6 search hits
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Analyse der Funktion und Regulation des ECF-Sigmafaktors YlaC aus Bacillus subtilis
(2005)
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Stephan Zellmeier
- Alternative Sigmafaktoren mit extracytoplasmatischer Funktion (ECF-Sigmafaktoren) repräsentieren eine sich kontinuierlich vergrößernde Gruppe an Regulatorproteinen, die an der differentiellen Genexpression in Bakterien beteiligt sind. Obwohl Gene, welche durch diese Regulatorproteine kontrolliert werden, ebenso wie die Regulationsmechanismen noch weitgehend unaufgeklärt sind, scheinen ECF-Sigmafaktoren in Bakterien eine Schlüsselrolle im Bereich der Stressantwort, der Differenzierung und der Pathogenese zu spielen. In dem Gram-positiven Modellorganismus B. subtilis konnten bislang sieben Gene mit Ähnlichkeiten zu ECF-Sigmafaktoren identifiziert werden (sigX, sigW, sigM, sigY, sigV, sigZ und ylaC), wobei hauptsächlich sigX, sigW und sigM im Zentrum durchgeführter Untersuchungen standen. Im Rahmen der vorliegenden Arbeit wurde der potentielle ECF-Sigmafaktor YlaC hinsichtlich seiner Funktion und Regulation untersucht. Es konnte gezeigt werden, dass ylaC Teil eines tetracistronischen Operons ist und mit dem Gen seines möglichen Anti-Sigmafaktors kotranskribiert wird. Zusätzlich kodiert das Operon für zwei weitere Membranproteine mit bislang unbekannter Funktion. Obgleich kein spezifischer Stressfaktor identifiziert werden konnte, wurde die positive Regulation des ylaA-Operons durch YlaC eindeutig gezeigt. Die Ergebnisse einer globalen Transkriptionsanalyse deuten darauf hin, dass YlaC ausschließlich sein eigenes Operon reguliert. Durch Primer-Extension konnte der Transkriptionsstartpunkt des ylaA-Operons bestimmt werden. Die ermittelte Promotorsequenz ähnelt bereits identifizierten Promotorsequenzen von ECF-Sigmafaktoren aus B. subtilis. Die für den ylaA-Promotor ermittelte Sequenz konnte vor keinem anderen Ges des B. subtilis-Genoms gefunden werden. Durch Northern-Blots und b-Galaktosidase-Tests mit einem DylaD-Stamm wurde gezeigt, dass YlaD als Anti-Sigmafaktor von YlaC wirkt. Gereinigtes YlaD bindet Zink in äquimolaren Mengen und wird deshalb einer neu-definierten Familie von Zink-bindenden Anti-Sigmafaktoren zugeordnet. YlaD ist ein Membranprotein, dessen C-terminus in der Cytoplasmamembran verankert ist. Nach Überexpression konnte YlaD in Western-Blots nur dann nachgewiesen werden, wenn das yluC-Gen, welches für eine intramembrane Protease kodiert, deletiert war. Eine YluC-Variante mit einer Mutation im aktiven Zentrum war nicht mehr in der Lage, das sW-Regulon aus B. subtilis zu aktivieren. In einer DyluC-Mutante reicherten sich auch verkürzte Formen der SigX und SigM Anti-Sigmafaktoren an, was auf eine generelle Rolle von YluC bzw. von intramembraner Proteolyse bei der Regulation von ECF-Sigmafaktoren in B. subtilis hindeutet. Eine mögliche Funktion der bislang uncharakterisierten Membranproteine YlaA und YlaB bei der Regulation der YlaC-Aktivität wurde durch die Konstruktion von Reporterstämmen, welche verschiedene Kombinationen aller vier Gene des ylaA-Operons enthielten, analysiert. Dabei konnte gezeigt werden, dass die Anwesenheit von YlaB einen Einfluss auf die Anti-Sigmafaktor-Aktivität von YlaD hat. Durch Western-Blots war ein stabilisierender Einfluss von YlaB auf YlaD nachweisbar. Eine direkte Interaktion zwischen YlaB und YlaD konnte durch in vitro-Experimente gezeigt werden. Diese Ergebnisse deuten darauf hin, dass YlaD, zusammen mit YlaB und eventuell auch mit YlaA, Teil eines membrangebundenen Proteinkomplexes ist, der YlaD vor dem Abbau durch YluC schützt.
