 3 Teaching & Events 100 4 Facts & Figures 112 Prof. Dr. Rolf Müller Prof. Dr. Helmholtz Institute for Julia Vorholt Pharmaceutical Research ETH Zürich, Switzerland Saarbrücken 8 9

1.1 Research Cellular Signal Processing and Regulation 10 research 11

Selected publications

Osmoprotectants in bacterial stress Hoffmann T, Bremer E (2011). Protec- tion of Bacillus subtilis against cold responses and cell integrity stress via compatible-solute acqui- sition. J Bacteriol 193, 1552-62.

Water is the foundation of life. It is essential for vital stress experienced by the cell. In Bacillus subtilis, the Pittelkow M, Tschapek B, Smits SH, processes like the replication and transcription of the model organism for Gram-positive that we Schmitt L, Bremer E (2011). The genetic material and biochemical reactions, but at study, important compatible solutes are glutamate, crystal structure of the substrate- the same time, these processes need protection from proline, and glycine betaine. Other Bacilli additionally binding protein OpuBC from Bacillus adverse environmental influences.T he development accumulate ectoine and its derivative 5-hydroxyec- subtilis in complex with choline. of semi-permeable lipid membranes that let the wa- toine. We have elucidated the synthesis pathways for J Mol Biol 411, 53-67. ter pass, but severely restrict the crossing of ions and these stress protectants and study the biochemical Becker J, Schäfer R, Kohlstedt M, P ROF. DR. Erhard Bremer most organic molecules therefore was a key step in and structural properties of key enzymes; e.g., that of Harder BJ, Borchert NS, Stöveken N, the evolution of proto-cells. Since the osmotic poten- the ectoine hydroxylase (Fig. 2) (Höppner et al, 2014). Osmotic Stress Responses of Bremer E, Wittmann C (2013). tial of the cytoplasm is considerably higher than that Compatible solutes can also be accumulated through Microorganisms Systems metabolic engineering of of the surrounding, water will flow along this osmotic import (Broy et al, 2015), and our genetic and struc- Corynebacterium glutamicum for Faculty of Biology, gradient into the cell and thereby generate an intra- tural analyses of the involved transport systems in production of the chemical chap- Philipps-Universität Marburg cellular hydrostatic pressure, the turgor. As the cyto- B. subtilis have broken new ground for their under- eron ectoine. Microb Cell Fact 12:110. Phone +49 6421 28 21529 plasmic membrane is a fragile structure that is unable standing in other microorganisms and their func- Email: [email protected] by itself to restrain turgor, microorganisms eventually tional annotation in genome sequencing projects. In Höppner A, Widderich N, Lenders M, Bremer E, Smits SH (2014). Crystal SYNMIKRO member since 2010 developed firm, yet elastic cell walls (Fig. 1). Turgor is Fig. 3, the crystal structure of the extracellular solute Figure 2. Crystal structure of a dimer of the ectoine hydroxylase (EctD) in complex with its co-substrate 2-oxoglutarate, the catalytically considered as the driving force for expansion of the receptor protein (OpuBC) for choline, the biosynthetic important iron atom and the reaction product 5-hydroxyectoine. structure of the ectoine hydroxy- sacculus during growth and is therefore con- precursor of the potent osmostress protectant glycine lase, a snapshot of the active site. J Biol Chem 289, 29570-83. sidered an essential attribute of most cells. betaine, is shown (Pittelkow et al, 2011). cells. We are studying the molecular biology and bio- but which might possess interesting novel proper- Broy S, Chen C, HoffmannT , Brock NL, Short CV Compatible solutes as effective chemistry of the biosynthetic routes for ectoine and ties. Structural analysis of the EctD enzymes (Fig. 2) Nau-Wagner G, Jebbar M, Smits SH, hydroxyectoine production. In collaboration with the (Höppner et al, 2014) helps us to design appropriate 1995– Professor of Molecular temperature stress protectants Dickschat JS, Bremer E (2015). Microbiology, Philipps-Universität group of Christoph Wittmann (TU Braunschweig), we substrates. Likewise, we found that synthetic deriva- Abiotic stress protection by eco- Marburg In addition to providing osmostress protection, our successfully transplanted a synthetically engineered tives of naturally occurring compatible solutes (e.g., logically abundant dimethylsulfo- 2013 Visiting Professor, Princeton work shows that compatible solutes also serve as ef- gene cluster for ectoine production into the industrial dimethlysulfoniopropionate; DMSP) can be smuggled niopropionate and its natural and University, USA fective temperature stress protectants for cells threat- workhorse Corynebacterium glutamicum (Becker et into osmotically stressed B. subtilis cells via a type of synthetic derivatives: insights from Figure 1. From a proto-cell to a cell-walled microorganism and Bacillus subtilis. Env Microbiol, doi: 1993–1995 Adjunct Professor, the concomitant development of turgor. ened by death both at the upper and at the lower al, 2013). In addition to this proof of principle, we are transport system (OpuC) that is widely distributed in Philipps-Universität Marburg boundaries of the temperature spectrum that B. sub- now exploring the possibility of exploiting the ectoine nature (Broy et al, 2015), opening up the way for novel 10.1111/1462-2920.12698 1992–1995 Independent Group In their natural ecosystems, free-living microorgan- tilis cells can tolerate (Hoffmann & Bremer, 2011; Broy hydroxylase (EctD) for the in vivo and in vitro synthe- applications of these remarkable compounds. Leader, Max Planck Institute for isms will experience fluctuations in the osmolarity et al, 2015). This occurs with a degree of effectiveness sis of ectoine derivatives that do not exist naturally Terrestrial Microbiology, Marburg that will either result in water influx (at low osmo- that is unmatched by other microbial temperature 1984–1992 Staff Scientist and larity) or water efflux (at high osmolarity).T o timely stress defense systems; e.g., the heat- and cold-shock Assistant Professor, University of respond to these environmental challenges, highly and the general stress system of B. subtilis. Konstanz 1989 Habilitation, University of integrated cellular stress responses are engaged. Konstanz Understanding these responses and the underlying Harnessing compatible solute systems 1982–1984 Postdoctoral Fellow, signal transduction mechanisms is the core of our re- for synthetic microbiology National Cancer Institute, Frederick, search efforts. Besides, as adequate turgor regulation MD, USA is an essential function for most microorganisms, Compatible solutes are not only excellent osmolytes 1982 Ph.D. in biology, Universität all efforts to synthetically construct proto-cells, or and temperature stress protectants, but also serve as Tübingen to design microbial cells with minimalized genomes stabilizers of macromolecules, ensure the function- 1980 Diploma in biology, Universität (“chassis”) must take care to implement systems for ing of biosynthetic processes and can even preserve Tübingen osmoregulation. Therefore, our group also embarked the integrity of entire cells. The function-preserving on the development of synthetic osmoprotection properties of compatible solutes have aroused con- gene clusters and of synthetic derivatives of natural siderable interest as they could be exploited for osmoprotectants with novel properties. biotechnological purposes, cosmetics, and medical Aa w rds & Honors applications. The compatible solutes ectoine and C ellular responses to high osmolarity 5-hydroxyectoine are currently produced on an indus- 2011 Elected as Member of the Euro- trial scale, and products containing them (in particu- pean Academy of Microbiology (EAM) When challenged by high osmolarity surroundings, lar those used in skin care) have reached consumer 1991 Young Investigator Award from bacteria engage in carefully timed stress responses markets. Production of these compounds relies on so- the Deutsche Gesellschaft für Hygiene und Mikrobiologie (DGHM) to curb the outflow of water. This cellular adjust- called bacterial milking, a procedure that involves the 1982-1984 Postdoctoral fellowships ment entails the accumulation of organic osmolytes, high-density fermentation of a highly salt resistant from the German Research Founda- so-called compatible solutes; these can be amassed bacterium (Halomonas elongata) followed by a severe to exceedingly high concentrations and in a fashion osmotic down-shock, which releases the ectoines tion (DFG) and the German Academic Figure 3. Crystal-formation of the purified OpuBC ligand binding protein (left) and its three-dimensional structure in complex with the Exchange Service (DAAD) that is sensitively linked to the degree of the osmotic via mechanosensitive channels from the producer ligand choline (right). 12 research 13

Selected publications

Structural biology of Geisselbrecht Y, Frühwirth S, Schröder C, Pierik AJ, Klug G, photoreception & yeast cell walls Essen LO (2012). CryB from Rhodobacter sphaeroides: A unique class of cryptochromes with novel Photoreceptors allow all forms of life to monitor F rom structures to evolution cofactors. EMBO Rep 13, 223-29. their environmental status in terms of light quality & quantity, and thus the time of day, or their location Photoreceptors of the photolyase-cryptochrome fam- Kiontke S, Geisselbrecht Y, Pokorny R, in soil, sediment, water column and hosts. We study ily mediate responses to blue light. Amongst other Carell T, Batschauer A, Essen LO the mechanisms of photoreceptors from different mi- things, these proteins catalyze light-driven repair of (2011). Crystal structures of an croorganisms by combining structural biology with UV-lesions in DNA, and act in blue-light dependent archaeal class II DNA photolyase and its complex with UV-damaged biochemical and biophysical techniques. When un- signaling, e.g., for the control of flowering or gene duplex DNA. EMBO J 30, 4437-49. Prof. Dr. derstanding how photon absorption triggers confor- expression. For this they utilize a U-shaped flavin ad- Lars-Oliver Essen mational changes and affects downstream signaling enine dinucleotide (FAD) chromophore together with a Maestre-Reyna M, Diderrich R, we can engineer these photoreceptors for optogenetic second chromophore, that harvests light like an anten- Structural Biochemistry Veelders M, Eulenburg G, Kalugin V, applications, e.g. the light-induced degradation of na. Our studies on class II photolyases, mostly found Brückner S, Keller P, Rupp S, Departments of Chemistry & Biology, proteins (Fig. 1) (Renicke et al, 2013) or the biosynthesis in animals and plants, and the recently discovered Moesch HU, Essen LO (2012). Struc- Philipps-Universität Marburg of second messengers. cryptochromes from proteo- and cyanobacterial spe- tural code for adhesin-mediated Phone: +49 6421 28 22032 The ultrastructure of fungal cell walls is a second fo- cies (CryPro) showed that the intramolecular transfer Figure 2. (A) Structure of the class II photolyase from Methanosarcina mazei in complex with UV-damaged DNA (Kiontke et al, 2011). invasion of host epithelia Email: [email protected] This enzyme is highly related to plant photolyases and has apparently been acquired by this archaeon via horizontal gene transfer. (B) by Candida glabrata. cus of our work. Yeasts are unicellular fungi, which can of electrons to this cofactor takes different routes than The biosynthesis route for its antenna pigment (8-HDF) was artificially introduced into Escherichia coli and allowed to characterize the Proc Natl Acad Sci USA 109, 16864-9. SYNMIKRO member since 2010 switch to a wide range of multicellular phenotypes previously known (Fig. 2). Likewise, the architectural recombinant photolyase in complex to 8-HDF. (C) The catalytic domain of photolyases and cryptochromes structurally resembles the large subunit of eukaryotic/archaeal primases (PriL), an enzyme class essential for DNA-replication. This raises the question of who such as biofilms, filaments, flocs and flors. Given their features relevant for DNA recognition of class I and II was first: photolyases or primases? Renicke C, Schuster D, Essen LO, lack of motility, these phenotypes enable yeasts to photolyases reflect different topological restraints im- Taxis C (2013). A LOV2 domain-based colonize various habitats and to escape for example posed by pro- and eukaryotic DNA packaging. In class II physical barrier against host defense, but also the homophilic Flo11-Flo11 and heterophilic Flo11-plastic optogenetic tool to control protein unfavorable nutrient conditions. A prerequisite for photolyases the antenna cofactor, 8-hydroxydeazafla- basis for cell-cell communication. Accordingly, a con- interactions just depend on the Flo11A domain. EM degradation and cellular function. multicellular phenotypes is cell adhesion via either vin, is utilized throughout the green lineage, but not cise understanding of yeast adhesion and its different 3D-reconstructions of yeast cell-cell contact sites in- Chem Biol 20, 619-26. cell-cell and/or cell-surface contacts. We analyze the in higher plants, which have developed a hitherto un- modes may pave the way for controlling the adhesive dicate that Flo11 acts as an unusual membrane-teth- N-terminal A-domains of fungal adhesins in terms known pigment for this function. properties of yeast cells, e.g., by the development of ered hydrophobin that mediates long-range cell-cell Veelders M, Brückner S, Ott D, of structure and specificity, because these domains The structure of Rhodobacter sphaeroides crypto- anti-adhesive antimycotics. interactions. Unverzagt C, Mösch HU, Essen LO determine the physical nature of cell-cell interactions. chrome B, a regulator of the photosynthetic appa- In bakers yeast, flocculins F( lo), a family of adhesins, (2010). Structural basis of the social function of flocculins. ratus genes, revealed not mediate aggregation of yeast cells into protective Proc Natl Acad Sci USA 107, 22511-16. Short CV only the photolyase-like flocs by self-recognition. Although flocculation plays fold and a third type of an an eminent role in and food industry 2001– Professor of Biochemistry, electron-transfer pathway Philipps-Universität Marburg (e.g., for beer & wine), the mode of flocculin-based 1996–2001 Research Group Leader, to FAD, but also two co- surface recognition and the nature of cognate ligands Max Planck Institute for Biochemis- factors only found in the remained elusive. By analyzing crystal structures of try, Munich CryPro subfamily: 6,7-di- the adhesion domain of flocculin 5 complexed to its 1995–1996 Postdoctoral Fellow, methyl-8-ribityl-lumazine cognate ligands, we found a PA14-like lectin fold that Laboratory of Molecular Biology, in its antenna-binding utilizes a unique DcisD calcium-binding motif for car- MRC, Cambridge, UK domain, and a [4Fe-4S]2+ bohydrate binding (Veelders et al, 2010). The structure 1995 Ph.D. in biochemistry, Max cluster within the catalytic allowed to engineer yeast strains, whose flocculation Planck Institute for Biophysics, domain (Geisselbrecht et is controlled by glucose and are hence applicable in Frankfurt al, 2012). Especially the biotechnology and the brewing industry. 1991 Diploma in biochemistry, ETH latter resembles the large The epithelial adhesins (Epa) from the human patho- Zürich, Switzerland primase subunit PriL, a key gen Candida glabrata are not only responsible for component of eukaryotic host-cell adhesion, but highly related to flocculin do- and archaeal DNA replica- mains. Therefore, we studied structures of the differ- tion. This finding implies ent Epa domains complexed to different carbohydrate that primases are evolu- ligands (Fig. 3). In doing so, we showed how the mu- tionary closely related to cins of human hosts are specifically discriminated by the CryPro subfamily of various Epa subtypes (Maestre-Reyna et al, 2012), and cryptochromes. that specificity is mainly governed by variation of two Figure 3. Structural analysis of the adhesion domain of Epa1 from Candida glabrata allowed to map its carbohydrate binding site, whereas specificity profiling by glycan chips allowed to identify human mucins as inner loops, CBL1 and CBL2. predominant interaction partners. F ungal adhesins Not all fungal adhesins utilize a lectin-based mode with different of interaction, though. Flocculin 11, which confers Aa w rds & Honors the formation of different multicellular structures Figure 1. The photon-sensitive degron developed in collaboration with the group of Christoph Tax- mechanisms 2012 Lee-Wee-Nam Visiting Professor, is, Philipps-Universität Marburg, allows light-dependent degradation of fused protein partners. such as biofilms, filaments or flors in bakers yeast, NTU, Singapore Top, structural and biophysical analyses found that upon illumination the end (yellow) of a light- A hallmark of fungi is their is one example: The Flo11A domain structure reveals sensitive domain (LOV, green) gets loosened and hence exposes a degron motif for proteasomal 1996 TMR Marie-Curie Fellowship degradation. Middle & bottom, quantitative modeling allows to predict its sensitivity to light thick cell wall that pro- a β-sandwich of the fibronectin typeIII type covered 1991 Silver medal of the ETH Zürich and can be manifold applied, for example to get yeast cells acting as a photographic medium. vides not only a strong by a unique hydrophobic apical region. We show that 14 research 15

that simple protein-protein interactions and protein- Selected publications DNA binding are sufficient, in principle, to implement Computational approaches to Domínguez-Escobar J, Wolf D, Fritz G, genetic circuits with the capabilities of memory de- HöflerC , Wedlich-Söldner R, vices, i.e., latches and flip-flops, known from digital Mascher T (2014). Subcellular local- microbial gene regulation electronics (Hillenbrand et al, 2013). However, while in ization, interactions and dynamics digital memory circuits the timing of all operations is of the phage-shock protein-like Lia Mathematical modeling and theoretical simulations While in many two-component systems the histi- rapidly updated by virtue of a synchronizing ‘clock’, response in Bacillus subtilis. have become valuable tools in the analysis of biologi- dine kinases perceive their stimuli autonomously, in biological memory circuit designs must compensate Mol Microbiol 92, 716–32. cal networks. In our group, we use such computation- Bce-like systems the ABC transporter is also strictly for their inherently slow and asynchronous dynamics. al approaches to elicit how microbes adapt to environ- required for stimulus perception. Recently, we used a In , such ‘sequential logic’ circuits Fritz G, Megerle JA, Westermeyer S, mental changes. For instance, we study the response combined experimental and theoretical approach to could be used, e.g., to memorize whether bacteria Brick D, Heermann R, Jung K, of Bacillus subtilis against antimicrobial peptides and discriminate between alternative models that might have encountered specific chemicals in the past. Rädler JO, Gerland U (2014). Single scrutinize the adaptation of Escherichia coli to fluc- explain this mutual requirement. In this work, we As a first step towards the implementation of novel cell kinetics of phenotypic switching in the arabinose utilization system Dr. Georg Fritz tuating sugar concentrations. Through quantitative could show that the regulatory dynamics in the Bce genetic modules and devices, we established a stan- of E. coli. PLoS ONE 9, e89532. modeling of the in vivo adaptation dynamics, our system is compatible with a model in which BceS and dardized and well-characterized genetic toolbox com- Computational Microbiology computational approach gives unprecedented insight BceAB form a sensory complex that detects transport prising a basic set of vectors, promoters, reporters and Hillenbrand P, Fritz G, Gerland U Faculty of Physics, into the mechanisms of signal transduction and gene activity, i.e., the flux perceived by individual affinity-tags for the Gram-positive model organism (2013). Biological signal processing Philipps-Universität Marburg regulation. On a more general level, this enables us to ABC transporters, rather than the external concentra- B. subtilis (Radeck et al, 2013). However - despite all with a genetic toggle switch. Phone: +49 6421 28 22582 link molecular regulation mechanisms to phenotypic tion of the antibiotic itself (Fig. 1). Such an unusual standardization efforts - the success of implementing PLoS ONE 8, e68345. Email: georg.fritz@synmikro. behavior and, hence, to provide model-driven hypoth- mode of stimulus perception might have evolved as complex circuits into microbes is challenged by the uni-marburg.de eses for the evolutionary consequences associated a cost-efficient ‘produce-to-demand’ strategy, which inherent interconnectivity of virtually all compounds Radeck J, Kraft K, Bartels J,C ikovic T, Dürr F, Emenegger J, Kelterborn S, SYNMIKRO member since 2014 with different regulation strategies. In addition to induces only if the antibiotic flux per within a living cell. Therefore, one goal of our ongoing these reverse engineering efforts, we apply computa- transporter exceeds a certain threshold and, hence, if work is to better understand the context-dependence Sauer C, Fritz G, Gebhard S, Mascher T tional strategies in the forward design of synthetic cir- an increased demand for detoxification exists. of synthetic biology building blocks, and to ultimately (2013). The Bacillus BioBrick box: Figure 2. Diffusionaltrapping of LiaI-GFP foci under cell wall an- Generation and evaluation of es- cuits, where our analyses serve as a theoretical guide engineer “orthogonal”, i.e., context independent syn- tibiotic stress. The upper panel shows tracks of individual LiaI- sential genetic building blocks for for the efficient implementation of synthetic gene -ex F rom modules to networks: The cell GFP foci under unstressed (left) and stressed (right) conditions. thetic circuits. The lower panel shows the results of our computational analysis standardized work with pression programs and help to decipher some of the envelope stress response in B. subtilis of these tracks, indicating highly sub-diffusive motion ( <1) of Bacillus subtilis. J Biol Eng 7, 29. design principles of biological signal processing. LiaI-GFP under antibiotic stress conditions. In addition to specific resistance modules, B. subtilis Modeling the anatomy of antimicrobial also mounts more unspecific systems to stabilize protein that acts as a membrane anchor for the resistance modules and protect its cell envelope against the action of phage-shock protein homologue LiaH (Dominguez- antimicrobial peptides. One example for such ‘pro- Escobar et al, 2014). Upon envelope stress, LiaI and In natural habitats with limited amounts of nutrients, tection modules’ is the liaIH operon that is strongly LiaH copy numbers increase drastically and co-localize many bacterial species produce a variety of antimi- induced by a wide spectrum of peptide . into almost static foci (Fig. 2). Their highly sub-diffu- Short CV crobial agents to impede their competitors. Key mol- We recently showed that LiaI is a small membrane sive motion suggests that heterooligomers of LiaI ecules in this “chemical warfare” are and LiaH become trapped – potentially at sites of cell 2014– Independent Research Group so-called peptide antibiotics that of- wall damage, where they might exert their putative Leader, Philipps-Universität Marburg 2012–2014 Postdoctoral Fellow, ten target important steps in cell wall role in maintaining the integrity of the cytoplasmic LMU Munich biosynthesis. Bacitracin, for instance, membrane. Currently, we elucidate how resistance 2006–2012 Ph.D. in theoretical binds undecaprenyl pyrophosphate modules and protection modules are coordinated and physics, LMU Munich (UPP), a lipid carrier of glycan bio- interconnected within the larger cell envelope stress 2007 Visiting Scientist, University of synthetic intermediates, and thereby response network of B. subtilis. California, San Diego, USA blocks its recycling. Hence, for bacte- 2006 Diploma in physics, Albert- ria to successfully thrive in such en- F orward design and implementation of Ludwigs-Universität Freiburg vironments, it is of vital importance synthetic genetic circuits to mount specific stress responses, which allow them to cope with endo- A central goal of synthetic biology is to rationally en- Figure 3. Sequential logic design using cis-regulatory transcrip- and exogenously produced peptide gineer living cells with new functions. To guide this tion elements. Bacteria can ‘memorize’ transient signals by using antibiotics. forward design process theoretically, we perform in bistable genetic circuits, such as the genetic toggle switch. If the output of a regulatory circuit depends both on the present inputs The BceRS-BceAB system of B. subti- silico simulations based on the in vivo characteris- as well as on the state of this internal ‘memory bit’, microbes can lis is one of these widely conserved tics of biological parts and thereby explore the sig- condition their responses upon signals perceived in the past. peptide sensing and detoxification nal processing potential of novel circuit modules. modules, conferring resistance to a For instance, it is well-established that bacteria can broad spectrum of compounds in- use their cis-regulatory transcription machinery to cluding bacitracin. It consists of the implement combinatorial logic gates (AND, OR, etc.), ABC transporter BceAB, which facili- allowing them to integrate multiple external stimuli tates removal of the antibiotic from to generate an appropriate transcriptional response. its target site by a so far unknown However, less is known about how microbes condition mechanism, and the two-component Figure 1. Need-based activation of antibiotic resistance by a flux-sensing mechanism. their responses upon signals perceived in the past, Aa w rds & Honors If the bacitracin flux per BceAB transporter exceeds a certain threshold (upper panel), system BceRS, which in turn induc- signaling via the two-component system BceRS leads to the up-regulation of bceAB that is, how and to what extent they can “memorize” 2008 CeNS Publication Award es the expression of BceAB (Fig. 1). expression levels, thereby reducing the bacitracin flux per transporter (lower panel). their environment. In a theoretical work, we showed 16 research 17

Selected publications

Mechanisms of Jonas K (2014). To divide or not to divide: control of the bacterial cell bacterial cell cycle control cycle by environmental cues. Curr Opin Microbiol 18, 54-60.

Important cellular processes are generally orches- manner. The second control module centers on the Jonas K, Liu J, Chien P, Laub MT trated by regulatory circuits, which are composed of response regulator CtrA that governs the asymmet- (2013). Proteotoxic stress induces a numerous genes and proteins. Our group studies the ric replicative fates of the two daughter cells, thereby cell cycle arrest by stimulating Lon operation principles, organization and evolution of acting as a spatial control module (Jonas et al, 2011; to degrade the replication initiator such circuits, focusing on the regulatory mechanisms Chen et al, 2011). Both DnaA and CtrA activities are DnaA. Cell 154, 623-36. that govern cell cycle progression in bacteria. In par- controlled by their own regulatory sub-circuits, which ticular, we are interested in understanding how cells allow them to function largely independently of each Jonas K, Chen YE, Laub MT (2011). Modularity of the bacterial cell cycle dr. Kristina Jonas adjust their cell division cycle in response to inputs other. Importantly, the genetic separability of spati- enables independent spatial and from the environment. As a primary model we use al and temporal control modules is reflected in their Cellular Circuits temporal control of DNA replication. the α-proteobacterium Caulobacter crescentus, which evolutionary history. DnaA is the central component Curr Biol 21, 1092-101. Faculty of Biology, Figure 2. Stress-mediated control of DNA replication. In optimal conditions the replication initiator DnaA accumulates and initiates divides asymmetrically and whose cell cycle can be of an ancient and phylogenetically widespread circuit DNA replication. Stress-induced protein unfolding causes the upregulation and activation of Lon, which induces DnaA degradation and Philipps-Universität Marburg easily synchronized. In addition, we also make use that governs replication timing in Caulobacter and a block of replication initiation. Chen YE, Tropini C, Jonas K, Tsokos CG, Phone: +49 6421 28 25321 of other model organisms such as Escherichia coli to most other bacteria. By contrast, CtrA, which is found Huang KC, Laub MT (2011). Email: kristina.jonas@synmikro. learn how the cell cycle circuit of Caulobacter relates only in asymmetrically dividing α-proteobacteria, was the levels of the conserved replication initiator DnaA and investigate how the degradation of specific pro- Spatial gradient of protein phos- uni-marburg.de to that of other bacteria. Based on our insight into the recruited later in evolution as an additional control (Fig. 2). We could show that a sophisticated network teins can be induced in response to certain input si- phorylation underlies replicative asymmetry in a bacterium. SYNMIKRO member since 2013 mechanisms governing cellular information process- layer to enforce replicative asymmetry of daughter encompassing a chaperone system as well as the gnals. In a next step, we aim to reprogram substrate ing, we develop novel tools for controlling cellular be- cells. Currently, we investigate the organization and AAA+ protease Lon tightly controls degradation of selectivity to trigger the destruction of pre-defined Proc Natl Acad Sci USA 108, 1052-57. haviors in synthetic biology settings. function of stress response circuits and study how DnaA in response to changes in the global protein proteins upon protease stimulation. Such controlla- Jonas K, Edwards AN, Ahmad I, they have become interlinked with the core modules folding state (Jonas et al, 2013). Under optimal growth ble degradation systems are critical for the develop- Romeo T, Römling U, Melefors Ö driving cell cycle progression during evolution. conditions, DnaA is relatively stable and accumulates ment of synthetic circuits, which so far largely rely on Modularity of (2010). Complex regulatory network complex regulatory systems at high abundance to initiate DNA replication with transcriptional control mechanisms. encompassing the Csr, c-di-GMP C ell cycle control by environmental cues maximal frequency. By contrast, stress conditions and motility systems of C omplex regulatory circuits are often built of simpler causing the unfolding of proteins, for example heat Salmonella typhimurium. parts or modules that carry out specific, but separable Research in the past has led to the identification of stress or chaperone failure, induce transcription of the Environ Microbiol 12, 524-40. functions. Such a modular architecture is thought to the key factors required for cell cycle progression and lon gene and stimulate activity of the Lon protease, Short CV enhance evolvability as it allows for the generation of we have by now a relatively detailed understanding of which then promotes degradation of DnaA (Jonas et 2013– Independent Reseach Group new functions by simply reusing existing modules in how these factors are wired in higher-order circuits to al, 2013). Leader, Philipps-Universität Marburg different contexts rather than inventing entire new drive cell cycle progression under optimal conditions. Currently, we are studying additional mechanisms 2009–2013 Postdoctoral Fellow, Mas- networks from scratch. Our previous work demonstra- However, it is only poorly understood how cell cycle that transduce environmental information into the sachusetts Institute of Technology, ted that the task of regulating DNA replication during progression is modulated in response to environmen- cell cycle machinery of C. crescentus. For example, we Cambridge, USA the C. crescentus cell cycle is distributed between two tal cues to ensure optimal adaptation and survival have evidence that in addition to DNA replication, the 2005–2009 Ph.D. in infection biol- genetically separable modules (Fig. 1) (Jonas et al, 2011). under changing conditions (Jonas, 2014). Our recent process of cell division is also subject to environmen- ogy, Karolinska Institute, Sweden One of them centers on DnaA, a positive regulator of work revealed that distinct stress conditions cause tal control. Our work shows that distinct stress condi- 2004 M.Sc. in biology, Uppsala University, Sweden DNA replication that dictates the periodicity of repli- C. crescentus to arrest the cell cycle with a single chro- tions cause a reversible block of cell division. We want 2004 Diploma in biology, University cation and hence regulates replication in a temporal mosome in G1-phase, which is mediated by a drop in to unravel the regulatory mechanisms leading to this of Tübingen stress-mediated cell division block and try to under- stand how a tight control of cell cycle processes can enhance the fitness of microorganisms in changing environments (Fig. 3).

T ools for the development of synthetic circuits

Understanding the general principles of bacterial decision-making processes will help us to reprogram Figure 3. Morphological responses of wild type C. crescentus to bacterial behaviors for custom-made applications. distinct stress conditions. (A) no stress, (B) heat shock, (C) carbon Aa w rds & Honors Based on our mechanistic insight into regulatory starvation, (D) salt stress, (E) long-term growth in stationary phase. processes, we aim to design and establish tools for 2011 Selected into the excellence manipulating and utilizing bacterial behaviors. For program Fast Track of the Robert instance, understanding the molecular mechanisms Bosch Foundation 2009 Postdoctoral fellowship from of inducible protein degradation will enable the en- the German Research Foundation (DFG) gineering of synthetic degradation systems, which 2005 Marie Curie Early Stage Training can eliminate proteins of interest in a highly regula- Figure 1. Modularity of the Caulobacter crescentus cell cycle. (A) Schematic of the asymmetric cell cycle of C. crescentus. (B) DnaA and Fellowship from the European Com- CtrA compose two control modules that separate the temporal and spatial control of DNA replication. (C) The genetic separability of ted manner. Therefore, we currently dissect the rules mission DnaA and CtrA functions is reflected in phylogeny. governing substrate selectivity of the protease Lon 18 research 19

Selected publications

Dynamics of signal transduction and Brückner S, Kern S, Birke R, Saugar I, Ulrich HD, Mösch HU (2011). The multicellular development in yeast TEA transcription factor Tec1 links TOR and MAPK pathways to coordinate yeast development. Biological signaling circuits and networks are often programs, for instance the central MAPK module and Genetics 189, 479-94. capable of processing several distinct stimuli, and the transcription factor Ste12 (Fig. 1B). It also harbors yet faithfully elicit appropriate cellular programs in a components that are required for only one of the two Brückner S, Mösch HU (2012). signal-specific manner.I n many cases, the wiring of programs, for example the TEA family transcription Choosing the right lifestyle: the individual signaling components and the accord- factor Tec1 that controls multicellular development adhesion and development in ing dynamic behavior of signaling pathways are not in combination with Ste12. We have previously shown Saccharomyces cerevisiae. yet well understood. We are studying the interplay of that in response to sexual pheromone, Tec1 is rapidly FEMS Microbiol Rev 36, 25-58. Prof. Dr. such pathways in the budding yeast Saccharomyces degraded to ensure efficient execution of the mating Heise B, van der Felden J, Kern S, Hans-Ulrich Mösch cerevisiae, which can choose between different life- program (Heise et al, 2010). More recently, we found Malcher M, Brückner S, Mösch HU styles (Brückner & Mösch, 2012) and, depending on the that Tec1 protein stability is also under control of the (2010). The TEA transcription factor correct environmental stimuli, undergoes for example nutrient-sensitive TORC1 signaling pathway, which Tec1 confers promoter-specifc gene Faculty of Biology, sexual cell fusion or multicellular development (Fig. plays a major role in cell growth and development in regulation by mechanisms depend- Philipps-Universität Marburg 1A). In doing so, we want to get detailed insights into most eukaryotes (Brückner et al, 2011). These findings ent and independent of Ste12. Phone: +49 6421 28 23013 (i) the topology and dynamic behavior of the respon- indicate that the MAPK and TORC1 signaling pathways Eukaryot Cell 9, 514-31. Email: [email protected] sible signal transduction pathways, and into (ii) the are linked via Tec1 to coordinate control of cellular SYNMIKRO member since 2010 precise spatial organization and temporal assembly development in response to sexual and nutritional Leinweber M, Bitter P, Brückner S, dynamics of the resulting multicellular growth forms. stimuli. In our attempt to further elicit the cellular Mösch HU, Lenz P, Freisleben B wiring of the MAPK pathway, we lately discovered that (2014). GPU-based simulation of yeast flocculation. PDP 22, 601-8. Cellular wiring and dynamic behavior of Tec1 and Ste12 can associate with the transcriptional Figure 2. Spatial analysis and simulation of yeast flocculation. (A) Illustration of cell-cell adhesion between two bearer cells that both co-regulators Msa1 and Msa2, which were previously present the mannobiose ligands and the Flo5 adhesin (green) or between a bearer cell and a cheater cell (red), which presents the ligand MAPK signaling pathways only. (B) Microscopic picture (left) and simulated 3D visualization (right) of a small floc consisting of a mixed population of bearer van der Felden J, Weisser S, found to control cell cycle specific transcription (van (green) and cheater (red) cells. (C) 3D real-time visualization of yeast flocculation with OpenCL showing screen shots after 0 (Start), Brückner S, Lenz P, Mösch HU (2014). 2.000 and 5.000 iteration steps, respectively. Lifestyle adaptation of S. cerevisiae is under control of der Felden et al, 2014). These findings add an addi- The transcription factors Tec1 and several evolutionary conserved signal transduction tional layer of complexity to the cellular wiring of the Ste12 interact with coregulators pathways including a MAPK cascade. This pathway MAPK cascade and the control mechanisms exerted Ste12 and Tec1 activity. In collaboration with the group detailed mathematical model for yeast flocculation, Msa1 and Msa2 to activate adhesion Short CV controls a number of cellular programs, for example by this signaling pathway. of Peter Lenz, we have also started to develop math- which allows us to analyze the dependence of this and multicellular development. multicellular development in response to nutrient In order to study the dynamic behavior of MAPK sign- ematical models to describe the activity of the tran- process on various relevant parameters in silico. To Mol Cell Biol 34, 2283-93. 2004– Professor of Molecular availability and cell fusion upon stimulation by sex- aling, we have established a fluorescence microscopy- scription factors in response to activation of the up- simulate our model process with acceptable runtimes, Genetics, Philipps-Universität ual pheromone. The MAPK pathway contains several based system for quantitative measurement of MAPK stream MAPKs. Our preliminary data indicate that our we have further developed a graphics processing unit Marburg signaling components, which are required for both signaling in vivo at single cell level with a focus on system is valid, because we find that the average data (GPU) implementation that allows us to track the cell 2003–2004 Professor of obtained by single cell measurements are comparable movement in a detailed manner by 3D visualization Microbiology, Georg-August- to previous biochemical measurements at population during execution (Leinweber et al, 2014). This GPU Universität Göttingen level. In our future efforts, we want to uncover signifi- implementation is up to a factor of 736 faster than 1996–2003 Junior Research Group Leader, Georg-August-Universität cant cell-to-cell variation of MAPK activity and to ana- a multithreaded C/C++ implementation and allows Göttingen lyze the contribution of different regulatory sub-cir- simulating up to 1.000.000 yeast cells in total. Initial 2001 Habilitation, Georg-August- cuits to the dynamic behavior of the MAPK pathway. simulations have led to several theoretical predictions Universität Göttingen that can now be tested by appropriate in vivo experi- 1993–1996 Postdoctoral Fellow, Spatial analysis and computer ments. Here, we will focus for example on the behav- Whitehead Institute, Cambridge, simulation of yeast flocculation ior of mixed populations, where defined fractions of MA, USA cells do no produce the adhesion proteins required for 1991–1993 Postdoctoral Fellow, ETH The multicellular development options of S. cerevisiae flocculation. Our preliminary data indicate that such Zürich, Switzerland include the process of flocculation, which is the adhe- cheater cells are not only pushed to the boundary of 1991 Ph.D. in microbiology, ETH sion-dependent interaction among yeast cells leading the flocs, but are also restricted to specific sites within Zürich, Switzerland to multicellular aggregates, so-called flocs.T he ability these protective multicellular structures. 1986 Diploma in biochemistry, ETH Zürich, Switzerland to form flocs can provide an evolutionary benefit to a yeast population, for instance by protecting individu- al cells from harmful conditions in the environment. Importantly, flocculation is not only relevant for the study of fundamental biological questions, but also for biotechnological applications, for instance in the Figure 1. Control of vegetative and sexual development in Saccharomyces cerevisiae. (A) Vegetatively growing single yeast cells (black) brewing and wine industry. can either (i) adhere to and fuse with sexual partner cells to undergo mating (orange pathway), or they can (ii) express adhesins for ag- Within SYNMIKRO, we have established a fluores- gregation with other vegetative cells to form flocs/biofilms where they then reproduce asexually via budding (green pathway). (B) MAPK cence microscopy-based system for the precise spa- Aa w rds & Honors signaling cascade for regulation of mating genes in response to pheromone and for control of adhesin gene expression. Components required for mating and adhesin gene expression are shown in black, mating-specific proteins are orange. The position of the mitogen- tial analysis of single cells within individual flocs activated protein kinases MAPKs in the signaling cascade is indicated. Tec1 is shown in green to indicate its specific requirement for 1991 Medal of the ETH Zürich for vegetative adhesion. Ste12-binding sites (PRE) and Tec1-binding sites (TCS) are shown. The control of Tec1 protein stability by the TORC1 (Fig. 2). In collaboration with the groups of Peter Lenz outstanding Ph.D. thesis pathway and complex formation between Ste12, Tec1, Msa1 and Msa2 are indicated. and Bernd Freisleben, we have further developed a 20 research 21

