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content Bad Bergzabern- map ................................................................................................................. 3 Program ...................................................................................................................................... 4 Poster Presentations.................................................................................................................... 8 Oral Presentations-Abstracts .................................................................................................... 12 Special Lectures .................................................................................................................... 12 Session 1: Quorum Sensing .................................................................................................. 14 Session 2: Regulation of Metabolism ................................................................................... 16 Session 3: Second Messengers, bacterial differentiation, and antibiotic production ........... 22 Session 4: Regulation by RNA ............................................................................................. 25 Session 5: Regulation in Biofilms ........................................................................................ 27 Session 6: Stress responses and Signal Transduction ........................................................... 29 Session 7: Diverse Regulators .............................................................................................. 34 Poster Presentations-Abstracts ................................................................................................. 39 2 Bad Bergzabern- map 3 Program Wednesday, 28.09. 2016 12:00 – 14:00 Arrival and Registration 14:30 Welcome Session 1: Quorum Sensing Chair: 14:45 Ralf Heermann, Ludwig-Maximilians-Universität München Silent chats - Communication among entomopathogenic Photorhabdus bacteria. 15:25 Nina Jagmann; Universität Münster Quorum sensing and pyocyanin production by Pseudomonas aeruginosa in a co-culture with Aeromonas hydrophila are co-regulated by the stringent response and other metabolic influences 15:45 coffee break Session 2: Regulation of Metabolism 16:10 Fabian Commichau, Universität Göttingen The Bacillus subtilis glutamate dehydogenases RocG and GudB play a double game. 16:50 Dominik Tödter, Universität Göttingen The interplay between an Asp23 protein family member and acetyl-CoA carboxylase in Bacilus subtilis 17:10 Miriam Dormeyer, Universität Göttingen Compensation for glutamate auxotrophy of a Bacillus subtilis gltC mutant by three independent mutational events 17:30 Andreas Schwentner, Universität Stuttgart Metabolic engineering to direct evolution in Corynebacterium glutamicum 17:50 Bingyao Zhu, Universität Göttingen SubtiWiki, an integrated database for model organism Bacillus subtilis 18:10 Jörg Stülke, Universität Göttingen Large-scale reduction of the Bacillus subtilis genome: Consequences for the transcriptional network, resource allocation, and metabolism 4 18:30 Dinner 20:00 Special Lecture I Josef Deutscher, Unité Expression Génétique Microbienne, CNRS, Paris The role of PTS components in catabolite repression of Listeria monocytogenes virulence genes 21:00 Poster and Wine Thursday, 29.09.2016 Session 3: Second Messengers, bacterial differentiation, and antibiotic production 8:30 Natalia Tschowri, Humboldt-Universität Berlin Cyclic di-nucleotide signalling in bacterial differentiation and antibiotic production 9:10 Ilka Bischofs, Universität Heidelberg Spore memory couples entry and exit from bacterial dormancy in Bacillus subtilis. 9:30 Carsten Volz, Helmholtz-Institut für Pharmazeutische Forschung, Saarbrücken Regulation of Secondary Metabolite Gene Clusters in Social Myxobacteria 9:50 coffee break Session 4: Regulation by RNA 10:20 Kai Papenfort, Ludwig-Maximilians-Universität München From Strings of Nucleotides to Collective Behavior: Lessons from Vibrio cholerae 11:00 Bernhard Remes, Universität Gießen Surprising small RNA features in Rhodobacter sphaeroides Session 5: Regulation in Biofilms Chair: 11:20 Ákos T. Kovács, Universität Jena Exploitation of the biofilm matrix to colonize a surface 12:00 Ramses Gallegos-Monterrosa, Universität Jena Cell-cell communications in Bacillus subtilis mixed-species biofilms 12:30 Lunch 5 14:00 Excursion to Burg Berwartstein, Erlenbach 18:15 Dinner 20:00 Special Lecture II t Erhard Bremer, Universität Marburg Osmotic forces at work – Stress responses to the front! 21:00 Poster / Flammkuchen/ New Wine Friday, 30.09.2016 Session 6: Stress Responses and Signal Transduction Chair: 8:30 Geraldine Laloux, Université catholique de Louvain, Brussels, Connecting cell wall homeostasis and a major envelope stress response in Escherichia coli 9:10 Emina Ćudić, Universität Osnabrück The Cpx-system of Escherichia coli analyzed by SRM and Superresolution Microscopy 9:30 Bianca Warmbold, Universität Marburg Uncovering the regulatory circuits of the glycine betaine synthesizing pathway in Bacillus subtilis 9:50 Diana Wolf, Technische Universität Dresden Characterization of the ABC-Transporter Associated Two-Component System YxdJK in Bacillus subtilis as Biosensor for Eukaryotic Antimicrobial Peptides 10:10 coffe break Session 7: Diverse Regulators Chair: 10:30 Kim Julia Kraxner, Forschungszentrum Jülich A new piece in the big puzzle of cell division: The transcriptional regulator FtsR regulates FtsZ in Corynebacterium glutamicum 10:50 Eugen Pfeifer, Forschungszentrum Jülich Silencing of cryptic prophages in Corynebacterium glutamicum 11:10 Aathmaja A. Rangarajan, Universität Köln Inverse correlation between the transcription repression in Escherichia coli rate and H-NS/StpA 6 11:30 Susann M. Fragel, Universität Köln Characterization of structural features controlling activity of LeuO, a pleiotropic transcriptional regulator and H-NS antagonist 11:50 Hannes Breddermann, Universität Köln Feedback control of leuO encoding a pleiotropic regulator and H-NS antagonist in Escherichia coli 12:10 Poster Price, Concluding Remarks 12:30 Farewell lunch 7 Poster Presentations Poster 1: Computational prediction of the regulatory interactions for an ECF sigma factor group with fused C-terminal domain and co-factor Hao Wu and Georg Fritz. Poster 2: Implementation, analysis and mathematical modeling of ECF sigma factor-based synthetic gene cascades in E.coli and B.subtilis Marco Mauri1, Daniela Pinto2, Stefano Vecchione1, Hao Wu1, Thorsten Mascher2, Georg Fritz1. Poster 3: Modulation of the behaviour of an Extracytoplasmic Function sigma factor (ECF)-based genetic switch Daniela Pinto1, Franziska Dürr1, Dayane Araújo1,2, Qiang Liu1,2 and Thorsten Mascher1. Poster 4: Theoretical models and FRET-assays for signal transduction via switchable allosteric modulator proteins (SAMPs) in Bacillus subtilis Heiko Babel and Ilka B. Bischofs. Poster 5: Regulation of gene expression by small sRNA Dipl.-Ing. Lyubov Kyselova Poster 6: Regulatory interactions between Corynebacterium glutamicum and the prophage CGP3 Max Hünnefeld, Eugen Pfeifer and Julia Frunzke. Poster 7: Deciphering the Response of Corynebacterium glutamicum to Oxygen Deprivation Julian Lange1, Tobias Busche2, Jörn Kalinowski2, Ralf Takors1, Bastian Blombach1 Poster 8: From substrate specificity to promiscuity: molecular analysis of a hybrid ABC transporter L. Teichmann, C. Chen & E. Bremer Poster 9: The cellular function and localization of the cyclic-di-GMP phosphodiesterase PdeL Cihan Yilmaz and Karin Schnetz Poster 10: Elucidation of the metabolic pathway for SDS degradation and its regulation in Pseudomonas aeruginosa Gianna Panasia and Bodo Philipp. 8 Poster 11: The role of the phosphodiesterase NbdA in NO-induced dispersal of Pseudomonas aeruginosa Martina Rüger1, Sabrina Heine2, Michael Entian2, Yi Li3, Karin Sauer3 and Nicole Frankenberg-Dinkel1, 2 Poster 12: Osmotic responsive transcription of ectoine biosynthetic genes from Pseudomonas stutzeri is transferable to a non-ectoine producing surrogate host Laura Czech, Philipp Hub, Florian Kindinger, Oliver Dähn, Nadine Stöveken & Erhard Bremer Poster 13: A special role for acetate kinase AckA in the regulation of CiaR activity in the absence of the cognate kinase CiaH. Anne Sexauer and Reinhold Brückner. Poster 14: Transport and regulation by the alternative anaerobic C4-dicarboxylate-transporters DcuA, DcuB and DcuC in Escherichia coli Alexander Strecker and Gottfried Unden Poster 15: The DxxxQ phosphatase motif in the O2 sensor kinase NreB of Staphylococcus carnosus Ann-Katrin Kretzschmar and Gottfried Unden Poster 16: The function of the ExxN motif of the C4-dicarboxylate sensor kinase DcuS of Escherichia coli in signal transduction Stefaniya Gencheva, Sebastian Wörner and Gottfried Unden Poster 17: Analysis of quinone mutants in respect to ArcA phosphorylation and product formation Annika Nitzschke and Katja Bettenbrock Poster 18: Analysis of the signal transduction by the heme-based sensor kinase MsmS from Methanosarcina acetivorans Fiege, K.1,2, Molitor, B.2, Blasius, L.1, Querebillo, C.3,4, Hildebrandt, P.3, Laurich, C.5, Lubitz, Poster 19: A TCS is involved in the regulation of the organohalide respiration in Sulfurospirillum spp. Jens Esken1, Tobias Goris1, Cynthia Sharma2, Torsten Schubert1, and Gabriele Diekert1. 9 Poster 20: Biochemical characterization of the iron responsive regulator RirA from Dinoroseobacter shibae Maren Behringer, Elisabeth Härtig and Dieter Jahn Poster 21: Adrenochrome – oxidation product of adrenaline and bacterial effector molecule Charlotte Toulouse, Kristina Metesch, Pit Engling, Bernd Michel and Julia Steuber. Poster 22: Mechanism and function of non-standard circadian clock systems in cyanobacteria Christin Köbler1, Anja Dörrich2, Anika Wiegard3, Annegret Wilde1 Poster 23: Characteristics of a SoxR-based single cell NADPH biosensor in Escherichia coli Alina Spielmann, Meike Baumgart and Michael Bott 10 11 Oral Presentations-Abstracts Special Lectures The role of PTS components in catabolite repression of Listeria monocytogenes virulence genes Josef Deutscher Unité Expression Génétique Microbienne, Centre National de la Recherche Scientifique, 13, Rue Pierre et Marie Curie, F-75005 Paris, France Listeria monocytogenes is a saprophyte well adapted to growth in the soil on decaying plants and other organic material. For this purpose, L. monocytogenes contains a large number of carbohydrate transport systems, which allow the bacterium to utilize the numerous carbon sources produced during plant decay. However, this bacterium has a dual lifestyle, because it is also a foodborne human pathogen causing the disease listeriosis (1). The infection process by L. monocytogenes has been intensively studied. Infection usually occurs via the digestive tract after ingestion of contaminated food. The pathogen is able to actively invade several types of human cells and to enter the bloodstream, where it can cause sepsis and after crossing the blood-brain barrier meningitis. It can also cross the blood-placental barrier and if women are infected during pregnancy, this can lead to abortion. Infection by L. monocytogenes requires a set of virulence factors, which have been intensively studied. These proteins allow the pathogen to adhere to and to enter into host cells via phagocytosis, to escape from the phagocyte, to proliferate in the cytoplasm of the host cell, to move within the host cell and to spread from one epithelial cell to another. Most of the genes encoding these virulence factors are located on two pathogenicity islands. The expression of these virulence genes is controlled by PrfA, a Crp-like transcription activator. A recent study revealed that if the virulence genes would be expressed when the bacterium is roaming in the environment, this would significantly reduce the fitness of L. monocytogenes and its competitiveness. L. monocytogenes therefore developed mechanisms aimed at repressing the expression of its virulence genes when it is present in the soil. One of these mechanisms is based on sensing the temperature of the environment. It has been observed that while the virulence genes are strongly expressed at 37°C, a relatively small shift to 30°C already strongly reduces their expression and therefore the synthesis of the virulence factors. This mechanism is based on a thermoswitch of a secondary structure formed by the RNA preceding the prfA gene. At temperatures at 30°C or lower, formation of this secondary structure prevents the access to the ribosome binding site preceding the prfA mRNA and hence synthesis of the transcription activator of virulence genes. A second virulence gene repression mechanism responds to the presence of efficiently metabolized carbon sources. Inside host cells, L. monocytogenes encounters glycerol and some glucose-6-P as carbon sources, which are less efficient than glucose and therefore allow the expression of its virulence genes. However, when exposed to glucose, fructose, cellobiose or other efficiently utilized carbon sources found in decaying plants, the virulence genes are strongly repressed. Repressing sugars are taken up by the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which phosphorylates its substrates before they enter the cytoplasm. Components of the PTS are involved in the general carbon catabolite repression mechanism. Certain components of a cellobiose-specific PTS and their state of phosphorylation play also a role in L. monocytogenes virulence gene repression. When an efficiently metabolized PTS sugar is present, the PTS components are dephosphorylated and one of them specifically inhibits PrfA activity by a yet unknown mechanism. Deletion of this component leads to a general relief from virulence gene repression by carbon sources. References 12 1. Freitag, N.E., Port, G.C. and Miner, M.D. (2009) Listeria monocytogenes - from saprophyte to intracellular pathogen. Nat. Rev. Microbiol. 7, 623-628. Osmotic forces at work – Stress responses to the front! Erhard Bremer Laboratory for Microbiology, Department of Biology, Philipps-University Marburg, Karlvon-Frisch Str. 8, D-35043 Marburg, Germany [[email protected]] The development of a semi-permeable cytoplasmic membrane through which water can pass freely, but ions, nutrients and waste products cannot, was a key event in the evolution of microbial proto-cells. Changes in the external osmolarity will inevitably trigger water fluxes along the osmotic gradient in or out of the microbial cell. As a consequence, the magnitude of vital turgor will be affected and the ensuing osmotic stress will negatively impact cell growth and integrity. Water influx at low external osmolarity will prompt a raise in turgor that eventually results in cell rupture. Conversely, high external osmolarity will trigger water efflux and thereby causes dehydration of the cytoplasm; growth will be arrested. It is obvious that if these negative consequences of fluctuations in the external osmolarity would not be counteracted, microbial cells will be placed under considerable strain and will eventually die. I will present an overview of the stress reactions of the ubiquitously distributed Grampositive bacterium Bacillus subtilis, which allows the cells to survive exposures to either low or high osmolarity souroundings. I will describe the synthesis pathways for compatible solutes (L-proline and glycine betaine), the structure of importers for these osmostress protectants and their operation at high salinity, and address the release of water-attracting ions and compatible solutes via MscS- and MscL-type mechanosensitive channels upon an osmotic down-shock. I will also focus on the genetics of osmostress-regulated gene expression. Finally, I will present data connecting the osmotic stress responses of free-living cells with key regulatory factors controlling biofilm formation. It will become clear from my presentation that the B. subtilis cell has to engage into a highly coordinated and systems-wide fight in order to survive osmotic fluctuations in its varied habitats. These stress responses encompass the SigB-controlled general stress response, adjustment processes that deal with management of water fluxes in or out of the cell, and genetic and cellular amendments that will allow life of B. subtilis in a biofilm. 