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Regulated intramembrane proteolysis in the control of the Bacillus subtilis anti-sigma factor RsiW.
(2010)
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Janine Heinrich
- The activity of the extracytoplasmic function (ECF)-sigma factor SigW of the Gram-positive soil bacterium Bacillus subtilis is modulated by a specific membrane-bound anti-sigma factor (RsiW). Initiated most likely by cell wall stress, RsiW is degraded by the mechanism of regulated intramembrane proteolysis (RIP). This process involves two site-specific proteolytic cleavage events in the extracytoplasmic part (site-1) and in the transmembrane domain (site-2) of RsiW. In consequence, SigW is released to interact with the RNApolymerase, and the transcription of SigW-controlled genes is initiated. In general, regulation of differential gene expression by RIP seems to play a major role in prokaryotic stress responses, pathogenesis and antibiosis. However, in most cases the molecular basis is not understood. The main objective of this work was to use B. subtilis SigW/ RsiW as a model to investigate the mechanism of RIP in detail. In particular, there are significant differences to the inducing stress-signal and the site-1 protease that are described for the well investigated Escherichia coli ECF sigma factor SigE-system. The basis of this work were different mutants with a defect in RIP of RsiW that were isolated in a transposon screen. First, PrsW (formerly YpdC) was identified as the site-1 protease. It belongs to a superfamily of potential membrane embedded metalloproteases (MEM) with so far unknown function in bacteria. Further characterization of PrsW in a reconstituted E. coli system showed that PrsW cleaves RsiW in a site-specific manner to form a protein truncated for 40 C-terminal extracytoplasmic amino acid residues. The tail specific protease Tsp was shown to further degrade the extracytoplasmic part of this RsiW site-1 cleavage product, which is crucial for subsequent RasP catalyzed site-2 clipping. Several other peptidases seem to be involved in trimming of RsiW downstream of PrsW and upstream of RasP in B. subtilis. Second, the transposon screen revealed that a defect of the ABC-transporter EcsAB impairs RsiW site-2 cleavage by RasP for unknown reasons. It is conceivable that an EcsAB substrate competitively inhibits RasP activity. In summary, a new model of two proteolytic modules involved in intramembrane proteolysis of RsiW could be established. Each module consists of a site-specific processing peptidase (site-1: PrsW, site-2: RasP) that subjects cleaved RsiW to degradation by unspecific proteases (site-1: Tsp-like, site-2: Clp-proteases).
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Development of Bacillus subtilis spores and cells for surface display of proteins
(2011)
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Quynh Anh Nguyen
- Surface display has attracted the attention of researchers in developing efficient display systems expressing heterologous polypeptides on the surface of bioparticles such as phages, bacterial and eukaryotic cells and bacterial spores. Among these bioparticles, the endospore from B. subtilis has advantages, including feasibility of production, safety feature, the robustness of the bacterial spore allowing storage in the desiccated form, a technological platform supported by extensive tools for genetic manipulation and less size restrictions of the displayed proteins compared to cell- and phage-based systems. A strategy to engineer B. subtilis spores to display heterologous protein on their surface is to use outer spore coat proteins (CotB, CotC, CotG) or an inner-coat protein (OxdD) with the coat genes’ transcriptional and translational signals as carriers (Isticato et al., 2001; Mauriello et al., 2004; Hinc et al., 2010; Zhou et al., 2008a; Potot et al., 2010; Kim et al., 2005a; Kwon et al., 2007). This strategy guarantees the timing for fusion protein synthesis during coat formation, but the amount of produced fusion proteins cannot be controlled. Therefore, the first aim of this doctoral thesis focused on construction of more effective expression systems for spore surface protein anchoring. A novel approach of substitution of native promoter by two different IPTG-inducible promoters to the increase the production of fusion protein is presented here. CotB was used and the expression of the cotB gene was regulated by either its own promoter, the Pgrac and the PSgrac promoter in a series of plasmids which can be integrated into or replicated independently of the B. subtilis chromosomal DNA. Two reporter proteins, α-amylase Q from B. amyloliquefaciens (AmyQ) (Palva, 1982) and GFPuv – an enhanced