Selected publications

Modulation of DNA sequences via Plagens A, Tripp V, Daume M, Sharma K, Klingl A, Hrle A, Conti E, prokaryotic immune systems Urlaub H, Randau L (2014). In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade Many bacteria and most archaea have simple immune and a minimal I-F variant (in Shewanella putrefaciens). interference complex. systems, the so-called CRISPR-Cas systems, which To investigate the processing of these subtypes, we Nucleic Acids Res 42, 5125-38. provide adaptive immunity against mobile genetic utilize a combination of computational, RNA-Seq and elements like phages and conjugative plasmids. In biochemistry approaches. E.g., RNA-Seq analyses veri- Daume M, Plagens A, Randau L these systems, specificity is conferred by theC RISPR fied transcription and maturation of crRNAs from all (2014). DNA binding properties of arrays, repeated DNA sequences that are interspersed investigated CRISPR loci. Furthermore, the cleavage the small Cascade subunit Csa5. with spacer sequences representing fragments of sites and the relative abundance of individual crRNA Figure 2. RNA-Seq profile of a CRISPR cluster. A long C. thermocellum cluster displays abundance variability of individual crRNAs. The PLOS ONE 9,e105716. Cas6b cleavage site within the repeats is indicated. Dr. Lennart Randau such foreign DNA (Fig. 1). Transcription and process- molecules were determined (Fig. 2) (Randau, 2012; Su Zöphel J, Randau L (2013). RNA-Seq ing of a CRISPR array yields small CRISPR RNA (crRNA) et al, 2013). Our differential NR A-Seq approach with se- Prokaryotic Small RNA Biology analyses reveal CRISPR RNA process- molecules that are then incorporated into a Cas ri- lective treatment of crRNAs with T4 polynucleotide ki- ing and regulation patterns. Max Planck Institute for Terrestrial bonucleoprotein complex termed Cascade (type I) or nase verified the presence of 5’-OH termini in crRNAs. Biochem Soc Trans 41, 1459-63. Microbiology Cas9 (type II). In case of a reinfection, these complexes Also, we identified the endonucleases responsible for Phone: +49 6421 178 600 utilize the base-complementarity between crRNA and crRNA maturation and reconstituted this reaction in Su AAH, Tripp V, Randau L (2013). Email: lennart.randau@mpi- foreign DNA to induce site-specific ND A cleavage. vitro. For subtype I-B, we discovered and biochemi- RNA-Seq analyses reveal the order marburg.mpg.de Only recently, the design of custom spacer sequences cally characterized a crRNA maturation endonuclease of tRNA processing events and the allowed for targeted DNA cleavage during genome that we termed Cas6b. M. maripaludis Cas6b can form maturation of C/D box and CRISPR SYNMIKRO member since 2010 RNAs in the hyperthermophile engineering, and the type II protein Cas9 is now fre- dimers in the presence of non-cleavable substrates Figure 3. DNA interference by a type I-A Cascade/crRNA complex. The T. tenax Cascade complex was reconstituted in the presence of quently used to change the sequence of bacterial and and harbors two catalytic histidine residues in a gen- crRNA and displayed target DNA cleavage (cleavage sites are indicated) in dependence of the correct protospacer-adjacent motif (PAM) Methanopyrus kandleri. Nucleic Acids Res 41, 6250-58. eukaryotic genomes in vivo. Our work within SYNMI- eral acid/base catalytic triad. and crRNA complementarity. KRO focuses on the processing, assembly and mecha- The profile of crRNA abundance for the fiveC RISPR loci Randau L (2012). RNA processing in nism of three different typeI Cascade complexes, and found in C. thermocellum revealed several surprises lum. Here, a second crRNA was identified that in turn from C. thermocellum was identified, and all five Cas the minimal organism Nanoarchae- on constructing a synthetic minimal variant as a po- (Zöphel et al, 2013): While promoters were already is complementary to the tRNA-targeting crRNA, and protein subunits can be produced as soluble recom- um equitans. Genome Biol 13, R63. tential genome editing tool, which would be a smaller known to exist within a leader region upstream of that might act as an RNA sponge to remove the harm- binant proteins. Finally, a minimal type I-F system can alternative to the Cas9 system. the CRISPR clusters, we could show that a spacer se- ful crRNA. The mechanism of antisense RNA-mediated be reconstituted from only four Cas protein subunits. quence with a potential promoter element can stimu- regulation of crRNA abundance will be investigated Future studies will address how these systems differ An anti-crRNA for regulation late internal transcription and dramatically raise the in the future - understanding the mechanisms that in their recognition of the DNA targets using differ- level of downstream crRNAs containing spacers which regulate crRNA abundance is pivotal to ensure that ent subtype-specificC as protein subunits. A minimal CRISPR/Cas systems are highly diverse – even though likely were acquired earlier in the organism’s evolu- synthetic crRNAs are functional in the cell. Cas3-guided interference complex might represent a Short CV they all contain crRNA and ribonucleoprotein com- tion. Moreover, spacer sequences were also shown viable alternative (with different target DNA specific- 2010– Max Planck Research Group plexes, both their Cas protein composition and the to promote anti-crRNA transcription which could be A minimal alternative to CRISPR/Cas9 ity and restrictions) to currently popular Cas9-mediat- Leader, Max Planck Institute for way the CRISPR arrays are processed differ significant- used to regulate the abundance of potentially harm- ed genome editing methods. Terrestrial Microbiology, Marburg ly. In our studies, we focus on the three CRISPR/Cas ful crRNAs. Indeed, we found one striking example of F urthermore, our group aims to establish the func- 2006–2010 Postdoctoral Fellow, Yale subtypes I-A (in Thermoproteus tenax), I-B (in Methano- such a problematic self-targeting crRNA that is com- tional reconstitution of Cascade effector complexes in University, USA coccus maripaludis C5 and Clostridium thermocellum), plementary to a tRNA-Proline of the host C. thermocel- vitro, and of a minimal Cascade effector complex in 2006 Ph.D. in microbiology, Techni- the heterologous host Escherichia coli. Here, the par- cal University of Braunschweig allel analysis of three ribonucleoprotein complexes 2002 Diploma in biology, Technical allows for the elucidation of mechanistic differences University of Braunschweig and similarities and enables us to investigate the function and evolution of individual Cas protein sub- units. Until now, we have successfully assembled the first complete typeI -A Cascade complex consisting of six Cas protein subunits and synthetic crRNAs (Pla- gens et al, 2014; Daume et al, 2014). This ribonucleo- protein complex was shown to be active and to cleave DNA in dependence of crRNA complementarity and a three nucleotide protospacer-adjacent motif (Fig. 3). Furthermore, we could show that the endonuclease responsible for target DNA cleavage, the Cas3 protein, is an integral part of the type I-A Cascade of T. tenax, whereas in the type I-E Cascade of, e.g., E. coli, Cas3 was shown to be a separate protein only temporarily recruited to the complex. Therefore, we hypothesize Aa w rds & Honors that this integral Cas3 is an adaptation to the mostly 2010 Max Planck Research Group Figure 1. Composition of CRISPR-Cas systems. A CRISPR-Cas system consists of genes coding for Cas proteins and a CRISPR array with hot environments of organisms with CRISPR-Cas sub- appointment repeat DNA elements and interspersed spacers that can be derived from previously encountered mobile genetic elements. types I-A. Also, the composition of a type I-B Cascade 22 research 23

Selected publications

Design principles of Kentner D, Sourjik V (2009). Dynamic map of protein interac- microbial networks tions in the Escherichia coli chemot- axis pathway. Mol Syst Biol 5, 238.

All complex functions in a cell are executed by groups nals – e.g., nutrients like sugars – are perceived and Oleksiuk O, Jakovljevic V, of interconnected proteins. Understanding the struc- processed by the chemosensory clusters and subse- Vladimirov N, Carvalho R, Paster E, ture and operation of these protein networks is one quently transmitted to flagellar motors F( ig. 1A). Over Ryu WS, Meir Y, Wingreen NS, of the next grand challenges in biology, and it is the the last years, we used fluorescence resonance energy Kollmann M, Sourjik V (2011). Ther- first step to the rational design of novel synthetic net- transfer (FRET) combined with computer modeling and mal robustness of signaling in works. In our group, we combine tools of quantitative simulations to quantify the function of the chemot- bacterial chemotaxis. Cell 145, 312-21. fluorescence microscopy with mathematical modeling axis pathway in Escherichia coli. We applied FRET (Fig. Neumann S, Grosse K, and Sourjik V Prof. Dr. Victor Sourjik in order to characterize the real-time functioning of 1B) to map all interactions within the pathway and (2012). Chemotactic signaling via networks in microorganisms. We are particularly in- to study their dynamics upon chemotactic stimula- Figure 2. Protein interactions and assembly of the flagellar motor in E. coli. Microbial Networks phosphotransferase systems terested in elucidating mechanisms behind evolution- tion (Kentner & Sourjik, 2009). These studies revealed (A) FRET-based interaction map of flagellar motor and export apparatus proteins. Positive FRET pairs that correspond to direct interactions are indi- in Escherichia coli. Department of Systems and ary selected properties that are common to most net- how chemotactic stimuli are amplified through allos- cated by solid lines, while those reflecting protein proximity are indicated Proc Natl Acad Sci USA 109, 12159-64. Synthetic Microbiology, works, such as the capability to function robustly in a teric interactions between receptors within the che- by dotted lines. (B) Sequential assembly pathway of the flagellar motor. Open arrow indicates promotion of assembly. (C) Motor stability, with grey Max Planck Institute for Terrestrial noisy environment, to detect and integrate multiple mosensory clusters. They also showed how multiple scale indicating exchange of protein at the flagellar motor measured by Press MO, Li H, Creanza N, Kramer G, Microbiology extra- and intracellular cues with high sensitivity, and signals are integrated within the pathway, including FRAP, with darker shading corresponding to slower exchange. Queitsch C, Sourjik V, Borenstein E Phone: +49 6421 28 21400 to self-organize into macromolecular complexes within such general physic-chemical stimuli as pH and tem- (2013). Genome-scale co-evolution- Email: victor.sourjik@synmikro. a cell. Ultimately, we would like to understand why the perature, and sugar-uptake related stimuli mediated metabolic requirements and growth conditions. We studied protein networks and structures in E. coli as ary inference identifies functions mpi-marburg.mpg.de observed network designs were evolutionary selected by the cytoplasmic phosphotransferase system (PTS) also utilize the potential of signal amplification and model systems. Thus far, we have studied the chem- and clients of bacterial Hsp90. PLoS Genet 9, e1003631. SYNMIKRO member since 2013 out of a large number of possibilities, and to use such (Neumann et al, 2012). Here, receptor interactions integration within the chemotaxis pathway to de- osensory complexes and flagellar motors, as well as established principles of “evolutionary design” for syn- serve an additional function to couple the response velop novel biosensors that can sensitively detect a the network of multiple chaperone systems that regu- Krembel AK, Neumann S, Sourjik V thetic biology applications. and adaptation to different stimuli, which is neces- variety of chemicals such as xenobiotics, and their lates refolding of protein aggregates. In all cases, we (2015). Universal response- sary for optimal navigation in chemoeffector gradi- combinations. used a combination of FRET with imaging to elucidate adaptation relation in bacterial Signal processing and integration by ents (Krembel et al, 2014). the order of the structure assembly and the interac- chemotaxis. J Bacteriol 197, 307-13. Short CV the bacterial chemotaxis network Currently, we investigate the relation between the Robustness of signaling in chemotaxis tions involved in this process (Fig. 2A). Furthermore, chemotactic and metabolic preferences of E. coli, elu- we applied fluorescence recovery after photobleaching 2014– Honorary Professor of Biology Bacterial chemotaxis is one of the best-studied model cidating mechanisms that enable cells to optimally Robustness is believed to be one of the major underly- (FRAP) to explore the dynamics of these assemblies. and Physics, Philipps-Universität systems for signal transduction. Chemotactic sig- adjust chemotactic signaling dependent on their ing properties of cellular networks, but its implemen- For all of these structures, we demonstrated that the Marburg tation in specific cases remains poorly understood. assembly proceeds through a well-defined sequence 2013– Director and Head of the Department of Systems and Synthetic We showed that signaling in chemotaxis is robust of steps, where formation of each subsequent struc- Microbiology, Max Planck Institute against stochastic variations in protein levels or gene ture stabilizes the entire assembly (Fig. 2B). We believe for Terrestrial Microbiology, Marburg expression noise, and demonstrated that this robust- that such combination of the hierarchical order with 2009–2013 Professor of Molecular ness is an evolved property of the pathway topology, gradual stabilization represents a general rule for the Biology, Zentrum für Molekulare being achieved through the balance of opposing en- assembly of large macromolecular structures. Biologie der Universität Heidelberg zymatic activities at the level of the pathway output. We further showed that both chemosensory clusters (ZMBH) More recently, we have analyzed the robustness of the and flagellar motors consist of several subcomplexes 2003–2009 Junior Group Leader, chemotaxis pathway against another common type of with different stability F( ig. 2C), which ensures that Zentrum für Molekulare Biologie der perturbation, namely variations in ambient tempera- the structure can be gradually recycled while retain- Universität Heidelberg (ZMBH) ture. We demonstrated that such thermal robustness ing its function. Recently, we have further demon- 1998–2003 Postdoctoral Fellow, is achieved through several compensatory mecha- strated that cellular assembly of the motor and of Harvard University, USA 1997 Ph.D. in genetics, Universität nisms, primarily because similar temperature effects the chemosensory clusters is assisted by a molecular Regensburg on opposing reactions cancel each other, but also due chaperone HtpG, a highly conserved member of the 1993 Diploma in physics and to the growth-temperature dependent adjustment Hsp90 chaperone family that up to now has not been engineering, and M.Sc. in molecular of the enzyme levels (Oleksiuk et al, 2011). Currently, assigned a clear physiological function in bacteria biology, Moscow Institute of Physics we apply a similar framework to analyze robustness (Press et al, 2013). Using FRET, we have also observed and Technology, Russia of the mating pathway in the budding yeast S. cerevi- physical interactions between the flagellar motor and siae. Finally, we have developed a general mathemati- several other systems, such as membrane-embedded cal framework for the analysis of network robustness energy complexes or second-messenger binding pro- based on topology, which will be useful for the future teins that directly tune bacterial motility dependent design of robust synthetic networks. on environmental or growth conditions (Fig. 2A). Aa w rds & Honors

2011 ERC Advanced Grant Assembly and dynamics of 2007 Chica und Heinz Schaller macromolecular complexes Research Award Figure 1. Spatial organization and interaction map of the chemotaxis network in Escherichia coli cells. (A) Localization of chemosensory 2006 EMBO Young Investigator clusters, labelled by a CheR-CFP (blue), and of flagellar motors, labelled by FliM-YFP (red). (B) FRET-based interaction map of the E. coli Another focus of our work is to understand general chemotaxis pathway. Positive FRET pairs correspond to direct interactions (solid lines) or proximities (dashed or dotted lines) between Award chemotaxis proteins, flagellar motor (FliM) and PTS components EI and EIIAGlc. Interactions that are independent of chemotactic stimu- principles behind assembly and dynamics of macro- 1990 DAAD Scholarship lation are indicated by open circles, those dependent on chemotactic stimulation by filled circles. molecular complexes in bacteria, using several well- 24 25

1.2 Research Metabolism 26 research 27

serves as an assembly Selected publications ATPase and, after a so far Synthetic microbiology in Archaea Wagner M, Wagner A, Ma X, Kort JC, unknown switch, it is es- Ghosh A, Rauch B, Siebers B, Albers SV The central theme of our research is the biology of generation biofuels. So far, strains were engineered sential for archaellum ro- (2013). Investigation of the malE thermoacidophilic archaea from the genus Sulfolobus that contain different cellulases from other Sulfolobus tation (Reindl et al, 2013). promoter and MalR, a positive ° which grow optimally at 75 C and a pH of 2-4. Mainly, strains, which enabled S. acidocaldarius to use extra- FlaH is a predicted ATP- regulator of the maltose regulon we use genetic approaches to identify systems in Sul- cellular cellulose for growth. Moreover, a transcrip- binding protein as it has a for the vector system in folobus that are involved in the assembly of cell sur- tional regulator, termed MalR, was identified which is Walker A motif, but a non- Sulfolobus acidocaldarius. face appendages like archaella, the “archaeal flagella”, involved in the regulation of the α-amylase and the canonical Walker B motif. Appl Environ Microbiol 80,1072-81. and to investigate how these appendages are regu- maltose ABC transporter gene expression (Wagner et As we could also show by lated. Furthermore, we biochemically characterize the al, 2013). We will now use the MalR controlled promot- single particle analysis Reindl S, Ghosh A, Williams GJ, subunits and their interplay in the assembly process. ers to manipulate the expression of genes important and biochemical interac- Lassak K, Neiner T, Henche AL, Albers SV, Tainer JA Tainer (2013). As the growth conditions preferred by our model or- for (ligno)cellulosic waste degradation. tion studies that FlaX, FlaH Insights on FlaI functions in ar- Prof. Dr. ganisms are used for the pretreatment of (ligno)cel- and FlaI interact with each Sonja-Verena Albers chaeal motor assembly and motility lulosic waste before fermentation, we also engineer An old structure with a new name: other with high affinity, we from structures, conformations and Molecular Biology of Archaea Sulfolobus acidocaldarius to streamline the fermenta- the archaellum propose that these three genetics. Mol Cell 49,1069-82. tion process. proteins form the cyto- Institute for Biology II, Like almost all other archaea, Sulfolobus has one cy- plasmic motor complex of Orell A, Peeters E, Vassen V, Albert-Ludwigs-Universität Freiburg Sulfolobus strains for biofuel production toplasmic membrane that is covered by a protein- the archaellum (Banerjee Jachlewski S, Schalles S, Siebers B, Phone: +49 761 203 2630 aceous layer, called the S-layer (Fig. 1). Inserted into et al, 2014). Albers SV (2013). Lrs14 transcrip- Email: [email protected] from (ligno)cellulosic waste the cytoplasmic membrane are the archaea’s organs The crystal structure of tional regulators influence biofilm freiburg.de Before (ligno)cellulosic waste can be fermented by en- of locomotion: the archaellum and two types of pili. the monotopic membrane formation and cell motility of Cre- Figure 2. Current model of the S. acidocaldarius archaellum. FlaI hexamerizes upon ATP binding narchaea. ISME J 7, 1886-98. SYNMIKRO member 2013 – 2014 gineered yeast strains, an expensive five step process Archaellins, the subunits of the archaellum, have class protein FlaF was dominat- and interacts with FlaH and FlaX in the motor complex. FlaF is probably acting as a stator as it binds to the N-glycans of the S-layer and thereby anchors the archaellum in the cell envelope. ensures that it has the right composition to sustain III signal peptides. The ATPase FlaI and the integral ed by b-sheets. As we had Banerjee A, Ghosh A, Mills DJ, Kahnt J, yeast growth. One of these is an acid treatment at membrane protein FlaJ in turn share high homologies so far only identified cyto- Vonck J, Albers SV (2013). FlaX, a high temperature. However, S. acidocaldarius can grow with the respective components from bacterial type plasmic components of the archaellum, we hypoth- regulators as possible players in this process. S. acido- unique component of the crenar- at the conditions used for this pretreatment, and vola- IV pili assembly machineries and type II secretion sys- esized that FlaF might be involved in anchoring the caldarius contains six Lrs14 regulators in its genome, chaeal archaellum, forms oligomeric tile products like alcohol would directly evaporate at tems, whereas the bacterial flagellum is assembled by archaellum in the cell envelope. As almost all extracel- four of which had an effect on biofilm formation. By ring-shaped structures and inter- acts with the motor ATPase FlaI. Short CV such high temperature, which would circumvent the a type III secretion system. This and other marked dif- lular proteins in archaea are N-glycosylated, we tested far the highest influence had AbfR1 that acts as an need to isolate alcohol resistant strains of S. acido- ferences led Ken Jarrell and our group to propose the FlaF for sugar binding and could demonstrate that it inhibitor of biofilm formation by inducing archaella J Biol Chem 287, 43322-30. 2014– Professor of Microbiology, caldarius. Therefore, synthetic Sulfolobus strains will name “archaellum” instead of “archaeal flagellum” binds chitobiose, the central sugar of the N-glycan of biosynthesis and inhibiting EPS production (Orell Jarrell KF & Albers SV (2012). University of Freiburg be constructed which can directly grow on (ligno)cel- (Jarrell & Albers, 2012). S. acidocaldarius, with high affinities.T herefore, FlaF et al, 2013). Deletion of AbfR1 therefore led to a high The archaellum, an old structure 2008–2014 Max Planck Research lulosic waste at high temperature and can simultane- Using thermomicroscopy, we could demonstrate that might act as the stator of the archaellum. increase of EPS production and subsequent commu- with a new name. Group Leader, Max Planck Institute ously convert it to volatile products. This would be a S. acidocaldarius uses its archaellum for swimming nity formation. Moreover, it was shown that AbfR1 is for Terrestrial Microbiology, Marburg Trends Microbiol 20, 307-12. cost-effective alternative to the hitherto used process. with speeds up to 15 µm/s. When tethered to a surface phosphorylated in vivo, and ongoing experiments will 2003–2008 Project Group Leader, Role of UV induced pili in gene transfer In our lab, different cellulolytic enzymes and sugar via their archaella, cells rotate, implying that the ar- University of Groningen, clarify whether this phosphorylation is involved in the Netherlands transporters are thus tested for their capability to en- chaellum is rotating in the cell envelope. This is espe- All Sulfolobales have a UV inducible pili system that regulatory mechanism of AbfR1. 2001–2002 Postdoctoral Fellow, able S. acidocaldrius to efficiently degrade (ligno)cel- cially interesting in view of the fact that the archael- causes the cells to aggregate after UV irradiation – in University of Groningen, Netherlands lulosic waste and take up the obtained carbohydrates lum is structurally type IV pilus-like. S. acidocaldarius, up to 90% of the cells can be found 2001 Ph.D. in molecular microbiol- to channel the energy into the production of second To this point we have characterized four of the seven in aggregates 3 hours after UV stress. Using a con- ogy, University of Groningen, subunits of the S. acido- jugation assay, we could demonstrate that the cells Netherlands caldarius archaellum bio- in these aggregates exchange DNA. Therefore, we 1996 Diploma in biology, University chemically and structur- hypothesized that these systems might be part of a of Würzburg ally (Fig. 2). The soluble community effort to repair ND A damage by homolo- domain of the monotopic gous recombination. Using specificFI SH probes, we membrane protein FlaX demonstrated that the aggregation in mixed cultures was shown to form very containing different Sulfolobus species is strain-spe- stable ring-like oligomers cific.T his fact of course makes sense if UV-induced with a diameter of 30 nm DNA exchange is indeed used as an alternative DNA in single particle analysis repair mechanism. Aa w rds & Honors (Banerjee et al, 2013). The structure of FlaI, the only Biofilm formation by Sulfolobus species 2013 ERC Starting Grant active ATPase in the ar- 2012 Research Award of the chaellum operon, showed Although it is known that archaea are present in vir- Association for General and a hexameric organization tually all environmental niches and also have been Applied Microbiology (VAAM) with large conformational described in natural mats or communities, surpris- 2008 Max Planck Research Group appointment changes dependent on the ingly little is known about the molecular details of 2006 VIDI grant by the Dutch nucleotide-bound state. community formation in archaea. Therefore, we have Science Organization (NWO) In contrast to other type established Sulfolobus species as a model system Figure 1. Model of the cell envelope of S. acidocaldarius. After insertion into the membrane, the 2003 VENI grant by the Dutch archaellins or pilins are processed by PibD, a class III signal peptidase, and assembled into either IV pili assembly ATPases, to identify the molecular drivers of archaeal biofilm Science Organization (NWO) the archaeal adhesive pili (aap), the UV induced pili (ups) or the archaellum. FlaI is bifunctional: It first formation. An –omics approach indicated Lrs14-like 28 research 29

cancer contains high levels Selected publications of (R)-2-hydroxyglutarate, Anaerobic biofactories for Buckel W, Kung JW, Boll M (2014). which most likely inhib- The benzoyl-coenzyme A reductase its demethylation of the and 2-hydroxyacyl-coenzyme A valuable carboxylic acids DNA. In these patients a dehydratase radical enzyme family. mutation has converted ChemBioChem 15, 2188-94. The amino acids occurring in proteins are the essen- Recombinant production of the isoenzyme 1 of isoci- tial building blocks of life and are synthesized by al- glutarate and adipate trate dehydrogenase to a Djurdjevic I, Zelder O, Buckel W (2011). most all organisms. Removal of the amino group from (R)-2-hydroxyglutarate de- Production of glutaconic acid in a amino acids yields carboxylic acids, which are used We engineered Escherichia coli into a glutaconate hydrogenase. Hence this recombinant Escherichia coli strain. as fungicides (propionate) and antioxidants (indole- producer by introducing six genes from strict an- mutant enzyme catalyzed Appl Environ Microbiol 77, 320-2. propionate), or as technically important precursors aerobic glutamate fermenting clostridia, one gene the reduction of 2-oxo- for the syntheses of polymers and pharmaceuticals for (R)-2-hydroxyglutarate dehydrogenase, two genes Figure 2. Schematic presentation of electron bifurcation. Etf, electron transferring flavoprotein; glutarate with NADPH Parthasarathy A, Pierik AJ, Kahnt J, Bcd, butyryl-CoA dehydrogenase. The figure shows how the two electrons from NADH bifurcate, Zelder O, Buckel W (2011). Substrate Prof. Dr. (succinate, glutaconate, glutarate, adipate, 3-hydroxy- for glutaconate CoA-transferase, and three genes for whereby the ‘energy rich’ reduced ferredoxin is formed. rather than the oxidation Specificity of 2-Hydroxyglutaryl-CoA Wolfgang Buckel propionate, 4-hydroxybutyrate). To produce carboxylic (R)-2-hydroxyglutaryl-CoA dehydratase (Fig. 1) (Djurd- of (2R,3S)-isocitrate with Dehydratase from Clostridium sym- + Biochemistry of Anaerobes acids from the renewable resource glucose, the amino jevic et al, 2011). Using its own enzymes, E. coli con- NADP . We speculated biosum: Toward a Bio-Based acid biosynthesis route has to be diverted in such a verts glucose to 2-oxoglutarate, which is then taken Flavin based electron bifurcation therefore that a similar mutation could convert ho- Production of Adipic Acid. Faculty of Biology, way that the 2-oxoacid precursor is not converted to over by the recombinant enzymes to be reduced and moisocitrate dehydrogenase to the desired highly Biochemistry 50, 3540-50. Philipps-Universität Marburg the amino acid, but reduced to the desired carboxylic dehydrated to glutaconate. Alternatively, reduction We studied this novel energy conserving process in specific (R)-2-hydroxyadipate dehydrogenase (2-ox- & Max Planck Institute for acid. Thus, the pathway from glucose via oxaloacetate of the intermediate glutaconyl-CoA would lead to more detail and characterized the bifurcating Etf oadipate reductase) (Parthasarathy et al, 2011). Based Chowdhury NP, Mowafy AM, Terrestrial Microbiology Af to the amino acid aspartate has been redirected to glutaryl-CoA and further to glutarate (Fig. 1). The same and butyryl-CoA dehydrogenase (Bcd ) from Acida- on our idea, this experiment was indeed successfully Demmer JK, Upadhyay V, Koelzer S, Phone: +49 6421 28 22088 Af succinate by the chemical company BASF, already on enzymes may be applied to convert 2-oxoadipate to minococcus fermentans which couple the exergonic performed by Reitman et al. in 2012. The Süddeutsche Jayamani E, Kahnt J, Hornung M, Email: [email protected] a 1000 tons scale. However, enzymes that catalyze the adipate, one constituent of Nylon® (Parthasarathy et reduction of crotonyl-CoA to butyryl-CoA (equation 1) Zeitung reported this result, and a Californian journal- Demmer U, Ermler U, Buckel W SYNMIKRO member since 2010 reduction of oxaloacetate to succinate are widespread, al, 2011). The reductions of glutaconyl-CoA to glutaryl- to the endergonic reduction of ferredoxin (Fd, equa- ist asked me for an interview. (2014). Studies on the mechanism of electron bifurcation catalyzed by whereas the production of the other carboxylic acids, CoA and of homoglutaconyl-CoA to adipyl-CoA could tion 2) both with NADH (Fig. 2) (Chowdhury et al, 2014). electron transferring flavoprotein which have no essential biological function, requires be catalyzed by glutaryl-CoA dehydrogenase from Propionate, 3-hydroxypropionate (3HP) (Etf) and butyryl-CoA dehydro- + = + enzymes that only occur in some strict anaerobic Syntrophus aciditrophicus (GdhSa). The reaction re- (1) Crotonyl-CoA + NADH + H Butyryl-CoA + NAD ; and indolepropionate genase (Bcd) of Acidaminococcus -1 clostridia. The key enzymes of these transformations quires the bifurcating electron transferring flavopro- DG’ = -52 kJ mol . fermentans. J Biol Chem 289, 5145-57. - + + -1 are the oxygen-sensitive (R)-2-hydroxyacyl-CoA dehy- tein (EtfSa) to direct the electrons from NADH to GdhSa. (2) 2 Fd + NADH = 2 Fd + NAD + H ; DG’ = +42 kJ mol The introduction of (R)-lactyl-CoA dehydratase from dratases that need an unpaired electron for catalysis, We have produced recombinant Gdh and currently Clostridium propionicum converted E. coli either to Zhang J, Friedrich P, Pierik AJ, Mar- Short CV Sa which is introduced by an ATP-dependent electron try to improve the formation EtfSa in E. coli. EtfAf contains one flavin-adenin-dinucleotide F( AD) a propionate or to a 3-hydroxypropionate (3HP) pro- tins BM, Buckel W (2015). Substrate induced radical formation in 4-hy- 2008– Emeritus Professor, Philipps- transfer from reduced ferredoxin (Buckel et al, 2014). in subunit α (α-FAD) and a second FAD in subunit b ducer (Fig. 1). 3HP is a technically very important acid - droxybutyryl-CoA dehydratase from Universität Marburg (b-FAD). NADH reduces b-FAD to b-FADH which is because it can be polymerized or easily dehydrated to Clostridium aminobutyricum. Appl 1987–2008 Professor of Microbiology, considered as the bifurcating cofactor. One electron the very useful compound acrylate. The microflora of Environ Microbiol 81, 1071-84. Philipps-Universität Marburg goes to α-FAD and yields a stable anionic semiqui- the human intestine reduces tryptophan to indole- 1969–1987 Research Scientist, none, α-FAD●-, which donates this electron further to 3-propionate, which is excreted into the blood. The Universität Regensburg d-FAD of BcdAf. The remaining non-stabilized neutral propionate side chain allows indolepropionate to 1975 Habilitation, Universität semiquinone, b-FADH●, immediately reduces ferre- cross the blood-brain barrier and the indole moiety Regensburg doxin. Repetition of this process affords a second re- protects the brain from reactive oxygen species (ROS), 1970–1971 Postdoctoral Fellow, duced ferredoxin and d-FADH- of Bcd that converts a causative agent of Alzheimer disease. Recently we University of California, Berkeley, USA Af crotonyl-CoA to butyryl-CoA. The reduced ferredoxin have shown that Clostridium sporogenes, an inhabit- 1968–1969 Postdoctoral Fellow, Ludwig-Maximilians-Universität, is used for many reductive processes including the ant of the intestine, converts tryptophan to indolep- München production of molecular hydrogen or the formation of yruvate which is reduced and dehydrated further to + 1968 Ph.D. in biochemistry, Ludwig- an electrochemical Na gradient mediated by a mem- indolepropionate in an identical manner as pyruvate Maximilians-Universität, München brane bound ferredoxin-NAD+ reductase, whereby to propionate (Fig. 1). 1965 Diploma in chemistry, Ludwig- NADH is regenerated. If glutaryl-CoA dehydrogenase Maximilians-Universität, München together with EtfSa works like EtfAf + BcdAf and both 4-Hydroxybutyrate enzymes are introduced in the glutaconate producing E. coli strain, then the conversion of glucose to glutar- 4-Hydroxybutyryl-CoA dehydratase is a wide spread ate might follow the very simple equation: Glucose = radical enzyme that catalyzes the reversible dehydra- 2- + Glutarate + 2 H + CO2 + 2 H2. tion of 4-hydroxybutyryl-CoA to crotonyl-CoA. Since crotonyl-CoA is easily derived from acetyl-CoA, the Enzyme redesign guided by now recombinant available enzyme from Clostridium Aa w rds & Honors cancer-derived mutations aminobutyricum opens a way to synthesize 4-hy- droxybutyryl-CoA from acetate or glucose (Zhang et 2008–2017 Fellow of the Max For the synthesis of adipate, one essential enzyme al, 2015). 4-Hydroxybutyryl-CoA can be polymerized to Planck Society 2011 and 2008 Visiting Professor at has to be constructed by mutagenesis. It should poly-4-hydroxybutyrate or reduced to 1,4-butanediol the University of Kyoto, Japan catalyze the specific NAD(P)H-dependent reduction that is currently produced from oil on a million tons 1970 Postdoctoral fellowship from of 2-oxoadipate to (R)-2-hydroxyadipate without ac- scale. tion on 2-oxoglutarate. It was reported earlier that the German Research Foundation Figure 1. Biosynthesis of biotechnological valuable acids in recombinant Escherichia coli strains. The figure demonstrates the central (DFG) role of (R)-2-hydroxyacyl-CoA dehydratases in these conversions. the blood of patients suffering from various types of 30 research 31

