Regulation of secretion of the signalling protease PopC in Myxococcus xanthus [Elektronische Ressource] / Anna Konovalova. Betreuer: Lotte Søgaard-Andersen

Anna Fuller

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2011
• Nombre de lectures: 19
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

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 secr