13 Session 1: Quorum Sensing Silent chats - Communication among entomopathogenic Photorhabdus bacteria Ralf Heermann Ludwig-Maximilians-Universität München, Biozentrum, Bereich Mikrobiologie, Großhaderner Str. 2-4, 82152 Martinsried/München, Germany email: [email protected] It is well understood that bacteria communicate to coordinate their behaviour, a process termed quorum sensing (QS). Thereby, bacteria do not use voice, but a silent way for “chatting”: small diffusible molecules. The best understood way of bacterial communication is the use of acylated homoserine lactones (AHLs) as “language”. The prototypical of AHL using communication systems consists of a LuxI-like AHL synthase and a cognate LuxR-type receptor that senses the signal. However, many proteobacteria lack any LuxI-type synthase, and thus they cannot communicate via AHLs. Nevertheless, most of them have LuxR-type receptors, which are referred to as LuxR orphans or solos. Entomopathogenic bacteria of the genus Photorhabdus all harbor an extreme high number of LuxR solos, more than any other bacteria known so far. We have identified two novel ways for bacterial communication in Photorhabdus species to date, which the bacteria use for regulation of pathogenicity. P. luminescens and P. temperata -pyrones named photopyrones (PPYs) instead of AHLs, which are produced by the pyrone synthase PpyS and recognized by the LuxR solo PluR.[1] P. asymbiotica, a closely related insect and human pathogen, uses dialkylresorcinols (DARs) for communication, which are produced by the DarABC pathway and recognized by the LuxR solo PauR.[2] Upon sensing the specific signaling molecule, PluR as well as PauR activate expression of the pcf operon in P. luminescens and P. asymbiotica, respectively. This leads to cell clumping, which then contributes to the overall pathogenicity of the bacteria against insects. The PpyS/PluR as well as the DarABC/PauR systems are the first two examples of LuxR solo-based QS systems, which do not use AHLs as “language”. Since the different PPYs and DARs derivatives activate the respective QS response with different strength, these molecules as well as the different AHLs have been regarded as bacterial “dialects”.[3] In summary, our studies reveal that bacterial “silent chats” go far beyond AHL-signaling in nature. References [1] A.O. Brachmann, S. Brameyer, D. Kresovic, I. Hitkova, Y. Kopp, C. Manske, K. Schubert, H.B. Bode, R. Heermann (2013). Pyrones as bacterial signaling molecules, Nature Chem. Biol. 9(9):573-578. [2] S. Brameyer, D. Kresovic, H.B. Bode, R. Heermann (2015). Dialkyresorcinols as bacterial signaling molecules. PNAS 112(2):572-577. [3] S. Brameyer, H.B. Bode, R. Heermann (2015). Languages and dialects: bacterial communication beyond homoserine lactones. Trends Microbiol. 23(9):521-523. 14 Quorum sensing and pyocyanin production by Pseudomonas aeruginosa in a co-culture with Aeromonas hydrophila are co-regulated by the stringent response and other metabolic influences Nina Jagmann and Bodo Philipp Institute for Molecular Microbiology and Biotechnology, University of Münster, Corrensstr. 3, D-48149 Münster, Germany The opportunistic pathogen P. aeruginosa is a metabolically versatile bacterium that can adapt to different environments because of a complex regulatory network that is involved in sensing and responding to different environmental cues. Key regulatory elements of P. aeruginosa are its quorum sensing (QS) systems that control the production of virulence factors. Besides cell density, QS in P. aeruginosa is co-regulated by metabolic influences like nutrient limitation (1). Previously, we established a co-culture model system consisting of P. aeruginosa and the chitinolytic bacterium A. hydrophila with chitin as sole growth substrate, in which parasitic growth of P. aeruginosa is strictly dependent on the QS-controlled production of the virulence factor pyocyanin (2). This redox-active metabolite inhibits the enzyme aconitase of A. hydrophila through the formation of reactive oxygen species causing a block of the citric acid cycle and, thus, a massive release of acetate by A. hydrophila, which supports substantial growth of P. aeruginosa. The stringent response is a regulatory mechanism mediated by the alarmone (p)ppGpp, which leads to physiological adaptations to environmental stresses like nutrient deprivation. We could show that activation of QS under co-culture conditions is dependent on the stringent response as a relAspoT double mutant of P. aeruginosa did not produce pyocyanin in coculture anymore. To identify further genes that are involved in the co-regulation of QS by metabolic influences, we employed transposon mutagenesis and identified the gene gbuA encoding a guanidinobutyrase (3). Deletion of gbuA leads to a loss of pyocyanin production in cocultures and to a reduced pyocyanin production in single cultures. This is likely caused by an accumulation of 4-guanidinobutyrate (4-GB), the natural substrate of GbuA, as addition of 4GB to the mutant strain enhances the negative effect on pyocyanin production in single cultures. The gbuA mutant shows a reduced transcription of the pqsABCDE operon, which is part of the alkylquinolone-mediated QS system of P. aeruginosa controlling pyocyanin production. Production of pyocyanin by the gbuA mutant can be restored by the addition of alkylquinolone signal molecules and PqsE overexpression. The strong effect of gbuA deletion on the QS-controlled pyocyanin production in cocultures shows the value of this approach for the discovery of novel gene functions linking metabolism and QS in P. aeruginosa. References 1. Mellbye, B. and Schuster, M. (2014) Physiological framework for the regulation of quorum sensing-dependent public goods in Pseudomonas aeruginosa, J Bacteriol 196, 1155-1164 2. Jagmann, N., Brachvogel, H. P., and Philipp, B. (2010) Parasitic growth of Pseudomonas aeruginosa in co-culture with the chitinolytic bacterium Aeromonas hydrophila, Environ Microbiol 12, 1787-1802 3. Jagmann, N., Bleicher, V., Busche, T., Kalinowski, J., and Philipp, B. (2016) The guanidinobutyrase GbuA is essential for the alkylquinolone-regulated pyocyanin production during parasitic growth of Pseudomonas aeruginosa in co-culture with Aeromonas hydrophila, Environ Microbiol, doi: 10.1111/1462-2920.13419. 15 Session 2: Regulation of Metabolism The Bacillus subtilis glutamate dehydrogenases RocG and GudB play a double game Fabian M. Commichau Department of General Microbiology, University of Göttingen, Grisebach-Str. 8, D-37077 Göttingen, Germany Key metabolic intersections in the central metabolism of an organism have to be tightly regulated to make the most out of nutrients that are available in a given environment. The enzymatic reactions involved in glutamate synthesis and degradation constitute an important metabolic intersection because they connect carbon to nitrogen metabolism in the Grampositive model bacterium Bacillus subtilis and in other organisms (1). During growth with the carbon source glucose and ammonium as a source of nitrogen, the transcription factor GltC activates the expression of the glutamate synthase genes. By contrast, in the absence of glucose and in the presence of a source of glutamate, the glutamate synthase genes are not expressed, and glutamate is converted to ammonium and 2-oxoglutarate of which the latter is fed into carbon metabolism. Recently, we found that B. subtilis possesses the glutamate dehydrogenases RocG and GudB that are both trigger enzymes, active in glutamate degradation and in controlling gene regulation (2,3). We could show that the enzymes function as sensors that depending on the intracellular glutamate concentration control glutamate synthesis through an inhibitory interaction with the transcription factor GltC (2). Here, I will discuss the recent findings on the control of a key metabolic intersection in B. subtilis and provide an overview about the evolutionary stages of trigger enzymes. References 1. Gunka, K. and Commichau, F.M. (2012) Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation. Mol Microbiol 85, 213-224. 2. Stannek, L., Thiele, M.J., Ischebeck, T., Gunka, K., Hammer, E., Völker, U., Commichau, F.M. (2015) Evidence for synergistic control of glutamate biosynthesis by glutamate dehydrogenases and glutamate in Bacillus subtilis. Environ Microbiol 17, 3379-3390. 3. Commichau, F.M., Stülke, J. (2015) Trigger enzymes: coordination of metabolism and virulence gene expression. Microbiol Spectr 3, doi: 10.1128/microbiolspec.MBP-00102014 16 The interplay between an Asp23 protein family member and acetyl-CoA carboxylase in Bacilus subtilis Dominik Tödter and Jörg Stülke Department of General Microbiology, University of Göttingen, Germany Grisebachstr. 8, 37077 Göttingen More than 20% of the proteins in the PFAM database belong to domain of unknown function (DUF) families, indicating that a large fraction of proteins is in need of further investigation. This is especially the case for the Asp23 protein family (DUF322). The name giving alkaline shock protein 23 (Asp23) of Staphylococcus aureus is one of the most abundant proteins and members of this family are both highly conserved and highly expressed in Gram-positive bacteria (1). Despite the obvious importance of these proteins, almost nothing is known about the functions of Asp23 proteins. The aim of our work is the characterization of the so far unknown proteins YqhY and YloU, the representatives of the Asp23 protein family in Bacillus subtilis. In contrast to the previously reported essentiality of yqhY (2), we were able to delete the yqhY gene. The deletion of yqhY resulted in the rapid acquisition of suppressor mutations that affect the subunits of the acetyl-CoA carboxylase (AccABCD). This protein complex catalyzes the first committed step in fatty acid biosynthesis, the conversion of acetyl-CoA to malonyl-CoA. The observed genetic link between YqhY and the acetyl-CoA carboxylase as well as the location of the yqhY gene in the strongly conserved accBC yqhY operon suggest an involvement of YqhY in fatty acid synthesis. On the other hand, in some cases the suppressor mutations lead to the deletion of ctsR, a transcription regulator of clpC, clpE, clpP and clpX. These genes encode the proteases responsible for protein quality control by degrading unfolded or aggregated proteins. This indicates a participation of YqhY in protein degradation as well. The results of ongoing investigations about a possible interplay between the acetyl-CoA carboxylase, Clp-dependent protein degradation and YqhY will be discussed. References 1 Müller et al., 2014. Mol. Microbiology. 93: 1259–1268 2 Thomaides et al., 2007. J. Bacteriol. 189: 591-602. 17 Compensation for glutamate auxotrophy of a Bacillus subtilis gltC mutant by three independent mutational events Miriam Dormeyer, Anastasia L. Lübke and Fabian M. Commichau Department of General Microbiology, University of Göttingen, Grisebach-Str. 8, D-37077 Göttingen, Germany Glutamate is the most abundant metabolite in all organisms and it fulfills a variety of important functions such as the supply of nitrogen for anabolic reactions (1,2). The Grampositive model bacterium Bacillus subtilis can either use exogenous glutamate provided by the medium or synthesize it from glutamine and 2-oxoglutarate using a glutamate synthase, which is encoded by the gltAB genes (2). In the absence of exogenous glutamate, the LysRtype transcription factor GltC activates the expression of the GOGAT-encoding genes to meet the need for glutamate, and to achieve high growth rates. Thus, it is not surprising that a gltC mutant strain is auxotrophic for glutamate (3). Using a genetic screening system, we have isolated several mutants that had acquired the ability to synthesize glutamate, independent of GltC. Whole genome re-sequencing analyses revealed (i) mutations in the gltR gene, encoding the LysR-type transcription factor GltR (ii) mutations in the promoter of the gltAB genes and in a gene of unknown function, and (iii) massive amplification of the genomic locus containing the gltAB genes. Thus, high genome flexibility is the key to relieve glutamate auxotrophy of a B. subtilis gltC mutant. Currently, we are investigating how and to which extent the various mutations enable the bacteria to synthesize glutamate. References 1. Park, J.O., Rubin, S.A., Xu, Y.F., Amador-Noguez, D., Fan, J., Shlomi, T., Rabinowitz, J.D. (2016) Metabolite concentrations, fluxes and free energies imply efficient enzyme usage. Nat Chem Biol 12, 482-489. 2. Gunka, K. and Commichau, F.M. (2012) Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation. Mol Microbiol 85, 213-224. 3. Bohannon, D.E. and Sonenshein, A.L. (1989) Positive regulation of glutamate biosynthesis in Bacillus subtilis. J Bacteriol 171, 4718-4727. 4. Belitsky, B.R. and Sonenshein, A.L. (1997) Altered transcription activation of a mutant form of Bacillus subtilis GltR, a LysR family member. J Bacteriol 179, 1035-1043. 18 Metabolic engineering to direct evolution in Corynebacterium glutamicum Andreas Schwentner1, E. Hoffart1, T. Busche2, C. Rückert2, J. Kalinowski2, R. Takors1, B. Blombach1 1 2 University of Stuttgart, Institute of Biochemical Engineering, Stuttgart, Germany Bielefeld University, Center for Biotechnology (CeBiTec), Bielefeld, Germany Metabolic engineering to direct evolution (MEDE) is a novel evolutionary approach, which enables the identification and evolution of new targets for improving microbial producer strains. It consists of a first step of metabolic engineering, whereby evolutionary pressure is put upon the strain of choice, a second step of evolutionary growth phase, eventually yielding an evolutionary event, and a third step of comparative whole genome sequencing (WGS), to identify evolutionary targets. Growth as straightforward screening method and the minimal amount of mutations make MEDE a time-saving and easy to handle evolutionary method. In an applied approach, the genes ppc and pyc, encoding the anaplerotic enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase, were deleted in C. glutamicum ATCC 13032. The resulting strain C. glutamicum Δppc Δpyc showed strongly impaired growth and was cultivated in minimal medium containing 40 g l-1 glucose and 1 g l-1 yeast extract. Cells were sequentially transferred every third day for 14 days including concomitant screening for faster growing mutants. After an evolutionary event, WGS was performed to identify relevant mutations. In contrast to the initial strain C. glutamicum Δppc Δpyc, which showed a growth rate of 0.17 h-1, three independently evolved mutants yielded growth rates of about 0.32 h-1, indicating mutational events. Interestingly, the intersection of the mutations obtained by WGS revealed isocitrate dehydrogenase (ICD) as consistent target in these strains. Upon re-engineering in the basis strain, said point mutations led to diminished ICD activities and an activation of the glyoxylate shunt enzymes isocitrate lyase and malate synthase. Both enzymes are typically repressed during growth on glucose as sole carbon source (1). Product suitability of the re-engineered strains was demonstrated by introducing plasmid pJC4ilvBNCE (2), ensuring overexpression of the L-valine biosynthesis genes. These strains accumulated up to 8.1 ± 1.0 g l-1 L-valine (3.4 times more than the reference C. glutamicum pJC4ilvBNCE), corresponding to a product yield of 0.16 ± 0.01 g L-valine per g glucose. This proofs ICD mutations as a potent alternative to a pyruvate dehydrogenase complex with reduced activity (3) for the production of L-valine. References 1. Gerstmeir, R., Wendisch, V.F., Schnicke, S., Ruan, H., Farwick, M., Reinscheid, D., and Eikmanns, B.J. (2003). J. Biotechnol. 104, 99–122. 2. Radmacher, E., Vaitsikova, A., Burger, U., Krumbach, K., Sahm, H., and Eggeling, L. (2002). Appl Environ Microbiol 68, 2246–2250. 3. Buchholz, J., Schwentner, A., Brunnenkan, B., Gabris, C., Grimm, S., Gerstmeir, R., Takors, R., Eikmanns, B.J., and Blombach, B. (2013). Appl Environ Microbiol 79, 5566–5575. 19 SubtiWiki, an integrated database for model organism Bacillus subtilis Bingyao Zhu and Jörg Stülke Department of General Microbiology, University of Göttingen, Germany Grisebachstr. 