proteins with an amino acid sequence similarity larger Selected publications than 40% tend to have similar functions. Yet, below In silico methods for modeling, Leinweber M, Baumgärtner L, this threshold, results of sequence comparisons often Mernberger M, Fober T, Hüllermeier E, become uncertain. Klebe G, Freisleben B (2012). simulation and data analysis In this case, a structural comparison can provide fur- GPU-based Cloud computing for ther insights, especially when focusing on functionally comparing the structure of protein Mathematical modeling, computer-based simulation protein binding sites to predict protein function and important regions, such as protein binding sites. Sev- binding sites. 6th IEEE International and data analysis are useful for studying intra- and perform similarity retrieval, and (b) parallel simula- eral structural comparison algorithms are known, but Conference on Digital Ecosystems intercellular processes of microorganisms in order tion tools for studying the spatial organization and they have much longer runtimes than their sequence- Technologies (DEST), 1-6. to introduce novel synthetic variants. On the down- temporal assembly dynamics of multicellular growth based counterparts, severely limiting their use in side, these methods often require high computational forms. large-scale comparisons. One option to overcome this Leinweber M, Bitter P, Brückner S, runtimes and storage capacities. Our group develops obstacle is to pre-compute pairwise similarities of pro- Mösch HU, Lenz P, Freisleben B efficient algorithms to significantly speed up such Efficient structural analysis of tein binding sites in an all-against-all comparison, and (2014). GPU-based simulation of yeast cell flocculation. Prof. Dr. computations and reduce the complexity of handling to subsequently make them accessible to data analysis protein binding sites 22nd Euromicro International Bernd Freisleben “big data” in a biological context (Dalman et al, 2013; methods. Pairwise similarities can also be used to es- Conference on Parallel, Distributed and Haunschild et al, 2005). In collaboration with other A major goal in synthetic biology is the manipulation tablish a neighborhood structure to perform similarity C omputer Science - Distributed Network-Based Processing (PDP), 601-8. Systems SYNMIKRO research groups, we work on (a) parallel of the genetic setup of living cells in order to alter ex- retrieval more efficiently. Unfortunately, an all-against- software tools for large-scale structural analyses of isting and introduce novel biochemical pathways. A all comparison of the roughly 250.000 binding sites Haunschild M, Freisleben M, Faculty of Mathematics and prerequisite is a deep understanding of the biochemi- contained in CavBase, a database of protein-ligand Takors R, Wiechert W (2005). Computer Science, cal function of the proteins of interest. Since assessing data maintained by the Cambridge Crystallographic Investigating the dynamic behavior Philipps-Universität Marburg protein function experimentally is time-consuming Data Center, would require 297 years of computation of biochemical networks Phone: +49 6421 28 21568 and sometimes infeasible, protein function predic- time on a single CPU core of a contemporary computer. using model families. Email: [email protected] tion is a central task in computational biology. Typi- Selecting a biologically meaningful subset of about Bioinformatics 21, 1617-25. marburg.de Figure 1. GPU-based Cloud computing for structural analy- cally, the function of a protein is inferred from similar 144.000 binding sites would still require about 100 Dalman T, Dörnemann T, Juhnke E, SYNMIKRO member since 2010 sis of protein binding sites: sequential C++ program, paral- proteins with known functions, most prominently by years of computation time. lel OpenCL program, single GPU execution, multi GPU-based Weitzel M, Wiechert W, Nöh K, Amazon Cloud execution. sequence comparison, owing to the observation that By using a novel comparison algorithm, accelerating Freisleben B (2013). Cloud MapRe- the algorithm on graphical processing units (GPU) duce for Monte Carlo Bootstrap and parallelizing the computations in a GPU-based applied to metabolic flux analysis. Figure 3. Top: Microscopic image of a small floc consisting of a Cloud infrastructure of a set of Amazon EC2 Cluster mixed population of bearer (green) and cheater (red) cells. Mid- Future Generation Computer GPU Instances (Fig. 1) (Leinweber et al, 2012), 24 days dle and Bottom: 3D realtime visualization of yeast flocculation Systems 29, 582-90. showing screen-shots after the start, and after a few iteration instead of 100 years were required. This large-scale steps, respectively. Short CV study, performed in collaboration with the groups of Gerhard Klebe and Eyke Hüllermeier, resulted in a sim- 2002– Professor of Computer Science, ilarity database called CavSimBase (Fig. 2). Analyzing yeast flocculation depending on the cell surface ad- Philipps-Universität Marburg 1993–2002 Professor of Computer the similarity relation in its entirety requires methods hesin protein Flo5 has been proposed by the SYNMI- Science, Universität Siegen for dealing with “big data”. This is part of our current KRO research groups of Peter Lenz and Hans-Ulrich 1998–1999 Visiting Professor, North- work, and we invite the community to participate in Mösch. This kind of flocculation is mediated by the eastern University, Boston, USA this endeavor. CavSimBase can be accessed at: http:// heterophilic interaction between the Flo5 protein of 1993 Habilitation, Technische Uni- pc12872.mathematik.uni-marburg.de/. one yeast cell and mannobiose sugars in the cell wall versität Darmstadt of other yeast cells. The model can be used to perform 1985 Ph.D. in computer science, F ast computational modeling simulation studies in silico and recommend biotech- Technische Universität Darmstadt of yeast cell flocculation nological adjustments for optimizing the production 1981–1982 Research Scientist, IBM strains in vivo. Research and Development, C ells of the baker’s yeast Saccharomyces cerevisiae To significantly educer the runtimes of simulations, Böblingen interact with each other to form multicellular aggre- a GPU implementation of the model has been devel- 1981 M.Sc. in computer science, Pennsylvania State University, USA gates, called flocs.T his reversible asexual interaction oped in our work (Leinweber et al, 2014). Experimen- is calcium-dependent and provides an evolutionary tal results indicate that the GPU implementation is benefit for a yeast population, e.g., as a protection up to 736 times faster than a multithreaded C/C++ against environmental stress. Flocculation also is a implementation on a multi-core workstation for fast, cost-effective and environment-friendly way to simulations of up to 20.000 yeast cells. Moreover, it remove yeast cells at the end of fermentation process- requires only up to 225 milliseconds to simulate up to es in the production of beer, wine, ethanol, biodiesel 1.000.000 yeast cells. Finally, the quality of the simu- Aa w rds & Honors or pharmaceutical compounds. In addition, flocculent lation is improved, since the side effects of simula- yeast strains that effectively bind calcium ions have tions with limited cell numbers can be reduced, and 2007–2014 Best Paper Awards been used in bioremediation to remove other divalent second-nearest neighbor interaction can be modeled (IAS’07;CCGRID’08;AINA’09;CEC’11; ions, such as heavy metals, from contaminated sites. to increase the simulated level of detail (Fig. 3). WETICE’14) 2008 IBM Real-time Innovation Award Unfortunately, experimental optimization and control Our current work focuses on improving the model and 2007 IBM UIMA Award of flocculation is time-consuming and cost-intensive. finding optimal parameters to reproduce the natural 2005 IBM Eclipse Innovation Award Therefore, an understanding of the dynamics and behavior of yeast cells during flocculation.T he algo- Figure 2. Top: Embeddings of serine protease data sets based on structural information of binding site geometries (left) and se- 1986 IEEE Award for Outstanding quence alignments (right) for t-SNE. Bottom: Protein structure images related to two cavity-based t-SNE locations C2:2F6I (left) and structure of flocculation offers great benefits for -sev rithms for parameter optimization are designed to run Achievement C4:1TYF(right) taken from the protein data bank (http://www.rcsb.org/pdb/). eral industrial applications. A novel model describing in parallel on a large computing cluster of GPUs. 32 research 33

ther chemical synthesis. Selected publications It can also be synthesized Synthetic pathways for production Heider J & Schühle K (2013). Anaero- chemically, but is rather bic biodegradation of hydrocarbons costly and usually only ob- including methane. In: The prokar- of aromatic compounds tained as racemic mixture yotes-a handbook on the biology of both enantiomers. As of bacteria, 4th ed. (Rosenberg E, Microorganisms harbor a seen in Fig. 1, there are two DeLong EF, Thompson F, Lory S, multitude of biochemical principal ways of design- Stackebrandt E. eds), Chapter 80, pp. pathways which are involved ing a biochemical pathway 601-30, Springer-Verlag, Heidelberg. either in the biosynthesis or towards the synthesis of in the degradation of chemi- benzylsuccinate, using the Ebenau-Jehle C, Thomas M, Scharf G, cal compounds. The amount enzymes of anaerobic tol- Kockelkorn D, Knapp B, Schühle K, of naturally occurring chemi- uene metabolism: it can Heider J, Fuchs G (2012). Anaerobic Metabolism of Indoleacetate. Prof. Dr. Johann Heider cals produced or degraded by either be approached from Figure 2. Synthetic pathway for benzylsuccinate production in E. coli. The modules for benzoyl- CoA production and its conversion to benzylsuccinate are shown, assuming intrinsic succinate J Bacteriol 194, 2894-903. bacteria and archaea exceeds toluene and fumarate via Microbial Biochemistry generation via fermentative growth conditions. anything eukaryotic organ- the BSS reaction, or from Hilberg M, Pierik AJ, Bill E, Friedrich T, Faculty of Biology, isms are capable of. In addition benzoate and succinate, using the enzymes of the b- increased benzylsuccinate production was observed Lippert M-L, Heider J (2012). Philipps-Universität Marburg to this impressive metabolic oxidation module in reverse. Because of the extreme under anaerobic growth conditions. Therefore, it ap- Identification of FeS-clusters in the Phone: +49 6421 28 21527 variety, it is observed that mi- lability of BSS and our previous observations that all pears possible to further optimize this type of syn- glycyl radical enzyme benzylsucci- Email: [email protected] crobes can even adapt to the enzymatic reactions of benzylsuccinate degradation thetic pathways to redirect the metabolism of E. coli nate synthase via EPR and marburg.de degradation of most xenobi- are principally reversible in vitro, we tried to convert to produce suitable amounts of “alien” metabolites Mössbauer spectroscopy. J Biol Inorg Chem 17, 49-56. SYNMIKRO member since 2010 otics introduced into the en- the degradation pathway of benzylsucciante into a which may be as diverse as the different existing deg- vironment. Our group studies new backwards-moving synthetic pathway (Fig. 2). radation pathways in nature. the biochemistry behind the Because the intended new synthetic pathway starts Fuchs G, Boll M & Heider J (2011). Microbial degradation of aromatic bacterial degradation of re- Figure 1. Pathway of the anaerobic toluene metabolism. The first reaction is catalyzed by with benzoyl-CoA rather than benzoate, we con- F urther developments of benzylsuccinate synthase (BSS), followed by five enzymes of a b-oxidation module for ben- compounds – from one strategy to calcitrant substrates like aro- structed it in a modular fashion containing the genes zylsuccinate to benzoyl-CoA (bbs operon): BS-CT= benzylsuccinate CoA-transferase, BS-DH= synthetic pathways four. Nat Rev Microbiol 9, 803-16. matics or hydrocarbons under benzylsuccinyl-CoA dehydrogenase, PIH=phenylitaconyl-CoA hydratase, ADH=alcohol dehy- for benzoate uptake and activation as module I and drogenase, BST=benzoylsuccinyl-CoA thiolase. The successive metabolic modules for further anaerobic conditions. Whereas degradation of benzoyl-CoA are also indicated. the genes coding for the benzylsuccinate-degrading In our ongoing project, we are modifying further Leuthner B & Heider J (2000). Anaer- the presence of oxygen allows enzymes from Geobacter metallireducens as a second metabolic pathway modules in different directions. obic toluene catabolism of Thauera relatively easy degradation of these compounds even glycyl-radical enzyme benzylsuccinate synthase (BSS), module on two compatible inducible plasmids that The already established modules for synthesizing aromatica: a toluene-induced by eukaryotic organisms, their degradation under which represents the first module of the pathway and were introduced into Escherichia coli as heterologous benzoyl-CoA from externally supplied benzoate, e.g., operon codes for the enzymes of anaerobic conditions is limited to bacteria and ar- is encoded in a common operon with an activating host (Fig. 2). The transformed cells were grown under may be generally useful for the synthesis of many dif- b-oxidation of the intermediate ben- chaea. Since all known aerobic degradation pathways enzyme (BssD) necessary to create the active radical- either aerobic or anaerobic conditions with either ferent complex aromatic compounds via benzoyl-CoA zylsuccinate. J Bacteriol 182, 272-7. of these compounds include oxygen-dependent en- containing form of BSS (Fig. 1). The next module of this benzoate and succinate as precursor compounds, as building brick. To prove this point, we are trying to zymes, anaerobic degraders have developed com- anaerobic toluene metabolism is represented by five or benzoate only, as E. coli produces succinate in its combine this with biphenyl synthase (BIS), a different pletely new and surprising solutions to overcome successive enzymes involved in activation of benzyl- mixed-acid fermentation when grown anaerobically. benzoyl-CoA-metabolizing enzyme from rowan berry difficult reactions and have evolved novel pathways succinate and its subsequent degradation to benzoyl- Under aerobic conditions, very little benzylsuccinate trees. This enzyme belongs to the chalcone synthase connecting these unusual enzymic reactions. We are CoA and succinyl-CoA by a specialized b-oxidation was identified in the supernatant of induced cultures family and condenses a benzoyl-CoA starter molecule making use of the enzymes of these newly identi- pathway (Fig. 1). Benzoyl-CoA is the common inter- that were supplemented with benzoate and succinate with three malonyl-CoA units to a polyketide, which is fied pathways for the rational design of completely mediate of anaerobic degradation of any aromatics as precursor molecules, whereas an up to 1000-fold then converted to the secondary metabolite 3,5-dihy- new artificial biosynthetic pathways for interesting and is also generated from many other substrates. For droxybiphenyl (Fig. 3). bioproducts, using methods of synthetic biology. Our example, the metabolic module leading from benzo- In another extension proj- main resource organisms are denitrifying bacteria of ate to benzoyl-CoA consists of a benzoate transport ect, we intend to use en- the genera Thauera and Aromatoleum, Fe(III)-reducing system and a CoA-ligase or CoA-transferase as ac- zymes of the anaerobic Geobacter species or sulfate-reducing bacteria capa- tivating enzyme (Fig. 2). The further degradation of degradation pathway of ble of degrading aromatic or aliphatic hydrocarbons benzoyl-CoA involves benzoyl-CoA reductase, another indoleacetate to design a Short CV as sole substrates. highly unusual enzyme reducing the aromatic ring, similar synthetic pathway 2008– Professor of Microbiology, and several additional b-oxidation modules involved leading to phenylsuccinate Philipps-Universität Marburg Modularity of biochemical pathways in modifying and cleaving the dearomatised ring and instead of benzylsuccinate 2005–2008 Professor of Microbiology, further degrading the open-chain intermediates to (Fig. 3). This would show Technical University Darmstadt All biochemical pathways are designed in a modular acetyl-CoA and CO2 (Fig. 1). the general applicability of 1995–2005 Independent Research fashion and lead through a series of common interme- our strategy to use degra- Group Leader, Albert-Ludwigs-Uni- diates, which are interconverted by various cascades Re-design of the metabolic dation pathways based on versität Freiburg involving successive enzyme reactions. The enzymes routes of benzylsuccinate b-oxidation in reverse for 1999 Habilitation, Albert-Ludwigs- involved in such modules are very often encoded in the design of novel syn- Universität Freiburg common operons in bacteria, which eases their coor- We were attracted by the observation that the unusu- thetic routes. 1994–1995 Postdoctoral Fellow, Uni- versity of Georgia, Athens, GA, USA dinate regulation. As an example, the anaerobic deg- al metabolite benzylsuccinate occurs in the pathway 1992 Ph.D. in biology, Ludwig-Maxi- radation of toluene is initiated by an unusual reaction exclusively as the (R)-enantiomer. Like any derivative milians-Universität München adding the methyl group to a fumarate cosubstrate to of the important platform chemical succinate, benzyl- Figure 3. Additional planned synthetic pathways for aromatic compound synthesis from ben- yield the first intermediate (R)-benzylsuccinate. This succinate promises a variety of potential uses, e.g., for zoate. IaaL=thiolase, IaaP=hydratase/dehydrogenase, IaaF=acyl-CoA dehydrogenase, IaaGH=B12- 1988 Diploma in biology, Ludwig- dependent mutase, IaaL=CoA-transferase, all involved in anaerobic indoleacetate degradation; Maximilians-Universität München reaction is catalyzed by the extremely oxygen-labile the synthesis of polymers or as building block for fur- BIS=biphenyl synthase. 34 research 35

Selected publications

Structural basis of protein-ligand Klebe G (2015). Applying thermody- namic profiling in lead finding and protein-protein recognition and optimization. Nat Rev Drug Discov, doi:10.1038/nrd4486

The research of the Drug Design Group within SYN- The structural information is then linked via computer Fober T, Glinca S, Klebe G, Hüller- MIKRO is concerned with the question how small mol- simulations that try to predict binding properties and meier E (2011). Superposition and ecule ligands are recognized by macromolecular re- dynamics of protein-ligand complexes - the group has alignment of labeled point clouds. ceptors. Usually, these small molecules are substrates, developed a number of well-known tools used world- IEEE ACM TCOMPUT BI 8, 1653-66. inhibitors, regulators, or modulators of the function wide to analyze and predict binding characteristics. Fober T, Mernberger M, Klebe G, of the target proteins, and the understanding of the In particular, methods to analyze and compare pro- Figure 2. Fragment-based lead discovery starts with the spatial structure of a protein, determined by X-ray crystallography or NMR recognition processes involved is a prerequisite for teins and protein binding sites have been released. In spectroscopy. By use of either biophysical methods, computational analysis or crystal structure analysis, the binding of small molecu- Hüllermeier E (2012). Fingerprint lar probes (fragments with ≤200 Da) is characterized. Subsequently, the discovered fragments are optimized by design and synthesis kernels for protein structure Prof. Dr. Gerhard Klebe the rational design of ligands meant to influence the close collaboration with the Knowledge Engineering & from fragments-to-lead structures. comparison. J Mol Inf 31, 443-52. function of the addressed proteins. In pharmaceutical Bioinformatics Group of Eyke Hüllermeier, our group Pharmaceutical Chemistry research, such interactions are used to develop new developed a number of tools to compare protein bind- Biela A, Nasief NN, Betz M, Heine A, Faculty of Pharmacy, lead structures for putative drugs. In chemical biology, ing pockets (Fig. 1). Searches across the entire space of Hangauer D, Klebe G (2013). Dis- Philipps-Universität Marburg these techniques aim for the development of chemi- protein-exposed binding pockets (> 250.000 entries) secting the hydrophobic effect on Phone: +49 6421 28 21313 cal probes to interfere in a tailored fashion with the can now be accomplished within seconds (Fober et al, molecular level: the role of water, Email: [email protected] properties of bio-macromolecules. In synthetic micro- 2011; Fober et al, 2012). This approach allows us to com- enthalpy, and entropy in ligand binding to thermolysin. SYNMIKRO member since 2010 biology, the targeted interference with protein-ligand pare proteins with regard to their function, independ- recognition is oriented towards a better understand- ent of sequence and fold similarity. Such comparisons Angew Chem Int Ed 52, 1822-8. ing of metabolic pathways, the minimal requirement help to elucidate, e.g., putative cross-reactivities and of proteins necessary for cellular functions, and the undesired side effects of existing and putative drug Jakobi S, Nguyen TX, Debaene F, Metz A, Sanglier-Cianférani S, reprogramming of cellular networks. A second focus molecules in the field of pharmaceuticals.I n the area Reuter K, Klebe G (2014). Hot-spot of our work is the modification of enzyme active sites, of synthetic biology, proteins of unknown function analysis to dissect the functional Short CV which is of relevance, e.g., for the production of phar- can be annotated with respect to binding site similari- Figure 3. Protein-ligand complexes are exposed to a surrounding water environment. A layer of surface water molecules is formed which protein-protein interface of a tRNA- 1996– Professor of Pharmaceutical macologically interesting compounds by biological ties of catalytic centers, or proteins can be searched solvates the newly formed complex. By careful design, an optimal network of water molecules can be created that wraps around the modifying enzyme. Chemistry, Philipps-Universität system. In order to modify active sites in a way that for that possibly catalyze and process similar sub- exposed ligand functional groups and thus enhances binding affinity of the bound ligand towards its protein. Proteins 82, 2713-32. Marburg they can yield other products of relevance for a later strates. Such comparisons can indicate redundancies 2005 Visiting Professor, University pharmaceutical application, a detailed understanding in the function of proteins, an aspect that could be of initial fragment into the binding pocket (Fig. 2). Since of California, San Francisco, USA and appropriate modeling techniques to simulate and interest when genes are knocked out, but their loss- in all of these projects the influence and binding of 1984–1996 BASF-AG, Ludwigshafen predict the properties of enzyme variants with altered of-function is compensated by the up-regulation of water molecules takes an essential impact, the group 1991 Habilitation, Universität substrate specificity are required. redundant pathways. embarked onto a detailed study of water molecules Heidelberg and their role in ligand binding. These investigations 1983 Research Scientist, F ragment-based lead discovery and the require crystallographic studies at extremely high res- University of Bern, Switzerland In silico simulations of binding pockets olution with X-rays and with neutrons as well (Fig. 3). 1982 Research Scientist, University influence of water on protein interactions of Frankfurt/Main The Drug Design Group tries to assemble a solid Furthermore, molecular dynamics simulations help to 1981 Visiting Scientist, CNRS and knowledge base of how proteins recognize their li- In order to find new lead structures for the develop- understand the influence of water on binding (Biela Institute Laue-Langevin, Grenoble, gands, and particularly of thermodynamic data cor- ment of a drug, our group is heavily engaged in es- et al, 2013). France related with structural information (Klebe, 2015). As tablishing strategies for fragment-based lead discov- An additional focus of the group is put on the under- 1982 Ph.D. in physical chemistry, experimental techniques, crystallography and mi- ery (FBLD), a crystallographic approach starting with standing of protein-protein interfaces and how they University of Frankfurt/Main crocalorimetry are used along with other biophysical small probes (≤ 200 Da, so called ‘fragments’) as initial are stabilized energetically and kinetically. Strategies 1978 Diploma in chemistry, methods such as surface plasmon resonance, thermal entry points. Based on the crystal structure of such are developed to perturb and destabilize such inter- University of Frankfurt/Main stability measurements and micro thermophoresis. hits, more potent leads are developed by growing the faces by tailored mutations and by interference via small molecule binding. Apparently, also in this case water penetration takes decisive influence on the pro- tein-protein interface formation and stability (Jakobi et al, 2014). Aa w rds & Honors

2012 Carl Mannich Medal of the German Pharmaceutical Society 2011 ERC Advanced Grant 2008 Novartis Lecture Prize 1999 Literature price of the German chemical industry association (VCI) for the text book „Wirkstoffdesign“ together with Prof. Dr. H.-J. Böhm and Prof. Dr. H. Kubinyi Figure 1. A database holding more than 250,000 binding pockets has been created. Each pocket is characterized in terms of assigned pseudocenters that describe the exposed physicochemical properties of the binding pocket. The spatial distribution of the pseudocent- 1975–1982 Scholar of the „Studienstif- ers is subsequently used to compare individual binding pockets among each other. This approach allows to find functional relation- tung des deutschen Volkes“ ships or putative cross-reactivity between proteins independent of any sequence or fold similarity. 36 research 37

Selected publications

Structure-based prediction of protein Kolb P, Rosenbaum DM, Irwin JJ, Fung J, Kobilka BK, Shoichet BK ligands and enzyme substrates (2009). Structure-based discovery of β2-adrenergic receptor ligands. Proc Natl Acad Sci USA 106, 6843-8. Ever since the first X-ray structures of proteins were emerged, with the most potent compound binding

determined, they have been used to identify and op- with a Ki of only 9 nM (Fig. 1). Remarkably, two of the Kolb P, Phan K, Gao Z, Marko AC, timize small synthetic molecules that might bind compounds are chemically distant from known ligand Sali A, Jacobson KA (2012). Limits to them. One of the advantages of this approach is chemical space for this receptor, despite the fact that of ligand selectivity from docking that it does not require any known ligands and can it has been investigated in the pharmaceutical indus- to models: In silico screening for A adenosine receptor antagonists. thus discover ligands with novel chemistry, or even be try for the past 60 years. 1 PLOS ONE 7, e49910. applied to orphan targets. In particular, our lab uses Similarly, in the screen against the A1AR we were able Dr. Peter Kolb docking, a computational technology that screens to contribute 25 previously unknown ligands, one fifth Schmidt D, Bernat V, Brox R, large libraries of millions of molecules for those that with novel chemistry (Fig. 2) (Kolb et al, 2012). As no Computational Chemical Biology Tschammer N, Kolb P (2014). show steric complementarity to the binding site of a X-ray structure is available for this receptor, we calcu- Identifying modulators of CXC Faculty of Pharmacy, target protein. There are two main goals: one, to iden- lated four homology models based on a related recep- receptors 3 and 4 with tailored Philipps-Universität Marburg tify inhibitors or activators of proteins in order to use tor. Notably, three of the four models yielded ligands, selectivity using multi- Phone: +49 6421 28 25908 them to perturb biological systems; two, to identify which can be attributed to the extraordinary confor- target docking. Email: [email protected] potential substrates for enzymes of unknown func- mational flexibility of GPCRs. ACS Chem Biol, doi: 10.1021/cb500577j SYNMIKRO member since 2012 tion, assigning reactions to the thousands of proteins that have been crystallized in the various structural Ligands with tailored selectivity Xiang DF, Kolb P, Fedorov AA, genomics initiatives. Xu C, Fedorov EV, Narindoshivili T, The binding profile, i.e. its selectivity, is one of the Williams HJ, Shoichet BK, Almo SC, Raushel FM (2012). Structure-based most important features of a therapeutic molecule. Ligands with chemistry previously function discovery of an enzyme for Hence, it is extremely valuable to predict binding undescribed for a certain target the hydrolysis of phosphorylated patterns already at the screening and design stage. sugar lactones. The lab is focused on G protein-coupled receptors (GP- We recently did the proof of concept for two GPCRs Biochemistry 51, 1762-73. CRs), a ubiquitous receptor family whose members from the chemokine subfamily, peptide sensors that consist of seven membrane-spanning helices. They are involved in malignancies such as cancer, multiple sense a broad variety of agents (ranging from peptides sclerosis and HIV infections. We extracted both puta- down to individual photons) on the outside of a cell tively subtype-selective as well as dual binders from Figure 2. Four different conformations of the 1A AR used to identify 25 ligands. Each conformation yielded different and transmit the signal through the membrane. GP- our screen. Upon experimental verification, we found ligands, except for conformation C, which was unproductive. For comparison the X-ray crystallographic structure of Short CV CRs are the class of proteins most frequently targeted that more than 50% of the predictions were correct in the A2AAR, the template (PDB 3EML), is shown in black. (Copyright PLOS) 2011– Emmy Noether Independ- in pharmaceutical research: over 30% of currently each category (Schmidt et al, 2014). ent Junior Research Group Leader, marketed drugs interact with a GPCR. Philipps-Universität Marburg Through unbiased docking to a number of targets, Substrate identification for enzymes F uture directions 2007–2011 Postdoctoral Fellow, namely the β2-adrenergic receptor and the A1 adeno- of unknown function University of California, San Fran- sine receptor, we were able to find novel and potent T ogether with the Li lab, we are developing the appli- cisco, USA ligands in high numbers. In the screening cam- Through the efforts of structural genomics consortia, cation of prenyltransferases (PTs) for the modification 2006–2007 Postdoctoral Fellow, paign against the β2AR (Kolb et al, 2009), six ligands thousands of X-ray structures of putative proteins, of small molecule ligands. Initial hits can be chemi- University of Zurich, Switzerland some of them seemingly enzymes, from various mi- cally modified in a rapid and precise way by PTs, which 2006 Ph.D. in computational croorganisms are available. Yet, the majority of them allows us to explore their structure-activity relation- biochemistry, University of Zurich, Switzerland have been solved without any potential substrate in ship, i.e. how chemical structure relates to pharma- 2001 Diploma in chemistry, the binding site. These enzymes might catalyze use- cological potency. PTs are ideal tool enzymes in this University of Vienna, Austria ful reactions, however, so annotating them in a high- respect, because the prenyl-group that they transfer throughput manner is desirable. to a small molecule is an apolar moiety that is usu- We have focused on enzymes of the amidohydrolase ally associated with a gain in potency. In a first step, family, which are easily recognized by their character- we have cataloged and characterized the substrate

istic (β/α)8-barrel fold. They can catalyze a plethora of and reaction space of PTs. This knowledge is now used different reactions, and the precise subtype cannot to predict additional molecules that might be turned directly be deduced from the sequence. In silico, the over by a PT. Ultimately, PTs will be used to quickly highly labile active complex of putative substrates and efficiently modify small molecules emerging from can be constructed and docked. This is the species screens of large libraries against disease-related tar- that the enzyme is preorganized to stabilize and thus gets. Thus, the chemical space around a particular hit Figure 3. Docked poses of the substrates with Lmo2620. The position of the phosphate ion from the original docking screens using such reaction intermediates can be explored in a straightforward manner. structure is colored cyan; the protein and ligands are in grey and green carbons, respectively. (Copyright ACS) show a higher hit rate. This way, the substrate speci- ficity and precise reaction mechanism of five ami- Aa w rds & Honors dohydrolases could be elucidated (e.g., Lmo2620, an 2011 Emmy Noether Independent Figure 1. The six compounds identified by computationally dock- enzyme that dephosphorylates sugar lactones: Fig. 3

Junior Research Group appointment ing one million molecules to the X-ray structure of the β2AR. & Xiang et al, 2012). 38 research 39

Selected publications

Characterization and application of Li SM (2010). Prenylated indole deriv- atives from fungi: structure diversity, non-ribosomal peptide synthetases biological activities, biosynthesis and chemoenzymatic synthesis. Nat Prod Rep 27, 57-78. In the last years, availability of new genome sequences from diverse mi- Yin S, Yu X, Wang Q, Liu XQ, Li SM (2013). croorganisms, especially bacteria and Identification of a brevianamide F fungi, has led to the identification of reverse prenyltransferase BrePT from genes and gene clusters, which can be Aspergillus versicolor with a broad used for production of novel secondary substrate specificity towards trypto- metabolites by genetic manipulation. phan-containing cyclic dipeptides. Appl Microbiol Biotechnol 97, 1649-60. Prof. Dr. Shu-Ming Li These small molecules often show inter- esting biological and pharmacological Pharmaceutical Biology Mundt K, Li SM (2013). CdpC2PT, activities and therefore are important and Biotechnology a reverse prenyltransferase from Figure 3. Production of prenylated derivatives by coexpression of NRPS and prenyltransferase genes in Aspergillus nidulans. drug candidates or lead compounds in Neosartorya fischeri with a distinct Faculty of Pharmacy, drug development processes. The scaf- substrate preference from known Philipps-Universität Marburg fold of a large group of biologically ac- the amino acid level, expression constructs with sizes transferase genes into the genome was carried out by C2-prenyltransferases. Phone: +49 6421 28 22461 tive compounds from ascomycetes are of up to 15 kb under the control of the gpdA promoter PCR for overall 30 transformants. N1-regularly, C2- and Microbiology 159, 2169-79. Email: [email protected] often synthesized by modular multi- were created. Both constructs (pKM36 and pKM42) C3-reversely prenylated derivatives were isolated from Yin WB, Yu X, Xie XL, Li SM (2010). SYNMIKRO member since 2010 functional enzymes like non-ribosomal Figure 1. Map of an exemplary construct for expression of NRPS genes with gpdA were transformed separately into A. nidulans TN02A7, fungal cultures and identified by PH LC, NMR and MS promoter, trpC terminator and pyrG selection marker. peptide synthetases (NRPS), NRPS-like and complementation of the uracil auxotrophy was analyses (Fig. 3). Yields between and 5 and 30 mg l-1 were Preparation of pyrrolo[2,3-b]indoles synthases, polyketide synthases (PKS) used for the identification of recombinant transfor- calculated for these substances. Our results demon- carrying a beta-configured reverse C3-dimethylallyl moiety by or hybrids of PKS and NRPS. The generated fungal mium globosum, Neosartorya fischeri and Penicillium mants. Confirmation fo gene integration into the strate that different prenylated cyclic dipeptides can using a recombinant peptides or peptide-related products show remark- chrysogenum), 11 genes were subsequently cloned into genome of A. nidulans TN02A7 by PCR led to identifi- be produced by synthetic microbiology with scaffold- prenyltransferase CdpC3PT. able diversity both in their structures as well as in the cloning vector pGEM-T Easy to verify the sequence cation of three transformants for Pc21g15480 (A. nidu- forming and modification enzymes. Org Biomol Chem 8, 2430-38. their biological activities, and can even be further of each gene. Ten of these genes with own or artificial lans KM01-03) and 14 transformants for NFIA_074300 Short CV metabolized by different modification enzymes, e.g., promoter or terminator were finally cloned into the (A. nidulans KM45-58), respectively. HPLC analysis of Yin WB, Ruan HL, Westrich L, prenyltransferases (PTs) or cytochrome P450 (Li, 2010). expression vector pJW24 (pyrG+) (Fig. 1). Overall, 14 dif- all 17 transformants for production of newly accumu- Grundmann A, Li SM (2007). Cdp- 2008– Professor of Pharmaceutical Therefore, expressing NRPS, NRPS-like or PKS/NRPS ferent expression constructs were created, either with lated secondary metabolites showed two additional NPT, an N-prenyltransferase from Biology, Philipps-Universität genes in heterologous hosts and thereby generating own gene promoter or artificial A. nidulans gpdA pro- peaks in each transformant. Isolation on HPLC and Aspergillus fumigatus: overproduc- Marburg new “natural” products represent a novel strategy for moter, and with or without trpC terminator, and were structure elucidation by NMR and MS analyses proved tion, purification and biochemical 2006–2008 Professor of Pharmaceu- drug discovery and development. used for transformation into the fungal strain A. nidu- that both NRPSs are responsible for the biosynthesis characterisation. tical Biology, University of lans TN02A7 (pyrG-, pyroA-). of the cyclic dipeptides cyclo-l-Trp-l-His and cyclo-l- ChemBioChem 8, 1154-61. Düsseldorf -1 -l 2004–2006 Research Scientist, Production and identification of novel Trp-l-Pro. Yields of 6 mg l lcyclo-l-Trp- -His and 4 mg University of Tübingen secondary metabolites After PEG-mediated protoplast transformation of cyclo-l-Trp-l-Pro were achieved. The accumulation of 2004 Habilitation, University of A. nidulans TN02A7 using the expression construct such secondary metabolites provides experimental Tübingen After successful PCR-amplification of 12N RPS and pCaW28 bearing the NRPS gene NFIA_093690 (ftmPS) confirmation for our research approach of transform- 1995–2004 Assistant Professor of NRPS-like genes from six ascomycetes (Aspergillus nid- from N. fischeri under the control of gpdA promoter ing such large genes into heterologous hosts and Pharmaceutical Biology, University ulans, Aspergillus oryzae, Aspergillus terreus, Chaeto- and trpC terminator, complementation of the uracil thereby creating synthetic organisms with new fea- of Tübingen auxotrophy was used for selection. tures. 1992–1995 Postdoctoral Fellow, Uni- Integration of the gene ftmPS into the versities of Freiburg and Tübingen genome of A. nidulans was verified for T argeted production of 1992 Ph.D. in pharmaceutical four transformants by PCR (A. nidulans biology, University Bonn secondary metabolites CaW02-05). Analysis of secondary me- 1986 M.Sc. in medicinal chemistry, Beijing University, Beijing, China tabolites on HPLC showed the presence The first project focusing on the targeted gene coex- of one distinct product peak in com- pression was carried out with the NRPS gene ftmPS parison to its naïve host. Isolation and mentioned above and four prenyltransferase genes of structure elucidation of the newly ac- the DMATS superfamily, which were known to accept cumulated product by NMR and HR-MS cyclo-l-Trp-l-Pro as substrate, but with different fea- analyses revealed unequivocally that tures. For this purpose, coding sequences of the pre- the NRPS is responsible for the forma- nyltransferase genes brePT (Yin et al, 2013) from A. ver- tion of brevianamide F (cyclo-l-Trp-l- sicolor, cdpC2PT (Mundt & Li, 2013) and cdpC3PT (Yin et Aa w rds & Honors Pro). Yields of up to 36.9 mg breviana- al, 2010) from N. fischeri as well as CdpNPT (Yin et al, mide F per liter culture were calculated 2007) from A. fumigatus were cloned separately into 2009 PHOENIX Pharmaceutics + + + Science Awards for the transformants. a second expression construct (gpdA , trpC , pyroA4 ) 2004 PHOENIX Pharmaceutics For the two orthologous genes and transformed into the NRPS expression mutant Science Awards NFIA_074300 from N. fischeri and A. nidulans CaW03 carrying ftmPS (Fig. 2). After com- 1997 PHOENIX Pharmaceutics Figure 2. Map of an exemplary construct for expression of prenyltransferase genes Pc21g15480 from P. chrysogenum, which plementation of the additional pyridoxine auxotro- Science Awards with gpdA promoter, trpC terminator and pyroA selection marker. share a sequence identity of 60 % on phy, confirmation of the ectopic integration of prenyl- 40 research 41