version from the GFP protein of the jellyfish Aequorea victoria (Crameri et al., 1996), were fused downstream of the CotB protein. To assess the enhancement of GFPuv displayed on the spore surface, CotC and CotG were similarly examined. The results indicated that the Pgrac promoter is a suitable, hence recommended as a promoter of choice. Substitution of the native promoter by Pgrac promoter, the amount of proteins displayed per spore can be increased two-fold. Furthermore, the display of heterologous proteins on the spore surface when using different carriers is gene dosage dependent. And for the first time, the tendency of the three Cot proteins’ localization on the spore coat compartment is reported using the GFPuv tag. Second, a new B. subtilis spore-based system for protein expression and purification was developed. Using this system, proteins prone to form inclusion bodies can be anchored on the spore surface, separated by a mini-intein derived from the SSp DnaB, which was then used as self-cleaving tag for purification by shifting the pH and/or temperature conditions, with no addition of any proteases or thiol reagent (Mathys et al., 1999). To construct the system, the mini-intein was fused downstream of the CotB protein, followed by the reporter protein AmyQ. By changing the pH of the buffer, the mini-intein self-cleaving process was induced followed by the release of α-amylase into the supernatants. This observation suggests the use of the B. subtilis spores as an effective and low cost tool for protein purification. However, concerns related to premature of the pH-inducible mini-intein and auto-release of coat protein raise the question about the stability of the fusion coat-heterologous protein on the spore surface using the system. Hence, further investigation is needed to achieve a usable spore-based purification system. The last aim of the thesis was to apply the newly constructed B. subtilis spore display and the cell surface display systems (Nguyen and Schumann, 2006) to generate cellulose chips, in which enzymes were immobilized on the surface of microorganism cells or spores. The cellulase A (CelA) from C. thermocellum (Beguin et al., 1985) was utilized as a model enzyme. Unfortunately, the results showed an ineffective anchoring of CelA on the cell wall. This indicates the unsuccessful creation of cell-based cellulase chip when using the SrtA transpeptidase. In contrast, CelA was verified to be successfully displayed on the spore surface using CotB and CotG, but not CotC, as carriers. In general, a large volume of culture (up to one liter) must be prepared containing both cells and spores displaying CelA on the surface to assure sufficient CMC degradation. This might indicate a low activity of CelA. Further works should be done in selection of cellulase and improvement of the systems to generate the more effective cellulase chips.
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Construction of an efficient secretion system for recombinant proteins in Bacillus subtilis
(2011)
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Kelly Cristina Leite
- All proteins being translocated through the cytoplasmic membrane of bacteria cells as well as some proteins that are inserted into the cytoplasmic membrane contain a signal sequence at their N-terminus that is recognized by and targeted to the translocation machinery. Three translocation pathways have been described, so far in E. coli to allow secretion of proteins: The Sec, the Tat and the SRP (Signal Recognition Particle) pathway. While the Sec and the Tat pathway act post-translationally and accept unfolded and correctly folded polypeptides, respectively, the SRP pathway acts co-translationally. For proteins secreted through the cytoplasmic membrane via the Sec pathway, the ATP-dependent motor protein SecA is required for translocation. The translocation process of some proteins following the SRP pathway has also shown to be enhanced by the presence of SecA. The Sec and the SRP pathway share the heterotrimeric protein-conducting channel translocon complex composed of the SecYEG proteins. Based on the known characteristics of both pathways, the goal of this PhD project was to construct an efficient secretion system for recombinant proteins in Bacillus subtilis using an a-amylase as a reporter enzyme, which is secreted into the medium using the Sec pathway. Its gene amyQ was fused to an IPTG-inducible promoter. It turned out that increasing amounts of IPTG did not result in a concomitant increase of secreted a-amylase. Overproduction either formed aggregates within the cytoplasm or preproteins targeted to the translocon jammed the membrane. To release the accumulated protein within the cells two different experiments