8, 37077 Göttingen Information collecting and sharing has always been an important aspect of biological research. In this internet era, online platforms, including databases and other kind of online resources, are more preferred to the traditional paper-based publishing. SubtiWiki (http://subtiwiki.uni-goettingen.de) (Michna et al., 2016), as one of those online resources, has its main focus on the model organism Bacillus subtilis. It is a free collection of manually curated annotation and provides access to public. In SubtiWiki, information of individual genes/proteins is the center of our focus. Moreover, knowledge of association among genes and proteins, in form of interaction, regulation, and biochemical fluxes are also gathered and presented in diagrams. Furthermore, gene and protein expression data are compiled and displayed in interactive charts. With our iOS and Android App (available in App Store and Google Play Store), mobile access to our data is guaranteed. Along with the constant update of data, the database structure has also undergone significant changes. A new database layout is applied. Upon that, a PHP framework is developed to meet the needs to organize data. The source code of the framework is scheduled to go open source under MIT license. With the new database layout, better organized information, clear visualization of data, we will continue assisting the researchers who focus on B. subtilis and other Gram-positive bacteria. References Michna et al., 2016. Nucleic Acids Res. 44(D1):D654-62. 20 Large-scale reduction of the Bacillus subtilis genome: Consequences for the transcriptional network, resource allocation, and metabolism Daniel Reuß1, Josef Altenbuchner2, Ulrike Mäder3, Hermann Rath3, Till Ischebeck4, Bingyao Zhu1, Stefan Klumpp5, Fabian Commichau1, Uwe Völker3 and Jörg Stülke1 1 Dept. of General Microbiology, University of Göttingen, Grisebachstr. 8, 37077 Göttingen 2 Institute of Industrial Genetics, University of Stuttgart 3 Institute for Genetics and Functional Genomics, University Medicine Greifswald 4 Dept. of plant Biochemistry, University of Göttingen 5 Institute for Nonlinear Dynamics, University of Göttingen Understanding cellular life requires a comprehensive knowledge of the essential cellular functions, the components involved, and their interactions. Minimized genomes are an important tool to gain this knowledge. We have constructed strains of the model bacterium Bacillus subtilis whose genomes have been reduced by about 36%. These strains are fully viable and their growth rates in complex medium are comparable to those of wild type strains. An in-depth multi-omics analysis of the genome reduced strains revealed how the deletions affect the transcription regulatory network of the cell, translation resource allocation, and metabolism. A comparison of gene counts and resource allocation demonstrates drastic differences in the two parameters, with 50% of the genes using as little as 10% of translation capacity whereas the 6% essential genes require 57% of the translation resources. Taken together, the results are a valuable resource on gene dispensability in B. subtilis, and they suggest the roads to further genome reduction to approach the final aim of a minimal cell in which all functions are understood. 21 Session 3: Second Messengers, bacterial differentiation, and antibiotic production Cyclic di-nucleotide signalling in bacterial differentiation and antibiotic production Natalia Tschowri1, Maria A. Schumacher2, Susan Schlimpert3, Naga babu Chinnam2, Richard G. Brennan2 and Mark J. Buttner3 1 Institut für Biologie - Mikrobiologie, Humboldt-Universität zu Berlin, Philippstraße 13, 10115 Berlin 2 Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA 3 Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK The multi-talented bacteria Streptomyces are „Microbe of the Year 2016“ (VAAM) and have been awarded the Nobel Prize twice (1952 and 2015) for their exceptional ability to produce diverse medically-useful natural products. The synthesis of these secondary metabolites is genetically and temporally tightly interlinked with the developmental life cycle of Streptomycetes, and facing the urgent need for new antibiotics it is of particular significance to understand the signals and pathways that control development in these bacteria. In our recent study, we have shown that the bacterial second messenger cyclic di-GMP (c-di-GMP), which is produced by GGDEF-type diguanylate cyclases and degraded by EALor HD-GYP-type phosphodiesterases, determines the timing of differentiation initiation in S. venezuelae by regulating the activity of the highly conserved developmental master regulator BldD. Our structural and biochemical analyses revealed that a tetrameric form of c-di-GMP activates BldD DNA-binding by driving a unique form of protein dimerization, leading to repression of the BldD regulon of sporulation genes during vegetative growth (1, 2, 3). Currently, we aim to understand which of the 10 putative c-di-GMP-metabolising enzymes encoded by S. venezuelae contribute to c-di-GMP pool(s) sensed by BldD and how the BldD-c-di-GMP complex is assembled. Our initial data indicate that a distinct set of GGDEF / EAL proteins influences the developmental programme progression in S. venezuelae and that loading of BldD with tetrameric c-di-GMP is a two-step process. Altogether, our work will greatly improve our understanding of Streptomyces physiology and c-di-GMP signalling in multicellular differentiation and secondary metabolite production and can contribute to a better exploitation of genetic engineering in Streptomyces for the production of antibiotics. References 1. Tschowri N, Schumacher MA, Schlimpert S, Chinnam NB, Findlay KC, Brennan RG, Buttner MJ. (2014) Tetrameric c-di-GMP Mediates Effective Transcription Factor Dimerization to Control Streptomyces Development. Cell, 2014 Aug 28;158(5):1136-47 2. Bush MJ, Tschowri N, Schlimpert S, Flärdh K, Buttner MJ. c-di-GMP signalling and the regulation of developmental transitions in streptomycetes. (2015) Review. Nature Reviews Microbiology, 2015 Dec; 13(12):749-60 3. Tschowri N. (2016) Cyclic dinucleotide-controlled regulatory pathways in Streptomyces. Review. Journal of Bacteriology, 2016 Jan;198 (1):47-54 22 Spore memory couples entry and exit from bacterial dormancy in Bacillus subtilis Alper Mutlu, Stephanie Trauth, Marika Ziesack, Sonja Schulmeister and Ilka Bischofs Bioquant Center, University of Heidelberg Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany In bacteria, entry into and exit from dormancy are controlled by regulatory networks with little known overlap, indicating that the two processes operate independently from each other. Here we show that both processes are linked by phenotypic memory. Using B. subtilis as a model we developed an advanced time-lapse microscopy assay and a fluorescent marker that reports on a spore’s differentiation history to study the effect of variable sporulation timing on nutrient-induced spore revival. Early spores were kinetically favored over late spores on different levels of the revival pathway and in response to different stimuli. We furthermore show that spore outgrowth is controlled by phenotypic memory and can be reprogrammed with alanine dehydrogenase. As a consequence of the simple coupling provided by phenotypic memory, genetic changes that affect sporulation timing also affect the spore revival properties. We therefore suggest that phenotypic memory contributes to the emergence of complex adaptive traits. 23 Regulation of Secondary Metabolite Gene Clusters in Social Myxobacteria Carsten Volz and Rolf Müller. Department Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland, University Campus E8 1, D-66123 Saarbrücken, Germany Motile predatory myxobacteria are producers of multiple secondary metabolites and, on starvation, undergo concerted cellular differentiation to form multicellular fruiting bodies. These abilities demand myxobacterial genomes to encode sophisticated regulatory networks which are not satisfactorily understood. One approach in our efforts to identify and characterize new natural compounds exhibiting interesting biological activities is to elucidate the transcriptional regulation of the respective secondary metabolite gene clusters. The genetic manipulation of such regulatory components should enable an increased production of interesting natural compounds or should enable the induction of the production of unknown secondary metabolites. We here give an overview on interesting new regulatory mechanisms we have been able to elucidate but also on the drawback of such an approach working with myxobacteria. 24 Session 4: Regulation by RNA From Strings of Nucleotides to Collective Behavior: “Lessons from Vibrio cholerae” Kai Papenfort Faculty of Biology I, Ludwig-Maximilians-University, Munich Quorum-sensing (QS), is a process of bacterial cell-to-cell communication that relies on the production, release, and population-wide detection of extracellular signal molecules. Processes controlled by QS are unproductive when undertaken by an individual bacterium but become effective when undertaken by the group. QS controls many important microbial processes including bioluminescence, secretion of virulence factors, competence and biofilm formation. In this study, we identified and characterized of a novel bacterial communication system present in Vibrio cholerae. This system consists of a transcriptional regulator, VqmA and a small regulatory RNA (sRNA), VqmR. VqmR is activated by VqmA and functions as a trans-acting regulator through base-pairing with multiple target mRNAs. Among these targets are key factors for biofilm formation and virulence factor expression in V. cholerae indicating that VqmA/R could participate in the regulation of complex behaviors. Indeed, our results show that VqmA binds to and is activated by an extracellular signal, which we determined as the novel autoinducer molecule, DPO. DPO is a new molecule to biology and is produced by diverse pro- and eukaryotes. Further, we obtained evidence that the signaling molecule is produced by commensal species of the host microbiota and that VqmA/R plays an important role during V. cholerae pathogenesis and the communication with other bacteria. 25 Surprising small RNA features in Rhodobacter sphaeroides Bernhard Remes, Katrin Müller, Lennart Weber and Gabriele Klug. Department of Microbiology and Molecular Biology, University of Gießen, Heinrich-Buff-Ring 26-32, 35390 Gießen, Germany Among the strategies developed by bacteria to adapt to environmental changes is the use of sRNAs, which usually act at the post-transcriptional level via base-pairing with the targeted mRNA. Since trans-encoded sRNAs are located in another chromosomal location and are only partially complementary to their target mRNAs, most of them require Hfq for their stability in the cell and their regulatory function [1, 2]. In Rhodobacter sphaeroides RNAseq-based studies identified amongst others the sRNAs RSs0827 [3] and UpsM [4]. RSs0827 is the gene with the highest increase in expression after 60 h of stationary phase (Remes et al., submitted). UpsM is the most abundant orphan sRNA of R. sphaeroides and represents about 60% of all Hfq bound sRNAs [5]. Upon several stress conditions, RNase E cleaves UpsM in an Hfq- and target-dependent manner (Weber et al., submitted). The sRNA is located in the 5’ UTR of mraZ, the first gene of the dcw (division and cell wall) gene cluster. Transcription of mraZ depends on the UpsM promoter, which implicates that the terminator of UpsM allows read-through in order to guarantee transcription of mraZ. However, an answer for the regulatory function of the two sRNAs or the target responsible for processing of UpsM remained unclear. Using in vitro and in vivo experiments, we characterized UpsM as an atypical target for RSs0827. Indeed, RSs0827 base pairs in the 5’ region of upsM and triggers the decay of UpsM, henceforth renamed StsR (sRNA targeting sRNA). Moreover, cells lacking StsR showed increased read-through of the UpsM terminator, leading to mraZ expression and in turn to continuous growth even in late stationary phase. Collectively, we present the first interaction between two sRNAs and further widen our knowledge about the interplay between sigma factors, Hfq and sRNAs, as well as their role in controlling cell division in response to external stresses. References 1. Vogel, J. and B.F. Luisi, Hfq and its constellation of RNA. Nat Rev Microbiol, 2011. 9(8): p. 578-89. 2. Valentin-Hansen, P., M. Eriksen, and C. Udesen, The bacterial Sm-like protein Hfq: a key player in RNA transactions. Mol Microbiol, 2004. 51(6): p. 1525-33. 3. Remes, B., et al., Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides. BMC Genomics, 2014. 15: p. 794. 4. Berghoff, B.A., et al., Photooxidative stress-induced and abundant small RNAs in Rhodobacter sphaeroides. Mol Microbiol, 2009. 74(6): p. 1497-512. 5. Berghoff, B.A., et al., Contribution of Hfq to photooxidative stress resistance and global regulation in Rhodobacter sphaeroides. Mol Microbiol, 2011. 80(6): p. 1479-95. 26 Session 5: Regulation in Biofilms Exploitation of the biofilm matrix to colonize a surface Ákos T. Kovács Terrestrial Biofilms Group, Institute of Microbiology, Friedrich Schiller University Jena, Neugasse 23, D-07743 Jena, Germany Multicellular biofilm formation and surface motility are bacterial behaviors considered as mutually exclusive. Moreover, the basic decision to move over or stay attached to a surface is poorly understood in bacteria. It is well established that flagellum based individual cell based motility is required for founding an air-liquid interface biofilm (1). In Bacillus subtilis, the key sporulation- and biofilm-controlling transcription factor, Spo0A~P governs the flagellaindependent mechanism of social sliding motility (2). Microarray experiments and subsequent genetic characterization revealed that the machineries of sliding and biofilm formation, share the same secreted components (i.e. surfactin, the hydrophobin BslA, and exopolysaccharide). Sliding proficiency is transduced by the Spo0A-phosphorelay histidine kinases KinB and KinC, while potassium is the specific sliding-activating signal through a cytosolic domain of KinB, which resembles the selectivity filter sequence of potassium channels. Interestingly, the gradual increase in Spo0A~P orchestrates the sequential activation of sliding, followed by sessile biofilm formation and finally sporulation in the same population (2,3). Similar to sliding, the secreted matrix benefits colony biofilm expansion, while its production is costly for the individuals. Mutant strains lacking matrix production have a higher fitness under well mixed planktonic conditions. However, matrix producers have an advantage when cultivated in a spatially structured environment (4). The density of cells at the onset of biofilm growth on a solid surface affects pattern formation and high assortment facilitates cooperation during biofilm growth (4,5). In sum, I will present how multicellular behaviors (sessile biofilm development and surface spreading) are coordinately activated and highlight the impact of spatial assortment (6) and diffusion on privatization of secreted components prerequisite for sliding. References 1. Hölscher, T., Bartels, B., Lin, Y.-C., Gallegos-Monterrosa, R., Price-Whelan, A., Kolter, R., Dietrich, L.E.P. and Kovács, Á.T. (2015) Motility, chemotaxis and aerotaxis contribute to competitiveness during bacterial pellicle biofilm development. Journal of Molecular Biology 427, 3695-3708. 2. Grau, R., de Oña, P., Kunert, M., Leñini, C., Gallegos-Monterrosa, R., Mhatre, E., Vileta, D., Donato, V., Hölscher, T., Boland, W., Kuipers, O.P. and Kovács, Á.T. (2015) A duo of potassiumresponsive histidine kinases govern the multicellular destiny of Bacillus subtilis. mBio 6, e00581-15. 3. Kovács, Á.T. (2016) Bacterial differentiation via gradual activation of global regulators. Current Genetics 62, 125-128. 4. van Gestel, J., Weissing, F.J., Kuipers, O.P. and Kovács, Á.T. (2014) Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms. ISME Journal 8, 2069– 2079. 5. Kovács, Á.T. (2014) Impact of spatial distribution on the development of mutualism in microbes. Frontiers in Microbiology 5, 649. 6. Hölscher, T., Dragoš, A., Gallegos-Monterrosa, R., Martin, M., Mhatre, E., Richter, A. and Kovács, Á.T. (2016) Monitoring spatial segregation in surface colonizing microbial populations. Journal of Visualized Experiments e54752. 