sideroblastic anemia and Selected publications cerebellar ataxia (XLSA/A). Mechanisms of iron-sulfur protein Lill R (2009). Function and We recently succeeded to purify biogenesis of iron-sulfur proteins. and crystallize yeast Atm1 (Srini- Nature 460, 831-8. biogenesis in eukaryotes vasan et al, 2014). The 3D x-ray structure revealed a novel fea- Netz DJ, Mascarenhas J, Stehling O, Iron-sulfur (Fe/S) clusters are evolutionary ancient, ited from bacteria during evolution (Fig. 1): Seventeen ture of ABC transporters (Fig. 3): Pierik AJ, Lill R (2014). Maturation inorganic cofactors of proteins with functions in ca- known ISC proteins first assemble aF e/S cluster on a The two fully resolved C-terminal of cytosolic and nuclear iron-sulfur talysis, electron transfer and regulation. Fe/S proteins scaffold protein and then move the cluster to trans- a-helices were shown to bind to proteins. Trends Cell Biol 24, 303-12. in turn participate in central cellular processes such as fer proteins, from where the cluster is finally inserted each other, thereby protecting DNA synthesis and repair, chromosome segregation, into apoproteins by dedicated ISC targeting factors. the protein from degradation. Netz DJ, Stümpfig M, Doré C, protein synthesis, and respiration. Their biogenesis Cytosolic and nuclear Fe/S protein assembly also de- Another important feature of Mühlenhoff U, Pierik AJ, Lill R (2010). is thus essential for cell viability, and impairment of pends on this machinery, yet additionally requires the Atm1 is its substrate binding cav- Tah18 transfers electrons to Dre2 in cytosolic iron-sulfur protein Prof. Dr. Roland Lill Fe/S protein biogenesis causes genome instability, a mitochondrial ISC export apparatus and the cytosolic ity which is composed of mainly Figure 2. A mitochondrial electron transfer chain is needed for de novo generation of Fe/S clusters. (A) A persulfide (-SSH) intermediate is generated by the mitochondrial cysteine biogenesis. Nat Chem Biol 6, 758-65. hallmark of cancer. Our group is interested in iden- iron-sulfur protein assembly (CIA) machinery. The ABC positively charged residues, sug- Molecular Cell Biology and desulfurase Nfs1-Isd11 on the scaffold protein Isu1. Reduction of the persulfide involves the tifying and characterizing the molecular machinery transporter Atm1 of the export system transports a gesting a net negative molecule ferredoxin reductase FdxR and the [2Fe-2S] ferredoxin Yah1, which get their reducing equiv- Biochemistry alents from NADPH. Yah1 binds to Isu1 preferentially in its reduced form. Frataxin Yfh1 is Srinivasan V, Pierik AJ, Lill R (2014). catalyzing the synthesis of Fe/S clusters and their in- still unknown sulfur-containing compound (X‑S) to is bound and exported. In one of important for stimulating sulfur transfer. (B) The 3D structure of Yah1 and the interaction Crystal structures of nucleotide-free Faculty of Medicine, sertion into apoproteins in eukaryotes. To date, some the CIA machinery, which then assembles a [4Fe-4S] our crystal structures we found surface for Isu1 (pink) as revealed by HSQC NMR experiments. and glutathione-bound mitochon- Philipps-Universität Marburg 30 biogenesis proteins are known, and they are con- cluster on a scaffold complex. SpecificCI A targeting a bound glutathione in this area drial ABC transporter Atm1. Phone: +49 6421 28 66449 served from yeast to man (Lill, 2009; Netz et al, 2014). proteins finally transfer the cluster to recipient apo- (Fig. 3). Since glutathione was earlier shown to be es- Science 343, 1137-40. Email: [email protected] While we have a good cell biological picture of Fe/S proteins. sential for the export process, we suspect that the Webert H, Freibert SA, Gallo A, SYNMIKRO member since 2010 protein biogenesis, the molecular mechanisms under- compound may be part of the exported substrate. lying the individual reactions are only now being un- The role of ferredoxin in The locations of the four residues mutated in XLSA/A Heidenreich T, Linne U, Amlacher S, raveled using biochemical, biophysical, bioinorganic suggest an impaired function in substrate binding or Hurt E, Mühlenhoff U, Banci L, Lill R the reduction of sulfur (2014). Functional reconstitution of and ultrastructural methods. Knowledge of these re- membrane transport in disease cells. mitochondrial Fe/S cluster synthesis actions will help in synthetic biology to construct ar- In vivo studies have suggested a role of the ferredoxin on Isu1 reveals the involvement of tificial pathways for the synthesis of products such as Yah1 in mitochondrial Fe/S protein biogenesis, but A distinct pathway for maturation ferredoxin. Nat Commun 5, 5013. isobutanol or isoprene derivatives, which in turn serve its precise role remained unclear. We biochemically of cytosolic [2Fe-2S] proteins as versatile starting materials for pharmaceuticals, reconstituted the synthesis of a [2Fe-2S] on the Isu1 Short CV cosmetics and fine chemicals.O ur SYNMIKRO project scaffold and thus showed that Yah1 and ferredoxin A novel mechanistic observation was made in our 1996– Professor of Cell Biology, is dedicated to the better understanding of the vari- reductase are essential for this process (Webert et al, studies of the construction of metabolic pathways Philipps-Universität Marburg ous pathways of Fe/S protein biogenesis in yeast to 2014). Yah1 in its reduced form specifically interacts for the engineered synthesis of Isobutanol as part 1990–1996 Senior Research render such proteins available as building blocks for with the scaffold proteinI su1 to deliver its electron, of our collaboration with Gevo Co, Englewood, USA. Assistant, Ludwig-Maximilians- synthetic microbiology. thereby possibly reducing the persulfide (‑SSH) inter- This process makes use of a bacterial [2Fe-2S] protein Universität München Generation of cellular Fe/S proteins in eukaryotes is mediate of Isu1 to the sulfide needed for Fe/S cluster in the yeast cytosol, the dihydroacid dehydratase 1995 Habilitation, Ludwig- accomplished by three conserved biosynthesis ma- formation (Fig. 2A). The interaction surface for Isu1 on IlvD. We found that the maturation of IlvD to an Maximilians-Universität München chineries in the mitochondria and the cytosol. Bio- Yah1 was revealed by NMR spectroscopy and indicates active enzyme occurs independently of the known 1987–1989 Postdoctoral Fellow, genesis is initiated by the mitochondrial iron-sulfur that Isu1 binds close to the [2Fe-2S] of Yah1 (Fig. 2B). components of the CIA machinery, discriminating University of California, Los Angeles, USA cluster (ISC) assembly machinery which was inher- The ongoing resolution of the structure of the entire this pathway from the biogenesis of CIA-dependent complex will be helpful for [4Fe-4S] proteins. However, maturation still requires 1986 Ph.D. in biochemistry, Ludwig- Figure 3. Crystal structure of the ABC transporter Atm1 of the Maximilians-Universität München understanding the mode the mitochondrial ISC assembly machinery and Atm1. mitochondrial inner membrane. Atm1 is a homodimer (red and 1981 Diploma in chemistry, Ludwig- of electron transfer. No- We then realized that all tested cytosolic [2Fe-2S] green subunits). The binding site for glutathione (GSH) is within the putative substrate binding cavity. The yellow regions in- Maximilians-Universität München tably, an electron transfer proteins follow this pathway (Fig. 1). With this knowl- dicate amino acid residues that are involved in ATP binding. chain was described in the edge, we currently explore which additional cytosolic Residues mutated in the human Atm1 orthologue ABCB7 in the iron storage disease XLSA/A are shown in blue. cytosol for Fe/S protein bi- biogenesis proteins are needed for the maturation of ogenesis, but it is unclear [2Fe‑2S] proteins in the cytosol. This example shows why reduction is needed that application-oriented experimental approaches (Netz et al, 2010). can lead to new discoveries in basic science, too. Aa w rds & Honors The 3D structure 2014 Albrecht Kossel Prize of the of Atm1 suggests German Chemical Society (GDCh), glutathione as part and Luigi Sacconi Medal of the Società Chimica Italiana (SCI) of the substrate 2010 Feldberg Foundation Prize The central component of 2009–2014 Fellow of the Max Planck the ISC export pathway Society is the mitochondrial in- 2007 Elected Member of the Figure 1. An overview of some functions and the biogenesis of eukaryotic Fe/S proteins. Genera- German National Academy of tion of all cellular Fe/S proteins depends on the mitochondrial ISC assembly machinery consisting ner membrane ABC trans- Sciences Leopoldina of 17 proteins. The mitochondrial ISC export machinery and the CIA machinery are specifically porter Atm1 (Fig. 1). Its involved in the maturation of cytosolic and nuclear Fe/S proteins. Red circles: iron ions, yellow cir- 2003 Leibniz Prize of the German cles: sulfide; X-S, an unknown sulfur-containing compound, which is exported from mitochondria human orthologue ABCB7 Research Foundation (DFG) by the ABC transporter Atm1 to support Fe/S-protein biogenesis in the cytosol. is mutated in X-linked 42 research 43

new member of the CDPS Selected publications family showing a formerly Biosynthesis and engineering of Hegemann JD, Zimmermann M, unknown product pro- Zhu S, Klug D, Marahiel MA (2013). file, while tailoring of the Lasso peptides from proteobacteria: bioactive cyclic peptides CDP scaffold is achieved Genome mining employing heter- through the combined ologous expression and mass spec- Peptide natural products play an important role in lasso peptide microcin J25, yielding a potent αvβ3 actions of a CDP oxidase trometry. Biopolymers 100, 527-42. modern medicine for the treatment of many life- integrin receptor antagonist (Hegemann et al, 2014). (CDO) and two distinct threatening diseases. A large fraction of these natural In general, lasso peptide precursors are matured SAM-dependent O-/N- Hegemann JD, De Simone M, products are either ribosomally synthesized and post- by two enzymes: enzyme B, a cysteine protease ho- methyltransferases (Gies- Zimmermann M, Knappe T, Xie X, translationally modified peptides (RiPP), or assembled molog, and enzyme C, an adenylate forming enzyme sen et al, 2013a). Di Leva FS, Marinelli L, Novellino E, independent of the ribosome on large multi-modular homolog. Interestingly, the B enzyme shares little In a second study, we per- Zahler S, Kessler H, Marahiel MA enzymes called nonribosomal peptide synthetases similarity to enzymes outside of lasso peptide bio- formed a bioinformatic (2014). Rational improvement of the affinity and selectivity of integrin Prof. Dr. (NRPS). In our group, we investigate the structure, synthetic gene clusters. This inspired us to perform a analysis of a CDPS-con- binding of grafted lasso peptides. Mohamed A. Marahiel function, biosynthesis and engineering of ribosomal- B protein-centric genome mining approach through taining gene cluster from J Med Chem 57, 5829-34. ly and nonribosomally assembled cyclic peptides that which we were able to identify 102 putative lasso pep- Actinosynnema mirum. We Biochemistry show complex structures and diverse bioactivities. tide biosynthetic gene clusters from a total of 87 pro- established the biosyn- Giessen TW, von Tesmar AM, Department of Chemistry, teobacterial strains. Ten of these clusters were then thetic pathway leading to Marahiel MA (2013a). Insights into Philipps-University Marburg Characterization of novel lasso peptides heterologously expressed in Escherichia coli BL21(DE3), two methylated ditrypto- the generation of structural diver- Phone: +49 6421 28 25722 and all twelve corresponding lasso peptides were de- phan CDPs (cWW) through sity in a tRNA-dependent pathway Email: [email protected] Figure 2. Overview of the general action of CDPSs and their connection to the primary metabo- Lasso peptides are a group of natural products that tected and characterized by high-resolution Fourier in vivo and in vitro analy- lism. CDPSs hijack aminoacyl-tRNAs and employ them in the formation of CDPs, thus diverting for highly modified bioactive cyclic marburg.de share a unique structural motif: All known lasso transform mass spectrometry (Hegemann et al, 2013). ses. This CDPS was the first the flow of loaded tRNAs away from the ribosomal machinery. (From Giessen & Marahiel, 2014) dipeptides. Chem Biol 20, 828-38. SYNMIKRO member since 2010 peptides consist of an N-terminal macrolactam ring, member of this enzyme which is 7 to 9 amino acids in size and is formed be- family able to form cWW and the first prokaryotic machinery of any organism – represents a major chal- Giessen TW, von Tesmar AM, Biosynthesis of modified cyclic dipeptides Marahiel MA (2013b). A tRNA- tween the N-terminus of a Gly, Ser, Ala or Cys residue CDPS whose main product constituents differ from lenge for the creation of functionalized peptides and dependent two-enzyme pathway for at position 1 and the side chain of a Glu or Asp residue In recent years, it has become apparent that aminoa- the four amino acids (Phe, Leu, Tyr and Met) usually proteins. In collaboration with the group of Gert Bange, the generation of singly and doubly at position 7, 8 or 9 (Fig. 1). Their distinguishing feature cyl-tRNAs are not only crucial components involved in found in these systems. We also characterized a SAM- we recently devised a fusion protein-based design for methylated ditryptophan is the threading of the C-terminal tail of the peptide protein biosynthesis, but are also used as substrates dependent N-methyltransferase that carries out two synthetic tRNA-aminoacylation catalysts by combin- 2,5-diketopiperazines. through the macrolactam ring, yielding a structure and amino acid donors in a variety of other important successive methylations at the diketopiperazine ring ing NRPS adenylation domains (A-domains, which Biochemistry 52, 4274-83. Short CV reminiscent of a lariat knot. This entropically unfa- cellular processes ranging from bacterial cell wall bio- nitrogens of cWW and showed that this enzyme is select and activate the monomers in NRPS pathways) vorable confirmation is sustained by the placement synthesis and lipid modification to protein turnover also able to methylate four other phenylalanine-con- with a small eukaryotic tRNA-binding domain (like Ar- Giessen TW, Altegoer F, Nebel AJ, 1990– Professor of Biochemistry, of sterically demanding residues (plug amino acids) and secondary metabolite assembly. Therefore, we taining CDPs (Giessen et al, 2013b). c1p-C from yeast) (Fig. 3). E.g., we fused the A-domain Steinbach RM, Bange G, Marahiel MA Philipps-University Marburg above and below the ring, entrapping the tail inside investigated tRNA-dependent biosynthetic pathways PheA with the Arc1p-C domain using flexible linkers (2015). A synthetic adenylation 1977–1989 Assistant Professor, and preventing its unthreading. that generate different bioactive modified cyclic and thus were able to achieve tRNA-aminoacylation domain-based tRNA-aminoacylation Technical University Berlin A synthetic tRNA-aminoacylation catalyst with both proteinogenic (L-Phe) and non-proteinogen- catalyst. Angew Chem, doi: 10.1002/ 1987 Habilitation, Technical anie.201410047 The incorporation of non-proteinogenic amino acids – ic (D-Phe) amino acids (Giessen et al, 2015). University Berlin 1986–1987 DFG Fellow, Harvard i.e., amino acids not naturally used by the translational University, Cambridge, USA 1977 Ph.D in microbial biochemistry, Max Planck Institute for experimen- a b tal medicine/Georg-August Univer- sity Göttingen 1973 Diploma in chemistry, Georg- August University Göttingen

Figure 1. NMR structures of lasso peptides showing the interactions between the C-terminal tail and the macrolactam ring of capistruin, caulosegnin I, lariatin and microcin J25. The surface of the rings is colored in orange, the surface of the side chains of the plug amino acids positioned below the ring is colored by elements. Steric hindrance caused by the depicted side chains prevents the unthreading of the lasso peptides. All lasso peptides are shown from below the ring. (From Hegemann et al, 2013) Aa w rds & Honors The majority of known lasso peptides possess various dipeptides (CDPs). The enzymes responsible for the 2014 David Gottlieb Memorial biological properties, ranging from antibacterial and initial biosynthesis of a CDP-scaffold are referred to Lecture, University of Illinois, USA inhibitory to receptor antagonistic activities (Hege- as cyclodipeptide synthases (CDPSs) and use loaded 2012 Honorary Professorship from mann et al, 2013). As lasso peptides are of ribosomal tRNAs as their substrates (Fig. 2). Wuhan University, China origin and as the enzymatic maturation machinery First, we described the identification of the first no- 2009 Elected Member of the Royal was shown to have a rather relaxed specificity towards cazine biosynthetic gene cluster in Nocardiopsis das- Society of Chemistry, UK substitutions of most of the residues in the lasso pep- sonvillei and the elucidation of the biosynthetic path- 2008 Max Bergmann Medal in gold Figure 3. Design strategy for the construction of synthetic adenylation domain-based fusion proteins for tRNA-aminoacylation based on structural insights and molecular models. for work on Non-ribosomal Peptide tide scaffold, they are also promising candidates for way leading to nocazine E and XR334 - nocazines are (a) Comparative overview of the two reactions catalyzed by aaRSs and NRPS A-domains, respectively. In both cases the first catalyzed reaction consists of the ATP-dependent adenyla- Synthesis epitope grafting approaches.T his was demonstrated a newly defined family of antibacterial and cytotoxic tion of a substrate monomer, generating a high-energy adenylate intermediate (aa-AMP). In the second reaction a nucleophilic attack, either by the 3’-adenosine of the respective tRNA (aaRS) or the 4’-PPant-thiol group of a downstream PCP (NRPS), takes place. Our approach aims to assemble a fusion protein that is able to combine the first reaction shared by 2004 Elected Member of the German only recently through the incorporation of the bio- CDPs produced by different actinobacterial species. aaRSs and NRPS A-domains with the transfer of an activated intermediate to a tRNA-acceptor. (b) Examplary catalytic cycle of A-domain-based tRNA-aminoacylation. Note that the Academy of Sciences Leopoldina active RGD peptide epitope in the loop region of the We showed that CDP-formation is carried out by a shown order of tRNA-binding and adenylate formation only represents one of several possibilities. (From Giessen et al, 2015) 44 45

1.3 Research Cellular Organization 46 research 47

transmembrane protein FlhA serves as the adaptor for Selected publications the coordinated delivery of different flagella building Biogenesis and design of Altegoer F, Schuhmacher J, Pausch P, blocks to the fT3SS (Bange et al, 2010). Now we want Bange G (2014). From molecular to find out how the Tf 3SS can select the correct export evolution to biobricks and synthetic bacterial nanomachines cargos at the right time, and to obtain an in-depth modules: a lesson by the mechanistic and structural view of this process. In bacterial flagellum. Nanomachines are macromolecular complexes that e.g., E. coli), this pattern is ensured by two nucleotide- the future, we aim at building a minimal T3SS, which Biotechnol Genet Eng Rev 30, 49-64. play important roles in all processes of life, ranging binding proteins named FlhF and FlhG (Bange et al, could then be used for the controlled secretion of any from protein synthesis to motility. A common feature 2013; Bange et al, 2011; Bange et al, 2007). We want to user-defined target protein from minimal cells. Bange G, Sinning I (2013). of these nanomachines is their complex subunit ar- further this molecular understanding of flagellation SIMIBI twins in protein chitecture in a specific three-dimensional structure. pattern control and, in doing so, also tackle one of The flagellar filament as a hub targeting and localization. Therefore, their biosynthesis must be highly regu- the most challenging questions in today’s biology: for synthetic applications Nat Struct Mol Biol 20, 776-80. lated in time and space Bange G, Kümmerer N, Grudnik P, Dr. Gert Bange to ensure correctness The extracellular filament of the flagellum is a long Lindner R, Petzold G, Kressler D, and reproducibility. Our tubular polymer composed of over 20’000 copies of Analysis and Design of Hurt E, Wild K, Sinning I (2011). group aims at understand- a single protein named flagellin (Fig. 3). Flagellin con- Metabolic Networks Structural basis for the molecular ing this spatio-temporal sists of a highly conserved part that is essential for its evolution of SRP-GTPase Faculty of Chemistry, regulation underlying the assembly into the flagellar filament, and a variable re- activation by protein. Philipps-Universität Marburg assembly of biological gion (VR) that shapes the surface of the filament and Nat Struct Mol Biol 18, 1376-80. Phone: +49 6421 28 23361 nanomachines, e.g., the differs significantly between the various bacterial spe- Email: gert.bange@synmikro. bacterial flagellum, at the cies (Altegoer et al, 2014). E.g., the VR of E. coli flagel- Bange G, Petzold G, Wild K, Parlitz RO, uni-marburg.de molecular level. Further- lin comprises two domains of approximately 25 kDa, Sinning I (2007). The crystal struc- ture of the third signal-recognition SYNMIKRO member since 2013 more, we want to use the while the VR of B. subtilis is virtually non-existent. acquired molecular under- Based on our crystal structures of different flagellins particle GTPase FlhF reveals a homodimer with bound GTP. standing for the rational and the molecular models of the respective filaments, Figure 2. Architecture of the bacterial flagellum in Gram posi- Proc Natl Acad Sci USA 104, 13621-5. design of several synthetic tive species. The basal-body is composed of integral membrane we are currently engineering filaments with a variety components including the cytosolic C-ring (blue). The extracel- tools, for instance for the lular hook (yellow) and the filament structures (brown) extend of artificial peptides and protein domains within the Bange G, Kümmerer N, Engel C, localization of protein from the peptidoglycan forming the outer part of the flagellum. VR of their flagellin molecules. The so-created syn- Bozkurt G, Wild K, Sinning I (2010). complexes within the cell thetic filaments will be FlhA provides the adaptor for envelope or for the secre- useful for the display of coordinated delivery of late flagella tion of target molecules peptides (e.g., for vaccine building blocks to the type III into the medium. production), scavengers secretion system. Short CV Many bacteria move by (e.g., for recovery of no- Proc Natl Acad Sci USA 107, 11295-300. rotating rigid, helical or- ble earths), the design of 2013– Independent Research Group ganelles, the flagella (Al- nano-wires and the design Leader, Philipps-Universität Marburg tegoer et al, 2014). These and arrangement of micro- 2007–2012 Postdoctoral Fellow Figure 1. The two nucleotide-binding proteins FlhG and FlhF control the correct establishment of flagella represent some bial communities. and Project Leader, University of the flagellation pattern in B. subtilis, S. putrefaciens and H. pylori as well as in many other species. Heidelberg of the tiniest complex 2007 Ph.D. in biochemistry, motors in the biosphere and enable bacteria both to How do living organisms reproducibly self-organize University of Heidelberg move through liquids (swimming) and through highly regular patterns? The ‘molecular answer’ includes the 2002 Diploma in biochemistry, viscous environments or on surfaces (swarming). In potential for synthetic modules that specifically local- Martin-Luther-Universität Halle addition, they act as virulence factors in many patho- ize biosynthetic protein complexes within bacteria or genic species, as motility is often required to reach artificial cells. the site of infection. Last but not least, flagella have only recently been recognized to contribute to biofilm A high-throughput secretion machine formation. Therefore, bacterial flagella seem to fulfill many more functions beyond their role as organelles Despite these different flagellation patterns, the archi- of locomotion. tecture of the bacterial flagellum is conserved and can be divided into three main parts: the transenvelope- Modules for self-organization spanning basal body with the motor subunits and the of regular patterns extracellular hook and filament structures F( ig. 2). To establish these extracellular structures, a flagellum- Already early in microbial research, it was noticed that specific type III secretion system (fT3SS) consisting of Aa w rds & Honors bacterial species differ in number as well as locali- an ~ 9-protein core machinery exports flagella build- zation of their flagella (named ‘flagellation pattern’ ing blocks to the growing tip of the flagellum with 2013 iGEM gold medal in the hereafter) F( ig. 1). Despite the enormous amount of both high speed and stringent substrate discrimina- European jamboree & admission to Figure 3. (A) Domain architecture of Flagellin. The variable region (green) differs significantly be- finals at MIT in Boston (as advisor bacterial species, there are only a handful of differ- tion. However, an in-depth molecular understanding tween the various bacterial species. (B) Crystal structures of Salmonella typhimurium, Sphingomo- nas sp. and Bacillus subtilis Flagellin. The disordered D0 domains are missing due to flexibility. of the team) ent flagellation patterns known.E stablishing the cor- of how the export apparatus recognizes its substrates D1 domains are conserved among these species. However, the variable region consists of 220 aa 2012 Peter & Traudl Engelhorn Fellow rect flagellation pattern is a prerequisite for efficient within the myriad of cytoplasmic proteins and en- forming two distinguishable domains D2 and D3 in S. typhimurium, whereas in Sphingomonas it is reduced to a ß-domain of 120 aa and in B. subtilis only a small loop is formed. (C) Cross sections 2012 F1000 Prime for Kressler motility and for the pathogenicity of many disease- sures the correct chronological sequence of export is of flagellar filaments of different species. The variable region is exposed on the surface of the fila- & Bange et al, Science causing flagellated bacteria.I n many species (except, crucially missing. Previously, we could show that the ment, thereby generating distinct properties. 48 research 49

Selected publications

Synthetic cell compartments Stehlik T, Sandrock B, Ast J, Freitag J (2014). Fungal peroxisomes as based on peroxisomes biosynthetic organelles. Curr Opin Microbiol 22, 8-14.

Within each cell, different Freitag J, Ast J, Linne U, Stehlik T, metabolic pathways com- Martorana D, Bölker M, Sandrock B pete for substrates and (2014). Peroxisomes contribute to enzymes. Therefore, nature biosynthesis of extracellular uses cellular compartmen- glycolipids in fungi. talization as a strategy to Mol Microbiol 93, 24-36. prevent unwanted cross- Stiebler AC, Freitag J, Schink KO, Prof. Dr. Michael Bölker talk in metabolism. Our Stehlik T, Tillmann BA, Ast J, Bölker M research group aims at Genetics (2014). Ribosomal readthrough at a the construction of syn- short UGA stop codon context trig- Faculty of Biology, thetic cell compartments gers dual localization of metabolic Philipps-Universität Marburg that can be used to chan- enzymes in fungi and animals. PLoS Phone: +49 6421 28 21536 nel metabolic reactions Genet 10, e1004685. Email: [email protected] for novel products. Fungal SYNMIKRO member since 2010 peroxisomes are perfectly Ast J, Stiebler AC, Freitag J, Bölker M suited for this purpose (2013). Dual targeting of peroxisomal since they are not essen- proteins. Front Physiol 4, 297. tial. Thus, it is possible to Figure 3. Diverse secondary metabolic pathways are located in peroxisomes. Feitag J, Ast J, Bölker M (2012). redesign the peroxisomal Cryptic peroxisomal targeting via proteome and to engineer We were able to demonstrate that even in human cells In Ustilago maydis, biosynthesis of mannositol eryth- Figure 1. Dual targeting of intracellular proteins can occur via different molecular mechanisms. alternative splicing and stop only selected enzymes into this read-through element is used to target metabolic ritol lipid (MEL), an extracellular glycolipid, which codon readthrough in fungi. peroxisomes that cooper- enzymes such as malate dehydrogenase and lactate serves as an effective biosurfactant, occurs partially in Nature 485, 522-5. ate in defined and novel pathways. We used synthetic in a single compartment, but are dually targeted to dehydrogenase to both the peroxisomes and the cy- peroxisomes. Two acyl transferases (Mac1 and Mac2) retargeting of enzymes involved in glycolipid biosyn- different organelles (for review, see Ast, Stiebler & Fre- tosol (Stiebler et al, 2014). Both enzymes depend on involved in acylation of the sugar moiety of MEL carry thesis to alter the chemical composition of a fungal itag, 2014). We have recently discovered that glycolytic NAD/NADH for their activity and are involved in redox peroxisomal targeting signals. Tagging with green glycolipid, which serves as a potent biosurfactant. Re- enzymes contain cryptic peroxisomal targeting sig- homeostasis via shuttling reduced and oxidized inter- fluorescent protein (GFP) demonstrates peroxisomal location of the respective enzymes from peroxisomes nals that are post-transcriptionally unveiled by differ- mediates across the peroxisomal membrane. localization of these key enzymes. Interestingly, gly- Short CV into the cytosol resulted in incorporation of hydroxy- ential splicing or ribosomal read-through (Freitag et To study dual targeting of peroxisomal proteins, we colipid production is not affected, if peroxisomes are 1997– Professor of Genetics, lated fatty acids into the glycolipid, which are not ac- al, 2012). This unexpected mechanism is evolutionary are currently characterizing the peroxisomal import deleted by mutagenesis. This indicates that biosyn- Philipps-Universität Marburg cessible within the peroxisomes. This demonstrates conserved in fungi and plays an important role in their system both in the phytopathogenic fungus Ustilago thesis can also occur in the cytosol. However, retar- 1996–1997 Assistant Professor, that intracellular localization of metabolic enzymes metabolism. Mutants that are unable to target these maydis and in the well-studied model organism Sac- geting of these enzymes to the cytosol results in the Ludwig-Maximilians-Universität determines the product spectrum of metabolic reac- enzymes to both compartments displayed a growth charomyces cerevisiae (baker’s yeast). In contrast to production of a different set of glycolipids. This can München tions. We are currently constructing cells where the defect and were affected in virulence. yeast, the basidiomycetous fungus U. maydis con- be explained by the fact that in the cytosol both en- 1996 Habilitation, Ludwig- hydroxylating enzyme is retargeted to peroxisomes. Characterization of the sequence elements required tains two receptors for import of peroxisomal type 1 zymes gain access to hydroxylated fatty acids (Freitag Maximilians-Universität München We will use such synthetic compartments with tailor- for dual targeting via ribosomal read-through re- proteins. Both contain a C-terminal domain, which et al, 2014). These are derived from a competing glyco- 1992–1996 Postdoctoral Fellow, made proteomes based on peroxisomes to rechannel vealed that a short stop codon context is sufficient consists of a number of tetratrico repeats (Fig. 2). They lipid biosynthetic pathway. We are currently trying to Ludwig-Maximilians-Universität further metabolic pathway for the production of new to trigger efficient read-through at this termination form a binding pocket for the short C-terminal peroxi- further enlarge the potential spectrum of engineered München metabolites that do not exist in nature. codon. Remarkably, this element is conserved from somal targeting signal typical for most peroxisomal glycolipids by introducing additional enzymatic ac- 1991 Ph.D. in biochemistry, Freie Universität Berlin fungi to men and apparently serves as a basal read- matrix proteins. We could show that both receptors tivities into peroxisomes. 1988 Diploma in biochemistry, Freie Dual targeting of intracellular proteins through mechanism in a wide variety of species. recognize a largely overlapping but not identical set Universität Berlin of targeting signals. Currently, we are constructing Eukaryotic cells are organized into defined compart- chimeric receptors to alter the peroxisomal proteome. ments that differ in their chemical composition and environment. Among these are the nucleus harbor- Engineering metabolic pathways by ing the genetic information, and the mitochondria retargeting of biosynthetic enzymes that provide energy for cellular metabolism. Beside these organelles, all eukaryotic cells also contain per- It has become apparent that in fungi many metabo- oxisomes that are involved in fatty acid degradation lites, in particular those of secondary metabolism, and peroxide detoxification. Remarkably, peroxisomal are generated, at least partially, in peroxisomes matrix proteins are transported into these compart- (Fig. 3). Among these are antibiotics like penicillin ments after translation and in a fully folded and co- produced by Penicillium chrysogenum, or toxins like Figure 4. Peroxisomal localization of MEL acyltransferases Mac1 and Mac2 in U. maydis. Aa w rds & Honors factor-bound state. Correct targeting of peroxisomal aflatoxin secreted by diverse Aspergillus species. 2012– Vertrauensdozent of the proteins depends on short amino acid sequence mo- Therefore, this organelle constitutes an interesting Figure 2. Predicted molecular structure of the recognition „Studienstiftung des Deutschen tifs that act as intracellular zip codes. domain of Pex5b, one of the peroxisomal import receptors from compartment for the production of bioactive sub- Volkes“ Many important cellular proteins are found not only Ustilago maydis. stances (Stehlik et al, 2014). 50 research 51

Selected publications

Mathematical modeling of Dahlke S, Fornasier M, Raasch T, Stevenson R, Werner M (2007). microbiological systems Adaptive frame methods for elliptic operator equations: the steepest descent approach. Within SYNMIKRO, the research group Numerical sion phenomena can, e.g., be observed in the context IMA J Numer Anal 27, 717-40. Analysis is focusing on the mathematical modeling of protein translocations on nucleic acids. Recent ex- of diverse microbiological systems such as diffusion periments in the context of single-molecule spectros- Chegini N, Dahlke S, Friedrich U, phenomena in living cells or the regulatory processes copy performed in the group of Peter Graumann for Stevenson R (2013). Piecewise tensor underlying cell polarity and flagella localization.I n or- instance strongly indicate the occurrence of subdiffu- product wavelet bases by exten- der to gain a deeper understanding of these complex sion phenomena in living cells. By a statistical data sions and approximation rates. systems, we are developing and studying simplified, analysis, we could confirm this conjecture in many Math Comput 82, 2157-90. Prof. Dr. Stephan Dahlke but nevertheless appropriate mathematical models. cases. The mathematical modeling of anomalous dif- Rashkov P, Schmitt BA, Søgaard- The aim is to make reliable predictions based on these fusion phenomena leads to so-called nonstandard Numerical Analysis Andersen L, Lenz P, Dahlke S (2012). models that help in the design of future wet lab ex- fractional diffusion equations, which are highly non- A model of oszillatory protein Faculty of Mathematics and periments: In an iterative process, the mathematical local. Usually, the exact solutions to these equations dynamics in bacteria. Computer Science, models will be validated by experiments, and the find- cannot be computed explicitly, so that efficient nu- Bull Math Biol 74, 2183-203. Philipps-Universität Marburg ings from the mathematical analysis of the models merical schemes for their constructive approximation Phone: +49 6421 28 25474 will lead to optimized designs of experiments. Besides up to a predefined tolerance are needed. Due to the Rashkov P, Schmitt BA, Email: [email protected] the quantitative and qualitative analysis of the result- nonlocality, the numerical treatment of nonstandard Søgaard-Andersen L, Lenz P, Dahlke S marburg.de ing mathematical models, we are working on suitable diffusion equations is quite challenging. We are in Figure 2. Oszillations of a protein at one pole in a cell. We observe that stable protein oscillations (2013). A model for antagonistic discretization schemes for the numerical simulation particular interested in (adaptive) numerical schemes without external trigger are possible. protein dynamics. SYNMIKRO member since 2010 and visualization of the solutions. based on wavelets (Chegini et al, 2013), as wavelets Int J Biomath Biostat 2, 75-85. are particularly tuned to these kinds of equations, & 2) (Rashkov et al, 2012; Rashkov et al, 2013; Rashkov Rashkov P, Schmitt BA, Keilberg D, and the research group Numerical Analysis has a long et al, 2014). The models are based on so-called reac- Mathematical modeling of Søgaard-Andersen L, Dahlke S (2014). nonstandard diffusions standing expertise in this field (Dahlke et al, 2007). tion diffusion equations. We are working on the math- A model for spatio-temporal ematical analysis of these models (stability analysis dynamics in a regulatory network One central problem in cell biology is the understand- Dynamics of regulatory networks etc.) as well as on the numerical simulation of the for cell polarity. ing of diffusion phenomena.I n classical diffusion for cell polarity corresponding solutions. It turns out that the models Math Biosci 258, 189-200. models, which are based on variants of Brownian mo- are quite robust, and that a lot of different oscillation tions, the square of the distance covered by a particle Spatial-temporal oscillations of proteins in bacte- scenarios can be reproduced. is proportional to the time t. However, in many ap- rial cells play an important role in many fundamental plications in cell biology, e.g., in the context of mass biological processes. We study these processes in the Localization of flagella in bacteria transport through membranes or through heteroge- context of cell polarity in Myxococcus xanthus in close neous media inside the cell, one is faced with abnor- collaboration with the group of Lotte Søgaard-Ander- Only recently, our group embarked on the mathemati- mal diffusion processes. In these cases, the square sen. The motility of M. xanthus is due to two motility cal modeling of the mechanisms underlying bacterial Short CV of the distance is proportional to tα with α ≠ 1 (α > 1 systems: an A-motility system, and a type-IV pili sys- flagella localization. It has long been known that the 2002– Professor of Mathematics, superdiffusion, α < 1 subdiffusion).T ransient subdiffu- tem. The alignment of both motility systems on the localization of flagella as well as their number differs Philipps-Universität Marburg cell surface depends on significantly for the various different types of bacteria. 2001–2002 Lecturer and Research the correct localization of The formation of a flagellum is initiated by the locali- Group Leader, Zentrum für Techno- regulatory proteins at the zation of specific proteins at the cell membrane.O ur mathematik, University of Bremen cell poles which set up a aim is to derive new reaction-diffusion equations that 1998–2001 Research Scientist, polarity axis. In response describe these protein localizations and to determine RWTH Aachen to signaling by the Frz che- the underlying parameters. Within this project, we 2000 Interim Professor of Math- mosensory system, these collaborate with the research group of Gert Bange. ematics, University of Gießen 1996–1997 Interim Professor of regulatory proteins are re- Mathematics, RWTH Aachen leased from the poles and 1996 Habilitation, RWTH Aachen then rebind at the oppo- 1995–1996 Assistant Professor, site poles. Thus, over time, RWTH Aachen the regulatory proteins os- 1994–1995 Visiting Professor, cillate between the poles. University of South Carolina, The aim of our project is Columbia, SC, USA to derive mathematical 1992–1994 Assistant Professor, models that describe this RWTH Aachen behavior. Until now, we 1989–1992 Assistant Professor, have studied mathemati- Free University of Berlin 1989 Ph.D. in mathematics, cal models involving two University of Bremen and more regulatory pro- Figure 1. Visualization of the oscillations of two proteins in a Myxococcus xanthus cell. Both teins, with and without 1986 Diploma in physics, proteins stay at opposite poles of the cell for a relatively long time, followed by a very fast University of Bremen switch, as observed in practice. stochastic influences (Fig.1 52 research 53

Selected publications

Diffusive processes in Eckhardt B and Zammert S (2012). Non-normal tracer diffusion from and around bacterial cells stirring by swimming micro- organisms. Eur Phys J E 35, 96.