were carried out: i) a co-production and overexpression of SecA, and; ii) overexpression of an artificial secYEG operon. First, increased production of SecA showed significantly decrease in the total synthesis and secretion of a-amylase and did not reduce cytoplasmatic accumulation or membrane jamming. Second, the artificial operon enhanced expression of secY, secE and secG genes resulted in a higher amount of reporter enzyme secreted into the medium. Furthermore, two different experiments using the transposon mutagenesis strategy were carried out in order to screen for B. subtilis mutants able to increase secretion of α-amylase. Transposon mutagenesis was performed with the mariner-based transposon to inactivate gene(s) whose product might regulate directly or indirectly the secretion of α-amylase. No mutant strain presenting a higher secretion of α-amylase on indicator plates was found. In addition, I devised a modified transposon containing a xylose-expression cassette. To test the efficiency of the modified transposon, the promoter-less cat gene was used as a reporter gene and integrated into the B. subtilis chromosomal DNA. After transposon mutagenesis, mutants expressing the promoter-less cat gene were isolated. This result indicates that the modified transposon might lead to increased production of a gene in the presence of xylose and that this product might then enhance secretion of α-amylase to be detected on indicator plates. In the third part of my thesis, a terminator-test vector was constructed which should allow the identification of strong terminators acting as 5'-stabilizing element. This vector consists of an artificial bicistronic operon containing the two reporter genes bgaB and gfp allowing the insertion of the terminators between the two genes. Insertion of a terminator should lead to a reduction of the amount of GFP. The system was verified with the known sinIR transcriptional terminator. It turned out that the vector with the two reporter genes already exhibited instability in E. coli.
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Identification of substrate proteins of FtsH during sporulation of Bacillus subtilis
(2012)
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Hue Bach Thi Nguyen
- FtsH is an ATP- and Zn2+-dependent metalloprotease anchored in the cytoplasmic membrane by two transmembrane segments. It is the unique membrane-bound AAA-protease in bacteria that performs a variety of regulatory functions. In B. subtilis, an ftsH knockout exhibits a pleiotropic phenotype such as filamentous growth, sensitivity towards heat, osmotic shock and cells are unable to sporulate. Recently, it has been shown that ftsH knockout cells fail to entry sporulation stage II due to lack of a sufficient amount of Spo0A~P and the first substrate of FtsH identified in B. subtilis is the Spo0E phosphatase, a negative regulator that dephosphorylates Spo0A~P. However, the sporulation frequency in a spo0E ftsH double mutant strain was only partly restored, we hypothesized that FtsH might degrade additional substrate proteins involved in sporulation. To identify these proteins, two different strategies were applied. By using the 2D gel technique, the proteomes of an ftsH wild-type strain was compared with an ftsH null mutant. Several proteins were identified to be either up- or down-regulated in the absence of FtsH. One of them up-regulated about 4-fold was identified as Spo0M. Since ftsH did not interfere with transcription of spo0M, an in vitro proteolysis assay was established using purified components. It was shown that Spo0M was degraded by FtsH in an ATP- and time-dependent way. In the second strategy, an ftsHtrap mutant was constructed and tested for loss of its proteolytic activity. Protease trap mutants are still able to bind substrate proteins, but are unable to degrade them. By using FtsHtrap fused to a GST-tag, YwnF, a membrane protein, was trapped and identified as a substrate of FtsH by mass spectrometry. However, further experiments will be required to confirm YwnF as a target of FtsH. The last part of this thesis was focused on the eag gene, which forms a bicistronic operon with Spo0E. Construction and analysis of an eag insertion mutant exhibited a slight increase in the sporulation frequency and in the amount of Spo0A. A transcriptional fusion between the promoter of the spo0E-eag operon and the lacZ reporter gene revealed an increase in the beta-galactosidase activity from t0 when the cells were grown in sporulation medium. Since the Eag protein may be an integral membrane protein, it may bind excess Spo0E thereby preventing it from dephosphorylating Spo0A~P. Alternatively, Eag may bind Spo0E and present it as a modulator to FtsH for degradation.