27 Cell-cell communications in Bacillus subtilis mixed-species biofilms Ramses Gallegos-Monterrosa1, Stefanie Kankel1, Sebastian Götze2, Pierre Stallforth2, Ákos T. Kovács1 1 Terrestrial Biofilms Group, Friedrich Schiller University Jena. Neugasse 23, 07743 Jena, Germany 2 Leibniz Institute for Natural Product Research and Infection Biology. Beutenbergstraße 11/a, 07745 Jena, Germany Biofilm development in diverse bacteria has been shown to response to multiple environmental signals like small molecules secreted by microorganisms. These signals have been usually considered to be self-generated, i.e. quorum-sensing, but can also be produced by other organisms living in the vicinity, thus creating an interspecies communication network. Bacillus subtilis is a Gram-positive model bacterium for studying biofilm formation. It differentiates into several subpopulations of specialized cell types in response to different environmental cues thus making it an ideal model for studying complex signaling networks. Multiple soil bacteria can produce small signaling molecules that influence biofilm formation by B. subtilis. We aim to identify such organisms and to characterize the chemical nature and signaling pathway of novel signaling molecules that are able to modify the architecture of B. subtilis biofilms. Bacteria isolated from soil samples were screened for their ability to produce signaling molecules able to modify the structure of B. subtilis biofilms. Five soil isolates were selected for further characterization due to their ability to modify B. subtilis complex colony structures. These bacteria were identified through 16S rDNA sequencing. Four of the isolates were identified as either Lysinibacillus sp. or Bacillus pumilus, which are closely related to B. subtilis and thus may share similar signaling mechanisms. The culture supernatants of selected bacteria were submitted to chromatography, enzymatic, and biochemical analysis to discern the nature of the signaling molecules. The purine derivative hypoxanthine was identified as a signaling molecule produced by one of the Lysinibacillus sp. isolates. B. subtilis is able to recognize and respond to several signaling molecules produced mainly by members of closely related genus but also from non-related organisms that may share the same ecological niche. Hypoxanthine is one such molecule that has shown to increase wrinkle formation in complex colony biofilms of B. subtilis. The signalling pathway of hypoxanthine on B. subtilis is being studied through the use of knock-out mutants and fluorescent gene-expression reporter fusions. 28 Session 6: Stress responses and Signal Transduction Connecting cell wall homeostasis and a major envelope stress response in E.coli Geraldine Laloux Institut de Duve, Université catholique de Louvain Avenue Hippocrate, 75 - B1.75.08, 1200 Bruxelles The envelope of Gram-negative bacteria is an essential compartment that constitutes a protective and permeability barrier between the cell and its environment. The envelope also hosts the cell wall, a mesh-like structure made of peptidoglycan (PG) that determines cell shape and provides osmotic protection. Since the PG must grow and divide in a cell-cyclesynchronized manner, its synthesis and remodeling are tightly regulated. Here, we discovered that PG homeostasis is intimately linked to the levels of activation of the Cpx system, an envelope stress response system traditionally viewed as being involved in protein quality control in the envelope. We first show that Cpx is activated when PG integrity is challenged and that this activation provides protection to cells exposed to antibiotics inhibiting PG synthesis. By rerouting the outer membrane lipoprotein NlpE, a known Cpx activator, to a different envelope subcompartment, we managed to manipulate Cpx activation levels. We found that Cpx overactivation leads to aberrant cellular morphologies, to an increased sensitivity to b-lactams, and to dramatic division and growth defects, consistent with a loss of PG homeostasis. Remarkably, these phenotypes were largely abrogated by the deletion of ldtD, a Cpx-induced gene involved in noncanonical PG cross-linkage, suggesting that this transpeptidase is an important link between PG homeostasis and the Cpx system. Altogether our data show that fine-tuning of an envelope quality control system constitutes an important layer of regulation of the highly organized cell wall structure. 29 The Cpx-system of Escherichia coli analyzed by SRM and Super-resolution Microscopy Emina Ćudić1, Kristin Surmann2, Rainer Kurre3, Elke Hammer2 and Sabine Hunke1. 1 Department of Microbiology, University of Osnabrueck, Barbarastraße 11, D-49076 Osnabrueck, Germany 2 Department of Functional Genomics, University of Greifswald, Friedrich-Ludwig-Jahn Straße 15A, D-17475 Greifswald, Germany 3 Department of Biophysics, University of Osnabrueck, Barbarastraße 11, D-49076 Osnabrueck, Germany Bacteria rely on two-component systems (TCS) in order to sense and response to environmental changes and thus to different stimuli [1]. These systems make use of a phosphorylation cascade from a transmembrane sensor kinase (SK) to a cytoplasmic response regulator (RR) and are set back to the initial state via desphosphorylation of the RR [1]. For a better understanding of the functionality and the dynamics of a TCS it is important to know the absolute amounts of the proteins and their localization within living cells. Here, we used the Cpx-envelope stress TCS as a model. It consists of the inner membrane-spanning SK CpxA, the cytosolic RR CpxR and the periplasmic accessory protein CpxP, which inhibits the autophosphorylation activity of CpxA [2, 3]. We investigated absolute protein amounts by single reaction monitoring (SRM) and the localization of CpxA and CpxP by super-resolution microscopy. We could determine absolute amounts for CpxA, CpxR and CpxP being 41 molecules/cell (CpxA), 393 molecules/cell (CpxR) and 36 molecules/cell (CpxP) under Cpxnon affecting conditions [4]. However, although it is known that CpxA and CpxP interact in order to keep the Cpx-TCS in an OFF-state [5], the affinity between CpxP and CpxA is very low [6]. In combination with low absolute amounts per cell we asked how CpxA and CpxP might physically interact in living cells to promote inhibition of the Cpx-TCS. Therefore, we investigated the localization of CpxA and CpxP under different conditions in living cells and analyzed whether CpxA and CpxP co-localize as a prerequisite for functional interaction. We generated chromosomal fusions of CpxA with the SNAP-Tag® and CpxP with the HaloTag®. These fusions enable native protein levels and covalent staining with different combinations of fluorescent dyes for detection by Total Internal Refraction Fluorescence (TIRF)-Microscopy. We quantified co-localization, close-together localization and no colocalization events and found the biggest shift from close-together localization to colocalization after inhibition of the Cpx-TCS by cpxP-overexpression. Altogether, our results demonstrate the importance of a combination of in vitro and in vivo studies to get a deeper insight into the function of a TCS. References 1. Stock, A. M.; Robinson, V. L. and Goudreau, P. N. (2000). Annu. Rev. Biochem. 69, p. 183-215. 2. Fleischer, R; Heermann, R.; Jung, K, and Hunke, S. (2007). J. Biol. Chem. 282, p. 85838593. 3. Hunke, S.; Keller, R., and Müller, V.S. (2012). FEMS Microbiol. 326, p. 12–22. 4. *Surmann, K.; *Ćudić, E.; Hammer, E., and Hunke, S. (2016). MicrobiologyOpen doi: 10.1002/mbo3.353 5. Tschauner, K.; Hörnschemeyer, P.; Müller, V.S.; Hunke, S. (2014) PLoS ONE 9(9). e107383. doi:10.1371/journal.pone.0107383 30 6. Hörnschemeyer, P.; Liss, V.; Heermann, R.; Jung, K. and Hunke, S. (2016) PLoS ONE 11(2). e0149187. doi:10.1371/journal.pone.0149187 Uncovering the regulatory circuits of the glycine betaine synthesizing pathway in Bacillus subtilis Bianca Warmbold, Stefanie Ronzheimer, Tamara Hoffmann , Erhard Bremer. Laboratory for Microbiology, Department of Biology, Philipps-University Marburg, Karl-von-Frisch Str. 8, D-35043 Marburg, Germany Confronted with hyperosmotic stress, the soil bacterium Bacillus subtilis accumulates compatible solutes to maintain cell turgor, and to sustain growth under osmotically unfavorable circumstances. Glycine betaine is such a compatible solute and it can either be taken up from the environment via several Opu-transporters or it can be synthesized from the precursor choline. Oxidation of choline is mediated by the dehydrogenases GbsB and GbsA, whose structural genes are transcribed as an operon (1, 2). Upstream of the gbsAB gene cluster, the gbsR gene is located which encodes a choline-responsive repressor regulating the expression of the gbsAB operon as well as the opuB operon, which encodes a substratespecific ABC transporter for choline (3). Bioinformatic analysis of the DNA-region upstream of the gbsAB operon and of the opuB operon revealed a palindromic repeat within the in silico predicted GbsR binding site (4). We showed that mutations targeting these regions abolish GbsR-mediated repression as shown by gbsA-treA and opuB-treA reporter fusion studies. Binding of the ligand choline to the GbsR regulator relieves repression of the gbsAB operon. An in silico model of the GbsR protein structure hints that four phenylalanines arranged in an aromatic cage probably form a binding pocket for the inducer choline. We constructed mutants of the GbsR homologue OpuAR of B. infantis NRRL B-14911 and determined their binding affinity for choline by fluorescence spectroscopy. With this approach we were able to show that indeed each of the phenylalanine residues is involved in choline binding. Since GbsR acts as repressor of the opuB but not of the closely related opuC operon (it encodes an ABC transporter for various osmostress protectants, including choline), we studied the networks involved in the regulation of both gene clusters at the transcriptional level. This study revealed a striking difference in the expression pattern between the two operons. Whereas the expression of the opuB operon increases with rising salt concentrations, the opuC operon shows the strongest expression at moderate salt concentrations. However, they share the essential activation by the biofilm activator RemA (5) and the regulation through the MarR-type repressor OpuCR. We uncovered the physiological role of OpuCR, since we were able to show that it is involved in the re-establishment of opuC repression under high salt concentrations, a function in agreement with the salt-induced expression of the opuCR gene. Taken together, our findings highlight a complex regulatory circuit of the pathways leading to the formation of the cellular pool of the cytoprotectant glycine betaine in osmotically stressed B. subtilis cells. References 1. Boch, J. et al. (1996) J. Bacteriol. 178:5121-56129 2 .Boch, J. et al. (1997) Arch. Microbiol. 168:282-289 3. Nau-Wagner, G. et al. (2012) J. Bacteriol. 194:2703-2714 4. Leyn, S. et al. (2012) J. Bacteriol. 195:2463-2473 31 5. Winkelman, J.T. et al. (2013) Mol. Microbiol. 88:984-997 32 Characterization of the ABC-Transporter Associated Two-Component System YxdJK in Bacillus subtilis as Biosensor for Eukaryotic Antimicrobial Peptides Katharina Sievers, Diana Wolf and Thorsten Mascher. Institute of Microbiology, Technical University Dresden, Zellescher Weg 20 b, Dresden, Germany The cell envelope of bacteria represents the primary target of many antimicrobial substances, especially antimicrobial peptides (AMPs). Therefore, an intact cell envelope integrity is crucial for survival of the cell. Bacteria evolved designated cell envelope stress response (CESR) mechanisms to constantly monitor and maintain the cell envelope integrity. The main part of CESRs is mechanistically mediated by signal transduction via twocomponent systems (TCS) that links extracellular signal perception by a histidine kinase to a corresponding cellular response realized by a response regulator [1]. In B. subtilis, the CESRmediating TCSs are mostly associated to ABC-transporters which confer resistance to antimicrobial compounds. The Bce-system that responds highly specific to bacitracin and several other structurally related AMPs has been investigated very well [2]. The genome of B. subtilis contains two further Bce-like systems, the Psd- and the Yxd-system which are also involved in mediating resistance to peptide antibiotics. Regarding the Yxd-system, the expression of the ABC-transporter YxdLM controlled by the response regulator YxdJ that binds to PyxdL after induction caused by the human immune peptide LL-37 has already been described [3,4]. Recently, we observed a 1000 to 10000-fold activation of the Yxd-system after treatment with larvae extracts of the black soldier fly Hermetia illucens. Microarray studies emphasized the expression of diverse AMPs in H. illucens larvae. Accordingly, we investigated the Yxd-system induced with H. illucens larvae extracts in more detail and found that the system confer resistance to larvae extracts. Furthermore on basis that the Yxd-system responds specifically to AMPs produced by eukaryotes, an application of the system as a powerful biosensor for the identification of eukaryotic AMPs is feasible. References [1] Schrecke et al., 2012, In: Gross R, Beier D (eds) Two component systems in bacteria. Horizon Scientific Press, Hethersett, Norwich, UK, pp. 199-229 [2] Rietkötter et al., 2008, Mol Microbiol 68(3):768-85 [3] Joseph et al., 2004, Microbiology 150(8):2609-2617 [4] Pietiäinen et al., 2005, Microbiology 151(5):1577-1592 33 Session 7: Diverse Regulators A new piece in the big puzzle of cell division: The transcriptional regulator FtsR regulates FtsZ in Corynebacterium glutamicum Kim Julia Kraxner, Meike Baumgart, Michael Bott. IBG-1: Biotechnology, Institute of Bio- and Geosciences Forschungszentrum Jülich, Germany Corynebacterium glutamicum is a non-pathogenic, aerobic, Gram-positive soil bacterium which is used for the large scale production of several L-amino acids and other industrially relevant compounds [1]. Moreover, it is a useful model organism for the Corynebacteriales, including pathogenic species such as Corynebacterium diphtheriae and Mycobacterium tuberculosis. The inhibition of microbial cell division serves as an attractive target for the development of new antimicrobial drugs. Whereas the key steps in cell division and their regulation are well understood in other model bacteria such as E. coli and B. subtilis, knowledge about positive and negative regulators of cytokinesis in Actinobacteria is very limited [2]. In our studies we analyzed FtsR, a so far uncharacterized transcriptional regulator of C. glutamicum. A ftsR deletion mutant showed growth defects and a drastically altered cell morphology, suggesting a malfunction of cell division or cell wall synthesis. The wild-type phenotype could be restored by re-integrating ftsR at a different position in the chromosome. Additionally, full complementation of the growth defect and of the morphological phenotype was achieved by plasmid-based heterologous expression of the ftsR homolog from C. diphtheriae. To determine potential target genes of FtsR, chromatin affinity purification with subsequent next generation sequencing (ChAP-Seq) was performed, which revealed a region upstream of ftsZ as a major target. Using the ChAP-Seq results and the motif-based sequence analysis tool MEME-ChIP, a putative DNA-binding motif could be identified for FtsR. DNA microarray experiments revealed significantly reduced ftsZ mRNA levels in the ftsR mutant compared to the wildtype. Furthermore, electrophoretic mobility shift assays (EMSAs) and reporter gene studies confirmed ftsZ to be an FtsR target gene. In summary, a novel transcriptional regulator of C. glutamicum was identified, which serves as an activator of ftsZ expression and is required for normal growth and cell morphology. References 1. Eggeling, L., Bott, M. (2005) Handbook of Corynebacterium glutamicum. CRC Press, Boca Raton, USA 2. Donovan C., Bramkamp M. (2014) Cell division in Corynebacterianeae. Front Microbiol 5:132 34 Silencing of cryptic prophages in Corynebacterium glutamicum Eugen Pfeifer1, Max Hünnefeld1, Ovidiu Popa2, Meike Baumgart1, Tino Polen1, Dietrich Kohlheyer1 and Julia Frunzke1. 