The correct functioning of a cell requires that all its handle the exchange of signals and nutrients between Niedermayer T, Eckhardt B, molecular constituents are available at the right time the cell and its environment, others act as assembly Lenz P (2008). Synchronization, and at the right location. However, molecular diffu- platforms. In many cases, the details of their function phase locking, and metachronal sion acts against any localization efforts of the cell: are unknown. Since the membrane provides a fluid wave formation in ciliary chains. collisions with other molecules in the cytosol gradual- environment, the location of the proteins is not fixed, CHAOS 18, 037128. ly reduce gradients until the concentration is uniform. and their diffusive motion complicates their study. The effects of diffusion are reduced for larger assem- High-resolution STED microscopy allows to visualize Vollmer J, Vegh AG, Lange C, Eckhardt B (2006). Vortex formation Prof. Dr. Bruno Eckhardt blies, and they also depend on the degree of molecu- and track fluorescently labeled proteins and to trace by active agents as a model lar crowding or geometrical structuring of space, e.g., their movements, in order to learn about their dy- Theoretical Physics for Daphnia swarming. by organelles or cytoskeletal elements. Beyond the namics and the interactions and colocalizations with Phys Rev E 73, 061924. Faculty of Physics, interior of the cell, diffusion is also a significant fac- other proteins in the membrane. Philipps-Universität Marburg tor in the cell’s environment, where it impacts nutri- Using live cell high-resolution STED microscopy, the Phone: +49 6421 28 21316 ent distribution and uptake, and thereby the cell’s motion of YFP-tagged flotillins, membrane proteins Email: bruno.eckhardt@synmikro. chemotaxis. that coassemble into discrete microdomains, can be uni-marburg.de The point of reference for diffusive processes is “nor- observed in Bacillus subtilis membranes (Fig. 1). For the Figure 2. Traces of small passive particles moved around by swimming Shewanella. Different colors mark different particles. SYNMIKRO member since 2010 mal diffusion”, with a Gaussian concentration profile proper interpretation of the observed motions one and a displacement Δx2 that grows linearly with time, has to take into account that they are projections of Δx2 ≈t. Interactions with the structural elements or the the actual motions into the 2D focal plane, but calcu- us to change the swimming speed and the direction- ability density function (pdf), one notices strong de- motion of cells can cause significant deviations, re- lations allow to undo these projections. They further ality of the motion, and to explore their impact on viations from the normal Gaussian distribution: long sulting in either faster or slower spreading. Studying show that the diffusion of flotillins in the membrane particle dispersion in the solvent. excursion are much more frequent, and their probabil- these deviations provides valuable information about is isotropic, i.e. with no directional preference. With With high frequency cameras and microscopes of suf- ity is much better described by an exponential than a the underlying mechanisms and the effects on their the imaging procedures established we can now study ficient resolution it is possible to track the motion of Gaussian distribution (Fig. 3). In Ref. (Eckhardt & Zam- function. We are interested in normal and non-normal other proteins as well, and can characterize their in- particles and cells in 2D and 3D. The trajectories of the mert, 2012), we have related this behavior to another Short CV diffusion in two particular settings: the motion of pro- teractions and their spatio-temporal dynamics within cells show straight segments during the run phase general class of diffusion processes, so-called contin- 1996– Professor of Theoretical teins in membranes, in a collaboration with the group the membrane. and sharp directional changes during the tumble uous time random walks, where long excursions and a Physics, Philipps-Universität Marburg of Peter Graumann, and the stirring of surrounding flu- phase. The particles in the solvent are affected both persistence of motion are allowed. The fit to the data 1992–1993 Professor of Theoretical ids by swimming microorganisms, together with the Stirring and dispersion in by the velocity fields surrounding the swimming cells is encouraging. In ongoing efforts, we now want to ex- Physics, Carl von Ossietzky groups of Gert Bange and Kai Thormann (JLU Gießen). bacterial solutions and the Brownian forces from the solvent. They fol- plore the effects of changes in the flagellar structure, Universität Oldenburg low rather ragged trajectories, with sometimes fairly number, and activity on the efficiency of swimming in 1988–1992 Research Assistant, Protein dynamics in the The motion of microorganisms introduces flows in elongated excursions (Fig. 2). When the positions of the environment. Philipps-Universität Marburg cytoplasmic membrane their environment that affect the dispersal of nutri- the particles after some time are collected in a prob- 1986–1988 Research Scientist, ents and other solutes. The flows can be visualized Forschungszentrum Jülich The cellular membrane contains many proteins that and measured by the tracking of passively advected 1986 Ph.D. in theoretical physics, are responsible for important cellular functions. Some particles, which in case of Escherichia coli and the Universität Bremen 1982 M.Sc. in physics, marine alga Chlamydomonas Georgia Institute of Technology, reinhardtii has revealed that Atlanta, Georgia, USA the spreading process does not follow the laws of normal diffusion: the probability den- sity functions are not Gauss- ian, and the spreading law can change from a normal dif- fusive law to a super-diffusive law. In order to further explore Aa w rds & Honors the origins and implications of this process, we study dis- 2006 Fellow of the European persion in suspensions of She- Mechanics Society wanella. 2004 Fellow of the American allows for the modification Physical Society of the swimming properties 2004 Fellow of the Institute of Physics (London) of Shewanella by changing 2002 Leibniz Prize of the German the number of flagella, their Research Foundation (DFG) biochemical composition and Figure 3. Probability density functions (pdf) for the displacement of passive particles in a bacte- 1994 Physik-Preis of the Göttinger Figure 1. Bright field image of a single Bacillus subtilis cell, with the initial location of fluorescent the operation of the chemo- rial bath. Experimental data of (Leptos et al, PRL 103 (2009) 198103) for different concentrations Akademie der Wissenschaften proteins overlayed in green and colored lines indicating movements of the respective proteins. tactic apparatus. This enables together with the fit to a continuous time random walk (Eckhardt & Zammert, 2012). 54 research 55

ogs (Fig. 1B). Ccrps do not need a Selected publications A D 0 s nucleotide cofactor for filament Bacterial cells in 3D A Graumann PL (2007). Cytoskeletal formation and form very stable 0.02 s elements in bacteria. Despite their apparent simplicity, bacteria have a so- shaped bacteria like Bacillus subtilis, the actin-like pro- polymers (Fig. 1D). 0.04 s Annu Rev Microbiol 61, 589-618. phisticated cell structure and shape. E.g., they can be tein MreB mediates the elongated form with its two In vivo, B. subtilis MreB forms 0.06 s round, rod-shaped, bent, or helical. Our group is in- defined cell poles and the consequential axes (Grau- filaments underneath the cell Waidner B, Specht M, DempwolffF , vestigating two bacterial species, the Gram-positive mann, 2007). As such, MreB is essential for viability, membrane demonstrated by our 0.08 s Haeberer K, Schaetzle S, Speth V, soil-dwelling bacterium Bacillus subtilis, and – in a and mreB depleted B. subtilis cells become spherical, group via super resolution fluo- B Kist M, Graumann PL (2009). A novel 0.10 s subgroup headed by Dr. Barbara Waidner – the Gram- enlarged and finally lysed F( ig. 1A). Gram-positive bac- rescence microscopy reaching system of cytoskeletal elements in negative human pathogen Helicobacter pylori. As B. teria often have multiple mreB homologs, and B. sub- a resolution of 50 nm in living 0.13 s the human pathogen Helicobacter subtilis is rod-shaped, while H. pylori is helical, we ana- tilis has three: MreB, Mbl and MreBH. However, MreB cells (Fig. 1A) (Reimold et al, 2013). pylori. PLoS Pathog 5, e1000669. 0.15 s lyse the differences and similarities of both organisms is not needed for cell shape maintenance and viability These structures have approxi- in generating their cell shape. Furthermore, we are in the helical Gram-negative human pathogen Helico- mately half-cell circumference 0.17 s Specht M, Schätzle S, Graumann PL, Waidner B (2011). Helicobacter py- Prof. Dr. interested in the general question how bacterial cells bacter pylori (Waidner et al, 2009; Specht et al, 2011). and lie in angles between 70° and Bacillussubtilis 0.19 s lori possesses four coiled-coil-rich Peter l. Graumann are organized in 3D. For instance, not only proteins, In this organism, four filament-forming coiled coil- 110° (i.e., centring around 90°) C 0.21 s proteins that form extended fila- Molecular and Cellular Biochemistry but also genes have a defined position within the cell rich proteins (Ccrps) influence cell curvature (Fig. 1C). relative to the longitudinal axis mentous structures and control cell of Microorganisms caused by a specific folding of the bacterial chromo- Purified MreB andC crps form extended filamentous of the cell (Fig. 1A). They move 0.23 s shape and motility. some. The unravelling of the molecular network un- structures in vitro that can be imaged by fluorescence back and forth around the cell’s J Bacteriol 193, 4523–30. Faculty of Chemistry, 0.25 s derlying this 3D organization is one of the most fasci- microscopy (Fig. 1B) or by electron microscopy (Fig. 1D). circumference, reaching almost Philipps-Universität Marburg nating questions of today’s cell biology. MreB polymers contain ATP or GTP, are dynamically 100 nm/s. How these dynamic single molecule tracks 0.27 s Reimold C, Defeu Soufo HJ, Demp- Phone: +49 6421 28 22210 self-remodelling structures, and MreB of B. subtilis filaments achieve their task in wolff F, Graumann PL (2013). Motion Email: [email protected] SMC ScpA 0.29 s of variable-length MreB filaments at marburg.de Maintenance of cell shape forms joint filamentous structures with its two paral- maintaining cell shape is not yet known; but as they interact mobile SMC fraction static SMC fraction 0.31 s the bacterial cell membrane influ- SYNMIKRO member since 2012 In the development of cell morphology, filament- with several membrane proteins - active in de novo - in complex with ences cell morphology. Helicobacter DNA binding static ScpAB fraction 0.33 s Mol Biol Cell 24, 2340-49. forming proteins play a key role. E.g., in many rod- involved in the enlargement of C) Ccrp59 in vivo the cell wall during growth, it is 0.36 s Kleine Borgmann LA, Ries J, Ewers H, hypothesized that they might 0.38 s Bacillus Ulbrich MH, Graumann PL (2013). The mediate the positioning of the A) MreB in vivo 0.40 s bacterial SMC complex displays two cell wall synthetic enzymes, or distinct modes of interaction with coordinate the movement of SMC-YFP 0.42 s the chromosome. Cell Rep 3, 1483-92. Short CV these enzymes. In a synthetic approach, we use these filament- 2013–2014 Visiting Professor, Univer- forming proteins to serve as pro- Figure 2. (A) B. subtilis cells, DNA stained green, membranes stained red. White triangles sity of California, San Diego, USA indicate cells whose sister chromosomes are almost entirely separated into opposite cell 2012– Professor of Biochemistry, tein scaffolds, onto which other halves. (B) Cartoon of a bacterial cell (cell membrane in blue), in which the chromosome Philipps-Universität Marburg proteins can be stably attached, (red) is folded in an ordered manner; two chromosomes are shown, after cell division in the middle of the cell, each daughter cell has an origin (0°) at one cell pole, a terminus (180°) 2004–2012 Professor of Microbio- thereby increasing the rate of in- at the other pole, and the chromosome arms in between (90° and 270° are indicated). (C) logy, Albert-Ludwigs-Universität teractions. Cartoon of a B. subtilis cell in which ScpAB-bound SMC (green tracks) are static within two Freiburg condensation centres, while free SMC (blue tracks) moves all over the genome. (D) Move- ment of a single SMC-YFP molecule in a single Bacillus cell, tracked every 20 ms; tracks are 2000–2004 Independent Reseach B) MreB and homologs in vitro Separation of sister indicated by blue line. Group Leader, Philipps-Universität CFP YFP chromosomes Marburg 2003 Habilitation, Philipps- Bacterial chromosomes are organized into “nucleoids” chromosomes is still unclear for all kinds of cells. Re- Universität Marburg (green in Fig. 2A), and have a relatively fixed arrange- cently, we have tracked individual SMC molecules in 1997–2000 Postdoctoral Fellow, Harvard University, USA ment (Fig. 2B). This specific folding results in defined living cells using “slim-field” fluorescence microscopy 1997 Ph.D. in biology/biochemistry, 3D positions of genes within the cell, and this specific (Fig. 2D, the “track” of the molecule is shown in blue). Philipps-Universität Marburg order is even kept during chromosome replication, Our findings reveal two distinct modes of interaction 1994 Diploma in biology, Philipps- segregation and cell division. How is the arrangement of SMC with the chromosome: ScpAB-bound SMC Universität Marburg mCherry overlay of the chromosome kept in such a “perfect” order? stays stationary in co-called condensation centres, D) Ccrp59 in vitro One pivotal protein family for this process are SMC while free SMC runs all over the chromosome (Fig. (structural maintenance of chromosomes) proteins. 2C) (Kleine Borgmann et al, 2013). Thus, whereas SMC These highly conserved proteins are essential for alone dynamically interacts with many sites on the chromosome organisation and segregation in bacte- chromosome, it forms static assemblies together with ria, and for mitosis and meiosis in eukaryotes. SMC ScpAB. How the two fractions of SMC contribute to proteins are very large (135-150 kDa) proteins and the compaction of the whole chromosome, and how Aa w rds & Honors form flexible dimers with two long arms and head the condensation centres are formed and turned over, domains at both ends enabling the dimerization in is subject of our ongoing studies. 2008 Research Award of the Figure 1. (A) Fluorescence microscopy images of B. subtilis PY79 membranes demonstrating the effect of mreB depletion (left, top an ATP-dependent manner (Fig. 2C, bottom). This way Association for General and Applied and bottom), and STED super resolution fluorescence microscopy Microbiology (VAAM) image of YFP-MreB in B. subtilis cells (right). (B) B. subtilis MreB they can form rings around DNA and hold individual 2004 Heisenberg Fellow and its two homologs Mbl and MreBH jointly form filamentous (C) Effect of ccrp59 deletion (KE-59PCAT) on H. pylori wt (KE) cells. DNA strands together. In B. subtilis, the SMC homodi- structures on a flat membrane as shown via fluorescence micros- (D) Electron microscopy of Ccrp59 forming bundles of parallel 2000–2004 Emmy Noether Indepen- copy images (using CFP, YFP and mCherry protein fusions, respec- filaments in vitro. mer associates with two regulatory proteins known dent Junior Research Group Leader tively) and the overlay of these. White bars 2 µm, black bar 50 nm. as ScpA and ScpB. However, how they compact entire 56 research 57

Selected publications

Physics in synthetic biology You C, Okano H, Hui S, Zhang Z, Kim M, Gunderson CW, Wang YP, A major insight of the post-genomic era is that the an analytic approach to determine the phase bound- Lenz P, Yan D & Hwa T (2013). complexity of biological organisms arises not only ary between the stripe and the no-stripe phases. From Coordination of bacterial proteome from the large number of their constituents, but this solution, in turn, we were able to make various with metabolism by cyclic AMP also from the interactions between these compo- predictions how the patterns could be tuned experi- signalling. Nature 500, 301-6. Figure 2. In our coarse-grained mathematical model, carbon and nitrogen influxes combine to synthesize amino acids which are in turn nents. Consequently, the analysis of living matter mentally (Liu et al, 2011). assembled into proteins by the ribosomes (R). The external carbon source is converted to the pool of carbon precursors that sense the has changed dramatically in the last decade. Instead difference between the nutrient fluxes. The carbon and nitrogen fluxes are coordinated by an integral feedback system regulating the Liu C, Fu X, Liu L, Ren X, Chau CK, catabolic enzymes (C) and anabolic enzymes (A) by the carbon precursors. Cyclic AMP is the messenger regulating the carbon-branch. of individual genes and proteins, now modules and Regulation of bacterial metabolism Li S, Xiang L, Zeng H, Chen G, Tang LH, networks are in the focus of many studies. In this Lenz P, Cui X, Huang W, Hwa T & context, theoretical descriptions become more and In all domains of life signaling pathways link percep- Spatial analysis and modeling mathematical model for yeast flocculation, which al- Huang JD (2011). Sequential estab- more important. Long-term goal of such approaches tion of environmental or intracellular cues to specific lows us to analyze the dependence of this process on lishment of stripe patterns in an of yeast flocculation expanding cell population. Prof. Dr. Peter Lenz is to obtain a quantitative understanding of complete adjustments in cellular function. In this project, we various relevant parameters, such as, e.g., cell density Science 334, 238-41. organisms. However, even for the best characterized have reanalyzed one of the best-studied systems of Multicellular development of Saccharomyces cerevisiae or strength of the bonds between cells. To simulate C omplex Systems bacteria many constituents and their interactions are microbiology, the cyclic AMP (cAMP) dependent path- includes the process of flocculation, which is the cal- our model process with acceptable runtimes, we de- Leinweber M, Bitter P, Brückner S, Faculty of Physics, still unknown. Therefore, smaller subsystems have to way in E. coli that mediates carbon catabolite repres- cium-dependent interaction among vegetative cells veloped a graphics processing unit (GPU) implementa- Mösch HU, Lenz P, Freisleben B Philipps-Universität Marburg be investigated and mathematically described. In our sion (CCR) (Fig. 2). In doing so, we could demonstrate leading to multicellular aggregates, so-called flocs. tion that allows us to track the cell movement in a de- (2014). GPU-based simulation Phone: +49 6421 28 24326 group, we do this using different physics-based ap- that cAMP has a much broader physiological role than It has been shown that the ability to form flocs can tailed manner by a 3D visualization during execution of yeast cell flocculation. Email: peter.lenz@physik. proaches that range from molecular models to effec- merely being the signaling molecule of carbon status provide an evolutionary benefit for a yeast popula- (Leinweber et al, 2014). This GPU implementation is up 22nd Euromicro International uni-marburg.de tive macroscopic descriptions of biological systems. that mediates CCR. Rather, the cAMP signaling path- tion, for instance by protecting individual cells from to 736 times faster than a multithreaded C/C++, en- Conference on Parallel, Distributed and Network-Based Processing (PDP), 601-8. SYNMIKRO member since 2010 way coordinates the allocation of proteomic resources environmental stress such as ethanol, fungicides abling us to simulate up to 1.000.000 yeast cells in to- Bacterial pattern formation in response to the global metabolic needs of expo- or other harmful conditions. In collaboration with tal. From these simulations, we have obtained several nentially growing bacterial cells. More precisely, we Hans-Ulrich Mösch and Bernd Freisleben, two other predictions that we are now testing experimentally. In Bünemann M & Lenz P (2007). Mechanical limits of viral capsids. Periodic stripe patterns are ubiquitous in living or- revealed surprising relations linking the expressions members of SYNMIKRO, we set up a fluorescence mi- our current efforts, we also focus on the behavior of PNAS 104, 9925-30. ganisms. In many cases, however, the underlying de- of catabolic and biosynthetic genes: the expression croscopy-based system for the precise spatial analy- mixed populations, where some cells do not express velopmental processes are complex and difficult to of catabolic genes increased linearly with decreas- sis of single cells within individual flocs. To interpret the relevant adhesion proteins required for floccula- Lenz P & Swain PS (2006). An entrop- disentangle. In a novel synthetic biology approach in ing growth rates upon limitation of carbon influx, but the experimental findings, we developed a detailed tion. Our preliminary data indicate that such cheater ic mechanism to generate highly collaboration with groups from San Diego and Hong decreased linearly with decreasing growth rate upon cells are not only pushed to the boundary of the flocs, cooperative and specific binding but also restricted to specific sites within the protec- from protein phosphorylations. Short CV tive flocs. Current Biol 16, 2150-5. 2006– Professor of Theoretical Physics, Philipps-Universität Marburg 2005–2006 Visiting Professor, Uni- versity of California, San Diego, USA 2002–2006 Junior Professor of Theo- retical Physics, Philipps-Universität Marburg 1999–2001 Postdoctoral Fellow, Harvard University and Stanford University, USA, and Institut Curie, , France 1998 Ph.D. in theoretical physics, Max Planck Institute for Colloids and Interfaces, and University of Figure 1. (a) The engineered bacterium cells execute random walks at low densities, but become immotile at high densities. (b) This Potsdam coupling between density and motility leads to the formation of sequential patterns with tunable number of rings. 1995 Diploma in physics, Universi- ties of Regensburg, Hamburg, and Kong, we have implemented a genetic circuit coupling limitation of nitrogen or sulfur influx. In contrast, the Heidelberg cell density and motility into the bacterium Escheri- expression of biosynthetic genes exhibited the oppo- chia coli (Fig. 1). These cells excrete a small signaling site linear growth rate dependence as the catabolic molecule acyl-homoserine lactone (AHL), such that at genes. This striking linear response in gene expres- low AHL levels, cells are motile, while at high AHL lev- sion to nutrient limitation can be explained quan- els, cells tumble incessantly and become immotile. On titatively by a coarse-grained mathematical model agar plates these engineered bacteria form periodic based on two powerful constraints governing cellular stripes of high and low cell densities sequentially and metabolism: the balance of metabolic fluxes and the autonomously. To theoretically study the origin and partitioning of the ribosomal load. Key feature of the mechanism of this process, we developed a kinetic model is the prediction that carbon precursors, whose Aa w rds & Honors model that includes growth and density-suppressed intracellular level reflects the difference between the 2013 Paula und Richard von Hertwig motility of the cells. In this model, we analyzed the catabolic and biosynthetic fluxes, implement an inte- Award (together with collaborators) onset of pattern formation by calculating the front gral feedback scheme by regulating the level of cAMP Figure 3. Comparison between theoretically calculated (top) and experimentally observed (bottom) flocs formed by S. cerevisiae cells. 1999 Otto Hahn Medal of the Max profile of a region of cells that spread into an initially which in turn controls the expressions of catabolic In both cases, cheater cells (i.e., cells that do not express the rele- Planck Society cell-free region. From this exact solution, we obtained enzymes through the regulator Crp (You et al, 2013). vant adhesion proteins required for flocculation) are shown in red. 58 research 59

Aa w rds & Honors

Synthetic microbiology & 2014 Elected Fellow of the American Academy of Microbiology modularity of cell polarity systems 2008 Elected Member of the German Academy of Sciences Leopoldina 1999 Research grant from the Dan- All living cells are polarized and contain proteins that the lagging pole, and these proteins switch poles dur- ish Research Councils via the Female localize asymmetrically to specific subcellular regions. ing reversals. In other words, type IV pili pole-to-pole Researchers in Joint Action program In bacteria as well as in eukaryotic cells, many of these switchings depend on the dynamic localization of PilB (FREJA-program) proteins are important for fundamental cellular pro- and PilT. 1995 Odense Kommune’s Research cesses such as cell division, growth, motility and dif- Our goal is to identify and characterize the compo- Award to the Advancement of Science ferentiation. Therefore, we have as a working hypoth- nents of the regulatory system that underlies the dy- 1992 The Danish Academy of Natural esis that streamlined natural cells as well as synthetic namic polarity of motility proteins in M. xanthus. On Sciences award for best Ph.D. thesis 1991/1992 Prof. Dr. cells depend on some level of subcellular organization the basis of this system, we aim to define a minimal Lotte Søgaard-Andersen for optimal function. The overall goal of our work in module for regulation of dynamic cell polarity in bac- Bacterial Development and synthetic microbiology is to generate a module for teria and to establish this module in other microor- Differentiation regulating dynamic cell polarity in streamlined natu- ganisms as well as in synthetic cells. As part of this ral cells as well as in synthetic cells. research, we are addressing the more fundamental Department of Ecophysiology, question whether cell polarity systems are indeed Max Planck Institute for Terrestrial Dynamic regulation of modular. In other words, can these systems be trans- Microbiology ferred between organisms and still function? Or are Phone: +49 6421 178 201 type IV pili polarity they so tightly integrated with host cell Email: sogaard@mpi-marburg. mpg.de For most dynamically localized proteins, the localiza- that function is restricted to the original host? tion pattern changes in a cell cycle-dependent man- SYNMIKRO member since 2010 ner. An exception to this general rule are the proteins The polarity module of the type IV pili-dependent motility system in the Selected publications rod-shaped cells of Myxococcus xanthus that localize We recently showed that the regulatory system con- dynamically to the cell poles in a cell cycle-independ- trolling the polarity of motility proteins in M. xanthus Leonardy S, Miertzschke M, Bulyha I, ent manner. In the case of this motility system, the is built around the MglA, MglB and RomR proteins, Sperling E, Wittinghofer A, Søgaard- machinery assembles at the leading pole of a mov- which interact to define the leading/lagging polarity Andersen L (2010). Regulation of ing cell, and during a reversal it disassembles at the axis of the cell (Fig. 2). MglA is a small Ras-like GTPase dynamic polarity switching in bac- old leading pole and reassembles at the new leading that functions as a nucleotide-dependent molecular Figure 2. Left panel, MglA, MglB and RomR establish the leading/lagging cell polarity axis. Right panel, the Frz chemosensory system teria by a Ras-like G-protein and its signals to the RomR response regulator for reversals. cognate GAP. EMBO J 29, 2276–89. pole. We have shown that 8 of the 10 proteins required switch to regulate motility in M. xanthus (Leonardy for type IV pili function localize to both cell poles and et al, 2010; Miertzschke et al, 2011; Keilberg et al, 2012; Miertzschke M, Koerner C, Vetter IR, remain stationary during reversals (Fig. 1) (Friedrich Bulyha et al, 2013). The MglB protein functions as a pole, also together with RomR. Between reversals, Mg- Keilberg D, Hot E, Leonardy S, et al, 2014). Conversely, the PilB and PilT ATPases that GTPase activating protein (GAP) and converts active lA-GTP sets up the correct polarity of the dynamical Søgaard-Andersen L, Wittinghofer A energize extension and retraction of type IV pili, re- MglA-GTP to the inactive MglA-GDP. In a moving cell, motility proteins PilB and PilT by an unknown mecha- (2011). Mechanistic insights into spectively, localize to opposite poles with PilB pre- MglA-GTP localizes to the leading cell pole together nism. In response to signaling activity of the Frz che- bacterial polarity from structural Short CV dominantly at the leading and PilT predominantly at with RomR, while MglB localizes to the lagging cell mosensory system, MglA, MglB and RomR are released analysis of the Ras-like G protein from the poles and then relocate to the respective op- MglA and its cognate GAP MglB. 2008– Professor of Microbiology, posite poles. In total, this results in an inversion of the EMBO J 30, 4185–97. Philipps-Universität Marburg leading/lagging polarity axis and the relocation of dy- 2004– Director and Head of the namic motility proteins. Keilberg D, Wuichet K, Drescher F, Søgaard-Andersen L (2012). A re- Department of Ecophysiology, In our ongoing research, we focus on defining the Max Planck Institute for Terrestrial sponse regulator interfaces between parts of this cell polarity system and on elucidating Microbiology, Marburg the Frz chemosensory system and 2002–2004 Professor of Molecular how the various proteins interact to understand how the MglA/MglB GTPase/GAP module Microbiology, University of South- they can localize correctly to the cell poles and how to regulate polarity in Myxococcus ern Denmark, Denmark they switch poles in a coordinated manner. In parallel, xanthus. PLoS Genet 9, e1002951. 1996–2002 Associate Professor, we are attempting to establish the system in heter- University of Odense, Denmark ologous hosts. Bulyha I, Lindow S, Lin L, Bolte K, 1992–1996 Assistant Professor, Wuichet K, Kahnt J, van der Does C, University of Odense, Denmark Thanbichler M, Søgaard-Andersen L 1994–1995 Visiting Scientist, (2013). Two small GTPases act in con- Stanford University, USA cert with the bactofilin cytoskeleton 1991 Ph.D. in molecular biology, to regulate dynamic bacterial cell University of Odense, Denmark polarity. Dev Cell 25, 119–31. 1990 Visiting Scientist, Institute Pasteur, Paris, France Friedrich C, Bulyha I, Søgaard- 1988 M.D., University of Odense, Andersen L (2014). Outside-in as- Denmark sembly pathway of the type IV pili 1984 M.Sc. thesis in molecular biol- Figure 1. Type IV pili pole-to-pole switching depends on the dynamic polar localization of PilB (red, at leading pole) and PilT system in Myxococcus xanthus. ogy, University of Odense, Denmark (blue, at lagging pole). J Bacteriol 196, 378-90. 60 research 61

consequence, cells are able to adapt faster to environ- Selected publications mental changes that require the de novo synthesis of Mechanisms of spatial Kiekebusch D, Michie KA, Essen LO, proteins. Moreover, they need to produce less protein Löwe J, Thanbichler M (2012). to achieve optimal working levels, thereby gaining a Localized dimerization and nucleoid organization in bacteria fitness advantage in the oligotrophic environment binding drive gradient formation by that C. crescentus inhabits. In the future, this machin- the bacterial cell division inhibitor Life depends on the proper arrangement of macromol- ATPase MipZ, which dynamically interacts with pole- ery could be adapted for generating artificial cellular MipZ. Mol Cell 45, 245-59. ecules in time and space to coordinate the complex associated complexes of the chromosome segrega- compartments that may facilitate the storage of toxic and highly dynamic processes that underlie the func- tion protein ParB (Fig. 1). As a consequence, MipZ biosynthetic products. Kiekebusch D, Thanbichler M (2014). tion of biological cells. Unlike eukaryotes, bacteria forms a gradient within the cytoplasm, with its con­ Spatiotemporal organization of usually do not make use of membrane-bounded orga- centration being highest at the tips of the cell and microbial cells by protein concentration gradients. nelles to segregate cellular components into distinct lowest at the cell center. MipZ acts as a direct inhibitor Trends Microbiol 22, 65-73. functional units. However, despite the apparent con- of FtsZ polymerization, thus limiting formation of the Prof. Dr. tinuity of their cytoplasmic space, they have evolved division apparatus to the midcell region. Kühn J, Briegel A, Mörschel E, Martin Thanbichler a variety of mechan­isms to recruit proteins and DNA Steady-state protein gradients are a well-known regu- Kahnt J, Leser K, Wick S, Jensen GJ, to specific subcellular locations, thereby generating latory strategy in eukaryotic cells, usually established Figure 2. Model for the recruitment of the cell wall synthase PbpC Thanbichler M (2010). Bactofilins, a Prokaryotic Cell Biology to membrane-associated bactofilin polymers in C. crescentus. pseudo-compartments with distinct biological­ func- by localized protein synthesis followed by diffusion ubiquitous class of cytoskeletal pro- Faculty of Biology, tions. Our research focuses on the mechanisms that and protein degradation.­ However, such diffusional teins mediating polar localization of Philipps-Universität Marburg provide the spatial information required to position gradients are extremely unstable at the much smaller I n parti­cular, we focus on the delta-proteobacterium a cell wall synthase in Caulobacter. Phone: +49 6421 28 21809 cellular components, with an emphasis on essential scale of prokaryotic cells, suggesting that formation Myxococcus xanthus, an organism containing four EMBO J 29, 327-39. Email: [email protected] cellular processes such as cell division, chromosome of the MipZ gradient is based on a different principle. bactofilin homologs. In collaboration with the group Schlimpert S, Klein EA, Briegel A, SYNMIKRO member since 2010 segregation, and morphogenesis. In particular, we aim Indeed, we have recently shown that MipZ serves as of Lotte Søgaard-Andersen, we have shown that three Hughes V, Kahnt J, Bolte K, Maier UG, at deconstructing the complex machineries involved a molecular switch that, similar to small GTPases, of these proteins co-polymerize into extended bipolar Brun YV, Jensen GJ, Gitai Z, Than- in these processes, and at characterizing individual uses nucleotide binding and hydrolysis to alternate filaments. These structures, on the one hand, medi- bichler M (2012). General protein functional modules using synthetic biological and between two different func­tional states with distinct ate the subpolar localization of a small GTPase (MglA) diffusion barriers create compart- reverse-engineering approaches. Such in-depth know­ interaction networks and diffusion rates. This behav- regulating the dynamics of proteins involved in cell ments within bacterial cells. ledge of the organizing principles at work in native ior drives a unique localization cycle, with MipZ mol- motility. On the other hand, they contribute to proper Cell 151, 1270-82. systems will be critical for engineering machineries ecules oscillating back and forth between the polar chromosome segre­gation by controlling the subcel- that control the function and propagation of synthet- ParB complexes and pole-distal regions of the nucle- lular localization of the chromosome segregation ic cellular units in the future. oid. Our results for the first time shed light on the machinery (unpublished). Collectively, these findings Short CV basis of steady-state gradient formation in bacteria sug­gest that bactofilins serve as multifunctional mo- Regulation of division site placement and might provide a general mechanistic framework lecular scaffolds that ensure the proper arrangement 2014– Professor of Microbiology, for other gradient-forming systems in both prokary- of proteins within bacterial cells. It will be interesting Philipps-Universität Marburg In most bacteria, cell division is mediated by a multi- otic and eukaroytic cells (Kiekebusch & Thanbichler, to harness these structures for the development of 2008–2014 Junior Professor of protein complex called the divi­some, which assem- 2014). They thus form the basis for designing regula- synthetic molecular landmarks that enable the spe- Microbiology, Philipps-Universität bles on a ring-like polymeric structure formed by the tory modules for the positioning of protein complexes cific targeting of cellular components in artificial or Marburg bacterial tubulin homo­log FtsZ. We have previously in synthetic contexts. heterologous cellular systems. 2007–2014 Max Planck Research Group Leader, Max Planck Institute identified a novel regulatory mechanism that con- for Terrestrial Microbiology, Marburg trols the positioning of the divisome in the model Bactofilins: a new cytoskeletal scaffold Bacterial protein diffusion barriers 2002–2006 Postdoctoral Fellow, bacterium Caulobacter crescentus. It is founded on the Figure 3. The StpABCD diffusion barrier complex. (A) Localization of fluorescently tagged Stp complexes in live C. crescentus cells. Stanford University, USA In addition to the cytokinetic FtsZ ring, there are sev- Whereas eukaryotic cells contain a variety of mem- (B) Model of the Stp complex. (C) Model showing how diffusion 2002 Ph.D. in microbiology/bio- eral other cytoskeletal elements with a role in the spa- brane-bounded subcellular compartments, bacteria barriers (red) retain newly synthesized proteins (green) in the cell chemistry, Ludwig-Maximilians- tiotemporal organization of bacterial cells. Our group have long been thought to lack mechanisms to seg- body, thereby reducing the effective cell size. Universität München has recently identified a novel class of cytoskeletal regate their cytoplasmic space. However, we have re- 1998 Diploma in biology, Ludwig- proteins named bactofilins. We demonstrated that, cently discovered a protein-based diffusion barrier in Maximilians-Universität München in C. crescentus, two bacto­filin para­logues (BacAB) as- C. crescentus that subdivides cells into physiolo­gically semble into membrane-associated polymeric sheets distinct compartments (Schlimpert et al, 2012). that specifically localiz­ e to the old cell pole during de- The C. crescentus stalk is an extension of the cell body fined stages of the cell cycle (Kühn et al, 2010). These that largely consists of cell envelope surrounding a structures mediate the polar localization of a cell wall thin thread of cytoplasm. It is interrupted at regular biosynthetic enzyme involved in polar morpho­genesis intervals by so-called crossbands, disc-shaped elec- (Fig. 2). Bacto­filins polymerize spontaneously and in tron-dense struc­tures whose nature and function had the absence of nucleotide cofactors, forming long, remained unknown. We found that these crossbands Aa w rds & Honors biochemically inert filaments. This behavior is remi- are polymeric complexes composed of at least four niscent of inter­mediate filaments, even though there different proteins (StpABCD), which extend from the 2011 Research Award of the Associa- is no evolutionary or structural relationship between cytoplasmic core of the stalk to its outer membrane tion for General and Applied Micro- biology (VAAM) these two groups of proteins. layer (Fig. 3). Blocking the diffusion of both mem- 2007 Max Planck Research Group Interestingly, bactofilins are almost universally con- brane and soluble proteins in the cell envelope, they appointment Figure 1. Positioning of FtsZ by the MipZ•ParB•origin complex. served among bacteria. To clarify the spectrum of physiologically separate the stalk from other parts (A) In vivo localization of ParB (red) and FtsZ (green) in C. cres- 2003–2005 EMBO long-term post- centus cells. (B) Schematic representation of the components functions they can perform, we have set out to study of the cell and thus ensure that newly synthesized doctoral fellowship involved in the MipZ system. the role of bactofilins in other bacterial species. envelope proteins are retained in the cell body. As a 62 63

