Regulation of glutamate dehydrogenase in Corynebacterium glutamicum [Elektronische Ressource] / vorgelegt von Eva Hänßler

De
Regulation of glutamate dehydrogenase in Corynebacterium glutamicum Der Naturwissenschaftlichen Fakultät der Friedrich-Alexander-Universität Erlangen-Nürnberg zur Erlangung des Doktorgrades vorgelegt von Eva Hänßler aus Aachen Als Dissertation genehmigt von der Naturwissen- schaftlichen Fakultät der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 15.02.2008 Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. Andreas Burkovski Zweitberichterstatter: Prof. Dr. Reinhard Krämer Content Content 1 Zusammenfassung/Summary........................................................1 2 Introduction.....................................................................................3 2.1 Corynebacterium glutamicum.............................................................................3 2.2 Uptake and assimilation of nitrogen sources....................................................4 2.3 Nitrogen-dependent regulation...........................................................................6 2.4 GDH at the interface between nitrogen and carbon metabolism...................11 2.4.1 Glutamate dehydrogenase of E. coli ................................................................12 2.4.2 B. subtilis...........................................................13 2.4.
Publié le : mardi 1 janvier 2008
Lecture(s) : 42
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2008/857/PDF/EVAHAENSSLERDISSERTATION.PDF
Nombre de pages : 140
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Regulation of glutamate dehydrogenase in
Corynebacterium glutamicum











Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades






vorgelegt von
Eva Hänßler
aus Aachen







Als Dissertation genehmigt von der Naturwissen-
schaftlichen Fakultät der Universität Erlangen-Nürnberg



















