9 research outputs found
Regulatory mechanisms of the acid stress response of Escherichia coli on the transcriptional, posttranscriptional and proteolytic level and their role in the kinetics of the system
Die Bedeutung posttranskriptionaler Regulation in Stressantwortsystemen und
Differenzierungsprozessen in Prokaryoten wurde in den letzten Jahren immer
mehr erkannt und erforscht. Die Suche nach noch unbekannten
Proteolysesubstraten ist schwierig, da Regulatoren meist in geringen
zellulÀren Konzentrationen vorliegen und daher durch direkte FÀrbeverfahren
nicht detektierbar sind. Daher wurde hier eine Methode zur Identifikation von
Proteolyse-kontrollierten Regulatoren entwickelt mithilfe von
Transkriptomanalyse von Proteasemutanten (lon und clpP) versus Wildtypzellen
mit DNA-Microarrays in Escherichia coli. So war es möglich, mehrere Regulons
zu bestimmen, welche differenziell in den Mutanten exprimiert werden, z.B.
viele ÏSâabhĂ€ngige Gene und, besonders markant, Gene der SĂ€urestressantwort,
welche im lon Hintergrund verstÀrkt und in der clpP Deletionsmutante
vermindert transkribiert werden. Das Glutamat-abhÀngige SÀureresistenz-System,
kodiert von gadA/BC, ist essentiell fĂŒr Escherichia coli, um die Passage durch
den hochsauren Magen zu ĂŒberleben. Das System wird induziert bei niedrigem pH
und bei Eintritt in die stationÀre Phase. Diese Regulation benötigt den
Masterregulator ÏS, welcher die Synthese von GadE und GadX antreibt, die als
essentieller, zentraler Aktivator bzw. als Modulator der Expression dieser
Gene agieren (Weber et al., 2005). Ein anderer regulatorischer Schaltkreis
beinhaltet das EvgA/S Zweikomponenten-System und YdeO, welche einen positiven
Feedforward Loop bilden, um gadE zu regulieren (Foster, 2004). WĂ€hrend der
Masterregulator ÏS unter proteolytischer Kontrolle steht (Hengge-Aronis,
2002), wurde in dieser Arbeit ermittelt, dass auch GadE auf den Ebenen der
Expression und des Abbaus kontrolliert wird. In-vivo-Abbausexperimente
zeigten, dass GadE konstitutiv von der Lon Protease abgebaut wird. Immunoblot-
und Reportergenfusions-Daten bezeugen, dass der schnelle Anstieg des GadE-
Gehaltes nach SĂ€ureshift aufgrund posttranskriptionaler Kontrolle stattfindet,
die kleine RNA DsrA involvierend, wÀhrend die transkriptionale Induktion die
langsame Reaktion auf andauernden SĂ€urestress liefert. Die Lon-vermittelte
Proteolyse von GadE ist entscheidend fĂŒr die schnelle Termination der Antwort,
gezeigt durch GadE Immunoblot und gadA/BC Northern Blots in lon+/-. Andere in-
vivo-Abbauexperimente und GFP-Fusions-Studien weisen auf weitere Kandidaten
proteolytischer Kontrolle, wie YdeO und GadW, innerhalb dieses Systemes hin,
damit eine Basis fĂŒr zukĂŒnftige Studien schaffend. In Microarray-Studien und
LacZ-Reportergenfusions-Studien wurden voneinander abgegrenzte Gengruppen
identifiziert, die entweder von GadE und GadX zusammen oder nur von GadX
kontrolliert werden. Die komplexe regulatorische Architektur konnte in den
prinzipiellen Signalwegen aufgeklÀrt werden und ein Modell wurde entwickelt,
welches die Regulationsmechanismen und Dynamiken des SĂ€urestressantwort-
Netzwerkes integriert.The impact of posttranscriptional regulation in stress response systems and
differentiation processes in prokaryotes are increasingly recognized and
investigated in the recent years. The search for yet unknown regulatory
substrates of proteolysis faces the difficulty that regulators are usually
present in small amounts in bacteria, difficult to detect through direct
staining procedures. Therefore a method for identifying regulators subjected
to proteolysis was developed using transcriptomic analysis of protease mutants
(lon and clpP) versus wildtype cells via DNA microarrays in Escherichia coli.
This way it was possible to determine several regulons which are
differentially expressed in the mutants, e.g. many ÏS-dependent genes, and, as
the most prominent, genes of the acid stress response, which are upregulated
in the lon background and downregulated in the clpP deletion strain. The
Glutamate-dependent acid resistance system, encoded by the gadA/BC genes, is
essential for Escherichia coli to survive the passage through the highly
acidic stomach. The system is induced at low pH and during entry into
stationary phase. This regulation involves the master regulator ÏS, which
drives the expression of GadE and GadX, which act as the essential key
activator and as a modulator, respectively, for the expression of these genes
(Weber et al., 2005). Another transcriptional regulatory circuit implies the
EvgA/S two-component system and YdeO, which form a positive feedforward loop
regulating gadE (Foster, 2004). While the master regulator ÏS has long been
known to be under proteolytic control (Hengge-Aronis, 2002), it is established
here that also GadE is controlled both at the levels of expression and
degradation. In-vivo degradation experiments show that GadE is constitutively
degraded by the Lon protease. Immunoblot and reporter fusion data indicate
that the rapid increase in GadE levels upon pH downshift is due to
posttranscriptional regulation involving the small RNA DsrA, while
transcriptional induction provides the slow reactions to enduring acid threat
and during entry into stationary phase. Lon-mediated proteolysis of GadE is
crucial for the rapid termination of the response shown by GadE immunoblot and
gadA/BC Northern experiments in lon+/-. Other in-vivo degradation experiments
and GFP fusion studies strongly support additional candidates for proteolytic
control within this system, e.g. YdeO and GadW, providing a basis for future
studies. In microarray studies and LacZ reportergen fusion studies we
identified different groups of genes either controlled by GadE and GadX
together or by GadX alone. The complex regulatory architecture could be
assessed in the principal pathways and a model was developed, integrating the
regulatory mechanisms and dynamics of the acid stress response network
Genome-Wide Analysis of the General Stress Response Network in Escherichia coli: Ï(S)-Dependent Genes, Promoters, and Sigma Factor Selectivity
The Ï(S) (or RpoS) subunit of RNA polymerase is the master regulator of the general stress response in Escherichia coli. While nearly absent in rapidly growing cells, Ï(S) is strongly induced during entry into stationary phase and/or many other stress conditions and is essential for the expression of multiple stress resistances. Genome-wide expression profiling data presented here indicate that up to 10% of the E. coli genes are under direct or indirect control of Ï(S) and that Ï(S) should be considered a second vegetative sigma factor with a major impact not only on stress tolerance but on the entire cell physiology under nonoptimal growth conditions. This large data set allowed us to unequivocally identify a Ï(S) consensus promoter in silico. Moreover, our results suggest that Ï(S)-dependent genes represent a regulatory network with complex internal control (as exemplified by the acid resistance genes). This network also exhibits extensive regulatory overlaps with other global regulons (e.g., the cyclic AMP receptor protein regulon). In addition, the global regulatory protein Lrp was found to affect Ï(S) and/or Ï(70) selectivity of many promoters. These observations indicate that certain modules of the Ï(S)-dependent general stress response can be temporarily recruited by stress-specific regulons, which are controlled by other stress-responsive regulators that act together with Ï(70) RNA polymerase. Thus, not only the expression of genes within a regulatory network but also the architecture of the network itself can be subject to regulation