1.4 Research Chassis and Genomes 64 research 65

Selected publications

Engineering of Charoenpanich P, Meyer S, Becker A, McIntosh M (2013). Temporal expres- alpha-rhizobial genomes sion program of quorum sensing- based transcription regulation in Sinorhizobium meliloti. The size of bacterial genomes is highly variable, rang- their gene expression in response to local population J Bacteriol 195, 3224-36. ing from less than 0.15 mega base pairs (Mb) in ob- density. Communication occurs via small hormone- ligate symbionts to more than 10 Mb in free-living like molecules called QS signals. The most common Charoenpanich P, Soto MJ, Becker A, soil-dwelling bacteria. Studying naturally occurring QS signals in Gram-negative bacteria are N-acyl- McIntosh M (2014). Quorum sens- large bacterial genomes – which are usually found in homoserine lactones (AHLs), which are composed ing restrains growth and is rapidly bacteria subjected to frequently changing, highly di- of a fatty acid chain of 4 to 18 carbons attached to a inactivated during domestication verse environmental conditions - reveals strategies homoserine lactone ring (Fig. 2). QS systems provide of Sinorhizobium meliloti. Environ Microbiol Rep, doi: Prof. Dr. Anke Becker of genome expansion and provides insights into the parts and serve as blueprints for building intercellular 10.1111/1758-2229.12262. upper limit of genome size, as well as into concepts communication modules to control and coordinate C omparative Genomics of regulation and genome maintenance. A prominent cellular behavior in a population. of Microorganisms Figure 2. (a) Scheme of the S. meliloti Sin quorum sensing regulatory system. Expression of the AHL synthase gene sinI requires the Krol E, Becker A (2014). Rhizobial example of bacteria with large genomes is the group of We have investigated the function of a simple QS sys- transcription activator SinR and is strongly enhanced by the regulator ExpR in the presence of AHLs, resulting in positive feedback. homologs of long-chain fatty Faculty of Biology, a-rhizobial species that are able to form a root nodule tem in S. meliloti. This system consists of only three ExpR represses transcription of the sinI activating gene sinR at high AHL concentrations, leading to negative feedback regulation of acid transporter FadL facilitate sinI. Furthermore, ExpR controls expression of a multitude of target genes. (b) 3D structure model of the outer membrane protein FadL Philipps-Universität Marburg Sm symbiosis with leguminous host plants. The nodules protein components implementing positive and facilitating uptake of long-chain AHLs. The extracellular loop L5 conferring specificity to these signal molecules is labeled. OM, outer perception of long chain Phone: +49 6421 28 24451 are invaded by these a‑proteobacteria which in the en- negative feedback regulation to balance its activity membrane; IM, inner membrane. N-acyl-homoserine lactone signals. Email: [email protected] dosymbiotic bacteroid state fix molecular nitrogen to (Fig. 2): AHL signal molecules with acyl chains of 14 Proc Natl Acad Sci USA 111, 10702-7. marburg.de the benefit of the plant.C onsistent with changing life- or 16 carbons (long-chain AHLS) are produced by the transporter. By building hybrid FadL proteins we dem- at single cell level in colonies of S. meliloti growing in Schlüter JP, Reinkensmeier J, SYNMIKRO member since 2011 styles between free-living and endosymbiotic states, synthase SinI. sinI expression is controlled by an in- onstrated that the extracellular loop 5 (L5) of a-rhizo- a microfluidics system. QS signals and exopolysac- multipartite large genomes are prevalent in a-rhizo- terplay of two transcriptional regulators, SinR and bial FadL proteins contains determinants of specificity charides are secreted products that serve as common Barnett MJ, Lang C, Krol E, Giegerich R, Long SR, Becker A (2013). Global bia. These genomes often consist of one chromosome the AHL receptor ExpR. Furthermore, ExpR has a broad to long-chain AHLs (Krol & Becker, 2014) (Fig. 2). These goods, and monitoring the expression of their biosyn- mapping of transcription start sites and two to six large plasmids. The Sinorhizobium meli- regulatory role, including repression of motility genes experiments delivered a toolbox of L5 sequences that thetic genes therefore allows us to study bet hedging and promoter motifs in the symbi- loti genome is composed of a chromosome (3.65 Mb), and activation of genes responsible for biosynthesis can be used to modify the specificity of native FadL strategies and division of labor. In collaboration with otic alpha-proteobacterium Short CV and the megaplasmids pSymA (1.35 Mb) and pSymB of secreted sugar polymers (exopolysaccharides) (Cha- proteins to mediate uptake of long-chain fatty acids Prof. Peter Pfaffelhuber (University ofF reiburg), we de- Sinorhizobium meliloti 1021. (1.68 Mb) (Fig. 1). We use synthetic biology approaches roenpanich et al, 2013; Charoenpanich et al, 2014). and/or long-chain AHLs, which is a requirement for veloped a novel mathematical strategy for classifica- BMC Genomics 14, 156. 2011– Professor of Comparative to investigate the molecular requirements for the dif- A widespread mechanism of AHL perception involves the implantation of long-chain AHL-based QS circuits tion of the gene expression status in heterogeneous Genomics of Microorganisms, ferent lifestyles and explore the potential of a-rhizobi- binding of the signal molecules by cytosolic transcrip- into a heterologous host. bacterial populations based on the time course of Schlüter J-P, Czuppon P, Schauer O, Philipps-Universität Marburg Pfaffelhuber P, McIntosh M, 2008–2011 Professor of Systems al genomes for synthetic biology applications. tional regulators such as ExpR, which requires uptake signal intensities of individual cells. Heterogeneous Becker A (2015). Classification of Biology of , University of external AHLs. However, the outer membrane is Population heterogeneity expression of exopolysaccharide biosynthesis and phenotypic subpopulations in of Freiburg Regulatory circuits supposed to be an efficient barrier for diffusion of quorum sensing genes in S. meliloti populations was isogenic bacterial cultures by triple 2007 Adjunct Professor, Bielefeld long-chain AHLs. We found that sensing of these AHLs The ability of clonal cell populations to split into observed (Fig. 3). With respect to exopolysaccharide promoter probing at single cell level. University Quorum sensing (QS) is a widespread mechanism of is facilitated by the outer membrane protein FadL , subtypes displaying different phenotypes is a wide- biosynthesis, cells in the population were classified Sm J Biotechnol, doi: 2000–2007 Assistant Professor cell-cell signaling that allows bacteria to coordinate a homolog of the E. coli FadL long-chain fatty acid spread phenomenon among prokaryotes that has into non-contributors, weak contributors, and strong Ec 10.1016/j.biotec.2015.01.021 and Head of Junior Group, Bielefeld recently intrigued the scientific community and has contributors (Schlüter et al, 2015), showing that only University wide-ranging implications both for microbial ecology one third of the population strongly contributed to 2000 Habilitation, Bielefeld and biotechnology. Such phenotypic heterogeneity is the production of exopolysaccharide. University the basis for strategies like bet hedging and division 1999 Visiting Scientist, Massachu- of labor, prominent examples being the occurrence setts Institute of Technology, USA Genome engineering 1995–2000 Research Group Leader, of persister cells and cell differentiation in biofilms. Bielefeld University Mechanisms frequently involve variation in gene ex- T o facilitate genome engineering in a-rhizobia, novel 1994 Ph.D. in microbial genetics, pression, caused by fluctuations either in intrinsic or methods and tools were developed. These include sin- Bielefeld University in environmental factors. gle copy plasmids and controllable gene expression 1991 Diploma in biology, Bielefeld We have developed a versatile fluorescent reporter systems. Genome modifications were simplified by University cassette that allows for simultaneous monitoring of development of a cloning-free PCR-based method for activities of three promoters in single bacterial cells. gene disruption, deletion and replacement, and inser- This construct was applied to observe promoter activ- tion of heterologous DNA. ities related to QS and exopolysaccharide biosynthesis

Aa w rds & Honors

2002–2005 Heisenberg Fellow 1998–1999 Lise Meitner Fellow, Min- istry for Science and Research, NRW Figure 1. (a) Medicago sativa plants nodulated by S. meliloti. (b) Root nodule section showing plant cells infected by S. meliloti 1992–1994 Ph.D. scholarship, German labeled with the green fluorescent protein. Size bar, 500 µm. (c) National Merit Foundation Macroscopic view of root nodules. Size bar, 2 mm. (d) Electron 1994 Outstanding Dissertation micrograph of bacteroids. Size bar, 2 µm. (e) S. meliloti genome architecture. (f) Subcellular localization of the replication ori- Award, Westfälisch Lippische gins of the S. meliloti chromosome, pSymB megaplasmid and Figure 3. Time lapse of a S. meliloti colony grown in a microfluidics chamber. Cells carry fluorescence reporter gene fusions to specific promot- Universitätsgesellschaft the DNA polymerase III component DnaN. Size bar 1 µm. ers of exopolysaccharide biosynthesis and quorum sensing genes. Images represent an overlay of three fluorescent markers. Size bar, 5 µm. 66 research 67

acid sequence of these effectors.T herefore, we are Aa w rds & Honors currently unable to predict which of the 300 novel se- Adaptation of a pathogen 2011 TUM Distinguished Affiliated creted effectors are translocated.T o end this ambigu- Professorship, Technical ity, we have recently established an assay that allows University Munich to the plant host to discriminate apoplastic and cytoplasmic effectors. 2011 Honorary of the This assay is based on the ability of the biotin ligase Hebrew University, Jerusalem In our SYNMIKRO project, we aim to elucidate how BirA to biotinylate a 15 amino acid long sequence tag, 2008 Elected Member of the secreted proteins can cross membranes: The biotro- the Avitag (Fig. 4). To conduct the assay, effectors are German National Academy of phic plant pathogenic fungus Ustilago maydis (Fig. 1) fused with the Avitag and expressed in U. maydis. Such Sciences Leopoldina secretes a cocktail of about 300 novel protein effec- strains are then used to infect transgenic maize plants 1998 Medal of Merit (am Bande) of tors that modulate host defense responses and repro- expressing BirA in the cytosol. Biotinylated effectors the Federal Republic of Germany gram the host metabolism in favor of the pathogen are enriched by streptavidin affinity purification and 1993 Leibniz Prize of the German (Djamei & Kahmann, 2012; Lo Presti et al, 2015). After visualized by western blot. Biotinylation is observed Research Foundation (DFG) Prof. Dr. Dr. hc. secretion, effectors can either remain and function in only if the respective effector is translocated, while Regine Kahmann the interaction zone that is established between fun- apoplastic effectors do not get in contact with BirA. Molecular Phytopathology gal hyphae and host plasma membrane (apoplastic This assay does not only visualize translocation, but effectors), or they can be translocated to the host cell also allows to determine the uptake efficiency and will Department of Organismic (translocated effectors).N either the features/motifs in future permit to screen all 300 effectors for uptake Interactions, required for this translocation nor the uptake mecha- by maize plants (Lo Presti & Kahmann, unpublished). Max Planck Institute for Terrestrial nism are currently known. We expect that elucidating Microbiology these requirements and mechanism will provide an Phone: +49 6421 178 501 important tool for the manipulation of protocells in Infection: wild type tin2 mutant Email: kahmann@mpi-marburg. synthetic microbiology. mpg.de Figure 2. When transiently expressed in maize cells, a Cmu1- Figure 3. Maize plants infected with U. maydis wildtype and tin2 mCherry fusion protein spreads from the transformed cell (*) to mutant, respectively. Whereas the wildtype induces tumors and SYNMIKRO member since 2010 adjacent non-transformed cells to prepare these for infection by anthocyanin production (red patches), leaves infected with the T ranslocated effectors lowering salicylic acid levels in infected tissue. tin2 mutant develop smaller tumors and remain green.

We have identified the first translocated effector of U. maydis by electron microscopy after immunogold Cmu1 is initially secreted to the interaction zone be- Tin2 version lacking the signal peptide in maize cells labeling (Djamei et al, 2011). This effector, a virulence tween fungal hyphae and host plasma membrane using the red color of anthocyanin as readout. Tin2 determinant, is an active chorismate mutase (Cmu1) and is then taken up by plant cells. Inside plant cells interacts with and stabilizes the maize protein kinase that modulates the metabolism of maize by lower- Cmu1 resides in the cytosol and can spread to neigh- ZmTTK1 by masking a ubiquitin–proteasome degra- Selected publications ing the level of salicylic acid in infected tissue (Fig. 2). boring cells through plasmodesmata to prepare them dation motif in ZmTTK1. Active ZmTTK1 controls the for the upcoming infection. Cmu1 is then speculated activation of genes in the anthocyanin biosynthesis Djamei A, Schipper K, Rabe F, Ghosh A, to increase channeling of chorismate from the chlo- pathway and is presumed to lower the metabolite flow Vincon V, Kahnt J, Osorio S, Tohge T, roplasts to the cytosol together with the cytoplasmic into the lignin biosynthesis pathway, which otherwise Fernie AR, Feussner I, Feussner K, Short CV maize chorismate mutase, by converting chorismate would have negative consequences for fungal spread Meinicke P, Stierhoff Y, Schwarz H, Macek B, Mann M, Kahmann R (2011). to prephenate, which might return to the plastids to and nutrition (Brefort et al, 2014; Tanaka et al, 2014). 2001– Professor of Genetics, Metabolic priming by a secreted serve the biosynthesis of aromatic amino acids. This Philipps-Universität Marburg fungal effector. Nature 478, 395-8. 2000– Director and Head of would lower chorismate levels in chloroplasts und Establishment of an assay for the Department of Organismic thus decrease the amounts of precursor that could effector translocation Djamei A, Kahmann R (2012). Usti- Interactions, Max Planck Institute go into salicylic acid biosynthesis. The plant hormone lago maydis: Dissecting the Molecu- for Terrestrial Microbiology, Marburg salicylic acid can induce cell death associated immune We are unable to detect a conserved sequence motif lar Interface between Pathogen and 1992–2001 Professor of Genetics, responses which would be detrimental for a biotro- in the Cmu1 and Tin2 effectors, suggesting that such Plant. PLoS Pathog 8, e1002955. Ludwig-Maximilians-Universität, phic fungus like U. maydis that relies on maintain- a motif may not be apparent in the primary amino Munich ing its host cells alive. Thus, by translocating Cmu1 Tanaka S, Brefort T, Neidig N, Djamei A, 1987–1992 Independent Research Kahnt J, Vermerris W, Koenig S, to plant cells, U. maydis is reprogramming these cells Group Leader, Institut für Feussner K, Feussner I, Kahmann R metabolically for its own needs. Genbiologische Forschung Berlin (2014). A secreted Ustilago maydis GmbH, Berlin We have also shown that a catalytically inactive ver- effector promotes virulence by 1982–1986 Independent Research sion of Cmu1 (Cmu1KR) causes a dominant negative targeting anthocyanin biosynthesis Group Leader, Otto Warburg phenotype when overexpressed by U. maydis, a phe- in maize. eLife 3, e01355. Laboratory, Max Planck Institute for notype which we now use as an indicator for uptake. Molecular Genetics, Berlin In collaboration with the group of Gert Bange, the Brefort T, Tanaka S, Neidig N, 1980–1982 Research Associate, Max structure of Cmu1 was recently solved and now serves Doehlemann G, Vincon V, Kahmann R Planck Institute for Biochemistry, as a blueprint for defining and mapping the uptake (2014). Characterization of the larg- Martinsried motif, and for site-directed mutagenesis (Bange et al, est effector gene cluster of Ustilago 1974–1980 Postdoctoral Fellow and maydis. PLoS Pathog 10, e1003866. unpublished). Junior Staff ember,M Cold Spring Harbor Laboratory, USA In addition to Cmu1, we have identified a second trans- Lo Presti L, Lanver D, Schweizer G, Figure 1. (A) Corn smut caused by an U. maydis infection. The 1974 Ph.D. in genetics, Free kernels of the infected maize cob are much-enlarged and form located effector, Tin2. This effector induces anthocy- Tanaka S, Liang L, Tollot M, Zuccaro A, University, Berlin distorted tumors. (B) An U. maydis hypha (pink) infecting maize anin biosynthesis and also promotes virulence (Fig. 3). Figure 4. Model illustrating our translocation assay. Transgenic Reissmann S, Kahmann R (2015). (green). The hypha secretes effector proteins that either stay in maize cells express the E. coli biotin ligase BirA (orange). U. may- 1972 Diploma in microbiology, the interaction zone between pathogen and host cell (pink trian- In this case, we have demonstrated a function of Tin2 dis effector proteins are fused with the Avitag (red) and, if trans- Fungal effectors and plant suscepti- Georg-August-Universität Göttingen gles) or are translocated into the host cell (blue stars). inside plant cells indirectly, by transiently expressing a located to the host cells, biotinylated (purple). bility. Annu Rev Plant Biol, in press. 68 research 69

Selected publications

Solar-powered microfactories Bozarth A, Maier UG, Zauner S (2009). Diatoms in biotechnology: for fibers and therapeutics tools and applications. Appl Microbiol Biotechnol 82, 195-201.

In the last years, microalgae attracted much atten- Hempel F, Bozarth AS, Lindenkamp N, tion as resources of valuable natural products, such Klingl A, Zauner S, Linne U, as lipids for biodiesel production. Furthermore, micro- Steinbüchel A, Maier UG (2011a). algae are expected to have an enormous potential as Microalgae as bioreactors for biotechnological expression platforms for the produc- bioplastic production. tion of important products such as fibers or thera- Microbial Cell Factories 10, 81. peutics. In order to use diatoms as a new expression Hempel F, Lau J, Klingl A, Maier UG Prof. Dr. Uwe Maier system for recombinant proteins, our group estab- (2011b). Algae as protein factories: lished these unicellular organisms as solar-powered, Cyell Biolog expression of a human antibody and CO2-neutral microfactories in a SYNMIKRO project the respective antigen in the diatom Faculty of Biology, (Bozarth et al, 2009). Accepting this challenge, the Phaeodactylum tricornutum. Philipps-Universität Marburg diatom expressed portfolio includes production of a PLoS ONE 6, e28424. Phone: +49 6421 28 21543 bioplastic and spider silk as examples for fibers as well Email: [email protected] as human monoclonal antibodies and a vaccine. Hempel F, Maier UG (2012). An engi- marburg.de neered diatom acting like a plasma SYNMIKRO member since 2010 Bioplastic cell secreting human IgG antibodies with high efficiency. If possible, petroleum-based production of synthetic Microb Cell Fact 11, 126. materials should be reversed into production plat- Peschke M, Moog D, Klingl A, forms using regenerative resources as the basic ma- Maier UG, Hempel F (2013). Evidence terials. Synthesizing and using bioplastics might be for glycoprotein transport into the attractive aims to reduce the need of oil products and complex plastids. minimizing non-degradable waste. A naturally occur- Proc Natl Acad Sci USA 110, 10860-65. ring type of a bioplastic is PHB (poly-3-hydroxybu- tyrate). Starting with acetyl-CoA, only three enzymes are necessary to synthesize PHB. First attempts to change diatoms into PHB-producing organisms led to a strain synthesizing PHB to up to 11% of its dry Short CV Figure 2. Expression of the Hepatitis B virus surface antigen in the diatom Phaeodactylum tricornutum. The viral protein is tagged to weight (Fig. 1) (Hempel et al, 2011a). Very promising is the green fluorescent protein GFP, the plastid autofluorescence is depicted in red. 1997 Professor of Cell Biology, that the mentioned production rate can be obtained Philipps-Universität Marburg in 7 days and that the production strain was geneti- 1995–1997 Professor, University cally stable for years. toms for such an aim led to the efficient expression Of course there are several other products, which of Bayreuth of a human monoclonal antibody directed against a might be useful to be expressed in microlagae. How- 1989–1994 Assistant Professor, Spider silk Hepatitis B virus protein within the secretory system ever, increasing the quality of the expressed products University of Freiburg of the diatom (9% of total soluble protein) (Fig. 2). As and designing of useful molecules not known from 1988 Ph.D. in genetics, University of Spider silk has enormous physical capacities and is shown, the antibody can be purified from the cells and natural systems might be the major new challenges. Freiburg very interesting for diverse technical applications. is functionally active (Hempel et al, 2011b). Although In this respect, the posttranslational protein-modi- 1986 Diploma in biology, University In order to test if this amino acid-based fiber can be the expression rate in the diatom is smaller than in fying systems of microalgae, such as glycosylations of Freiburg produced in diatoms, we expressed spider silk mono- comparable mammalian expression systems, the an- (Peschke et al, 2013), have to be humanized for using mers. Up till now, spider silk molecules of more than tibody production in diatoms is, additionally to the synthesized molecules therapeutically. 150 kDa can be produced at different cellular localiza- advantages mentioned, cheap and quick. However, tions, highlighting diatoms as an efficient expression to further decrease production costs and to facilitate platform for huge proteins in biochemically isolated the purification process, a protocol was developed to compartments. produce the antibody in a way that it is secreted func- tionally active into the medium by the diatom (Hemp- Monoclonal antibodies/vaccines el and Maier, 2012). In order to scale up the production, an antibody-secreting strain was successfully grown F or analytic but also therapeutic applications, mono- in flat panel reactors (together with the Fraunhofer clonal antibodies are powerful tools. Although their IGB Stuttgart). Diatoms can be modified to produce expression in mammalian cell lines is generally es- antigens interesting for vaccination as well. This was tablished, production of antibodies in other expres- exemplarily shown by the expression of the target of Figure 1. Bioplastic production in the diatom P. tricornutum. (A) sion platforms is expected to dramatically decrease the monoclonal, anti-Hepatitis B virus protein anti- Algal cell culture. (B) Light microscopic picture of P. tricornutum the production cost. In addition, the synthesis of an- body. Thus for the virus example, an antibody as well Aa w rds & Honors cells. (C) Electron microscopic picture of a cell producing the bioplastic PHB. The bioplastic accumulates in granula-like tibodies in microalgae excludes potential and harm- as the respective antigen can be produced efficiently 1995 Heisenberg Fellow structures throughout the cytosol (marked with an arrow). ful human pathogenic contaminations. Testing dia- by the diatom. 70 research 71

Selected publications

F rom natural to synthetic Messerschmidt SJ, Kemter FS, Schindler D, Waldminghaus T (2015). chromosomes and back Synthetic secondary chromosomes in Escherichia coli based on the replication origin of Vibrio cholerae We are entering an era in life science in which syn- want to understand more about the role of DNA mo- chromosome II. thesis of chromosome-sized molecules becomes tif distribution in chromosome biology. The template Biotechnol J, doi: 10.1002/ technically feasible. This leads to the question if we for our synthetic secondary chromosome is Vibrio biot.201400031. actually understand construction rules of natural cholerae, a bacterium that carries a natural secondary chromosomes completely. That is not the case, but chromosome. Based on the replication origin of this Messerschmidt SJ, Waldminghaus T Figure 2. The synthetic secondary chromosome synVicII in E. coli based on the replication origin of the natural secondary chromosome (2015). Dynamic organization: Chro- such a thorough understanding would be essential to secondary chromosome, we have constructed a 10kb of V. cholerae. (A) Replicon map with relevant features. (B) Copy number analysis of synVicII in relation to the E. coli primary chromo- design and build synthetic chromosomes for organ- “mini-chromosome” called synVicII (Fig. 1+2) (Messer- some using comparative genomic hybridization with a custom microarray. mosome domains in Escherichia coli. J Mol Microbiol Biotechnol, in press. Prof. Dr. isms with new capabilities, e.g., for biotechnological schmidt et al, 2015). This synthetic chromosome rep- Torsten Waldminghaus purposes. Therefore, our group studies bacterial chro- licates in E. coli and represents the chassis for more Stokke C, Waldminghaus T, mosome biology using a variety of methods with em- synthetic secondary chromosomes to come. Initial Chromosome Biology Skarstad K (2011). Replication phasis on synthetic biology approaches. characterization suggests that the replication tim- patterns and organization of repli- Faculty of Biology, ing of synVicII in the heterologous host E. coli is ac- cation forks in Vibrio cholerae. Philipps-Universität Marburg Our philosophy: tually similar to that of the secondary chromosome Microbiology 157, 695 – 708. Phone: +49 6421 28 23338 1. You have to understand chromosome biology to in V. cholerae, indicating a certain independence of Email: torsten.waldminghaus@ build synthetic chromosomes. chromosome maintenance systems of the biological Waldminghaus T, Weigel C, synmikro.uni-marburg.de 2. You have to build synthetic chromosomes to under- background. One object of our future work is the opti- Skarstad K (2012). Replication fork movement and methylation SYNMIKRO member since 2011 stand chromosome biology. mization of synVicII regarding its stability within the cell. A second goal is to use synVicII as a backbone for govern SeqA binding to the Escherichia coli chromosome. building larger chromosomes with different arrange- The synthetic secondary Nucleic Acids Res 40, 5465-76. chromosome synVicII ments of chromosome maintenance motifs. A sub- sequent characterization is expected to provide new Figure 3. Model of the SeqA treadmilling. The protein SeqA leaves the DNA at the trailing end of a multimer and rebinds at the leading Waldminghaus T, Skarstad K (2009). Many studies in the field of synthetic genomics focus insights into chromosome maintenance. end near the replication machinery. The Escherichia coli SeqA protein. on the question which and how many genes are need- Plasmid 61, 141-50. ed for a minimal genome. However, chromosomes Replication of the two are much more than arrays of genes. They need to or- Vibrio cholerae chromosomes simplest possible system to study general questions end and shrinking in the trailing end (Fig. 3) (Wald- ganize the genetic material and guarantee its proper of multi-chromosome systems. For all these aspects, a minghaus et al, 2012). The movement of SeqA behind Short CV transfer to daughter cells – features that are sum- While the genome of eukaryotic organisms is divided deep understanding of the V. cholerae chromosome bi- the replication fork must be based on the combina- marized by the term “chromosome maintenance”. onto multiple chromosomes, bacteria usually have ology is essential. E.g., we and others have found that tion of DNA polymerase progression, re-methylation 2011– Professor of Synthetic In order to learn more about the biology of bacterial only one chromosome. An interesting exception to the secondary chromosome of V. cholerae starts to of the DNA by the Dam methyltransferase, and SeqA Microbiology, Philipps-Universität chromosome maintenance, we establish a synthetic this rule is Vibrio cholerae, the causative agent of the replicate later than the primary chromosome (Stokke binding and leaving the DNA. We want to uncover the Marburg secondary chromosome in Escherichia coli as experi- cholera disease. This bacterium carries a secondary et al, 2011), leading to a synchronized termination of causality and the details of this molecular treadmill- 2007–2011 Postdoctoral Fellow, mental system. chromosome (about 1 Mbps) in addition to its primary the replication of both chromosomes. Now we study ing mechanism using a combination of molecular bi- Norwegian Institute for Cancer Chromosome maintenance mechanisms usually con- chromosome (3 Mbps). Over the last years, this two- the mechanistic basis for this phenomenon using a ology, simulation and bioinformatics. Research, Oslo, Norway 2007 Ph.D. in molecular microbiology, sist of a DNA binding protein and the respective DNA chromosome system came into focus of scientists for wide range of molecular biology and bioinformatics Ruhr-University Bochum sequence motif (Messerschmidt & Waldminghaus, three reasons: First, targeting the unique replication approaches. 2004 M.C.Sc. in bioinformatics, 2015), and the distribution of such DNA motifs on of the secondary chromosome could open up possible University of Applied Science Berlin natural chromosomes is often crucial for the function routes to therapeutics development specific for Vibrio The role of SeqA in E. coli DNA replication 2002 Diploma in biology, Ernst- of the system. On a synthetic secondary chromosome, species. Second, the additional Vibrio-chromosome and chromosome segregation Moritz-Arndt-University Greifswald in contrast, the motif distribution can be assigned can be used as a template for synthetic secondary according to any design. By construction and charac- chromosomes in heterologous hosts (see above). One important player in chromosome maintenance is terization of such synthetic chromosome variants, we Third, the two chromosomes in V. cholerae present the SeqA, a fascinating protein with the unique ability to bind hemi-methylated GATC sites in a highly specific Figure 1. Construction of the synthetic sec- fashion (Waldminghaus and Skarstadt, 2009). Such ondary chromosome synVicII (yellow) in Es- cherichia coli based on the replication origin of hemi-methylation is characteristic for newly repli- Vibrio cholerae Chromosom II (red). The repli- cated DNA: While the old DNA strand is methylated, cation origin of the secondary Vibrio cholerae chromosome including neighboring genes en- the new strand will be un-methylated until the Dam coding the initiator protein and two segrega- methyltransferase adds the methyl-group at the N6 tion proteins was amplified by PCR. The DNA fragment together with other overlapping position of the adenine of the GATC sequence. Binding fragments of different origin was transformed of SeqA proteins to such hemi-methylated GATCs at Aa w rds & Honors in the yeast Saccharomyces cerevisiae. Homol- ogous recombination in yeast led to assembly the replication origin hinders immediate re-initiation of the overlapping fragments into a circular of DNA replication, an effect that is critical to guar- 2008 Ruth Massenberg Prize of replicon. Successful transformation to Escheri- Ruhr-University Bochum for out- chia coli showed the general functionality of antee proper timing of replication rounds. In addi- standing Ph.D. thesis the synthetic secondary chromosome synVicII. tion, SeqA follows the newly replicated DNA behind Figure from Messerschmidt and Waldming- 2008 Postdoctoral fellowship from the haus, BIOSpektrum 2015 with permission from the replication fork, potentially in a treadmilling-like German Research Foundation (DFG) Springer. fashion, with a SeqA structure growing at the leading 72 73

1.5 Research Microbial Communities 74 research 75

Drivers of microbial community structure in insect guts

Termite guts are tiny bioreactors that convert wood al, 2014). Comparative analysis documented strong and other lignocellulosic matter to microbial fermen- changes in the patterns of bacterial community struc- tation products, which then fuel the metabolism of ture that coincided with major events in termite evo- the host. Our research group studies the role of the lution (Fig. 1). gut microbiota in symbiotic digestion, focusing on Although certain bacterial lineages are represented the structure and function of the gut ecosystem, the among all termites of the same feeding guild, impli- biology of the prokaryotic and eukaryotic symbionts cating environmental factors as strong determinants Prof. Dr. Andreas Brune and their interactions, and the evolution of the intes- of community structure, the individual taxa typically Figure 2. The cockroach Shelfordella lateralis (A) as model for gnotobiotic studies. Germ-free juveniles are generated by surface sterilization of oothecae (B). After inoculation (C), tinal microbial community. Our studies with germ- cluster according to their respective host groups. juveniles are raised under axenic conditions (D). Panel (E) shows a germ-free juvenile two months after hatching. Microbial Ecology and Symbiosis free cockroaches are a novel approach to understand Evidence of true cospeciation, however, is scarce and Department of Biogeochemistry, fundamental mechanisms governing the assembly of rarely spans the entire host range. Rather, the coevo- Max Planck Institute for Terrestrial microbial communities and to assess the effects of lution of termite gut microbiota and host seems to encountered also in the guts of mammals, indicating activities in the gut environment. To our surprise, Selected publications Microbiology gut environment and organismic interactions on the be based on both the selective forces of microhabitat their general adaptation to the intestinal environ- both strict and facultative anaerobes successfully col- Brune A (2014). Symbiotic digestion Phone: +49 6421 178 701 activities of individual microbial populations in the and ecological niche and the faithful transmission of ment. onized the gut in high density if present alone. How- Email: [email protected] of lignocellulose in termite guts. digestive process. symbionts across host generations, which is facilitat- Elucidating the fundamental mechanisms of com- ever, when co-inoculated, the facultative anaerobe Nat Rev Microbiol 12, 168–80. SYNMIKRO member since 2010 ed by the social lifestyle of termites (Mikaelyan et al, munity assembly and succession in insect guts re- outcompeted the strict anaerobe, probably owing to The termite gut microbiota in preparation). quires an experimental approach. Since termites are a higher growth yield under the microoxic conditions Schauer C, Thompson CL, Brune A reflects host evolution obligately dependent on their intestinal symbionts, at the hindgut wall (Tegtmeier et al, in preparation). (2012). The bacterial community Germ-free cockroaches as a model we established the cockroach Shelfordella lateralis as Controlled inoculation with bacterial strains of natu- in the gut of the cockroach While the general role of the termite gut microbi- for community assembly a germ-free model to investigate the drivers of com- ral and foreign microbiota will also allow us to study Shelfordella lateralis reflects the ota in lignocellulose digestion is slowly emerging, munity structure and for gnotobiotic studies – i.e., potential host–microbe interactions, including a dif- close evolutionary relatedness of the factors shaping the structure of the intestinal A fundamental question in microbial ecology — not studies of hosts associated only with certain known ferential immune response of the host to colonization cockroaches and termites. Short CV community and the functions of many members only of intestinal ecosystems — concerns the mecha- microorganisms (Fig. 2). Germ-free juveniles of these by gut symbionts or potential pathogens and, recipro- Appl Environ Microbiol 78, 2758–67. are still mostly in the dark. When termites evolved nisms of community assembly. In contrast to the cockroaches develop poorly but can be rescued by in- cally, adaptations of the gut microbiota to the host’s Dietrich C, Köhler T, Brune A (2014). 2005– Honorary Professor, Philipps- from cockroaches more than 150 million years ago, distinct patterns observed in termites, the microbial fection with their natural gut microbiota (Tegtmeier et immune defense. Universität Marburg The cockroach origin of the termite the acquisition of cellulolytic flagellates as gut sym- communities in cockroach guts are quite similar, less al, in preparation). 2003– Research Group Leader, gut microbiota: patterns in bacterial bionts created new habitats for symbiotic bacteria, affected by diet, and highly variable among individu- Controlled inoculation of germ-free cockroaches with Max Planck Institute for Terrestrial community structure reflect Microbiology, Marburg which now colonize the surface and cytoplasm of als of the same species (Schauer et al, 2014; Bauer et termite microbiota confirmed that the gut microbial major evolutionary events. 2000 Habilitation, Universität these protists in all lower termites. These flagellates al, 2015). Many of the bacterial lineages common to community is indeed shaped by the host environ- Appl Environ Microbiol 80, 2261–9. Konstanz were lost again during the evolution of higher ter- both omnivorous and wood-feeding cockroaches are ment: Irrespective of the source of the foreign inocu- 1993–2003 Research Associate and mites, whereby wood par- lum, the bacteria colonizing the germ-free gut came Schauer C, Thompson CL, Brune A Group Leader, Universität Konstanz ticles became available from lineages that are abundant also in conventional (2014). Pyrotag sequencing of the 1991–1993 Visiting Research for bacterial colonization, cockroaches. However, the foreign strains were rapidly gut microbiota of the cockroach Associate, Michigan State providing niches for fiber- replaced by autochthonous microbiota upon exposure Shelfordella lateralis reveals a highly University, USA digesting species (Brune, to conventional cockroaches, indicating that host dynamic core but only limited effects of diet on community 1990–1991 Postdoctoral Fellow, 2014). specificity is caused by differences in competitiveness Eberhard-Karls-Universität Tübingen structure. PLoS One 9, e85861. In the first comprehensive of the bacteria in their particular habitat (Thompson 1990 Ph.D. in microbiology, study of a cockroach gut et al, in preparation). Universität Tübingen Bauer E, Lampert N, Mikaelyan A, 1986 Diploma in biology, Philipps- microbiota, we detected Köhler T, Maekawa K, Brune A (2015). Universität Marburg numerous bacterial line- Synthetic microbial communities Physicochemical conditions, me- ages that so far had been tabolites, and community structure found exclusively in ter- The complexity of the gut microecosystem with its of the bacterial microbiota in the mite guts, suggesting a metabolic networks and the multitude of possible in- gut of wood-feeding cockroaches common evolutionary ori- teractions among its biotic and abiotic components (Blaberidae: Panesthiinae). gin of the gut microbiota make it challenging to identify fundamental mecha- FEMS Microbiol Ecol, doi: 10.1093/ of termites and cockroach- nisms using conventional insects and their micro- femsec/fiu028 es (Schauer et al, 2012). We biota. Therefore, we chose the germ-free cockroach tested this hypothesis by model also to study metabolic activities of synthetic Aa w rds & Honors deep sequencing of the microbial communities under in situ conditions, intro- 16S rRNA genes of the ducing representative members of different microbial 2001 Research Award of the German Society for Hygiene and Microbiology bacterial communities in guilds that were isolated from insect guts. (DGHM) the hindgut of 34 termite We successfully inoculated germ-free cockroaches 1991–1993 Feodor Lynen Fellowship of and cockroach species, Figure 1. Phylogenetic patterns in the bacterial gut microbiota of termites and cockroaches. Com- with various strains of primary fermenters from the munity similarities are visualized by non-metric multidimensional scaling (NMDS). Colors indi- the Alexander von Humboldt Foun- representing the major cate clusters of samples from the same major host lineages; symbols indicate different feeding normal gut microbiota of S. lateralis and compared dation host groups (Dietrich et groups (Dietrich et al, 2014). their metabolic profiles in pure culture with their 76 research 77

Selected publications

Studying methane production with Brandt FB, Martinson GO, Pommerenke B, Pump J, Conrad R synthetic microbial communities (2015). Drying effects on archaeal community composition and methanogenesis in bromeliad Wetlands are an important source of the greenhouse tanks. FEMS Microbiol Ecol, in press. gas methane, but the mechanisms of formation and in particular the role of the microbial species involved Conrad R, Klose M, Noll M, Kemnitz D, are still not very well understood. For a long time, the Bodelier PLE (2008). Soil type links main approach for improving our knowledge has been microbial colonization of rice roots the rather descriptive analysis of structure and func- to methane emission. Global Change Biol 14, 657-69. tion of the microbial communities in these wetlands. Prof. Dr. Ralf Conrad Recently, however, we have started to manipulate Ke X, Angel R, Lu Y, Conrad R (2013). these systems in a synthetic approach in order to con- Methanogenic Degradation and Niche differentiation of ammonia firm, extend or refute the theories that have emerged Microbial Metabolism of Trace Gases oxidizers and nitrite oxidizers based on analytical studies. As model systems for this in rice paddy soil. Department of Biogeochemistry, manipulation, we have chosen rice roots and brome- Environ Microbiol 15, 2275-92. Max Planck Institute for Terrestrial liad tanks. Figure 3. Tank bromeliad with leaf rosettes forming a water-filled Microbiology tank. (Photo: Guntars Martinson) Martinson GO, Werner FA, Scherber C, Phone: +49 6421 178 801 Conrad R, Corre MD, Flessa H, Wolf K, Email: [email protected] Methanogenic archaea on rice roots Klose M, Gradstein SR, Veldkamp E the abundance of methanogenic archaea on the roots (2010). Methane emission from tank Rice cultivation is of central importance for feeding (Pump et al, 2014). We are presently testing whether SYNMIKRO member since 2010 bromeliads in neotropical forests. the human population, especially in Asia. Unfortu- the species composition of this methanogenic com- Nature Geoscience 3, 766-69. nately, flooded rice fields are also an important source munity is also of importance for CH4 emission. for the greenhouse gas methane, which is mainly In addition to methanogens, rice roots are also a suit- Pump J, Conrad R (2014). Rice produced on the roots of rice plants by the anaero- able habitat for other archaea, for example those that biomass production and carbon 13 bic degradation of photosynthesized organic mat- oxidize ammonia and thus control the cycling of ni- cycling in CO2 pulse-labeled Short CV ter (Fig. 1). Empirical evidence suggests, though, that trogen (Ke et al, 2013). Altogether, the microbial colo- microcosms with different soils under submerged conditions. 2006– Adjunct Professor at the the extent of methane formation may be dependent nization of roots plays a major role for the function Plant & Soil 384, 213-229. China Agricultural University, on the extent and speciation of root colonization by of the rice field ecosystem. We are presently studying Beijing, China methanogenic archaea (Conrad et al, 2008). In our to which extent this colonization is controlled by the Pump J, Pratscher J, Conrad R (2014). 1993– Adjunct Professor of SYNMIKRO project, we are testing this hypothesis by plant and/or by the soil microbiota. Colonization of rice roots with Microbiology, Philipps-Universität manipulating the colonization of the rice roots in rice methanogenic archaea controls Marburg microcosms. The plants are then pulse labeled with Microbial communities photosynthesis-derived 1991– Director and Head of the 13CO , and the amount of methane emitted by the methane emission. Department of Biogeochemistry, 2 in tank bromeliads microcosms is measured (Fig. 2). Furthermore, the mi- Environ Microbiol Reports, doi: Max Planck Institute for Terrestrial crobial colonization of the rice roots is assessed using T ank bromeliads are common epiphytes in neotropi- 10.1111/1462-2920.12675 Microbiology, Marburg 1986–1990 Professor of Microbiology, molecular techniques that target both the composi- cal forests and early successional vegetation. Their University of Konstanz tion of microbial communities and the abundance of leave rosettes form a tank, in which not only rainwa- 1978–1985 Postdoctoral Fellow, Max different microbial species in these communities.O ur ter, but also all kinds of detritus accumulate (Fig. 3). Planck Institute for Chemistry, Mainz studies demonstrated close ties between plant physi- These small wetland systems constitute another sig- 1976 Ph.D. in microbiology, Georg- ology, soil properties and microbial turnover of car- nificant source in the methane budget of the atmo- August-Universität Göttingen bon (Pump & Conrad, 2014). They further showed that sphere (Martinson et al, 2010). The bromeliad plants 1973 Diploma in biology, Georg- the rate of methane formation was indeed related to provide themselves with water and nutrients from the August-Universität Göttingen tank, which actually contains a di- verse community of methanogenic archaea, analogously to rice roots, but of different composition and structure. The manipulation of the Aa w rds & Honors content of the bromeliad tanks can easily be done. Therefore, we decid- 2010 Einstein Distinguished Chair ed to establish tank bromeliads as a Professorship, Chinese Academy of second model system for studying Sciences the interaction between plant and Figure 2. Device for pulse-labeling of rice microcosms with 13CO and measuring the flux of CO and CH between the microcosms and 2005 Fellow of the American Geo- 2 2 4 microbial community. For example, the atmosphere. (Photo: Judith Pump) physical Union 2003 Francis Clark Distinguished we recently studied the effect of Lectureship, Soil Science Society of desiccation on the tank microbial America community by manipulating the 1997 Division N Lecture, American water content in the tanks (Brandt Figure 1. Rice field in the Philippines with flux chambers for measuring 4CH emission. Society for Microbiology (Photo: Ralf Conrad) et al, 2015). 78 research 79

exhibited a reverse expression (Fig. 3). Greatest re- Selected publications sponse to increasing ammonium concentrations Methanotrophic bacteria and their Baani M, Liesack W (2008). Two showed two genes that encode hypothetical proteins isozymes of particulate methane (log fold changes of 6.1 to 9.3). 2 monooxygenase with different response to environmental changes While the expression of pmoCAB1 was unaffected, methane oxidation kinetics are pmoCAB2 was significantly downregulated (log2 found in Methylocystis sp. strain SC2. fold changes of -5.0 to -6.0) in response to increas- Proc Natl Acad Sci USA 105, 10203-8. ing ammonium concentrations. Among nitrogen metabolism-related processes, genes encoding hy- Dam B, Dam S, Kube M, Reinhardt R, droxylamine oxidoreductase (haoAB) were highly up- Liesack W (2012a). Complete genome regulated, while those for assimilatory nitrate/nitrite sequence of Methylocystis sp. strain reduction, high-affinity ammonium uptake, and nitro- SC2, an aerobic methanotroph with high-affinity methane oxidation Figure 2. Differential expression of strain SC2 genes in response gen regulatory protein PII were downregulated. to different nitrogen conditions. The histogram indicates the potential. J Bacteriol 21, 6008-9. PD Dr. Werner Liesack change in expression levels for the complete set of 4,058 genes Ammonia competitively inhibits pMMO, which in turn identified in the genome of strain SC2. High-quality non-rRNA leads to the production of hydroxylamine, a highly Methanotrophic Bacteria reads were mapped against the concatenated sequence of the Dam B, Kube M, Dam S, Reinhardt R, chromosome and the two plasmids of strain SC2. RNA-Seq ex- toxic molecule that must be transformed or removed Liesack W (2012b). Methanotroph- Department of Biogeochemistry, pression data were presented as RPKM (Reads Per Kilobase of quickly to prevent cellular damage. Competitive inhi- specific repABC-containing plasmids CDS model per Million mapped reads) values. Log2 fold changes Max Planck Institute for Terrestrial of RPKM values were compared for NMS/AMS (green), NMS/NH4 bition and, in consequence, hydroxylamine poisoning from Methylocystis sp. strain SC2. Microbiology (red), and AMS/NH4 (blue). The inset shows the same graph with would be of ecophysiological relevance particularly Appl Environ Microbiol 78, 4373-9. Phone: +49 6421 178 720 the y-axis zoomed in on the range from 0 to 10. Figure was taken from Dam et al, 2014. in low-methane environments, where only pMMO2, Email: liesack@mpi-marburg. but not pMMO1, is functional. This may be the rea- Dam B, Dam S, Blom J, Liesack W mpg.de (2013). Genome analysis coupled pmoCAB2 was validated, and all other genes required son why the expression of pmoCAB2, but not that of with physiological studies reveals for a methanotrophic lifestyle were identified. The pmoCAB1, is affected by ammonia.T he upregulation SYNMIKRO member since 2010 a diverse nitrogen metabolism in absence of genes encoding the soluble methane mo- of haoAB expression with increasing ammonium con- Methylocystis sp. strain SC2. nooxygenase was confirmed. Furthermore, a large rep- centration also fits well into such an ecophysiological PLoS ONE 8, e74767. ertoire of genes involved in nitrogen metabolism was perspective, assuming that the primary role of HAO detected (Dam et al, 2012a,b). is to detoxify hydroxylamine that has been produced Dam B, Dam S, Kim Y, Liesack W To verify that strain SC2 has diverse nitrogen metabo- by the pMMO isozymes. Methylocystis is widely dis- (2014). Ammonium induces differ- lism capabilities, we performed several physiological tributed in upland soils. The differential expression of ential expression of methane and experiments. For example, strain SC2 was found to pmoCAB2 thus provides some explanation as to why nitrogen metabolism-related genes grow with atmospheric N as the sole nitrogen source, ammonium fertilizers have a strong inhibitory effect in Methylocystis sp. strain SC2. 2 Environ Microbiol 16, 3115-27. preferably at low oxygen concentrations. Denitrifi- on atmospheric methane oxidation in such soils. 30 cation-mediated accumulation of 0.7 nmol N2 /hr/ Methylocystis sp. strain SC2 is one of the very few mg dry weight of cells under anoxic conditions was methanotrophs for which a finished genome se-

detected by tracer analysis. N2 production was re- quence exists that enables whole-genome expression lated to the activities of plasmid-borne nitric oxide studies. While the above study already provided im- Figure 1. Whole-genome plot of Methylocystis sp. strain SC2. The circles represent from outside to inside: circle 1, DNA base position (bp), base 1 to 3,773,444 are for the chromosome, followed by plasmids pBSC2-1 and pBSC2-2; circle 2, protein-coding regions transcribed and nitrous oxide reductases. The presence of a com- portant insights into the response of strain SC2 to dif- on the plus strand (clockwise); circle 3, protein-coding regions transcribed on the minus strand (anticlockwise); circle 4, tRNA genes; plete denitrification pathway in strain SC2, including ferent nitrogen sources and concentrations, we want circle 5, G+C content plotted using a 10-kb window (sea-green and magenta indicate values greater than and less than the average G+C content, respectively); circle 6, GC skew ([G+C]/[G-C]) plotted using a 10-kb window (blue indicates values above average and red indi- the plasmid-encoded nosRZDFYX operon, is unique to further improve our understanding of the genome- cates values below average). The whole-genome plot was generated using DNAPlotter version 1.4 from Artemis 12.0, Sanger Institute. among known methanotrophs. The exact ecophysio- wide transcriptome response of strain SC2 to environ- logical role of this pathway still needs to be elucidated mental change in the future. Another line of research Methane-oxidizing bacteria, so-called methano- locystis sp. strain SC2 as a model system. This strain (Dam et al, 2013). is the development of reverse genetic tools for func- Short CV trophs, are aerobic microorganisms that have the can adapt to a wide range of methane concentrations tional analysis of candidate genes in strain SC2 and unique ability to grow on methane as their only source due to its ability to produce two pMMO isozymes with Differential expression of methane and other methanotrophic bacteria. 2011– Research Group Leader, Max of carbon and energy. Their key enzyme, particulate different methane oxidation kinetics: The pmoCAB1 Planck Institute for Terrestrial nitrogen metabolism-related genes Microbiology, Marburg methane monooxygenase (pMMO), converts methane genes encode the well-known type of pMMO (pMMO1) 1997–Privatdozent, Philipps- to methanol and thereby attenuates methane emis- and are expressed only at methane mixing ratios >600 We used Illumina RNA-Seq to identify strain SC2 genes Universität Marburg sions from major sources, such as wetlands, rice pad- parts per million by volume (ppmv). The pmoCAB2 that respond to low (10 mM, AMS) and high (30 mM, + 1997–2011 Group Leader, Max Planck dies, and landfills, and constitutes the only biological genes encode pMMO2 that oxidizes methane at lower NH4) NH4 concentrations in the growth medium, com- - Institute for Terrestrial Microbiology, sink for atmospheric methane in upland soils. Metha- mixing ratios, even at the trace atmospheric mixing pared to 10 mM NO3 (NMS). Strain SC2 cells were incu- Marburg notrophs are therefore crucial players in the global ratio of 1.8 ppmv (Baani & Liesack, 2008). bated under high methane concentrations (20%, v/v) 1992–1997 Research Associate, Max cycle of the greenhouse gas methane; nevertheless, in the different nitrogen treatments (Dam et al, 2014). Planck Institute for Terrestrial their molecular biology is poorly understood. In addi- A unique denitrification pathway The majority of the strain SC2 genes showed no sig- Microbiology, Marburg tion to methane availability, nitrogen source and con- among methanotrophs nificant changes in the expression level. Based on log2 1996 Habilitation, Philipps- centration are major determinants of methanotrophic fold changes of ≥ 2 or ≤ -2, a total of only 198 genes Universität Marburg activity. In particular, ammonia is known to act both T o provide a basis for further research into the molec- were identified as differentially expressed in NMS/AMS 1990 Research Fellow, University of Queensland, Australia as a nutrient for growth and as a competitive inhibitor ular biology of strain SC2, we sequenced its genome and NMS/NH4 (Fig. 2). A set of 56 genes was differen- Figure 3. Correlation between the log2 fold changes of genes 1988 Ph.D. in microbiology, of pMMO. and found it to comprise a 3.77 Mb circular chromo- tially expressed in response to both treatments. Their that were differentially expressed in response to both standard + University of Kiel Our research aims to improve our understanding of some and two large plasmids of 229.6 kb (pBSC2-1) expression levels in AMS and NH4 showed a strong (10 mM) and high (30 mM) NH4 concentrations. Comparison was made between NMS/AMS and NMS/NH4. Genes that showed log2 1984 Diploma in biology, how methanotrophs respond at the molecular and and 143.5 kb (pBSC2-2) (Fig. 1). The presence of two linear correlation. Fifty-two genes were either up- fold changes of ≥ 2 or ≤ −2 in both treatments were included in University of Kiel cellular levels to environmental change, using Methy- nearly identical copies of pmoCAB1 and one copy of or downregulated in a similar way, while four genes the comparison. Figure was taken from Dam et al, 2014. 80 research 81

Aa w rds & Honors

C onstruction of a Methanosarcina 2013 Medal of Merit of the German Academy of Sciences Leopoldina strain that can grow on glucose 2008 Carl Friedrich Gauß Medal of the „Braunschweigische Wissen- schaftliche Gesellschaft“ In anoxic environments, cellulose, a polymer of glu- required for glucose import (glucose transport facili- 2001 Honorary doctorate of the ETH

cose, is fermented to CO2 and methane via a syn- tator glf from Zymomonas mobilis) and glucose acti- Zürich, Switzerland trophic association of anaerobic bacteria, protozoa vation to glucose-6-phosphate (glucokinase glk from 1987 Leibniz Prize of the German Research Foundation (DFG) and fungi that ferment cellulose to acetic acid, CO2 Escherichia coli) into Methanosarcina acetivorans, a 1984 Otto Warburg Medal of the and H2, and of methanogenic archaea that convert methanogen lacking only these genes for methano- „Gesellschaft fur Biologische acetic acid, CO2 and H2 to methane (see also: Thauer genesis from glucose. Indeed we found that cell sus- RK, Kaster A, Seedorf H, Buckel W & Hedderich R pensions of recombinant M. acetivorans are capable Chemie“ (GBM) Prof. Dr. Dr. hc. mult. (2008). Methanogenic archaea: ecologically relevant of methanogenesis from glucose, albeit presently at Figure 2. Methanogenesis from glucose (0.2 mM) in cell suspen- Rudolf Thauer differences in energy conservation. Nat Rev Microbial only very low specific rates F( ig. 2). sions of recombinant Methanosarcina acetivorans with the gene 6, 579-91) (Fig. 1). There is no organism known that Next, the rate limiting steps in this fermentation will glk for glucokinase from Escherichia coli and the gene glf for the Microbiology and Biochemistry glucose transport facilitator from Zymomonas mobilis. can ferment glucose to 3 CO2 and 3 CH4 on its own, have to be identified and to be overcome. One of the Max Planck Institute for Terrestrial although the free energy change associated with such problems already encountered is that, within the re- Microbiology an overall fermentation would allow for the largest combinant strain, glucose-6-phosphate accumulates which may well be the case, they will have to be char- Phone: +49 6421 178 100 ATP-gain (mol ATP per mol substrate). to toxic concentrations when glucose is present in acterized on the genome and metabolome level via Email: [email protected] A kinetic theory relating growth rates to the length the medium. Another consequence of the apparently single cell techniques, some of which are still under SYNMIKRO since 2010 of metabolic pathways and the number of coupling too rapid glucose phosphorylation is the fact that the development. sites can explain these finding for energy substrate- ATP concentration, the phosphorylation potential and From 2013 on, Michael Rother and Christian Sat- limited planktonic cells (see also: Pfeiffer T, Schuster energy charge drop to values below those required to tler, the Ph.D. student involved, have continued the S & Bonhoeffer S (2001).C ooperation and Competition sustain growth. project alone at the Technical University of Dresden, in the Evolution of ATP-Producing Pathways. Science In the future, nano-SIMS (secondary ion mass spec- where they moved end of 2011. 292, 504-7). The same theory predicts, however, that trometry) ion imaging will be used to screen biofilms

methanogens capable of fermenting glucose to CO2 for methanogens that can metabolize glucose or oth- and methane should exist in biofilms.I n collaboration er easy fermentable substrates. Once such methano- Selected publications with Prof. Dr. Michael Rother, University Frankfurt, we gens have been identified, efforts will have to be made set out to test this hypothesis. We cloned the genes to isolate and cultivate them. If this is not possible, Thauer RK, Kaster A, Goenrich M, Schick M, Hiromoto T, Shima S (2010). Hydrogenases from metha- nogenic archaea: structure and function, nickel regulation,

and H2- storage. Ann Rev Biochemistry 79, 507-36.

Short CV Scheller S, Goenrich M, Boecher R, Thauer RK, Jaun J (2010). The key 2007– Professor Emeritus, Max nickel enzyme of methanogenesis Planck Institute for Terrestrial catalyses anaerobic oxidation of Microbiology, Marburg methane. Nature 465, 606-9. 1991–2007 Director and Head of the Department of Biochemistry, Max Kaster AK, Moll J, Parey K, Thauer RK Planck Institute for Terrestrial (2011). Coupling of ferredoxin- and Microbiology, Marburg heterodisulfide reduction via 1976–2005 Professor of Microbiol- electron bifurcation in hydrogeno- ogy, Philipps-Universität Marburg trophic methanogenic archaea. 1999 Visiting Professor, Stanford Proc Natl Acad Sci USA 108, 2981-6. University, CA, USA 1991 Visiting Professor, University Shima S, Krueger M, Weinert T, Frankfurt Demmer U, Kahnt J, Thauer RK, 1972–1976 Professor of Biochemistry, Ermler U (2012). Crystal structure of Ruhr-University Bochum a methyl-coenzyme M reductase 1972 Postdoctoral Fellow, Case West- from Black Sea mats that oxidize ern Reserve University, Cleveland, anaerobically methane. Ohio, USA Nature 481, 98-101. 1971 Habilitation, University of Freiburg Huang H, Wang S, Moll J, Thauer RK 1968 Ph.D. in biochemistry, (2012). Electron bifurcation involved University of Freiburg in the energy metabolism of the Figure 1. The global microbial methane cycle. More than 70% of the net primary production (NPP) of plants are lignocelluloses, of which 1966 Diploma in biochemistry, acetogen bacterium Moorella ther- about 2% end up each year in anoxic environments where the cellulose is fermented to CH4 and CO2 via a syntrophic association of University of Tübingen anaerobic bacteria, protozoa and fungi with methanogenic archaea. moacetica. J Bacteriol 194, 3689-99. 82 83

1.6 Research Bioethics 84 research 85

cell’s behavior - and no influence on the environment, Eckhardt, Prof. Dr. Dr. hc. Regine Kahmann, and Prof. Selected publications since the experiments “only” serve fundamental re- Dr. Friedemann Voigt. The publication of the lectures Ethical assessment of Hacker J, RendtorffT , Cramer P, Hallek search, and the modified cells are not released. If the is scheduled for 2015 (Voigt (Ed.), 2015). M, Hilpert K, Kupatt C, Lohse M, cell’s own chromosome should eventually be fully re- Müller A, Schroth U, Voigt F, Zichy M. synthetic biology placed, the project must be re-assessed. Metaphors in the debate (2009). Biomedizi­nische Eingriffe on synthetic biology am Menschen. Ein Stufenmodell zur The Bioethics group deals with the ethical aspects, both those responsible for and those affected by the Public discussions on synthetic biology ethischen Bewertung von Gen- und normative uncertainness and public questions re- research can be identified and addressed. On the basis The ethical debate on synthetic biology is character- Zellthera­pie, De Gruyter, Berlin. garding synthetic biology. Based on a consequential- of this description, the second step develops criteria In collaboration with SYNMIKRO and the Graduate ized by metaphors such as “engineering life”, “living ist approach, these ethical conflicts must be confront- for the assessment of current research projects in syn- Center for Life and Natural Sciences of MARA (Marburg machines”, “playing god”, etc. These metaphors il- Voigt, F (Ed.) (2012). Freiheit der ed with research through differentiated identification, thetic biology. These criteria must be realigned with University Research Academy), the Bioethics Group lustrate how synthetic biology touches culturally and Wissenschaft. Beiträge zu ihrer Bedeu- description and classification. Our goal is to develop a each case and are presented in three dynamic dimen- organized a series of public events called “Synthe- normatively charged distinctions of living and not tung, Normativität und Funktion, De model for the ethical assessment of synthetic biology sions of assessment: The dimension identifiability and tische Biologie im Dialog” in 2013/14 (see also p. 108). living matter, and thereby can cause irritation and Gruyter, Berlin/New York. Prof. Dr. that gives a realistic description of the actual state of complexity of the research object, the dimension range The purpose of these events was to illustrate the dif- discomfort. The dissertation project of Daniel Falkner Voigt, F (2013). Komplexität. Zukunft Friedemann Voigt research and grades it into different stages from ethi- and reversibility of consequences, and the dimension ferent approaches, assumptions and beliefs in the cur- seeks to critically analyze the ethical dimensions of der biomedizinischen Wissenschaften cally unproblematic via more problematic and riskier ethical meaning and scientific purpose of research. rent debates about modern life sciences. Based on the metaphors in debates on modern life sciences and Bioethics (ed. by Trutz Rendtorff), 63-5. procedures up to ideas that are entirely speculative The model concludes the ethical assessment through central terms and concepts “life”, “complexity” (Voigt, new techno-scientific areas, for which synthetic biol- Faculty of Protestant Theology, and ethically unjustifiable. This ethical assessment a third step of classifying the particular research in 2013) and “natural/synthetic”, the different perspec- ogy is a prime example. Voigt, F (Ed.) (2015). Synthetische Philipps-Universität Marburg model is developed in close collaboration with all re- five stages of ethical accountability. The starting point tives were reflected and presented in short lectures As exemplified by the metaphor of the “genetic code”, Biologie im Dialog, Karl Alber (Series: Phone: +49 6421 28 24277 search areas of SYNMIKRO. Through interdisciplinary is stage 1, the ethically unproblematic and widely ac- and subsequent panel discussions with experts of this approach leads to a reassessment of synthetic „Lebenswissenschaften im Dialog“, Email: [email protected] events and publications, the Bioethics Group also ini- cepted case. From this basis, every other stage will be both natural sciences and humanities. Speakers were biology as a science between artificial life and living edited by Kristian Köchy and Stefan marburg.de tiates a dialogue with external experts both from life developed, which allows a classification of concrete Prof. Dr. Nedilijko Budisa, Prof. Dr. Dr. hc. mult. Jörg art, and to a new ethical dimension which is in the Majetschak), Freiburg i.Br., in press. SYNMIKRO member since 2011 sciences and human sciences, and addresses a wider application cases by their ethical problem sustain- Hacker, Prof. Dr. Gerald Hartung, Prof. Dr. Dr. Kristian fundamental relationship of responsibility and trust public. ability and with regard to the expectable uses and the Köchy, Prof. Dr. Klaus Mainzer, and Prof. Dr. Sven Panke. between science and society (Voigt, 2012). The ethi- Falkner, D (2014). Was ist Leben – eine ethische Frage? TTN Edition probable risks. Research classified on stage 2 is ethi- Hosts were Prof. Dr. Michael Bölker, Prof. Dr. Bruno cal question then is no longer: „What is life?“, but: 3/2014, München (in press). F rom describing to classifying: cally responsible but, due to increasing uncertainty „How do we want to live a stage model factors, there is an increasing need for ethical reflec- together and what role tion. Stage 3 describes research that is ethical prob- should synthetic biology With our “stage model for the ethical assessment of lematic, but can be justified under certain conditions play in our social life?” synthetic biology”, a proposal is made to objectify and and requirements which must be precisely formulat- (Falkner, 2014). A part of de-escalate the debate on ethical risks and changes of ed. Stage 4 includes applications that are not respon- the answer probably lies synthetic biology (Cf.: Hacker et al, 2009). The model sible at the moment. On stage 5 are extreme positions in the metaphors we use includes the methodological steps describing, as- and absurd scenarios that are obviously not subject to talk about life and sessing, and classifying (Fig. 1), and thereby provides of a realistic ethical assessment that remains close to synthetic biology. a dynamic and multidimensional criteriology that the actual state of research. corresponds to the complexity and distinctiveness of In reality, the transitions between the stages become synthetic biological research. In the first step, levels of blurred. The demarcation necessary for the assess- descriptions offer a differentiated classification of the ment between ethically favorable and acceptable re- research field of synthetic biology, determine domains search and not anymore justifiable experiments runs of possible actions and ethically relevant consequenc- between steps 3 and 4. It should be noted, however, es, and present levels of ethical responsibility, to which that new findings and developments in science can lead to modifications in the criteriological assess- ment. The ethical evalu- ation must be aware of these dynamics and tran- sitions. For example, in the research project of synthetic chromosomes of the group of Torsten Short CV Waldminghaus, there is a strong intervention and a 2011– Professor of Social Ethics, high grade of complexity Philipps-Universität Marburg at the molecular level, due 2006–2011 Head of the BMBF Junior to its relatively new, con- Research Group “Religion in bioe- structed character. Howev- thischen Diskursen”, LMU München 2006 Habilitation, LMU München er, introduced into the cell 1996 Doctoral degree in protestant in addition to the natural theology, LMU München chromosome, the synthet- 1992 Exam in protestant theology, ic chromosome has only University of Frankfurt/Main Figure 1. The methodological steps of the stage model: describing, assessing, and classifiyng. a small influence on the 86 87

2u infrastr cture 88 infru astr cture 89

Dr. Javier Serrania

Scientific Manager

Phone: +49 6421 28 24452 Email: [email protected] marburg.de

Short CV

2012– Scientific Manager, SYNMIKRO Laboratory Automation Unit 2008–2011 Research Scientist, University of Freiburg 2008 Ph.D. in molecular genetics, Bielefeld University 2005 Diploma in molecular biotech- nology, Bielefeld University

The Tecan Freedom Evo automation platform with its numerous integrated laboratory devices, such as PCR machine, multimode reader, sealer and centrifuge. Di pl. Ing. Patrick Manz

Technical Manager

Phone: +49 6421 28 22001 Laboratory Automation Unit Email: patrick.manz@synmikro. uni-marburg.de In synthetic biology, high-throughput screens and tory Automation Unit at its Department of Microbial forms integrating various liquid handling and colony combinatorial experiments are powerful strategies to Comparative Genomics which has been operational picking options with laboratory devices, such as incu- Short CV support the design–build–test engineering process since summer 2013. The unit provides access to this bator, shaker, centrifuge, plate sealer, PCR machine, of gene circuits and biosynthetic pathways. How- technology to the members of the center and is a multimode reader, and fluorescence microscope. 2014 - Technical Manager, SYNMIKRO ever, manual execution of such experiments is error- competent partner both for the design and the execu- Laboratory Automation Unit prone and often not feasible because of the required tion of automated experiments. In addition, the unit’s Applications 2006–2014 Project Manager, throughput. Only recently, advances in computer- mission is the development and application of novel Tecan, Switzerland aided design and laboratory automation technologies experimental methods and processes in the field. The portfolio of applications comprises 2004–2006 System and Application enable such innovative and highly complex experi- • high-throughput screens as well as combinatorial Engineer, Tecan, South Africa 1998–2004 Various positions ments: Instead of building and testing of a handful Equipment constructions and performance tests of biological at Tecan, Germany of genetic constructs manually, hundreds to thou- parts and modules including DNA assembly 1997 Diploma in biomedical sands of constructs can now be generated and char- The unit operates state-of-the-art laboratory automa- • automated microscopy of microorganisms includ- engineering , University of Applied acterized in an automated way. To tap the potential tion platforms. Its current infrastructure is based on ing screening of prokaryotic cells for morphology Sciences Gießen of these approaches, SYNMIKRO established a Labora- one Tecan Genesis and three Tecan Freedom EVO plat- phenotypes and subcellular protein localization 90 infru astr cture 91

Coo-w rker: Flow Cytometry and Protein Silvia Gonzales Sierra Technical Manager, Flow Cytometry

Diffusion & Interactions Unit Phone: +49 6421 28 21493 Email: silvia.gonzalessierra@ Fluorescently-tagged proteins are ideal reporters for Flow cytometry is a laser-based technology that al- synmikro.mpi-marburg.mpg.de studying the physiological status of living cells. For lows the simultaneous measurement of fluorescence cells synthetically endowed with new modules or in single cells at different wavelengths reporting the Short CV circuits, measuring their properties via relevant fluo- cellular concentration of several fluorescently-tagged rescent reporters helps to characterize the effects of molecules, and of other physical or biological param- 2014- Technical Manager, Flow Cytometry these modifications and to optimize the synthetic eters such as cell size or granularity in a statistically 2010-2014 Technical Manager, constructs. Consequently, SYNMIKRO invested in robust fashion. In our instrument, up to thirteen Microscopy Laboratory, National Dr. Gabriele Malengo several devices with different key aspects regarding fluorescence channels can be monitored in parallel Research Center for Human the analysis of fluorescently-tagged molecules for its for every single cell, in thousands of cells per second. Scientific Manager Evolution, Burgos, Spain Flow Cytometry and Protein Diffusion & Interactions Typical applications include single-cell measure- 2009-2010 Technical Manager, Phone: +49 6421 28 21487 Unit launched in October 2014. ments of gene expression, analysis of transcriptional Scanning Electron Microscopy Labo- Email: gabriele.malengo@synmikro. reporters, and cell cycle studies in ratory, Institute of Nanoscience, mpi-marburg.mpg.de prokaryotic and eukaryotic micro- Technical Manager Silvia Gonzales Sierra operating the BD Fortessa Flow Cytometer. University of Zaragoza, Spain organisms. 2004-2009 Specialist Technician, Short CV Protein diffusion plays a crucial role in determining • Fluorescence lifetime imaging microscopy Microscopy and FACS Facility, University of León, Spain 2014– Scientific Manager, SYNMIKRO Imaging-based fluorescence exper- what function a protein serves within the cell and • Fluorescence correlation and cross-correlation spec- 2003-2004 M.Sc. in biomedicine, Flow Cytometry and Protein Diffu- iments, on the other hand, allow how, when and where it may physically interact with troscopy for measurement of protein diffusion and University of León, Spain sion & Interactions Unit us to get more insights into living other proteins and macromolecules in response to ex- protein-protein interactions 2003 Diploma in biology, 2008–2014 Postdoctoral Fellow, cells, e.g., by measuring protein ternal stimuli. Protein diffusion in living cells is effec- • Confocal imaging University of León, Spain Zentrum für Molekulare Biologie, properties such as interactions or tively measured by fluorescence correlation spectros- • Measurements of single-cell gene expression in po- University of Heidelberg diffusion. As the design and func- copy (FCS), a fluctuation-based approach that allows pulations, including analysis of gene expression noise 2008 Ph.D. in cellular and molecular tion of signaling networks de- to carry out measurements at a physiological protein • Measurements of dose-dependent activation of biology, The Open University, Milton pends on protein-protein interac- expression level. transcriptional reporters in multi-well plates Keynes, UK and University Vita- tions, which in turn are affected by • Cell cycle studies in prokaryotic and eukaryotic Salute San Raffaele, Milan,I taly 2004 Research Associate, University protein diffusion, resolving such Equipment microorganisms of Milano Bicocca, Italy Autocorrelation functions of proteins of different molecular weight diffusing in the cyto- properties in space and time helps plasm of Escherichia coli cells obtained from fluorescence correlation spectroscopy data. to understand signaling up to sys- The Flow Cytometry and Protein Diffusion & Inter- 2003–2004 Visiting Scientist, ISS The arrows in the inset pictures points to the cell volume were the measurements were inc., Champaign, IL, USA, and Labo- acquired. In black: normalized autocorrelation function of CFP-CheR-eYFP. In blue: nor- tem-level properties such as signal actions Unit is part of the Max Planck Department ratory for Fluorescence Dynamics, malized autocorrelation function of eYFP. integration and amplification. of Systems and Synthetic Microbiology, which is UIUC, Urbana, IL, USA equipped with several fluorescence microscopes dedi- 2003 M.Sc. in physics, University of cated to automatic imaging and FRET applications. Milan, Italy The unit helps with the design and execution of pro- tein interactions and protein diffusion experiments. The measurements are run on a Nikon widefield fluo- Coo-w rker: rescence microscope customized for automatic ac- Dr. christelle rosazza ceptor photobleaching and ratiometric FRET measure- Technical Manager, ments, and on a Zeiss LSM880 confocal microscope Protein Diffusion & Interactions dedicated to fluorescence correlation spectroscopy and fluorescence lifetime imaging. Phone: +49 6421 28 21487 Protein-protein interactions in liv- The flow cytometry measurements are carried out on Email: christelle.rosazza@synmikro. ing cells are monitored in a quan- a customized BD Fortessa Flow Cytometer with 4 laser mpi-marburg.mpg.de titative time- and space-resolved lines (445, 488, 514 and 561 nm) and 13 fluorescence fashion by the microscopy-based Short CV channels. Laser lines were selected in order to maxi- Förster Resonance Energy Trans- mize the fluorescence excitation of fluorescence pro- 2014- Postdoctoral Fellow and fer (FRET) approach. This method teins such as CFP, GFP, YFP and mCherry. The cytometer Technical Manager, relies on the efficiency of energy is endowed with a high throughput sampler that al- Protein Diffusion & Interactions transfer between different fluores- lows to perform automatic measurements of 96-well 2014 Ph.D. in biology/chemistry, cently-tagged proteins as an indi- plates in only 15 minutes. University of Konstanz, and Insti- cator for protein proximity. Several tute of Pharmacology and Structural technologies are available in our Biology (IPBS), Toulouse, France Applications 2008 M.Sc. in pharmacological inno- unit for measuring FRET, ranging vation, University of Toulouse, France from the robust and fast acceptor • Automatic screening of protein-protein interactions 2007 Engineering degree in biochem- Examples of flow cytometry measurements for Saccharomyces cerevisiae strains contain- photobleaching FRET approach to by population-based acceptor photobleaching FRET ing a GFP reporter stimulated by different concentration of pheromone. (A) Density scat- the highly informative fluores- Ratiometric FRET measurements acquired on Escherichia coli cells expressing CheY-eYFP and CheZ-eCFP fluores- istry, National Institute of Applied ter plots showing GFP intensity versus forward scatter (proportional to cell size) for the • Ratiometric and acceptor photobleaching single- cent fusions. Plots show data analysis at the population and single cell level reporting chemotaxis response to Sciences (INSA), Toulouse, France wild type. (B) GFP distributions in wild type and two different mutants. cence lifetime imaging approach. cell FRET addition and subsequent removal of Methyl-Aspartate. 92 IunfRastr cture 93

Super Resolution Microscopy Unit The accurate spatio-temporal visualization of cellular components is of great importance for the under- standing of natural cells, which in turn is indispen- sable for the design and construction of synthetic modules, circuits or entire synthetic cells. Moreover, the ability to visualize synthetic constructs and their effects allows us to test our models and thus brings us closer to a true understanding of the inner work- ings of biological systems. Modern fluorescence mi- croscopy is the tool that facilitates all of these tasks: Dr. Barbara Waidner It allows the visualization of several types of proteins in parallel, e.g., in the membrane, next to the cell wall Scientific Manager and in the cytoplasm. Furthermore, the bacterial cell Phone: +49 6421 28 22212 wall itself, the membrane and the chromosome can Email: barbara.waidner@synmikro. also be visualized simultaneously – all of this both in uni-marburg.de live or in fixed cells, with striking spatial resolution and even in a time-dependent manner.

Dynamics of protein structures. Overlay of a bright field image of a single Bacillus subtilis cell with the first frame of fluorescence (green dots) of a YFP tagged protein and of trajectories, each hav- ing a different color, displayed over a time course of 8 seconds.