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Regulation der Aktivität des Anti-Sigmafaktors RsiX aus Bacillus subtilis
(2011)
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Katharina Schäfer
- Der Gram-positive Modellorganismus Bacillus subtilis besitzt 17 alternative Sigmafaktoren, von denen 7 zur Gruppe der ECF (extra-cytoplasmic function) -Sigmafaktoren gehören. Einer dieser ECF-Sigmafaktoren, SigX, und sein entsprechender Anti-Sigmafaktor, RsiX, sind Gegenstand dieser Arbeit. Dabei handelt es sich bei RsiX um ein Transmembranprotein, während SigX im Cytoplasma lokalisiert ist und von der N-terminalen RsiX-Domäne von einer Interaktion mit der RNA-Polymerase abgehalten wird. SigW, ein weiterer ECF-Sigmafaktor, und RsiW, der dazugehörende Anti-Sigmafaktor, sind bereits sehr gut untersucht. Aufgrund struktureller Ähnlichkeiten zwischen SigX/RsiX und SigW/RsiW wurde angenommen, dass die Aktivierung des SigX-Regulons in vergleichbarer Weise erfolgt, wie es für das SigW-Regulon aufgrund intensiver Analysen sehr gut untersucht ist. Die in dieser Arbeit verwendete Methode der Transposonmutagenese lieferte entscheidende Hinweise über die Regulation des sigX-rsiX-Operons. Es konnte gezeigt werden, dass das sigX-rsiX-Operon einer Regulation durch den Transkriptionsterminator Rho unterliegt. Damit wurde das erst dritte Beispiel für eine Termination der Transkription durch den Faktor Rho in B. subtilis bekannt. Als wichtiges Element für die faktorabhängige Termination der Transkription gilt das Vorhandensein einer rut-site, der Bindestelle des Faktors Rho in der naszierenden mRNA. In dieser Arbeit wurden verschiedene Analysetechniken angewandt, um mit Hilfe eines GFP-RsiX-Reporters erstmals eine rut-site in B. subtilis zu kartieren. Die hier identifizierte rut-site von rsiX ist am 5´-Ende dieses Gens lokalisiert. Bei diesen Analysen wurde außerdem deutlich, dass es im sigX-rsiX-Operon zu einer faktorabhängigen intragenischen Termination der Transkription kommt, bei welcher nicht nur die Transkription von rsiX, sondern auch die Expression des stromaufwärts liegenden sigX beeinflusst wird. Punktmutationen in der Sequenz für die rut-site von rsiX zerstörten den Einfluss des Faktors Rho auf das sigX-rsiX-Operon, so dass keine Termination der Transkription mehr stattfand und die detektierbare Menge an sigX-rsiX-Transkript deutlich anstieg. Nur so war es möglich, einen rsiX-Knockout zu komplementieren. Messungen der beta-Galaktosidase-Aktivität eines lacZ-Reporters belegten zudem die Funktion von RsiX als Anti-Sigmafaktor von SigX. Der Verlust des Einflusses von Rho auf das sigX-rsiX-Operon aufgrund einer mutierten rut site machte auch immunologische Nachweise des Anti-Sigmafaktors möglich. Infolgedessen konnte der Frage einer möglichen RsiX-Proteolyse nach dem Vorbild der RsiW-Proteolyse nachgegangen werden. Für RsiW wurde bereits gezeigt, dass ein bestimmtes Stress-Signal den Abbau des Anti-Sigmafaktors einleitet. Die Proteolyse von RsiW erfolgt dann nach dem Mechanismus der regulierten intramembranen Proteolyse (RIP) unter der Beteiligung mehrerer Proteasen. Neben der Beteiligung der Protease RasP an der RsiW-Proteolyse konnte hier auch die Beteiligung von RasP an der RsiX-Proteolyse nachgewiesen werden. Gleichzeitig wurde aber ausgeschlossen, dass die Protease PrsW, welche auch an der RsiW-Proteolyse beteiligt ist, in die RsiX-Proteolyse involviert ist. Demnach können die einzelnen Schritte der RsiW-Proteolyse nicht einfach auf die RsiX-Proteolyse übertragen werden, obwohl es sich bei beiden Transmembranproteinen um Anti-Sigmafaktoren ihrer jeweiligen ECF-Sigmafaktoren handelt.