Tag der mündlichen Prüfung: 15.02.2008

Vorsitzender der
Promotionskommission: Prof. Dr. Eberhard Bänsch

Erstberichterstatter: Prof. Dr. Andreas Burkovski

Zweitberichterstatter: Prof. Dr. Reinhard Krämer




Content
Content

1 Zusammenfassung/Summary........................................................1
2 Introduction.....................................................................................3
2.1 Corynebacterium glutamicum.............................................................................3
2.2 Uptake and assimilation of nitrogen sources....................................................4
2.3 Nitrogen-dependent regulation...........................................................................6
2.4 GDH at the interface between nitrogen and carbon metabolism...................11
2.4.1 Glutamate dehydrogenase of E. coli ................................................................12
2.4.2 B. subtilis...........................................................13
2.4.3 Glutamate dehydrogenase of C. glutamicum ...................................................15
2.5 Objectives ...........................................................................................................17
3 Materials and methods.................................................................19
3.1 Bacterial strains and plasmids .........................................................................19
3.2 Cultivation of bacteria........................................................................................22
3.2.1 Culture medium for E. coli ................................................................................22
3.2.2 ia for corynebacteria .....................................................................22
3.2.3 Antibiotics .........................................................................................................23
3.2.4 Growth conditions.............................................................................................24
3.3 Genetic manipulation of bacteria......................................................................25
3.3.1 Preparation of competent E. coli cells and transformation ...............................25
3.3.2 C. glutamicum cells and transformation..................26
3.4 Working with DNA ..............................................................................................26
3.4.1 Isolation of plasmid DNA from E. coli ...............................................................26
3.4.2 Gel electrophoresis and extraction of DNA from agarose gels.........................27
3.4.3 Preparation of chromosomal DNA from C. glutamicum....................................27
3.4.4 Purification and enrichment of DNA .................................................................27
3.4.5 Polymerase chain reaction (PCR) ....................................................................28
3.4.6 Two-step PCR ..................................................................................................28
3.4.7 Restriction of DNA............................................................................................29
3.4.8 Ligation of DNA fragments ...............................................................................29
3.4.9 Sequencing of DNA..........................................................................................29
3.5 Working with RNA ..............................................................................................30
3.5.1 Isolation of total RNA and RNA gel electrophoresis.........................................30 Content
3.5.2 Synthesis of digoxigenin-labeled RNA probes .................................................31
3.5.3 Northern blot analysis.......................................................................................31
3.5.4 Dot blot analysis ...............................................................................................33
3.5.5 Reverse transcriptase (RT) PCR......................................................................34
3.5.6 Quantitative real time RT PCR .........................................................................34
3.5.7 Primer extension analysis ................................................................................35
3.6 Working with proteins........................................................................................37
3.6.1 Protein purification............................................................................................37
3.6.2 Quantification of proteins..................................................................................38
3.6.3 SDS polyacrylamide gel electrophoresis (PAGE).............................................38
3.6.4 Staining with Coomassie Brilliant Blue .............................................................39
3.6.5 Western blotting ...............................................................................................39
3.6.6 Determination of enzyme activity .....................................................................41
3.6.6.1 GDH activity measurements41
3.6.6.2 Glutamyltransferase test...........................................................................42
3.6.7 Determination of promoter activity....................................................................43
3.6.8 Gel shift assays and competition assays .........................................................43
4 Results...........................................................................................45
4.1 Purification and characterization of glutamate dehydrogenase....................45
4.1.1 Purification of GDH45
4.1.2 Characterization of GDH in lysine-producing strains........................................47
4.1.3 Gradual expression of gdh ...............................................................................49
4.2 Transcriptional regulation of gdh .....................................................................51
4.2.1 Mutational analyses of the gdh promoter region ..............................................51
4.2.2 Determination of the transcription start.............................................................57
4.2.3 Nitrogen-dependent transcription60
4.2.3.1 Function of AmtR......................................................................................61
4.2.3.2 Influence of putative regulators on gdh transcription................................66
4.2.4 Studies on sigma factor-dependent gdh expression ........................................71
4.2.5 Investigation of the putative orf Cg2281...........................................................73
4.3 Identification of AmtR and FarR target genes .................................................75
4.3.1 Identification of FarR target genes ...................................................................75