On these grounds, SYNMIKRO invested in several state-of-the-art fluorescence microscopes for its Su- per Resolution Microscopy Unit located at the Depart- ment of Biochemistry and Cell Biology of Microorgan- isms. The unit comprises several types of fluorescent microscopes with different features. The most ad- vanced techniques are stimulated emission depletion (STED) and structured illumination (SIM) super resolu- tion microscopy, achieving a resolution of 50 and 125 nm, respectively – several fold lower than the diffrac- Short CV tion limit of conventional light microscopy of about The stimulated emission depletion super resolution scanning confocal microscope (Leica, TCS SP8 STED). 2012– Scientific Manager, SYNMIKRO 250 nm. Thus, using super resolution microscopy, Super Resolution Microscopy Unit, more details become visible in cells, whose protein and head of the Helicobacter molecules are in the range of 5 to 10 nm, and whose subgroup at the Department of protein complexes in the range of 15 to about 100 nm. Applications Biochemistry and Cell Biology of Microorganisms Equipment The portfolio of applications comprises 2011 Interim Professor of Microbiol- • Tracking of single molecules in millisecond time ogy, University of Freiburg F or super resolution microscopy, a STED super reso- scales (SMM) 2007–2010 Independent Junior Re- lution scanning confocal microscope (Leica, TCS SP8 • Total internal reflection (TIRF) microscopy search Group Leader funded by the STED) and a SIM/PALM (Zeiss, Elyra) microscope are • STED (stimulated emission depletion DFG, University Hospital Freiburg available. The current infrastructure of the unit fur- microscopy) 2004–2006 Postdoctoral Fellow, University Hospital Freiburg ther comprises several epifluorescence microscopes, • Spinning disk confocal microscopy 2000–2003 Ph.D. in biology/microbi- some of which are modified for single molecule mi- • SIM (structured illumination microscopy) ology, University of Karlsruhe croscopy (SMM), for total internal reflection fluores- • PALM (photoactivation light microscopy) 1999 Diploma in biology, University cence (TIRF) microscopy and for fluorescence recovery • FRAP (photo recovery after photobleaching) Localization of Bacillus subtilis proteins in live cells by STED microscopy. Left panel Nomarski DIC, central panel YFP fluorescence, of Karlsruhe after photobleaching F( RAP). • Flow cell set up right panel overlay of Nomarski and fluorescence. Protein structures (FloA-YFP) are on average 75 nm large. White line 2 µm. 94 infru astr cture 95

Electron Microscopy Unit The Electron Microscopy Unit offers researchers in synthetic microbiology the opportunity to literally see the outcome of their work and - so to say - to check the “shape” of created “building blocks” per se or in cellular context. Beyond and more profound, electron microscopy can provide essential structural understanding. Thus, it contributes to setting up the foundation the “bricks” are built upon.

Equipment Dr. Thomas Heimerl The unit operates a 200kV (JEOL JEM2100) and a 80 kV Scientific Manager (Zeiss CEM902) transmission electron microscope. In Phone: +49 6421 28 21509 addition, we can help in advanced structural investi- Email: thomas.heimerl@synmikro. gations via focus ion beam scanning electron micros- uni-marburg.de copy (JEOL JIB4610F).

Applications Localization of the A1AO ATP synthase of the hyperthermophilic crenarchaeon Ignicoccus hospitalis by immunolabeling on a 50 nm ultrathin section. Beside structural investigations of cells and proteins in 2D and 3D, localization of proteins in cells is pos- sible. For this purpose, immunogold labeling repre- • Freeze substitution (Leica AFS2) sents a powerful technique, especially when limita- • Embedding in resins (e.g., Epon, Lowicryl) tion in resolution by fluorescent labeling approaches • Ultramicrotomy (Leica EM UC7, Reichert Ultracut-E) is an issue. To satisfy all these demands, we provide • Immunogoldlabeling on ultrathin sections or freeze a variety of conventional and advanced methods and fracture replica techniques: • 3D analysis by electron tomography of resin embed- • Negative staining (e.g., uranylacetate, phospho- ded samples tungstic acid) • 3D analysis by FIB/SEM tomography • Carbon coating (Balzers BAE 121) • Processing and visualization of 2D and 3D data • Gold coating (Technics Hummer Sputter Coater) (ImageJ, IMOD, AMIRA) Freeze fracturing, freeze etching, platinum/carbon shadowing (Balzers BAF301) Thus, we can address a broad spectrum of questions • High pressure freezing (Wohlwend HPF Compact 02) in house.

Short CV

2014– Scientific Manager, SYNMIKRO Electron Microscopy Unit 2014 Ph.D. in microbiology, University of Regensburg 2011 Visiting Scientist, Oak Ridge National Laboratory, Tennessee, USA 2009 Diploma in biology, University of Regensburg 3D model of two (half) cells of the diatom Phaeodactylum tricornutum based on a FIB/SEM dataset comprising 971 consecutive images. The JEOL JEM-2100 Transmission Electron Microscope. 96 IunfRastr cture 97

MarXtal Structural Biology Unit The determination of the 3D structures of molecules helps in elucidating the molecules’ biological func- tions. This, in turn, is impor- tant for the understanding and manipulation of, e.g., disease mechanisms, or the production of biotechno- logically relevant metabo- Dr. Vasundara Srinivasan lites. X-ray crystallography enables such structural Scientific Manager insights at atomic resolu- Phone: +49 6421 28 23390 or -22138 tion, and since there is no Email: [email protected] size limit for its objects of investigation, small mol- Crystals of CIA1, a protein involved in the cytosolic Iron-sulfur cluster assembly. ecules as well as huge mac- romolecular complexes like ribosomes or whole vi- A liquid-handling system Lissy is available for creating ruses can be analyzed with this method. The MarXtal these screening solutions in 96-well deep well blocks. Structural Biology Unit localized at the Department A COY anaerobic glove box is at the user’s disposal of Chemistry, Structural Biochemistry Group, assists for proteins/cofactors/ligands that are sensitive to its users in every step of the process, from the design oxygen. Furthermore, light-sensitive proteins can be of suitable expression constructs for protein produc- dispensed in the dark to maintain stability. tion via the optimization of crystals and the collection of data up to the determination and interpretation of Once the drops are set, crystallization experiments the crystal structure. can be incubated at 4°C and/or at 18°C and automati- cally monitored for crystal growth using two Formula- trix Rock ImagersTM. These imagers record high-resolu- One of the Formulatrix Rock Imagers™ with incubator (left) and imaging unit (right). Short CV tion pictures of the drops that the user can access via 2014– Scientific Manager, SYNMIKRO internet from their own computer. A home rotating Structural Biology Unit anode X-ray generator and high-energy synchrotron Applications 2012–2013 Research Scientist, beamlines at ESRF in Grenoble, BESSY in Berlin and SLS Philipps-Universität Marburg in Villigen (CH) are then used to check the diffraction • Crystallization trials using ‘nanoliter’ drops by 2007–2012 Research Group Leader, properties of the crystals and record images in order sitting drop vapour diffusion method Free University Brussels & VIB, to finally solve the atomic resolution crystal struc- • Automated dispensing of thousands of screening Brussels, Belgium tures. solutions 2001–2007 Postdoctoral Fellow, • Monitoring of crystal growth both at 4°C and at 18°C Max Planck Institute of Biophysics, • Anaerobic and light-sensitive proteins Frankfurt 2000 Ph.D. in biochemistry, Univer- • Membrane proteins sity of Georgia, USA • Data collection and structure solution of molecules 1995 M.Sc. in biophysics, University of any size of Madras, India

A section of the electron density map of the CIA1 protein at 1.7Å resolution, clearly showing one of the β-sheets and the side chain amino acids.

Equipment Technical Assistant Ralf Pöschke operating the Honeybee 963™.

T o achieve this goal, the facility provides state-of-the- art instrumentation such as a Honeybee 963TM and a Cartesian Microsys TM SQ 4000 robot for setting up high-throughput crystallization experiments within minutes. Routinely, up to a thousand different crys- tallization conditions are screened in parallel, with a Coo-w rker: Crystal structure of CIA1 determined to 1.7Å resolution. The wide range of buffers and precipitating agents pro- structure depicts a seven bladed β-propeller fold with water Ralf Pöschke (Technical Assistant) vided both for soluble and for membrane proteins. molecules (green spheres) that can be seen at this high resolution. 98 IunfRastr cture 99

C ore Facility for Mass Spectrometry The detailed analysis of both small molecules and Synapt with IMS, ETD High-resolution mass (bigger) biopolymers is a keystone of today’s biology, and HDX automation spectrometer mainly and mass spectrometry (MS) is the method of choice (Waters) used for HDX mass to address these issues: In Metabolomics, qualitative spectrometry and mass and quantitative analysis of metabolites by means determination of intact of HPLC-MS or GC-MS gives insights into the func- proteins tion and catalytic properties of enzymes or even into the metabolic flux within complex systems.I n Prot- AccuTOF GCv (JEOL) High-resolution GC-MS eomics, such qualitative or quantitative analysis by coupled with a 7890 system mainly used for means of NanoHPLC-MS/MS identifies proteins within GC (Agilent) small molecule applica- Dr. Uwe Linne mixtures, quantifies expression levels under different tions conditions, or gains information on post-translational Scientific Manager modifications; the latter, in turn, can be useful to un- 1100 Single Quadru- Low-resolution HPLC-MS Phone: +49 6421 28 25618 or -25526 derstand enzymatic mechanisms on the molecular pol HPLC-MS system system mainly used for Email: [email protected] level or to get insights into the regulation or modu- (Agilent) method development lation of protein function or activity. For structural investigations, HDX MS is a powerful and emerging method that is used to address protein dynamics and 5973N/6890N GC-MS Low-resolution GC-MS to map protein-protein interfaces at the molecular system (Agilent) system mainly used for level. quantification of small molecules Such an in-depth understanding of natural systems is a prerequisite for the design and construction of syn- Element2 ICP-MS High-resolution ICP-MS thetic modules or circuits. Moreover, these methods system (Thermo system used for analysis can be used both to track the impact of modifications Scientific) of trace elements made to natural systems, and to test the function of actual synthetic systems. Therefore, SYNMIKRO sup- Short CV ports the University’s Core Facility for Mass Spec- 2010– Scientific Manager,C ore trometry – most recently, e.g., the center participated Facility for Mass Spectrometry in the purchase of a new mass spectrometer with 2007–2010 Research Scientist, Core an HDX-automation platform, which will be used for Facility for Mass Spectrometry “high-throughput” analysis. As one of the first insti- 2001–2007 Research Scientist, tutions worldwide, we want to implement this tech- Filling liquid nitrogen into the supra-conducting magnet of the LTQ-FT Ultra mass spectrometer. The nano-RSLC seen in the back is coupled to an Orbitrap Velos Pro mass spectrometer. Philipps-Universität Marburg nology to support crystallization efforts, i.e., to detect 2001 Ph.D. in chemistry, “weak spots” of proteins that are highly dynamic and Philipps-Universität Marburg unstructured and thus difficult to crystallize. 1998 Diploma in chemistry, Applications Philipps-Universität Marburg Equipment T he Core Facility for Mass Spectrometry covers almost • HDX mass spectrometry of proteins or protein I n the Core Facility for Mass Spectrometry localized at the whole range of conceivable applications: complexes the Department of Chemistry, a number of “state-of- • Qualitative or quantitative analysis of small mole- • Ion Mobility Separation (IMS) coupled to classical the-art” mass spectrometers are operated: cules (Metabolomics) mass spectrometry • Qualitative or quantitative analysis of biopolymers, • Analysis of trace elements by ICP-MS especially of proteins (Proteomics), including protein LTQ-FT Ultra (Thermo Ultra-high-resolution identification, analysis of post-translational modi- Scientific) coupled to mass spectrometer main- fications, and mass determination of intact proteins an 1100 HPLC (Agilent) ly used for the analysis of or a TLC/MS-Interface small molecules (CAMAG) Adjustment of the nanospray ion source at the Orbitrap Velos Pro mass spectrometer. Coo-w rkers: Orbitrap Velos High-resolution mass Jan Bamberger (Chemical Engineer) coupled to a nano- spectrometer mainly Tina Krieg (Laboratory Technician) RSLC (both Thermo used for all kinds of pro- Yvonne Ullrich (Laboratory Technician) Scientific) tein analysis, especially Heike Mallinger (Laboratory Technician) of digests Martina Gerlach (Laboratory Technician) 100 101

3c Tea hing & Events 102 Teaching & events 103

at the Giant Jamboree in Boston. They also started an intense collaboration with the Blindenstudienanstalt (BLISTA) in Marburg and introduced visually impaired people to synthetic biology. Judges at the Giant Jam- boree especially honored this approach.

The center’s Graduate School

Over 40 doctoral students receive an intense and pro- fessional training in the SYNMIKRO Graduate School. The curriculum involves weekly lectures and seminars in synthetic biology offered by external guests or in- house experts and, every other year, a retreat for all The 2013 iGEM team at the MIT in Boston. (Photo: SYNMIKRO) students enrolled. In addition, our graduate students participate in at least two workshops to advance searchers to deepen their knowledge and broaden their methodological and soft skills. Soft skill train- their methodological expertise. The outgoing fellow- ing is offered through an intense collaboration of the ship allows a three-month stay at another research center with the Marburg University Research Acade- institution in Germany or abroad. The incoming fel- my (MARA). Moreover, the members of the SYNMIKRO lowship enables national or international researchers Graduate School profit from activities within theI n- a three-month visit at SYNMIKRO to profit from the ternational Max Planck Research School for Environ- theoretical and methodological expertise available at mental, Cellular and Molecular Microbiology (IMPRS- the center. Consequently, the exchange program has Mic) and the Graduate School of the SFB 987. already promoted a significant number of scientific Thesis Advisory Committees that consist of three PIs collaborations with other national and international from SYNMIKRO or other research institutions sup- research institutions and represents a spring-board port each graduate student in annual consultations for synthetic microbiologists at an early stage in their to ensure optimal progress and development. Where career. relevant for their research projects, our graduate stu- In addition to these short-term fellowships, the center dents perform internships in other SYNMIKRO groups also established a long-term fellowship that allows or visit labs abroad through the center’s short-term excellent postdoctoral researchers to establish an in- fellowships. Moreover, graduate students are obliged dependent research program and prepare first grant to participate in international scientific conferences. proposals. Since 2013, six long-term fellowships were Social and scientific meetings further intensify inter- awarded. Building a vibrant community actions – prominent examples being the “Missions for Synthetic Biology” club where our students and for front line synthetic biology external guests discuss challenges and opportunities of this new emerging scientific discipline, the Postdoc SYNMIKRO provides training of synthetic biologists at Engineered Machine (iGEM) competition founded by Symposium, or the Twin Talks, pairing up two SYN- the undergraduate, graduate and postdoctoral level. the Massachusetts Institute of Technology (MIT). Eve- MIKRO graduate students from different disciplines All our educational programs engage with technology ry year, student teams from universities all over the to present current research topics. and knowledge at the frontiers of the field. world engage in creative scientific projects with an emphasis on synthetic biology. Moreover, the teams Promoting early careers Mastering synthetic microbiology are encouraged to present their work to the general public, promoting a dialogue between science and F urthermore, SYNMIKRO established an easy-to- Within the Master program Molecular and Cellular society. After about nine months, they compete with access exchange program that provides short-term Biology of the university’s Faculty of Biology, SYN- hundreds of other teams at the iGEM jamboree. The funding to graduate students and postdoctoral re- MIKRO members offer a specialization in synthetic challenges associated with this concept lead to an microbiology. In this specialization, our experts guide impressive progress in the students’ scientific skills, hands-on education, combining classic and synthetic their level of independent thinking and their personal biology approaches with modern high-throughput confidence. robotics, high-resolution imaging, structural biology In 2013, the Marburg iGEM team used the microalga and modeling techniques. A major goal is the educa- Phaeodactylum to synthesize and secrete an antibody tion of our students towards an in-depth understand- against a Hepatitis B virus protein (PHAECTORY). This ing of the function and organization of microbial cells approach constitutes an elegant and cost-effective as the basis for successful synthetic approaches. way of producing a high-value molecule important for medical applications, earning the team a gold medal SYNMIKRO at the iGEM competition in the European competition and the admission to the world finals at the MIT in Boston. In 2014, our iGEM With the support of the center, undergraduate and team engineered synthetic scaffolds and catalysts by graduate students of the Philipps-Universität suc- creating synthetic units for redirecting functionalities cessfully participated in the international Genetically (SURF). Again the team won a gold medal, this time SYNMIKRO students advertising the Master program. 104 Teaching & events 105

The Schools: interdisciplinary training of early career scientists

SYNMIKRO aims to foster the international and inter- supported by the German Academic Exchange Service disciplinary culture of synthetic biology research at all (DAAD). Building on the great success of these events, career levels. To this end, our seasonal schools provide early career researchers are invited to the next SYN- expert training on cutting-edge research and a stimu- Marburg Summer School „From Microbial Cell Biology lating environment for graduate students and young to Complex Communities“ taking place from 7th to 18th postdocs to broaden their knowledge and develop of September, 2015. their network of contacts. International Autumn School: SYNMarburg Summer School Biology featuring Engineering

The first two international SYNMarburg Summer SYNMIKRO also joined forces with the Helmholtz Schools were organized by SYNMIKRO in 2012 and 2013, Initiative on Synthetic Biology to organize the Inter- focusing on „Microbial Cell Biology and Synthetic national Autumn School: Biology feat. Engineering in Signaling Systems“. In 2013, 22 German and interna- November, 2014. Its realization was a shared effort “Laboratory Automation” workshop at SYNMIKRO, International Autumn School. (Photo: SYNMIKRO) tional Master and doctoral students as well as young of the DKFZ Heidelberg, Forschungszentrum Jülich, postdoctoral researchers from 11 nations attended Karlsruhe Institute of Technology (KIT) and SYN- This practical part was followed by joint activities this 2-week school to learn about the emerging field MIKRO, all offering hands-on computational and/ in Marburg to foster the exchange of knowledge: of synthetic microbiology. Symposia with lectures by or laboratory workshops in Heidelberg, Jülich and Internationally renowned experts in synthetic biol- invited experts and laboratory courses covered vari- Marburg during the first week of the school. The 35 ogy from academia and industry delivered lectures. ous aspects of the field, and the participants enjoyed participants from 9 countries could choose among Moreover, the attendees had the opportunity to pre- a diverse social program in Frankfurt and Marburg. eight courses on topics such as “Synthetic regulatory sent their own research as well as the knowledge and One evening contained a lecture on ethics in biology, circuits in microbial and mammalian cells”, “Rewir- expertise acquired during the workshops to the other which triggered a lively discussion among the partici- ing cells to explore logic, function and evolution”, or participants. This special concept allowed the young pants. The SYNMarburg Summer Schools were kindly “Single cell analysis in microfluidic chips”. researchers to get to know leading institutes in the synthetic biology landscape in Germany and to meet invited national and international speakers. A number of social events supported networking activities. The International Autumn School: Biology feat. En- gineering received major financial support from the BMBF and the Helmholtz Initiative; further support came from Carl Roth GmbH, Nikon, Life Technologies, and Hessen Trade & Invest.

“Single cell analysis in microfluidic chips” workshop at the Forschungszentrum Jülich, International Autumn School. (Photo: Forschungszentrum Jülich) “Synthetic regulatory circuits in microbial and mammalian cells” workshop at the DKFZ Heidelberg, International Autumn School. (Photo: DKFZ) 106 Teaching & events 107

Bringing scientists together to stimulate new ideas and concepts

from all over the world to discuss systems biology, bi- ochemistry, structural and cell biology, and the genet- ics of Archaea. In 2014, the first meeting on “Microbial Cell Biology” of the respective section of the German Association for General and Applied Microbiology (VAAM) highlighted new concepts and ideas follow- ing recent developments in molecular and imaging techniques. Later that year, almost 90 scientists from 18 countries met in Rauischolzhausen for the EMBO workshop on “Stalked alpha-Proteobacteria and Rela- tives: from Genes to Structure”, at which advances in the field of bacterial cell biology, ranging from genom- ics and chromosome dynamics over cell cycle regula- tion, morphogenesis and cell division to modeling and biotechnological applications were presented. The most prominent outcome of this commitment, though, is the annual SYNMIKRO Symposium, a one- Stimulating discussions during the lunch break in the courtyard of the Old University in downtown Marburg. day-conference in spring that brings together scien- tists from academia and industry. This format, for Prof. Kristala Jones Prather (MIT) presenting her research at the SYNMIKRO Symposium 2014. which the center found a strong partner in Hessen speakers both from academia and industry, e.g., Prof. Trade & Invest GmbH (HTAI), was initiated by Erhard Bärbel Friedrich (HU Berlin), Prof. Wilfried Weber (U P art of SYNMIKRO’s self-assigned mission is to foster Bremer, who together with his co-organizers arranged Freiburg), Dr. Holger Zinke (BRAIN AG), Prof. Oskar the exchange between scientists. For this, the center up-to-date programs with prominent speakers that Zelder (BASF SE), Dr. Markus Schwab (Evolva SA), and regularly organizes and supports workshops, meet- attracted more and more participants over the years, Prof. Ralf Wagner (GENE ART AG). In 2012 the sympo- ings, and conferences on various topics related to syn- filling the assembly hall of the scenicO ld University sium addressed “Design of Antibiotics – Innovation thetic biology. In 2012, the conference “Mathematical in downtown Marburg to the last seat with close to Potential of Synthetic Microbiology”, with Prof. Jörg Modeling of Microbiological Systems” brought close 400 attendees. Hacker (Leopoldina), Prof. Helge Bode (U Fankfurt), Dr. to 70 experts from 11 countries to Marburg. In the This conference was first organized in 2011, then Claus Lattemann (Sanofi-Aventis GmbH),P rof. Helga same year, the third conference on “Molecular Biology dealing with “Synthetic Microbiology – Perspectives Rübsamen-Schaeff (AiCuris GmbH) and Dr. Wolfgang of Archaea” attracted approximately 150 participants for Biotechnology and Pharmacy”, with prominent Mutter (Hyglos GmbH) among the speakers. The 2013 conference “From Biological Diversity to Microbial Cell Factories” dealt with the question how modern -om- ics technologies and synthetic approaches could im- prove the exploitation of the diverse catalytical prop- erties developed by microorganisms during evolution; speakers included Dr. Guido Meurer (BRAIN AG), Prof. Alfred Pühler (U Bielefeld), Dr. Jan Van den Brulle (Mor- phoSys AG), and Dr. Esther Gabor (BRAIN AG). The most recent conference – „Microbial Formation of Biofuels and Platform Chemicals“ in 2014 – brought experts on metabolic engineering to the podium, including Prof. Jay Keasling (UC Berkeley), Dr. Thomas Haas (Evonik Industries), Prof. Kristala Jones Prather (MIT), Dr. An- drea Herold (BASF SE), and Prof. Ferdi Schüth (MPI Mühlheim). The 2015 SYNMIKRO Symposium on April 22nd will fo- cus on “Microbial Biosensors and Regulatory Circuits”.

A mesmerized audience (from left): Prof. Peter Dürre (Ulm), Prof. Volker Müller (Frankfurt), Prof. Jay Keasling (Berkeley), Prof. Rudolf Thauer (Marburg), Dr. Rainer Waldschmidt (HTAI), and Prof. Erhard Bremer (Marburg). 108 Teaching & events 109

Art & Science

The local artist Ingrid Hermentin was repeatedly in- Kunstverein, the artist adapted the iGEM approach by spired by SYNMIKRO research. For her exhibition “Tran- assembling her “synthetic images” (modules) in the skriptionen_TATA-Box” at the Lutherische Pfarrkirche given art space (chassis), in order to achieve a change Marburg in 2013, Hermentin created “synthetic pic- of perspectives. tures” based on images of the microalga Phaeodac- The exhibitions were each accompanied by public tylum tricornutum and phylogenetic trees, visualizing events with talks of SYNMIKRO members on scientific her view on “synthetic evolution”. For “Transkrip- and/or ethical aspects. tionen_BioBricks”, presented 2014 at the Marburger

Panel discussion on “Complexity” in December, 2013. From left: Prof. Bruno Eckhardt, Prof. Sven Panke, Prof. Friedemann Voigt, and Prof. Klaus Mainzer.

A dialogue between synthetic biology and humanities

Since the early days of the center, SYNMIKRO paid at- Gerald Hartung, Universität Wuppertal, hosted by tention to the political and ethical aspects of synthet- Prof. Friedemann Voigt and Prof. Michael Bölker. The ic biology. In 2010, ethicist Prof. Peter Dabrock, at that second event addressed the “Complexity” of biologi- time member of the Faculty of Protestant Theology cal systems, with Prof. Sven Panke, biotechnologist in Marburg and of the center, hosted a panel discus- from the ETH Zurich, and Prof. Klaus Mainzer, philoso- sion on the chances and risks of the field.I n 2013 and pher from the Technical University Munich, as speak- 2014, a whole series of public events was organized ers, and Prof. Bruno Eckhardt and Prof. Friedemann in order to promote the mutual understanding of life Voigt as hosts. The last event dealt with the opposites sciences and humanities in the recurrent debates. At „Natural/Synthetic“ and, e.g., the observation that each event, short lectures of prominent experts from most advances in synthetic biology are still based on both disciplines first demonstrated their different ap- natural molecules or building blocks, whereas a lot of proaches and points of view. According to Prof. Friede- what is perceived as natural in our environment – like mann Voigt, present leader of the SYNMIKRO Bioeth- the European cultural landscape – already is the result ics group and initiator of the series, these different of human intervention; speakers were Prof. Nedilijko perspectives often lead to misunderstanding and even Budisa, chemist from the Technical University Berlin, distrust. Therefore, subsequent panel discussions of- and Prof. Kristian Köchy, philosopher from the Univer- fered an opportunity for the experts to enter into a sity Kassel, hosted by Prof. Regine Kahmann and Prof. F urther reading dialogue, and for the audience to ask questions. Friedemann Voigt. Voigt, F (Ed.) (2015). Synthetische Biologie im Dialog, Karl Alber The first event was dedicated to “Life”, its definitions, All events were organized by SYNMIKRO and the (Series: „Lebenswissenschaften im and the question whether or not scientists are allowed Graduate Center for Life and Natural Sciences of the Dialog“, edited by Kristian Köchy to rebuild or even change it; speakers were biologist Philipps-Universität Marburg. and Stefan Majetschak), Freiburg Prof. Jörg Hacker, President of the German Academy Top: The exhibition „Transkriptionen_TATA-Box“ 2013 at the Lutherische Pfarrkirche Marburg. Bottom: The exhibition „Transkriptionen_BioBricks“ 2014 at the Marburger Kunstverein. i.Br., in press. of Natural Sciences Leopoldina, and philosopher Prof. (Photos: Bildarchiv Foto Marburg) 110 Teaching & events 111

Informing and inspiring society

Work hard, party hard

Members of SYNMIKRO and the university’s iGEM team regularly show-case interesting features of synthetic biology to the general public, e.g., at the „Hessentag“, an annual event of the federal state of Hessen with more than one million visitors, and the annual city festival „3 Tage Marburg“. (Photos: SYNMIKRO)

Left: Training for the dragon boat race at the town festival “3 Tage Marburg”. Right: Halloween and Christmas Parties. (Photos: SYNMIKRO) 112 113

4 FACts & Figures 114 Fac ts & Figures 115

Financial and infrastructural resources

SYNMIKRO was established following the successful application for the excellence program LOEWE of the state of Hessen: For its first three years the center was awarded a total of € 22.4 m, and for the second fund- ing period up to € 21.7 m. This support has been used to build and shape the center via (i) the appointment of two new professors at the university, (ii) the found- ing of a fourth department at the Max Planck Insti- tute, (iii) the appointment of four temporary groups at the university to attract junior researchers to Mar- burg, (iv) the provision of seed money to initiate proj- ects that can then be carried on with external funding, and (v) major investments in infrastructure.

External funding

Over the years, these investments in the infrastruc- ture and in the projects paid off and the center at- tracted additional external funding: By the end of 2014, The Mehrzweckgebäude on the science campus Lahnberge, temporary home for six SYNMIKRO groups, the Super researchers at the center had been awarded grants The recently completed building for the new Max Planck Department, a junior research group, an independent Max Planck Research Group, and the Flow Cytometry and Protein Resolution Microscopy Unit, the Laboratory Automation Unit, and the Head Office. (Photo: Reinhold Eckstein) totaling more than € 15 m, a major contribution Diffusion & Interactions Unit. being the Sonderforschungsbereich 987, which alone LOEWE funding was granted € 7.058 m over its four years of funding. The largest fraction of grants comes from the German many microscopes, the building offers a safety-level 2 lecture hall will be located in the new building, pro- Research Foundation (DFG), followed by the EU via the laboratory and room for a laboratory automation plat- viding SYNMIKRO with excellent working conditions European Research Council and a EU-Cost action. The form. Currently, about 100 people work here. and the long-sought integrating home. We expect the aim for the next few years is to expand on funding Space for the fourth Department at the Max Planck building to be finished by 2019. from the BMBF and industrial collaborations. Among Institute was provided in a newly constructed build- coordinated programs, another Sonderforschungsbe- ing, located on the Lahnberge between the Meh- reich and a Graduiertenkolleg are planned. Overall, the rzweckgebäude and the Max Planck Institute (and center is well on its way to establish an annual funding marked “Zentrum für Synthetische Mikrobiologie” on that amounts to at least half of the annual seed fund- the map on p. 119). Funded through an extra grant by ing by the state of Hessen. This, together with the ba- the state of Hessen, the new building was commis- sic support for the Departments at the Max Planck In- sioned in 2011 and finished in August 2014. Its 1200 m2 External funding stitute for Terrestrial Microbiology and for the research lab and office space are occupied by Victor Sourjik and and infrastructure at the Philipps-Universität Marburg, his group, a junior research group and an independent secures the long-term perspective of the center. Max Planck Research Group, as well as the new Flow Cytometry and Protein Diffusion & Interactions Unit. T emporary facilities...... and a future home The seed money provided by the LOEWE program enabled us to establish more than 130 positions in F rom the very beginning, the aim has been to bring research and infrastructure. Some of the new per- together the key research groups and infrastructural sonnel were integrated into the existing groups, but units in one building so as to intensify interactions for the largest part new labs and offices had to be and, in particular, interdisciplinary collaborations. provided. The dedicated support of the construction The decision by the German Council of Science and department of the university allowed us to establish Humanities (Wissenschaftsrat) in spring 2014 to fund Sources of the external funding temporary, but nevertheless state-of-the-art labo- the respective proposal finally paved the way for this ratories and offices within the Mehrzweckgebäude SYNMIKRO building. With about 6000 m2 lab and of- on the science campus Lahnberge. Occupying four fice space, it will house the Max Planck Department levels around stairway C, the groups of Anke Becker, of Systems and Synthetic Microbiology, five groups Peter Graumann, Torsten Waldminghaus, Gert Bange, from the university’s Faculties of Biology and Chem- Kristina Jonas and Georg Fritz, and the Head Office of istry, four junior groups and two groups in biological SYNMIKRO make use of about 1100 m2 lab space and modeling. Furthermore, most of the center’s infra- 450 m2 of offices.I n addition to special spaces for structural units, a high-resolution cryo-TEM and a 116 Fac ts & Figures 117

Awards & Honors

2014 Roland Lill Albrecht Kossel Prize, German Chemical Society (GDCh) Luigi Sacconi Medal, Società Chimica Italiana (SCI) Mohamed A. Marahiel David Gottlieb Memorial Lecture, University of Illinois, Urbana, IL Lotte Søgaard-Andersen Elected Fellow, American Academy of Microbiology

2013 Sonja- Verena Albers ERC Starting Grant, European Research Council Peter Lenz Paula and Richard von Hertwig Award for interdisciplinary cooperation Rolf Thauer Medal of Merit, German Academy of Sciences Leopoldina

2012 A moment of success. The speaker of the SFB 987 Mohamed Marahiel (right) and the vice speaker Erhard Bremer celebrate the decision of Sonja- Verena Albers Research Award, Association for General and Applied Microbiology (VAAM) the DFG to establish and fund the Collaborative Research Center Microbial Diversity in Environmental Signal Response. (Photo: SYNMIKRO) Gerhard Klebe Carl Mannich Medal, German Pharmaceutical Society Mohamed A. Marahiel Honorary Professorship, Wuhan University, China SFB 987: Microbial Diversity in 2011 Erhard Bremer Elected Member, European Academy of Microbiology Regine Kahmann Distinguished Affiliated Professorship, TU München Environmental Signal Response Honorary Doctorate, Hebrew University, Jerusalem

Microorganisms successfully colonize almost every The SFB 987, which will receive 7 058 000 € from the Gerhard Klebe ERC Advanced Grant, European Research Council possible ecological niche, regardless of its conditions. DFG over the initial four years, began its work in July, Victor Sourjik ERC Advanced Grant, European Research Council A key factor for this success is their enormous bio- 2012. Within this framework, 15 microbiology-focused Martin Thanbichler Research Award, Association for General and Applied Microbiology (VAAM) chemical, physiological, and cellular adaptability: In research teams from the Faculties of Biology, Chemis- the course of evolution, microorganisms have devel- try and Medicine of the Philipps-Universität and from oped an array of mechanisms that enables both in- the Max Planck Institute for Terrestrial Microbiology 2010 dividual cells and cellular communities to recognize combine their research activities and expertise. 14 of Ralf Conrad Einstein Distinguished Chair Professorship, Chinese Academy of Sciences environmental cues with high sensitivity, precision these groups are also members of SYNMIKRO, and and specificity, to adapt to changing circumstances, the research activities of the SFB and of SYNMIKRO Roland Lill Feldberg Foundation Prize and to exploit new opportunities. The scientists that are thus tightly interwoven. Furthermore, SYNMIKRO joined forces within the framework of the DFG-funded attracted strong new research teams to Marburg, pro- Collaborative Research Center 987 (SFB 987) focus on viding the members of the SFB 987 with even more interesting partners for future collaborations. On the Logo of the SFB 987. these key features of microorganisms and structure their work around the encompassing theme Microbial other hand, a thorough understanding of the genet- Diversity in Environmental Signal Response. ics, biochemistry, cell biology, and physiology of mi- The title of the SFB 987 heralds one of the strength croorganisms is needed to exploit them for synthetic Former Members of microbial research in Marburg: Instead of concen- microbiological purposes and to devise cells and pro- trating on just a few model organisms, the research cesses that do not exist naturally. This expertise is the Ekaterina Kostina accepted an offer from U Heidelberg (2015) groups work with a large variety of Bacteria, Archaea, solid foundation of the activities of the SFB 987. Sonja-Verena Albers accepted an offer from U Freiburg (2014) and Fungi and study their properties both under controlled laboratory conditions and in their natural Erhard Bremer and Mohamed A. Marahiel Andreas Klingl accepted an offer from LMU Munich (2014) habitats. The central aim is to comprehensively under- Eyke Hüllermeier accepted an offer from U Paderborn (2014) stand specific cellular activities and signaling process- Alexander Böhm (†) was a member of Synmikro in 2012 es of defined species, the behavior of individual cells and complex microbial communities, the interactions Peter Dabrock accepted an offer from U Erlangen (2011) of microorganisms with each other and with their eu- karyotic hosts, and the countless contributions that microorganisms make to globally-acting cycles of nu- trients and gases operating on our planet. A breadth of approaches and techniques are applied to further these goals. 118 Fac ts & Figures 119

Science Campus Lahnberge

Bahnhof

Universitätsklinikum

Anneliese-Pohl- Kindertagesstätte Mensa

Biomedizinisches Forschungszentrum Fußgängerüberweg Marburg (BMFZ)

Head Office Zentrum für Tumor- und Immunbiologie (ZTI)

LOEWE Center for Synthetic Microbiology (SYNMIKRO) Neubau Philipps-Universität Marburg Chemie Hans-Meerwein-Straße 6, Mehrzweckgebäude Mehrzweckgebäude 35032 Marburg Hörsaalgebäude Phone: +49 6421 28 24401 Zentrum für Email: [email protected] Synthetische Mikrobiologie Geplantes SYNMIKRO-Gebäude www.synmikro.com

Max-Planck-Institut für terrestrische Mikrobiologie Biologie

Kindertagesstätte Zappel-Philipp

Botanischer Garten

The support team (from left): Sina Debus, Nadine Lang, Inge Langenbach (all Financial and Personnel Management), Dr. Gundula Meißner Zentrum/B3/Gießen (Scientific Coordinator), Vera Bettenworth (Public Relations), Karin Sievers (Secretary), Dr. Andres Schützendübel (Head of Administra- tion), Rolf Klamberg (IT-Coordinator). (Photo: Andreas Kautz) 120

Editor Vera Bettenworth, SYNMIKRO Head Office

Design Florian Conrads www.florianconrads.de

Photography Reinhold Eckstein (portraits), Philipps-Universität Marburg Rolf K. Wegst (portrait p. 3, and all other photographs, unless indicated) www.rolf-wegst.de Portraits p. 6, 26, 60 & 74: private Photo p. 82/83: Uwe Dettmar / Hessen schafft Wissen Small photo p. 115: SYNMIKRO

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February, 2015 www.synmikro.com/en/