1) Institute of Bio- und Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425 Jülich, Germany 2) Quantitative and Theoretical Biology, Heinrich-Heine-Universität Düsseldorf 40225, Düsseldorf, Germany Viral DNA and prophage-like elements can account for up to 20% of the entire bacterial genome and may significantly contribute to their host’s fitness [1]. However, acquisition of new genetic elements bears risks for their hosts since their activation or gene expression may cause high metabolic costs and can even lead to cell death by lysis. To enable controlled integration of newly acquired DNA into the host’s regulatory network organisms rely on the action of small nucleoid-associated proteins, which function as xenogeneic silencers of foreign DNA elements [2]. In our studies, we use the Gram-positive soil bacterium Corynebacterium glutamicum ATCC 13032 as model system to study prophage-host interactions. The genome of ATCC 13032 comprises three cryptic prophages (CGP1-3), of which CGP3 was shown to undergo spontaneous activation in a small fraction of cells [3]. However, the molecular factors controlling CGP3 activity are currently not known. Recently, we identified a small nucleoid-associated protein, named CgpS, which is encoded on the CGP3 prophage island and shares sequence similarity with the mycobacterial xenogeneic silencer Lsr2. In our studies, we could show that CgpS is an essential gene due to its function as a silencer of cryptic phage elements in C. glutamicum. Genome-wide binding analyses displayed the preferred association to AT-rich elements, especially to the CGP3 prophage, but also to other regions which have likely been acquired by horizontal gene transfer. Bioinformatical analysis revealed orthologous proteins in almost all Actinomycetes, but remarkably, also in several phage and prophage genomes. Our results emphasize CgpS as a key factor for the control of CGP3 activity and highlight the importance of small nucleoidassociated proteins for the control of foreign DNA in bacterial host strains. References [1] Nanda, A.M., K. Thormann & J. Frunzke, (2015) Impact of spontaneous prophage induction on the fitness of bacterial populations and host-microbe interactions. J Bacteriol 197: 410-419. [2] Pfeifer, E, M. Hünnefeld, O. Popa, T. Polen, D. Kohlheyer, M. Baumgart & J. Frunzke (2016). Silencing of cryptic prophages in Corynebacterium glutamicum. Nucleid Acid Res In Revision. [3] Helfrich, S., E. Pfeifer, C. Kramer, C.C. Sachs, W. Wiechert, D. Kohlheyer, K. Noh & J. Frunzke, (2015) Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations. Mol Microbiol 98: 636-650. 35 Inverse correlation between the transcription rate and H-NS/StpA repression in Escherichia coli Aathmaja A. Rangarajan and Karin Schnetz. Institut für Genetik, Universität zu Köln, Zülpicher Str. 47a, 50674 Cologne, Germany. The nucleoid-associated protein H-NS is an enterobacterial global repressor that inhibits transcription by forming extended DNA stiffening or bridging complexes. H-NS represses transcription at the level of initiation either by excluding RNA polymerase or by trapping it at promoters. StpA is a H-NS paralogue that presumably acts similarly as H-NS. H-NS (and StpA) mediated repression can be relieved locus specifically by binding of specific transcription factors, by perturbations of the DNA structure, and other mechanisms [1]. However, it is an open question whether H-NS (and StpA) complexes also interfere with transcription elongation. In vitro, at conditions that favour bridging, H-NS has been shown to enhance RNA polymerase pausing and to promote Rho-dependent termination [2]. Complementarily, it was shown that inhibition of Rho-mediated termination that results in increased transcription reduces H-NS binding [3]. Furthermore, our previous data revealed an inverse correlation between the efficiency of repression by H-NS when binding within the transcription unit and the strength of the promoter that is directing transcription across the HNS-bound DNA tract [4]. In this project we analyzed the effect of transcription elongation on repression by H-NS. In our experimental system we inserted a cassette consisting of a constitutive promoter and conditional transcriptional terminator (λtR1) upstream of H-NS (and StpA) loci, and varied the transcription rate by expressing anti-terminator protein λN. Our data show that transcription elongation across H-NS-bound DNA tracts relieves repression of H-NS and H-NS/StpA repressed promoters. In addition we observed a linear inverse correlation of transcription across the H-NS-bound DNA tracts and H-NS-mediated repression. The data suggest that the transcribing RNA polymerase is able to remodel the H-NS (and StpA) complex and/or dislodge H-NS (and StpA) from the DNA and thus relieve repression, while at low transcription rates the H-NS repression complex is stable. 36 Characterization of structural features controlling activity of LeuO, a pleiotropic transcriptional regulator and H-NS antagonist Susann M. Fragel and Karin Schnetz. Universität zu Köln, Institut für Genetik, Zülpicher Str. 47a, 50674 Köln LeuO is a conserved LysR-type transcriptional regulator of global function, controlling more than 100 genes in Escherichia coli and Salmonella enterica. LeuO is required in the control of pathogenicity, stress adaptation, and biofilm formation in various enterobacterial species. A vast majority of the LeuO regulated genes are regulated together with the nucleoidstructuring and global repressor protein H-NS and LeuO is considered a global H-NS antagonist. LeuO function and leuO gene regulation have been addressed in depth. Nonetheless, open questions are which signals induce leuO expression and whether the LeuO protein activity is modulated by a co-effector, similar to the control of other regulators of the LysR-family. In this project, we characterize structural features that control LeuO activity. In a first step, we analyzed the control of LeuO target promoters, among which the CRISPR-associated cas promoter turned out to be the target that is most specifically regulated by LeuO in E. coli. Using the cas promoter as reporter, we then performed site-directed and random mutagenesis screens for LeuO mutants with changed activity. By this approach we identified amino acid residue exchanges that render LeuO hyper-active. Mapping of these residues onto a predicted structure of the C-terminal effector-binding domain of LeuO suggests that these residues are all surface exposed, with two of them mapping at the entrance of the presumptive co-effector binding cleft. Additional substitutions of amino acid residues, located within the presumptive co-effector binding cleft, inactivate the LeuO protein. Preliminary data obtained of the crystal structure of the C-terminal effector-binding domain of LeuO support this interpretation. Our structural and functional characterization of LeuO suggests that LeuO activity is modulated by a co-effector. This is relevant in understanding the molecular mechanism of transcriptional regulation by LeuO, and the role of LeuO in the bacterial stress response, CRISPR-cas mediated immunity, and pathogenicity. 37 Feedback control of leuO encoding a pleiotropic regulator and H-NS antagonist in Escherichia coli Hannes Breddermann and Karin Schnetz. Institut für Genetik, Universität zu Köln, Zülpicher Str. 47a, 50674 Köln, Germany The enterobacterial LeuO protein is a pleiotropic LysR-type transcriptional regulator that is conserved in Enterobacteriaceae and plays an important role in pathogenicity, stress adaptation and the CRISPR/Cas immunity system. At standard growth conditions, expression of leuO is silenced by the master regulator H-NS and by its paralogue StpA. Expression of leuO can be activated by BglJ-RcsB and involves a double-positive feedback loop regulation. The leuO gene is activated by BglJ-RcsB [2], and LeuO activates expression of bglJ, encoded within the H-NS repressed yjjQ-bglJ operon [1]. Activation of leuO by BglJ-RcsB is in addition antagonistically controlled by LeuO [2] suggesting that the double-positive feedback regulation of leuO is tightly controlled. The activation dynamics of the leuO promoter by the antagonistic action of LeuO and BglJ-RcsB were characterized by a leuO promoter fluorescence reporter fusion in dependence of ectopically expressed LeuO and BglJ. The leuO promoter activity was analyzed by flow cytometry. Results suggest that the antagonistic control of the leuO promoter activity by LeuO and BglJ is controlled in dependence of their relative concentration. H-NS and StpA mediated silencing probably keeps leuO in an OFF state and dominates positive feedback regulation of leuO and bglJ at standard laboratory growth conditions. The data are in agreement with a straightforward model of antagonistic regulation by the two regulators that act independently of each other. Furthermore, screening for additional activators of leuO revealed LrhA as further regulator. LrhA activates a third and a fourth leuO promoter and shows direct DNA-binding to the leuO promoter region. The obtained data suggest a coregulation of the leuO promoter by BglJ-RcsB, LeuO and LrhA, reminiscent of complex regulation of leuO expression. This tightly controlled and complex regulation is likely to be important in the response to specific, virulence-related environments. References 1. Stratmann, T., Madhusudan, S., & Schnetz, K. Regulation of the yjjQ-bglJ operon, encoding LuxR-type transcription factors, and the divergent yjjP gene by H-NS and LeuO. J. Bacteriol. 190, 926-935 (2008). 2. Stratmann, T., Pul, Ü. Wurm, R., Wagner, R., & Schnetz, K. RcsB-BglJ activates the Escherichia coli leuO gene, encoding an HNS antagonist and pleiotropic regulator of virulence determinants. Mol. Microbiol., 83, 1109–1123 (2012). 38 Poster Presentations-Abstracts Uneven numbers please be at your poster Wednesday, even numbers Thursday. Poster 1: Computational prediction of the regulatory interactions for an ECF sigma factor group with fused C-terminal domain and co-factor Hao Wu and Georg Fritz. LOEWE Center for Synthetic Microbiology, Philipps University Marburg Hans-Meerwein Str. 6, D-35032 Marburg, Germany Evolutionary co-variation of amino acid residues has been extensively exploited to predict conserved intra- and inter-domain interaction of proteins. Some groups of extracytoplasmic function (ECF) sigma-factors contain a large C-terminal extension that might function as a fused anti-sigma factor. A previous study suggested a rather complex regulatory role of this C-terminal domain. Here we focus on ECF sigma factors of group 42, which, on top of a fused C-terminal domain, feature a highly conserved gene encoding a YCII-related protein in its genomic context. To gain first insights into potential regulatory interactions between these players, we here use a computational method to predict protein-protein contacts among the sigma-domain, the C-terminal domain and the YCII-related protein. Our results suggest a cluster of interactions interfacing between the sigma4 domain of ECF42 and the first alpha helix in the fused C-terminal domain. Moreover the YCII-related protein is also predicted to contact a number of residues at the same interface, suggesting a function as a co-factor to the regulation of the sigma factor. With this, our computational analysis provides intriguing hints to the regulatory mechanism employed by these underexplored signaling devices. 39 Poster 2: Implementation, analysis and mathematical modeling of ECF sigma factor-based synthetic gene cascades in E.coli and B.subtilis Marco Mauri1, Daniela Pinto2, Stefano Vecchione1, Hao Wu1, Thorsten Mascher2, Georg Fritz1. 1 LOEWE Center for Synthetic Microbiology, Philipps University Marburg, Hans-Meerwein-Str. 6C, 35043 Marburg, Germany 2 Institut für Mikrobiologie, Technische Universität Dresden Zellescher Weg 20b, 01217 Dresden, Germany Functionality of synthetic gene networks is often restricted by cross-reactions between network components and physiological processes within the host. In addition, to date most synthetic biology applications rely on a limited set of building blocks consisting of a handful of transcriptional regulators. To overcome these restrictions, we use Extracytoplasmic function sigma factors (ECFs) to build natural switches. ECFs are the largest group of alternative sigma factors in bacteria and represent ideal building blocks for synthetic network design because they are modular, universal and highly promotersequence specific. Here, we present a first implementation of gene cascade based on ECFs in E.coli and B.subtilis. After a quantitative study of simple ECF switches, we use a computational modeling approach to predict the function of more complex networks. We show that our ECFbased gene cascade sequentially activates a series of target genes with a defined time delay both in E.coli and in B.subtilis. The characterization and the theoretical modeling of such constructs serve as rational design principle for universal ECF-based switches in bacterial cells. 40 Poster 3: Modulation of the behaviour of an Extracytoplasmic Function sigma factor (ECF)-based genetic switch Daniela Pinto1, Franziska Dürr1, Dayane Araújo1,2, Qiang Liu1,2 and Thorsten Mascher1. 1 Institüt für Mikrobiologie, Technische Universität Dresden, 01062 Dresden, Germany Department Biology I, Ludwig-Maximilians-Universität München, Großhaderner Str. 2-4, 82152 Planegg-Martinsried, Germany 2 Bacteria rely on distinct signal transducing systems to monitor their environment and mount fast and adequate responses. These systems can be divided into three groups: onecomponent systems, two-component systems and extracytoplasmic function sigma factors (ECFs). While the first two systems have been extensively explored as tools for synthetic biology, ECFs have only been slightly exploited (1). Our starting point was a Bacillus subtilis ECF genetic switch based on the ECF41 of Bacillus licheniformis (2). We have systematically altered both the ECF as well as its cognate promoter and determined the behaviour of the switch. We have altered the copy number of the ECF and/or its target promoter, the nature of the inducible promoter driving the expression of the ECF, the stability of the ECF and the size of the DNA fragment containing the promoter. Additionally, we have explored the effects of antisense transcription driven by constitutive promoters of different strengths. In order to expand the toolbox of available ECF-based switches in B. subtilis we have additionally attempted to implement ECFs and cognate promoters from other sources. With the aim of covering a wide taxonomical range we focused on ECFs from Bacillus cereus, Escherichia coli, Sinorhizobium meliloti and Streptomyces venezuelae. Collectively, our efforts generated a collection of switches with different behaviours, e.g., different levels of maximal activity, background activity or activation thresholds. Additionally, this work highlighted a previously unknown limitation: that only ECFs from closely related organisms, e.g. Firmicutes, can be implemented into B. subtilis without further manipulation. References 1. Rhodius V.A., Segall-Shapiro T.H., Sharon B.D., Ghodasara A., Orlova E., Tabakh H., Burkhardt D.H., Clancy K., Peterson T.C., Gross C. A. and Voigt C. A. (2013), Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters. Mol. Syst. Biol. 9:702. 2. Wecke T., Halang P., Staroń A., Dufour Y.S., Donohue T.J. and Mascher T. (2012), Extracytoplasmic function σ factors of the widely distributed group ECF41 contain a fused regulatory domain. Microbiologyopen 1:194–213. 41 Poster 4: Theoretical models and FRET-assays for signal transduction via switchable allosteric modulator proteins (SAMPs) in Bacillus subtilis Heiko Babel and Ilka B. Bischofs. BioQuant, University of Heidelberg, Im Neuenheimer Feld 267, D-69120 Heidelberg, Germany Allosteric regulation is a common motif in prokaryotic signal transduction networks. Not only receptor-proteins are regulated allosterically by a molecular signal but also modulators can be subject to allosteric regulation. Proteins of the Rap-modulator family in Bacillus subtilis are regulated by a molecular peptide-signal; they are so-called switchable allosteric modulator proteins (SAMPs). The Phr-peptide signal, which is co-expressed with a cognate Rapmodulator, is processed by an import-export circuit and finally inhibits its Rap protein. Theoretically, the Phr-peptide could act as a Quorum-sensing signal and the Rap-modulator as its Quorum-sensing receptor. However it is currently not known how signals are processed via SAMP-based receptors. To address this issue we developed a mathematical model to systematically identify possible influencing parameters of SAMP-signal processing1. In enzymatic SAMPs two allosteric modes determine signal-transduction. They can implement diverse switching-behaviors that differ in amplitude, Hill-coefficient and half-maximal signal-concentration. Interestingly optimal information-processing in SAMPs also depends on an optimal modulator-to-regulator stoichiometry which is shaped by the pathway activity and the allosteric modes of the modulator. Because it is still unclear how Phr-peptides are processed in vivo, we developed a novel FRET-sensor for the RapA-modulator of B. subtilis. The sensor is able to detect the PhrAsignal selectively. In conjunction with the mathematical model, we find that the RapAmodulator forms a trimolecular complex with the PhrA-signal and the Spo0F-regulator. Also RapA seems to employ only one allosteric mode, which agrees with biochemical studies 2 and possibly could influence the optimal modulator-regulator ratio. References 1. Babel, H. & Bischofs, I. B. Molecular and Cellular Factors Control Signal Transduction via Switchable Allosteric Modulator Proteins (SAMPs). BMC Syst. Biol. (2016). 2. Diaz, A. R. et al. Bacillus subtilis RapA phosphatase domain interaction with its substrate Spo0F~P and inhibitor PhrA peptide. J. Bacteriol. 194, 1378–88 (2012). 42 Poster 5: Regulation of gene expression by small sRNA Dipl.-Ing. Lyubov Kyselova. Max Planck Institute for Dynamics of Complex Technical Systems, Research Group: Experimental Systems Biology Sandtorstr. 1, 39106 Magdeburg, Germany Many genes are essential for the cell growth and biosynthesis. Their deletion may lead to strong or complete growth inhibition. sRNA (small regulatory RNA) allow the dynamic switch off of genes. Small RNAs interact with the mRNA by direct base pairing, which leads in most cases to the destabilization of the mRNA and to the inhibition of translation. A major advantage of this approach in comparison to the chromosomal gene deletion is that sRNA genes can be regulated at any point in time, allowing dynamic process control [1]. Synthetic sRNA is cloned into an inducible plasmid [2]. The construct consists of the target sequence (reverse complementary to the target mRNA), a MicC scaffold sequence and a terminator site. By adding the inducer, the promoter is activated and the small RNA is synthesized. Several target sequences can be cloned in the plasmid, so that various genes can be controlled simultaneously. The gene lacZ was selected as target, because its activity is easy to determine. The activity of lacZ after induction was significantly smaller. This method was used to block translation of gene ackA in a next step. The expression of ackA was 4 times smaller in induced strain. Therefore the described method is a good possibility to decrease gene expression. Further task is to apply this method in respect to production of succinate in E.coli [3]. References 1. Na et al. Metabolic engineering of Escherichia coli using synthetic small RNAs. Nature Biotechnol. 31 (2):170-174), 2013 2. Yoo et al. Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nature Protocols 8, 1694-1707, 2013 3. Sánchez et al. Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metabolic engineering 7, 229-239, 2005 43 Poster 6: Regulatory interactions between Corynebacterium glutamicum and the prophage CGP3 Max Hünnefeld, Eugen Pfeifer and Julia Frunzke. Institute of Bio- und Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, 52425 Jülich, Germany Virus-derived DNA represents a predominant cause for strain-specific differences within a bacterial species. However, the integration of these DNA elements into the genome and into host regulatory circuits requires a stringent regulation. The genome of the Gram-positive soil bacterium Corynebacterium glutamicum ATCC 13032 contains three prophages (CGP1-3). Among those, the large, cryptic prophage element CGP3 covers almost 6 % of the entire genome (~187 kbp) and is still inducible [1]. Prophage activation can be triggered both spontaneously and in an SOS-dependent manner [2]. Hitherto, current studies focus on the investigation of the molecular mechanisms underlying the control of prophage induction in C. glutamicum and its regulatory interaction with the host. In recent studies we identified the small nucleoid-associated protein CgpS (CgpS: C. glutamicum prophage silencer), which was shown to act as an essential silencer of cryptic prophage elements in C. glutamicum [3]. ChAP-Seq experiments in combination with EMSA studies revealed that CgpS binds to AT-rich DNA and represses gene expression of mainly horizontally acquired genomic regions. Counteraction of CgpS activity by overexpression of the N-terminal oligomerization domain resulted in a severe growth defect and a highly increased frequency of CGP3 induction leading to cell death. In recent attempts, we aimed at the identification of further transcriptional regulators interacting with CGP3. Interestingly, DNA affinity chromatography using promoter regions of various prophage genes revealed several host regulatory proteins binding to the CGP3 element. Among those, we identified prominent regulators of global stress responses and central carbon metabolism. These proteins illustrate the tight regulatory interaction of the host and its prophage. Current studies are aiming at a further functional analysis of selected candidates and their impact on CGP3 control. References 1. 2. 3. Helfrich, S., et al., Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations. Mol Microbiol, 2015. 98(4): p. 636-50. Nanda, A.M., K. Thormann, and J. Frunzke, Impact of spontaneous prophage induction on the fitness of bacterial populations and host-microbe interactions. J Bacteriol, 2015. 197(3): p. 410-9. Pfeifer, E., et al., Silencing of cryptic prophages in Corynebacterium glutamicum. in revision, 2016. 44 Poster 7: Deciphering the Response of Corynebacterium glutamicum to Oxygen Deprivation Julian Lange1, Tobias Busche2, Jörn Kalinowski2, Ralf Takors1, Bastian Blombach1. 1 2 Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany Center for Biotechnology (CeBiTec), University of Bielefeld, Bielefeld, Germany Background. Bacteria encounter varying oxygen concentrations in manifold situations e.g. in their natural habitat and especially in large scale industrial processes evoking viability and production deficiencies (1). These fluctuations range from aerobic via micro-aerobic to anaerobic conditions. To adapt to the changing environment bacteria have to remodel their entire metabolism (2, 3). Despite its relevance for pathogenicity and pharmaceutical and biobased production processes, the molecular events during these transitions are poorly understood. To address this question, we systematically investigate the adaptation of the industrially relevant Corynebacterium glutamicum to such altering conditions. Methods. A “triple-phase” batch bioprocess with C. glutamicum was established that depicts the three successive phases (aerobiosis, micro-aerobic interface and anaerobiosis) in a single bioreactor. Throughout the process, samples were withdrawn and analyzed for substrate consumption, organic acid production, enzyme activities and intracellular metabolites and additionally used for whole transcriptome analysis by RNA-sequencing. Results. The bacterium’s physiological changes delineated a decreasing growth rate at increasing oxygen limitation. L-lactic acid, succinic acid and acetic acid were the main fermentation products secreted to the culture supernatant, interestingly in a manner, that their respective differential product yields clearly bordered each process phase. RNA Sequencing analysis revealed differential expression of 1421 genes. Notably, under anaerobic conditions translation as well as transcription itself was tightly downregulated, which was not yet prominent under micro-aerobiosis. Pentose phosphate pathway activity was found to be regulated not on a transcriptional but presumably solely on a metabolic level. Differential expression of 34 transcriptional regulators may include yet unknown oxygen sensors. Conclusion. The established bioprocess is an elegant approach for the systematic understanding of C. glutamicum’s adaptation to a progressive oxygen deprivation. Deeper analysis of the RNA-sequencing data especially focusing on regulators and the network’s hierarchy could reveal novel targets for strain optimization or even pharmaceutical applications. References 1. Blombach B, Riester T, Wieschalka S, Ziert C, Youn J-W, Wendisch VF, Eikmanns BJ. 2011. Appl. Environ. Microbiol. 77:3300–10. 2. Patschkowski T, Bates DM, Kiley PJ. 2000. p. 61–78. In Storz, G, Hengge-Aronis, R (eds.), Bacterial stress responses. ASM Press, Washington, D.C. 3. Bunn HF, Poyton RO. 1996. Physiol. Rev. 76:839–885. 45 Poster 8: From substrate specificity to promiscuity: molecular analysis of a hybrid ABC transporter L. Teichmann, C. Chen & E. Bremer. Laboratory for Microbiology, Department of Biology, Philipps-University Marburg, Karl-von-Frisch Str. 8, D-35043 Marburg, Germany The two closely related ABC transporters OpuB and OpuC (osmoprotectant uptake) are crucial for acquiring a variety of compatible solutes under osmotic and temperature stress conditions in Bacillus subtilis [1]. Whereas the substrate binding protein OpuCC recognizes a broad spectrum of compatible solutes, its 70% sequence-identical paralogue OpuBC only binds choline [2] [3]. This raises the question about the molecular determinants governing the strikingly different substrate specificities of the OpuB and OpuC systems. In this study we used molecular “micro-surgery” to genetically substitute the OpuBC substrate binding protein of the OpuB transporter with that (OpuCC) of the OpuC system in order to analyze whether the hybrid ABC-transporter OpuB::CC is functional and what its substrate specificity might be. Physiological experiments showed that the hybrid OpuB::CC ABC system is functional and able to import all compatible solutes previously transported exclusively by OpuC. Hence, by implanting the ligand binding protein of an ABC transporter with a broad substrate spectrum, we were able to synthetically convert OpuB into a promiscuous ABC transporter. OpuC exhibits high affinity towards glycine betaine (Km= 4.8 µM) and possesses substantial transport capacity (vmax= 100 nmol min-1 mg protein-1), whereas the hybrid transporter exhibits a weaker transport capacity for this substrate (vmax = 20.2 nmol min-1 mg protein-1) but shows the same high affinity (Km= 5.6 µM). By genetic enrichment, we screened for suppressor mutants that showed increased transport capacity via the hybrid OpuB::CC system by providing enhanced osmostress protection of B. subtilis at a very low external glycine betaine concentration. Indeed, several suppressor mutants were found that exhibited enhanced transport capacity (vmax= 92-99 nmol min-1 mg protein-1). The corresponding mutations were mapped to the coding region of gbsR, which encodes a repressor (GbsR) of the opuB operon. The studied mutant gbsR genes encode GbsR protein variants carrying only single amino acid substitutions. These mutant GbsR proteins are either not at all, or only partially functional and thereby enhance opuB transcription through a less tight negative transcriptional control. These mutant variants give important clues with respect to the functioning of this repressor protein when they were projected onto an in silico generated model of the GbsR structure. References [1] Hoffmann & Bremer (2011) J Bacteriol 193: 1552-1562 [2] Pittelkow et al., (2011) J Mol Biol 411: 53-67 [3] Du et al., (2011) Biochem J 436: 283-289 46 Poster 9: The cellular function and localization of the cyclic-di-GMP phosphodiesterase PdeL Cihan Yilmaz and Karin Schnetz. Institute for Genetics, University of Cologne, Zülpicher Str. 47a, 50674 Cologne Cyclic-di-GMP is a universal bacterial second messenger for the control of a variety of lifestyle parameters including the regulation of biofilm formation, motility, and virulence [1]. PdeL is a cyclic-di-GMP phosphodiesterase and putative transcription regulator in E. coli which harbors a N-terminal FixJ/NarL-type DNA-binding domain (PdeLHTH) and a Cterminal, enzymatically active EAL-domain (PdeLEAL) [2]. So far PdeL has been characterized in respect of its enzymatic activity and its role in cyclic-di-GMP hydrolysis. However, deletion of pdeL has no phenotype and does not cause high cellular levels of cyclicdi-GMP, unlike deletion of the major phosphodiesterase pdeH. Further, pdeL expression is repressed by the global transcription regulator H-NS. We performed a functional analysis of PdeL by different experimental approaches including analysis of the cellular localization as well as its role in motility and biofilm formation. We analysed the cellular localization of PdeL, by plasmidic expression of fusions of full length PdeL and its individual domains to the fluorescent protein mVenus. This demonstrated that the full length protein, but not the individual domains, co-localizes with the nucleoid. Nucleoid localization depends on dimerization of the C-terminal domain for which the native C-terminal domain can be substituted by another C-terminal dimerization domain to the N-terminal PdeLHTH DNAbinding domain. Alanine mutagenesis of PdeL supports the relevance of PdeLHTH in nucleoid association while mutagenesis of the PdeLEAL domain had only slight effects. Plasmidic expression of fluorescent PdeL fusion proteins affected cell growth and size as well as chromosomal organization. Further, PdeL expression decreases motility and increases biofilm formation independent of the PdeL phosphodiesterase activity, which is contrary to known effects that changes in cellular cyclic-di-GMP levels have on motility and biofilm formation in E. coli [3]. Taken together these findings indicate a possible role of PdeL as a cyclic-diGMP dependent transcription regulator that may control expression of genes involved in chromosome organization as well as motility and/or biofilm formation. References 1. Römling, U., M.Y. Galperin, and M. Gomelsky, Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger. Microbiology and Molecular Biology Reviews, 2013. 77(1): p. 1-52. 2. Sundriyal, A., et al., Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP. Journal of Biological Chemistry, 2014. 289(10): p. 6978-6990. 3. Serra, D.O. and R. Hengge, Stress responses go three dimensional - the spatial order of physiological differentiation in bacterial macrocolony biofilms. Environ Microbiol, 2014. 16(6): p. 1455-71. 47 Poster 10: Elucidation of the metabolic pathway for SDS degradation and its regulation in Pseudomonas aeruginosa Gianna Panasia and Bodo Philipp. Institute of Molecular Microbiology and Biotechnology, University of Münster, Corrensstr. 3, D-48149 Münster, Germany Pseudomonas aeruginosa is an ubiquitous environmental bacterium that can act as an opportunistic and nosocomial pathogen. Its metabolic versatility and pronounced resistance against toxic chemicals enables it to survive and grow in hygienic environments. Thereby, it can cause outbreaks in clinical settings. In this context, P. aeruginosa is able to use the common and toxic detergent sodium dodecyl sulfate (SDS) as growth substrate [1]. Previous studies demonstrated cell aggregation of P. aeruginosa during growth with SDS as a specific survival strategy [2]. In the current model, the proposed stress sensor SiaA, a putative ser/thr phosphatase, acts together with SiaD, a di-guanylate cyclase, as a signal transduction module regulating SDS induced cell aggregation in a c-di GMP and RsmA dependent manner [3,4]. However, little is known about the metabolic pathway for the SDS degradation and its regulation. Thus, we address the unknown enzymatic steps and the respective gene expression. Based on a DNA-microarray analysis comparing SDS- and succinate-grown cells several genes with a plausible function in SDS degradation were identified [3]. Thereby, a special alcohol dehydrogenase, encoded by exaA, belonging to the ethanol oxidation system and an unknown putative oxidoreductase, encoded by PA0364, are induced. Single deletion mutants of these genes still grow with SDS but have different phenotypes in cell aggregation behavior in comparison to the wild type. Remarkably, the respective double knockout mutant is not able to grow with SDS. Complementation with either exaA or PA0364 restored growth with SDS similar to the respective single mutants. This indicates a significant participation of both genes in SDS degradation. In general, the analysis of this catabolic pathway on the transcriptional and posttranslational level, will give insight in how P. aeruginosa copes to grow with SDS and potentially other similar toxic compounds. References [1] Klebensberger J. et al. (2006). Cell aggregation of Pseudomonas aeruginosa strain PAO1 as an energy-dependent stress response during growth with sodium dodecyl-sulfate. Arch. Microbiol. 185:417-2. [2] Klebensberger J. et al. (2007). Detergent-induced cell aggregation in subpopulations of Pseudomonas aeruginosa as a preadaptive survival strategy. Environ. Microbiol. 9(9):224759. [3] Klebensberger J. et al. (2009). SiaA and SiaD are essential for inducing autoaggregation as a specific response to detergent stress in Pseudomonas aeruginosa. Environ. Microbiol. 11(12):3073-86. 48 [4] Colley B. et al. (2016). SiaA/D interconnects c-di-GMP and RsmA signaling to coordinate cellular aggregation of Pseudomonas aeruginosa in response to environmental conditions. Front. Microbiol. 7:179 doi: 10.3389/fmicb.2016.00179. Poster 11: The role of the phosphodiesterase NbdA in NO-induced dispersal of Pseudomonas aeruginosa Martina Rüger1, Sabrina Heine2, Michael Entian2, Yi Li3, Karin Sauer3 and Nicole Frankenberg-Dinkel1, 2. 1 Department of Biology, Microbiology, Technical University Kaiserslautern, Kaiserslautern, Germany 2 Physiology of Microorganisms, Ruhr-University Bochum, Bochum, Germany 3 Department of Biological Sciences, Binghamton University, Binghamton, New York, USA Pseudomonas aeruginosa is an important opportunistic human pathogen causing a variety of nosocomial infections including pneumonia, sepsis, catheter and urinary tract infections. The bacterium has become a model system for biofilm research because of its resistance to conventional antibiotics, host antimicrobial effector mechanisms and its ability to form biofilms. Dispersal is considered as the last step of the biofilm life cycle being a process used by bacteria to transfer from sessil to motile lifestyle. Changes in c-di-GMP levels have been shown to be associated with biofilm detachment in a number of different bacteria. The signalling molecule nitric oxide (NO) induces biofilm dispersal through stimulation of c-diGMP (bis-(3‘-5‘)-cyclic dimeric guanosine monophosphate) degrading phosphodiesterase (PDE) activity. We characterised the membrane-bound proteins MucR and NbdA (NOinduced biofilm dispersion locus A) regarding their role in NO-induced dispersal. Both share an identical domain organisation consisting of MHYT-GGDEF-EAL. Inactivation of mucR impaired biofilm dispersal in response to glutamate and NO while deletion of nbdA only negatively affected biofilm detachment upon exposure to NO. Biochemical analyses of recombinant protein variants lacking the membrane-anchored MHYT-domain revealed NbdA being an active PDE. In contrast, MucR showed diguanylate cyclase and PDE activity in vitro [1]. Interestingly, a P. aeruginosa strain lacking both, nbdA and mucR displayed enhanced biofilm formation under tested conditions, whereas ΔnbdA and ΔmucR single mutants showed rather wild type like phenotype. The hyper biofilm formation phenotype of the ΔnbdA ΔmucR double mutant might be due to highly increased c-di-GMP levels caused by lacking PDE activity of NbdA and MucR. These results suggest either a functional redundancy or possible interdependence of both proteins. References [1] Li Y, Heine S, Entian M, Sauer K, Frankenberg-Dinkel N. (2013) NO-induced biofilm dispersion in Pseudomonas aeruginosa is mediated by an MHYT domain-coupled phosphodiesterase. J Bacteriol. 195:3531-3542. 49 Poster 12: Osmotic responsive transcription of ectoine biosynthetic genes from Pseudomonas stutzeri is transferable to a non-ectoine producing surrogate host Laura Czech, Philipp Hub, Florian Kindinger, Oliver Dähn, Nadine Stöveken & Erhard Bremer. Laboratory for Microbiology, Department of Biology, Philipps-University Marburg, Karl-von-Frisch Str. 8, D-35043 Marburg, Germany A large number of microorganisms posses the ability to produce and accumulate the compatible solutes ectoine and 5-hydroxyectoine in response to an osmotic upshift to maintain vital turgor and to sustain cell growth under osmotically unfavorable conditions. The production of these chemical chaperones [1] has already been described in the soil bacterium Pseudomonas stutzeri A1501 and depends on the salt-inducible ectABCD_ask operon [2]. Analysis of an P. stutzeri A1501 ectA-ectB-lacZ-reporter gene fusion in a non-ectoine producing Escherichia coli heterologous host strain showed a highly dynamic response of promoter activity, when cells faced step-wise increases in the external salinities. Activity of the ect promoter could not only be stimulated by high NaCl concentration but also by other osmolytes, demonstrating that this promoter responds to a true osmotic stress signal. And not just to salt. This expression pattern is neither modulated by the nucleoid-structuring protein H-NS, nor by the alternative sigma factor (RpoS) controlling the general stress response of E. coli. Successive truncations of the region up-stream of ectA resulted in a reporter gene fusion, that exhibits a hybrid promoter consisting of a weak -35 region stemming from the backbone of the plasmid and the original -10 region. This observation, together with sequence comparison of the regions in front of ectA derived from different Pseudomonas strains allowed the determination of the promoter consisting of a nearly perfect -35 region, an 18 bp spacer sequence and a -10 region unusually rich in C-G base pairs. We introduced different point mutations into the ect promoter in order to analyze its response to changes in external salinity. Creation of a perfect SigA promoter resulted in the loss of the salt-induction and a 16-fold increase in the basal activity. Collectively, our data suggest that the finely tuned osmotic control of the P. stutzeri ect promoter is dependent on its unusual 18 bp spacer and the abnormal -10 region. Changes in DNA supercoiling might be a contributing factor as well. Since osmotic control of ect gene expression also occurs in a non-ectoine producing surrogate host organism E. coli, it seems to us that no specific regulatory protein is required to drive osmostress-responsive ectABCD_ask gene expression. A possible mechanism will be discussed. References 1. Pastor, J. M. et al. (2010) Biotechnol Adv. 28: 782–801. 2. Stöveken, N. et al. (2011) J Bacteriol. 193: 4456–4468. 50 Poster 13: A special role for acetate kinase AckA in the regulation of CiaR activity in the absence of the cognate kinase CiaH. Anne Sexauer and Reinhold Brückner. Department of Microbiology, University of Kaiserslautern, Paul-Ehrlich Str. 24, D-67663 Kaiserslautern, Germany The two-component regulatory system CiaRH of Streptococcus pneumoniae is implicated in competence regulation, ß-lactam resistance, maintenance of cell integrity, bacteriocin production, host colonization, biofilm formation and virulence. A surface-exposed protease HtrA and five small noncoding csRNAs, all directly controlled by CiaR, are the major mediators of these phenotypes (4). Expression analyses indicated that the CiaR system is highly active under a variety of growth conditions, not showing an on-off switch typical for many other two-component systems. In addition, depending on the growth conditions, CiaR is active in the absence of its cognate kinase CiaH, although phosphorylation of CiaR is required for DNA binding and gene regulation (2). To determine if acetyl phosphate could be the alternative phosphodonor, genes involved in pyruvate metabolism were disrupted to alter cellular levels of acetyl phosphate. In a CiaH-deficient strain devoid of pyruvate oxidase SpxB, phosphotransacetylase Pta, and acetate kinase AckA, very low acetyl phosphate levels were observed, and in paralell, strongly reduced CiaR-mediated gene expression (3). These results clearly indicate that alternative phosphorylation of CiaR is dependend on acetyl phosphate. A surprising synthetic lethality was detected in CiaH-deficient strains producing high levels of acetyl phosphate. The ackA gene could not be inactivated. Furthermore, a strain producing half of the acetyl phosphate level of the wild type lacking AckA showed a 13-fold increase in CiaR-dependent promoter activation. In the absence of AckA, CiaR appears to be extremely activated, provided acetyl phosphate is present and the CiaH kinase is absent. It appears therefore, that alternative phosphorylation of CiaR is affected negatively by AckA. In a first step to determine if this negative regulation is direct, the adenylate reconstituion Escherichia coli two hybrid system (1) was applied to detect interaction between CiaR and AckA. The results of these experiments clearly demonstrated contact between these two proteins. The surprising link of the response regulator CiaR to a metabolic enzyme, AckA, adds another level of complexity to two-component regulatory system regulation. References 1. Battesti, A., and E. Bouveret. 2012. The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli. Methods 58:325-334. 2. Halfmann, A., A. Schnorpfeil, M. Müller, P. Marx, U. Günzler, R. Hakenbeck, and R. Brückner. 2011. Activity of the two-component regulatory system CiaRH in Streptococcus pneumoniae R6. J. Mol. Microbiol. Biotechnol. 20:96-104. 3. Marx, P., M. Meiers, and R. Brückner. 2015. Activity of the response regulator CiaR in mutants of Streptococcus pneumoniae R6 altered in acetyl phosphate production. Front. Microbiol. 5:772. 51 4. Schnorpfeil, A., M. Kranz, M. Kovács, C. Kirsch, J. Gartmann, I. Brunner, S. Bittmann, and R. Brückner. 2013. Target evaluation of the non-coding csRNAs reveals a link of the two-component regulatory system CiaRH to competence control in Streptococcus pneumoniae R6. Mol. Microbiol. 89:334-349. Poster 14: Transport and regulation by the alternative anaerobic C4-dicarboxylate-transporters DcuA, DcuB and DcuC in Escherichia coli Alexander Strecker and Gottfried Unden. Institute for Microbiology and Wine Research Johannes Gutenberg-University Mainz, Germany Escherichia coli metabolizes C4-dicarboxylates during aerobic and anaerobic growth. Under anaerobic conditions the uptake of C4-dicarboxylates is catalyzed by the three transporters DcuA, DcuB and DcuC [1]. DcuB forms a complex with DcuS and functions as a coregulator for DcuSR dependent gene expression under anaerobic conditions [2]. The deletion of DcuB causes a constitutive expression of DcuS regulated genes. The homologous transporters DcuA and DcuC show no regulatory effect on gene expression [3]. Complexome profiling of membrane proteins, mSPINE and BACTH indicates that the DcuA, DcuB and DcuC transporters interact and form heterocomplexes. A potential role of DcuA/DcuB or DcuC/DcuB heterocomplexes on DcuS function was characterized. [1] Unden, G., Strecker, A., Kleefeld, A., & Kim, O. B. (2016) EcoSal Plus, 7(1). doi: 10.1128/ecosalplus.ESP-0021-2015. [2] Wörner, S., Strecker, A., Monzel, C., Zeltner, M., Witan, J., Ebert‐Jung, A., & Unden, G. (2016). Environ Microbiol. doi: 10.1111/1462-2920.13418. [3] Kleefeld, A., Ackermann, B., Bauer, J., Krämer, J., & Unden, G. (2009) J Biol Chem. 284(1):265-75. 52 Poster 15: The DxxxQ phosphatase motif in the O2 sensor kinase NreB of Staphylococcus carnosus Ann-Katrin Kretzschmar and Gottfried Unden. Institute for Microbiology and Wine Research, Johannes Gutenberg-University Mainz Johann-Joachim-Becherweg 15, D-55128 Mainz, Germany In Staphylococcus carnosus the anaerobic nitrate respiration is regulated by the O2-sensitive two component system NreB-NreC and the nitrate sensor NreA [1, 2]. The sensor kinase NreB is autophosphorylated at His159 and the phosphoryl group is transferred to the response regulator NreC. NreA modulates NreB activity by nitrate dependent interaction resulting in a combined oxygen/nitrate sensing complex [1, 2]. NreB contains a DxxxQ motif adjacent to the phospho-accepting His159. In the nitrate sensor kinase NarX of E. coli this motif is crucial for kinase and phosphatase activity of the sensor [3, 4]. The significance of the Asp160 and Gln164 residues of the DxxxQ motif was tested in vivo and in vitro by mutation. The data suggest that the motif is important for autophosphorylation of NreB and dephosphorylation of NreC, both in response to O2 and to nitrate regulation by NreA. References: [1] Nilkens, S., Koch-Singenstreu, M., Niemann, V., Götz, F., Stehle, T., Unden, G. (2014) Mol Microbiol 91:381-393 [2] Niemann, V., Koch-Singenstreu, M., Neu, A., Nilkens, S., Götz, F., Unden, G., Stehle, T. (2014) J Mol Biol 426:1539-1553 [3] Huynh, T., Noriega, C., Stewart, V. (2010) Proc Natl Acad Sci USA 107: 21140-21145 [4] Hentschel, E., Mack, C., Gätgens, C., Bott, M., Brocker, M., Frunzke, J. (2014) Mol Microbiol 92: 1326-42 53 Poster 16: The function of the ExxN motif of the C4-dicarboxylate sensor kinase DcuS of Escherichia coli in signal transduction Stefaniya Gencheva, Sebastian Wörner and Gottfried Unden. Institute for Microbiology and Wine Research Johannes Gutenberg-University Mainz, Germany The two component system DcuSR of E. coli is composed of the membrane-bound histidine kinase DcuS and the cytoplasmic response regulator DcuR. Under anaerobic conditions DcuS forms a DcuS/DcuB sensor complex with the transporter DcuB. This complex formation is essential for conversion of DcuS to the C4-dicarboxylate responsive form and the activation of the kinase domain of DcuS [1, 2]. In vitro studies show a positive effect of non complexed DcuS on DcuR dephosphorylation [3]. The DHp domain of DcuS contains a conserved ExxN phosphatase motif [4]. In vivo and in vitro studies were performed with DcuS and ExxN mutants to study the role of the motif in DcuS phosphorylation and dephosphorylation. [1] Unden, G., Wörner, S., Monzel, C. (2016): In Adc Microbiol Physiol., Poole, Robert K., 68:139–167. [2] Steinmetz, P. A., Worner, S. Unden, G. (2014): Mol Microbiol. 94: 218–229. [3] Janausch, I. G., Garcia-Moreno, I. Unden, G. (2002):Biochim Biophys Acta.1553: 39-56. [4] Huynh, T. N., Noriega, C.E., Stewart, V. (2010): Proc Natl Acad Sci U S A. 107: 21140– 21145. 54 Poster 17: Analysis of quinone mutants in respect to ArcA phosphorylation and product formation Annika Nitzschke and Katja Bettenbrock. MPI for dynamics of complex technical systems, Sandtor Str. 1, D-39106 Magdeburg, Germany E.coli is able to respond to changes in the oxygen availability through regulation of metabolism. Two major transcription factors (TF) are responsible for this adaption, the twocomponent system ArcB/A und the global TF FNR. In contrast to FNR, the two-component system ArcB/A reacts only indirectly to the change in oxygen supply. Rather other signals seem to have an influence on the ArcB/A activation. The effect of the redox pool of the cell on the activation of ArcA has already been discussed in literature, whereby the focus has been on the redox ratio of the quinones (1). These function as electron carriers between the dehydrogenases and oxidases of the electron transport chain (ETC). E.coli possesses three different quinone species: ubiquinone (UQ), which is synthesized mainly during aerobic conditions and demethylmenaquinone (DMK) und menaquinone (MK), which are synthesized mainly during anaerobic respiration (2). To study in more detail the function of different quinone species, strains with deletions preventing UQ synthesis, as well as MK and/or DMK synthesis were cultured under aerobic as well as anaerobic conditions. A special focus was on deriving a correlation between the compositions of the quinone pool und the ArcA phosphorylation state. In contrast to the indirect measurements of ArcA phosphorylation by reporter genes, we determined the relative phosphorylation state of the TF directly by Phos-tag SDS-PAGE and Western Blot. The results from the characterization of the mutants compared to the wild type strain MG1655 showed that in contrast to the in vitro results (3), in vivo, no inhibitory effect from the UQ on the ArcA phosphorylation was observed. Furthermore gene expression analysis under aerobic conditions showed that the ubiquinone knockouts exhibit a “pseudo” fermentative state independent of the phosphorylation of ArcA. This indicates that it is a problematic and error-prone to deduce the phosphorylation state of ArcA from indirect measurements using reporter gene fusions. A comprehensive analysis of gene expression in the three quinone deletion strains under anaerobic conditions is currently performed. First results show, that the reduced phosphorylation of ArcA in the ubiquinone deletion strains AV33 and AV36 influences gene expression under anaerobic conditions. In addition the mutation preventing UQ synthesis was combined with an arcA deletion. This strain shows qualities of a production strains with a good glucose consumption rate in spite of not growth. Sequencing results indicate that this strain frequently acquires secondary mutations, namely a deletion of about 16 kBp, containing inter alia ubiE and metE. We are currently testing this strain in more detail. References 1. Malpica, R. and Franco, B. (2004) Identification of a quinone-sensitive redox switch in the ArcB sensor kinase (2004), Proc. Natl. Acad. Sci. USA 101(36), 13318-13323. 2. Bekker M. (2009), Respiratory electron transfer in Escherichia coli: components, energetics and regulation,(2009) 55 3. Georgellis D. and Kwon O. (2001), Quinones as the redox signal for the arc twocomponent system of bacteria (2001), Science 292(5525), 2314-2316. Poster 18: Analysis of the signal transduction by the heme-based sensor kinase MsmS from Methanosarcina acetivorans Fiege, K.1,2, Molitor, B.2, Blasius, L.1, Querebillo, C.3,4, Hildebrandt, P.3, Laurich, C.5, Lubitz, W.6, Rother, M.5, Frankenberg-Dinkel, N.1,2. 1 TU Kaiserslautern, Department of Microbiology, Kaiserslautern, Germany Ruhr University Bochum, Physiology of Microorganisms, Bochum, Germany 3 TU Berlin, Institute for Chemistry, Berlin, Germany 4 School of Analytical Sciences Adlershof, Humboldt-Universität zu Berlin, Berin, Germany 5 TU Dresden, Institute for Microbiology, Dresden, Germany 6 Max-Planck-Institute for Chemical Energy Conversion, Mülheim, Germany 2 The multidomain protein MsmS from Methanosarcina acetivorans is one of the first examples for the biochemical characterization of an archaeal sensor kinase with autophosphorylation activity. It consists of two alternating PAS and GAF domains and a Cterminal H_ATPase domain. The second GAF domain of MsmS covalently binds a heme cofactor via a cysteine residue. For MsmS, the redox state of the heme cofactor was shown to influence the autophosphorylation activity of the adjacent kinase domain [1]. For the investigation of the function of this archaeal signal transduction system and its redox sensory function, the heme coordination structure was analyzed using UV-vis and Resonance Raman spectroscopy. Therefore, several variants of truncated MsmS were analyzed to identify the heme coordinating residues. First UV-vis spectroscopic analysis identified a histidine residue as the proximal ligand for the heme cofactor. The gene msmS is encoded upstream of the regulator protein MsrG. It is assumed that these both proteins form a two-component system. Therefore, also the intermolecular interaction with the regulator protein was analysed. Finally, the presented results will be discussed in the light of the putative cellular function of the heme-based sensor kinase. [1] Molitor, B., Stassen, M., Modi, A., El-Mashtoly, S. F., Laurich, C., Lubitz, W., Dawson, J. H., Rother, M., and Frankenberg-Dinkel, N. (2013) A heme-based redox sensor in the methanogenic archaeon Methanosarcina acetivorans. J. Biol. Chem. 288, 18458-18472 56 Poster 19: A TCS is involved in the regulation of the organohalide respiration in Sulfurospirillum spp. Jens Esken1, Tobias Goris1, Cynthia Sharma2, Torsten Schubert1, and Gabriele Diekert1. . 1 Department of Applied and Ecological Microbiology, University of Jena, Philosophenweg 12, D-07743 Jena, Germany 2 Research Center for Infectious Diseases, Julius Maximilians University Würzburg, Josef Schneider Str. 2/ D15, 97080 Würzburg, Germany Organohalide respiration was studied in detail in the tetrachloroethene (PCE)-dechlorinating Sulfurospirillum multivorans and S. halorespirans. The organisms display an unusual type of long-term down-regulation of the PCE reductive dehalogenase gene (pceA) expression in the absence of PCE (1). In close proximity to pceA, open reading frames encoding twocomponent systems (TCS) were identified. As revealed by RNA sequencing, the 40-kbp organohalide respiration (OHR) gene region, which surrounds pceA, was transcribed only in the presence of PCE. The expression of eight transcriptional units ceased completely, when PCE was absent. As an exception, an operon encoding a two-component system (TCS) still displayed a low transcript level under these conditions. The histidine kinase (HK) of the TCS is predicted to contain seven transmembrane helices and an N-terminal domain putatively exposed to the periplasm. This domain might serve as PCE sensor. Since PCE is a very hydrophobic compound, its detection outside the cytoplasmic membrane appears effective. Upstream of the gene for the HK, a response regulator is encoded. The respective gene is disrupted by a transposase in the non-dehalogenating S. multivorans strain N and S. JPD1. The residual OHR gene region is almost 100% identical to that of S. multivorans and S. halorespirans. This and the results of the RNA sequencing led to the assumption that the TCS is involved in PCE-sensing and its malfunction in S. multivorans strain N and S. JPD1 might be the reason for the non-dehalogenating phenotype. References 1. John et al. (2009) J Bacteriol. 191:1650-5. 57 Poster 20: Biochemical characterization of the iron responsive regulator RirA from Dinoroseobacter shibae Maren Behringer, Elisabeth Härtig and Dieter Jahn. Institute of Microbiology, University Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany; The rhizobial iron regulator RirA from Dinoroseobacter shibae belongs to the Rrf2- family of transcription factors and is supposed to coordinate a Fe-S cluster and thereby measure iron availability. In Rhizobium leguminosarum RirA is acting as a repressor of iron uptake systems in the presence of iron. RirA of D. shibae was recombinantly produced and anaerobically purified. Ligation of an Fe-S cluster was detected by UV/Vis spectroscopy. Using electron paramagnetic spin resonance (EPR) spectroscopy and Fe-content determinations by atom absorbance spectroscopy (AAS) one [3Fe-4S]1+ cluster as cofactor per RirA molecule was determined. Supporting cyclic Voltammetry studies revealed, the cluster is not involved in electron transport and showed no redox potential. DNA binding of the anaerobically purified RirA wildtype and mutant proteins was analyzed using electro mobility shift assays (EMSA). The RirA protein of D. shibae contains four cysteine residues which are highly conserved in other RirA homolog proteins and might be important for Fe-S cluster formation [1]. To specify the role of these cysteine residues for the coordination of an Fe-S cluster as cofactor, each of the four conserved cysteine residues of RirA was changed to an alanine residue via site directed mutagenesis of the corresponding gene. UV/Vis spectroscopy using the anaerobically purified mutant proteins showed a reduction of the absorption at 420 nm, indicating a loss of the Fe-S cluster. To define the RirA regulon transcriptome analyses using DNA microarrays were performed. First results indicate, that RirA is able to regulate napF gene expression, encoding the ferredoxin-type protein NapF. Surprisingly, the RirA-dependent regulation was not dependent on iron. A β-galactosidase enzyme assay was established to proof, which promoters are under the control of the RirA regulator. [1] Bhubhanil, S, Niamyim, P, Sukchawalit, R, and S. Mongkolsuk (2013). Cysteine desulphurase-encoding gene sufS2 is required for the repressor function of RirA and oxidative resistance in Agrobacterium tumefaciens. Microbiology, 160:79-90 58 Poster 21: Adrenochrome – oxidation product of adrenaline and bacterial effector molecule Charlotte Toulouse, Kristina Metesch, Pit Engling, Bernd Michel and Julia Steuber. Department of Cellular Microbiology, University of Hohenheim, Garbenstr. 30, D-70599 Stuttgart, Germany Catecholamines such as adrenaline and noradrenaline are known to stimulate growth and swarming of some proteobacteria like EHEC [1] or Salmonella enterica Typhimurium [2] and also, recently shown by our group, Vibrio cholerae [3], the causative agent of the Cholera disease. Since catecholamines are known to undergo oxidative degradation [4], we investigated their stability during bacterial cultivation. Catecholamines can oxidize to their corresponding aminochromes by autoxidation with O2 or by superoxide. Their formation and effect on growth, swarming and virulence of V. cholerae was investigated. Adrenochrome was confirmed, by LC-MS, as oxidation product of adrenaline during cultivation of Vibrio cholerae. Isolated V. cholerae membranes contribute to the adrenochrome formation. The central respiratory membrane protein Na+-NADH:quinone oxidoreductase (Na+-NQR) of V. cholerae enhances the adrenochrome formation upon addition of NADH. We explain this through the production of superoxide during electron transfer, since no adrenochrome formation takes place under anaerobic conditions or when superoxide dismutase (SOD) was added. We suppose that the availability of O2 and particularly reactive oxygen species (ROS) in solution determines the amount of adrenochrome formed. Hence, not only the concentration of the signaling molecule adrenaline is affected by the O2 partial pressure during growth of V. cholerae, but also adrenochrome, a molecule with putative function in signaling, is formed. We assume that during the respiratory burst of immune cells, aminochrome formation is also enhanced. Finally, we show that adrenochrome alters swarming and growth of some proteobacteria including V. cholerae depending on the choice of medium. References 1. Clarke, M.B., et al., The QseC sensor kinase: a bacterial adrenergic receptor. Proceedings of the National Academy of Sciences of the United States of America, 2006. 103(27): p. 10420-5. 2. Moreira, C.G., D. Weinshenker, and V. Sperandio, QseC mediates Salmonella enterica serovar typhimurium virulence in vitro and in vivo. Infection and immunity, 2010. 78(3): p. 914-26. 3. Halang, P., et al., Response of Vibrio cholerae to the Catecholamine Hormones Epinephrine and Norepinephrine. Journal of bacteriology, 2015. 197(24): p. 3769-78. 4. Bors, W., et al., The involvement of oxygen radicals during the autoxidation of adrenalin. Biochimica et biophysica acta, 1978. 540(1): p. 162-72. 59 Poster 22: Mechanism and function of non-standard circadian clock systems in cyanobacteria Christin Köbler1, Anja Dörrich2, Anika Wiegard3, Annegret Wilde1. 1 Albert-Ludwigs-University Freiburg, Germany; 2Justus-Liebig-University Germany; 3Heinrich-Heine-University Duesseldorf, Germany Giessen, Through the rotation of the earth, all organisms are subjected to daily environmental changes. To facilitate their adaption, many organisms generate an internal rhythm with a period length of around 24 hours, referred to as circadian rhythm. Within cyanobacteria Synechococcus elongatus PCC7942 (hereafter Synechococcus) functions as model organism for the circadian clock. Its clock consists of three core proteins: KaiA, KaiB and KaiC. Phosphorylation and dephosphorylation of KaiC maintains the timing mechanism. The circadian system is rather well understood in Synechococcus, but can be quite diverse in other cyanobacteria. Some species lack kai genes, whereas others acquired additional kai homologs. Synechocystis sp. PCC 6803 (hereafter Synechocystis) for example, contains two additional homologs of the kaiB and kaiC genes. However, it is suggested that they form other time keeping mechanisms, but their function is still unknown. Using deletion mutants for each additional kai gene we want to elucidate their respective phenotype and uncover their function. Additionally, we want to explore a putative cross talk between the non-canonical Kai homologs via interaction studies. Furthermore, the core oscillator of Synechocystis seems to employ a different output signaling pathway than Synechococcus and until now, most of this pathway remains unsolved. We aim to identify new components of the output signaling pathway by establishing a fluorescence based gene reporter system for mutant screening, as well as, screening for the impaired growth of oscillator deficient strains under dark-light cycles. Finally, we want to determine interaction partners and position of the newly identified components within the regulatory network of the circadian clock. 60 Poster 23: Characteristics of a SoxR-based single cell NADPH biosensor in Escherichia coli Alina Spielmann, Meike Baumgart and Michael Bott IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich, D-52425 Jülich, Germany NADPH-dependent alcohol dehydrogenases and ketoreductases play an important role in industrial biotechnology, especially for the production of chiral alcohols. Therefore, the improvement of these enzymes for in-vivo and in-vitro applications is of high interest. Typically, screenings of mutagenized libraries of such enzymes involve dedicated assays for a certain substrate or product. We recently reported an alternative technology for highthroughput in-vivo screening of NADPH-dependent dehydrogenases using suitable Escherichia coli reporter strains. The screening system is based upon the genetically encoded NADPH biosensor pSenSox, which exploits the transcriptional regulator SoxR, its target promoter PsoxS, and the reporter gene eyfp, encoding a fluorescent GFP derivative [1]. SoxR activity is controlled by the redox status of its [2Fe2S] cluster: in the oxidized state SoxR is active and triggers eyfp expression, in the reduced state it is inactive. The enzymatic reduction and inactivation of oxidized SoxR is NADPH dependent. An increased cellular NADPH demand lowers SoxR reduction and causes increased expression of eyfp. In this way, the plasmid-based biosensor pSenSox is able to sense intracellular NADPH availability. For example, E. coli cells expressing an NADPH-dependent alcohol dehydrogenase of Lactobacillus brevis become fluorescent when the substrate methyl acetoacetate is reduced to (R)-methyl 3-hydroxybutyrate. Under suitable conditions, the specific fluorescence of the cells correlates with the activity of the NADPH-dependent dehydrogenase to be analyzed. In this way, the system allows high-throughput screening of large dehydrogenase libraries using fluorescence-activated cell sorting (FACS). As a prerequisite for further improvements of the screening system, the characteristics of pSenSox-based NADPH-sensing were further characterized. The redox-cycling drugs paraquat and menadion were found to trigger the pSenSox-based eyfp fluorescence. The proteins RseC and RsxABCDGE were reported to be involved in NADPH-dependent SoxR reduction [2] and should be relevant for the pSenSoxbased sensor response. Therefore, ΔrseC und ΔrsxABCDGE deletion mutants of E. coli were constructed as well as strains overexpressing rseC and rsxABCDGE. It could be shown that the eYFP fluorescence signal was increased in the deletion strains compared to the reference strain, whereas it was decreased in the overexpression strains. These results support the finding that SoxR is reduced by RseC and RsxABCDGE and that the levels of these proteins influence the ratio between oxidized active SoxR and reduced inactive SoxR. References 1. Siedler, S., Schendzielorz, G., Binder, S., Eggeling, L., Bringer, S., & Bott, M. (2014). SoxR as a single-cell biosensor for NADPH-consuming enzymes in Escherichia coli. ACS Synth Biol, 3(1), 41-47. 2. Koo, M.S., Lee, J. H., Rah, S. Y., Yeo, W. S., Lee, J. W., Lee, K. L., Roe, J. H. (2003). A reducing system of the superoxide sensor SoxR in Escherichia coli. EMBO J, 22(11), 26142622. 61 Organization: Dr. Reinhold Brückner and Anne Sexauer Microbiology, University of Kaiserslautern Paul-Ehrlichstrasse 24 67663 Kaiserslautern Germany 62