4.3.1.1 Characterization of arginine biosynthesis genes ......................................76
4.3.1.2 Determination of FarR and ArgR binding sites .........................................79
4.3.1.3 Transcriptional regulation of arginine biosynthesis genes........................81 Content
4.3.2 Identification of AmtR target genes ..................................................................83
5 Discussion.....................................................................................89
5.1 Characterization of GDH in the context of systems biology..........................89
5.2 Transcriptional regulation of gdh .....................................................................93
5.3 Identification of FarR and AmtR target genes ...............................................100
5.4 The interface between nitrogen and carbon metabolism.............................106
6 Appendix......................................................................................109
6.1 Regulation of glutamine synthetase in corynebacteria................................109
6.2 Plasmid constructions.....................................................................................112
References ........................................................................................117
Publications ......................................................................................131
Abbreviations and units...................................................................132 Zusammenfassung 1
1 Zusammenfassung
Die Glutamatdehydrogenase (GDH) aus Corynebacterium glutamicum, einem
Actinomyceten mit herausragender biotechnologischer Bedeutung, befindet sich an einer
wichtigen Position innerhalb des Stoffwechsels, da sie Stickstoffassimilation und den
Zentralstoffwechsel verbindet. Unter Überschussbedingungen ist die GDH an der
Ammoniumassimilation beteiligt und über das Substrat α-Ketoglutarat besteht eine direkte
Verknüpfung zum Citrat Zyklus. Aufgrund der NADPH-Abhängigkeit kann weiterhin der pool
an Reduktionsäquivalenten beeinflusst werden. Trotz dieser scheinbar bedeutsamen
Stellung konnte bis auf die mögliche Beteiligung zweier Transkriptionsregulatoren kein
detaillierter Regulationsmechanismus beschrieben werden.
Um die GDH genauer charakterisieren zu können, wurde das Protein gereinigt und in
definierten Lysinproduktionsstämmen untersucht. Hierzu wurde im Rahmen dieser Arbeit
2+ein Protokoll zur Überexpression in C. glutamicum, gefolgt von einer Kombination aus Ni
NTA Affinitätschromatographie und Gelfiltrationschromatographie etabliert. Dadurch wurde
die Grundlage für weiterführende kinetische Messungen gelegt. Weiterhin wurde GDH-
Aktivität, Proteinmenge und Transkriptlevel in Produktionsstämmen bestimmt.
Veränderungen von Stoffwechselflüssen, die auf eine erhöhte Lysinproduktion
zurückzuführen waren, zeigten keinen Einfluss auf die GDH im Vergleich zum Wildtyp.
Als Schwerpunkt der Arbeit wurde, auf Grund der widersprüchlichen und geringen
Informationen, die gdh Transkription näher betrachtet sowie die Regulatoren AmtR und
FarR hinsichtlich neuer Targetgene untersucht. Mit diesem Ansatz konnte durch
Mutagenese die putative -10-Region des gdh Promotors experimentell bestätigt und
Promotoren mit abgestufter Aktivität konstruiert werden. Im Rahmen dieser
Untersuchungen wurden zusätzlich zu dem bekannten Promoter ein zweiter Promotor
sowie ein bisher nicht bekannter Transkriptionsstartpunkt identifiziert. Es wurde erstmalig in
vivo nachgewiesen, dass die gdh Transkription unter Stickstoffmangel von beiden
Promotoren zunimmt und dass dieser Mechanismus AmtR-abhängig ist. Weitere putative
Regulatoren, OxyR und FarR, zeigten keine Effekte, so dass zusätzlich mit
Untersuchungen zur Regulation durch alternative Sigmafaktoren begonnen wurde.
Mit mez (kodiert für das Malat Enzym) und dapD (kodiert für die
Tetrahydrodipicolinatsuccinylase) wurden zwei bisher nicht bekannte AmtR Targetgene
inklusive Bindestellen identifiziert. Für FarR, einen weitern putativen Regulator der gdh
Transkription, wurde eine mögliche Beteiligung an der Regulation der Argininbiosynthese
nachgewiesen. Summary 2
1 Summary
Glutamate dehydrogenase (GDH) of the industrially highly relevant actinomycete
Corynebacterium glutamicum is located at an important branch-point of metabolism. On the
one hand, it is the enzyme primarily involved in ammonium assimilation under ammonium
surplus. On the other hand, it is connected to the tricarboxylic acid cycle by its substrate 2-
oxoglutarate and influences the intracellular pool of reductive equivalents due to its NADPH
dependency. Despite this crucial position and intense studies, only an incomplete model of
regulation has been proposed including two putative transcriptional regulators.
To characterize the GDH enzyme, within this work studies were performed that included
purification of GDH and investigation of GDH in lysine-producing strains. It was possible to
establish a protocol for overexpression of GDH in C. glutamicum followed by purification by
2+Ni NTA affinity chromatography and size exclusion chromatography, so that the
foundation for kinetic measurements has been laid. Examination of GDH activity, protein
level, and gdh transcript in lysine-producing strains revealed that altered metabolic fluxes
due to the enhanced production did not lead to changes compared to the wild type.
However, because of contrary results reported and an apparent lack of information, the
main focus was put on the reinvestigation of transcriptional control of gdh, which included
characterization and mutagenesis of the gdh promoter as well as further examination of the
two regulators AmtR and FarR. The predicted -10 region of the gdh promoter was verified
by experimental approaches. In the course of these studies, promoters varying in activity
were constructed. Besides the known promoter, it was possible to identify an additional
promoter including a so far not determined transcriptional start site. Transcription of gdh
was shown to be induced upon nitrogen deficiency from both promoters and for the first
time, AmtR-mediated regulation could be demonstrated in vivo. Studies on other putative
regulators of gdh transcription, OxyR and FarR, did not show any effects so that the focus
was put on transcription by alternative sigma factors as well.
Two novel AmtR target genes, namely, mez encoding malic enzyme and dapD encoding
tetrahydrodipicolinate succinylase, were additionally identified including respective binding
sites. Investigations on the second putative regulator of gdh transcription, FarR, suggested
a role in regulation of arginine biosynthesis. Introduction 3
2 Introduction
2.1 Corynebacterium glutamicum
During a screening program for glutamate-producing microorganisms, the Gram-positive
bacterium Corynebacterium glutamicum was isolated from soil samples taken at the Ueno
Zoo in Tokyo, