Regulation of secretion of the signalling protease PopC in Myxococcus xanthus Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Anna Konovalova aus Kamyanets-Podilskiy, Ukraine Marburg/Lahn, Dezember 2010 Die Untersuchungen zur vorliegenden Arbeit wurden von Oktober 2007 bis Oktober 2010 am Max-Planck-Institut für terrestrische Mikrobiologie unter der Leitung von Prof. MD, PhD Lotte Søgaard-Andersen durchgeführt. Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation am: ____________________ angenommen Erstgutachter: Prof. MD, PhD Lotte Søgaard-Andersen Zweitgutachter: Prof. Dr. Erhard Bremer Tag der mündlichen Prüfung: Die während der Promotion erzielten Ergebnisse sind zum Teil in folgender Orginalpublikation veröffentlicht: Konovalova, A., S. Wegener-Feldbrügge, S. Lindow, N. Hamann & L. Søgaard-Andersen, (2010) Proteins of unknown function are required for regulated secretion of the signalling protease PopC in Myxococcus xanthus. Submitted. Rolbetzki, A., M. Ammon, V. Jakovljevic, A. Konovalova& L. Søgaard-Andersen, (2008) Regulated secretion of a protease activates intercellular signalling during fruiting body formation in M. xanthus. Dev Cell15: 627-634. Die Ergebnisse dieser Arbeit, wie auch anderer Arbeiten auf demselben Gebiet, wurden während der Dissertation in folgendem Review diskutiert: Konovalova, A., T. Petters & L. Søgaard-Andersen, (2010) Extracellular biology of Myxococcus xanthus. FEMS Microbiol Rev34: 89-106. Ergebnisse aus in dieser Dissertation nicht erwähnten Projekten sind in der folgenden Orginalpublikation veröffentlicht: Kahnt, J., K. Aguiluz, J. Koch, A. Treuner-Lange, A. Konovalova, S. Huntley, M. Hoppert, L. Søgaard-Andersen & R. Hedderich, (2010) Profiling the outer membrane proteome during growth and development of the social bacterium Myxococcus xanthus by selective biotinylation and analyses of outer membrane vesicles. J Proteome Res9: 5197-5208. Table of contents 4 Table of contents Abstract ............................................................................................................ 5 Zusammenfassung .......................................................................................... 7 Abreviations ................................................................................................... 10 1. Introduction ................................................................................................ 11 1.1. The life cycle of Myxococcus xanthus ..............................................................11 1.2. Intercellular signalling during fruiting body formation ....................................13 1.3. Protein secretion in Gram-negative bacteria ....................................................16 1.3.1. Two-step translocation ...................................................................................................... 16 1.3.2. One-step translocation ...................................................................................................... 18 1.4. Scope ..................................................................................................................21 2. Proteins of unknown function are required for PopC secretion ............ 23 2.1. Results ................................................................................................................23 2.1.1. Bioinformatic analysis of putative secretion systems in M. xanthus ................................. 23 2.1.2. Inactivation of T1SS, T3SS and T6SS do not interfere with secretion of PopC ............... 31 2.1.3. Identification of genes required for secretion of PopC ...................................................... 33 2.2. Discussion ..........................................................................................................42 3. Identification of a regulatory cascade controlling PopC secretion ....... 51 3.1. Results ................................................................................................................51 3.1.1 Activation of PopC secretion depends on the RelA protein and is independent of de novo protein synthesis ......................................................................................................................... 51 3.1.2. PopDinteracts with directly PopC ...................................................................................... 54 3.1.3. PopD inhibits of PopC secretion and is essential in csgA+popC+genetic background ..... 59 3.1.4. RelA and PopD act in the same pathway to regulate PopC secretion.............................. 63 3.1.6. PopC secretion does not depend on LonD protease. ....................................................... 66 3.2. Discussion ..........................................................................................................67 4. Ectopic expression of the PopC protease bypasses the requirement for intercellular A-signaling during development ............................................. 73 4.1. Results ................................................................................................................73 4.2. Discussion ..........................................................................................................80 5. Materials and methods .............................................................................. 86 6. Supplementary data ................................................................................. 108 7. References ................................................................................................ 119 Acknowledgments ....................................................................................... 133 Curriculum Vitae .......................................................................................... 135 Abstract 5 Abstract In response to starvation Myxococcus xanthus initiates a developmental program that culminates in fruiting body formation. Completion of this developmental program depends on cell-cellcommunication involving at least two intercellular signals, the A-signal and the C-signal. The contact-dependent intercellular C-signal function to induce and coordinate the two morphogenetic events in fruiting body formation, aggregation and sporulation, temporally and spatially coordinated. The intercellular C-signal is a 17 kDa protein (p17), which is generated by proteolytic cleavage of the full-length 25 kDa csgA protein (p25), and is essential for fruiting body formation. p25 and PopC, the protease that cleaves p25, accumulate in the outer membrane and cytoplasm, respectively in vegetative cells. PopC is specifically secreted during starvation. Therefore, restriction of p25 cleavage to starving cells depends on a compartmentalization mechanism that involves the regulated secretion of PopC in response to starvation. In this report, the main focus is on understanding the mechanism underlying regulated secretion of the PopC protease. We first focused on the identification of proteins required for PopC secretion. PopC lacks a signal peptide and is secreted in an unprocessed form. We report that two incomplete type III secretion systems, a type VI secretion system and type I secretion systems are not involved in PopC secretion. From a collection of mutants generated by random transposon mutagenesis and unable to complete fruiting body formation, we identified seven mutants unable to secrete PopC. None of the insertions were in genes coding for known secretion systems. The mutations were divided into three classes based on the insertion sites. The class I mutation was in a gene cluster largely encoding proteins of unknown function, predicted to localize to the cell envelope, and with a narrow phylogenetic distribution except for a D,D-carboxypeptidase and two Ser/Thr kinases. The class II mutations were in two clusters encoding paralogous proteins of unknown function predicted to localize to the cytoplasm. Several of the class II genes are phylogenetically widely distributed and frequently present in gene clusters linked to genes encoding secretion systems. We speculate that the class I mutation affect a novel type of secretion system involved in PopC Abstract 6 secretion and that the class II mutations either affect proteins with accessory or regulatory functions in PopC secretion. Next, we focused on elucidating the molecular mechanism underlying the activation of PopC secretion in response to starvation. Our data demonstrate that PopC secretion is controlled at the post-translational level by a regulatory cascade involving the RelA and PopD proteins. Specifically, RelA is required for activation of PopC secretion in response to starvation and PopD, which is encoded in an operon with PopC, interacts directly with PopC and acts as an inhibitor of PopC secretion. On the basis of genetic and biochemical data we suggest that PopC and PopD form a cytoplasmic complex that blocks PopC secretion in the presence of nutrients. In response to starvation, RelA is activated resulting in induction of the stringent response. Activated RelA by an unknown mechanism induces the proteolytic degradation of PopD in the PopC/PopD complex in that way releasing PopC for secretion. On the basis of these data, we suggest that the generation of p17 depends on a two-step proteolytic cascade involving degradation of PopD and, subsequently, the specific cleavage of p25 by PopC. The current model for intercellular A-signaling in M. xanthus proposes that starvation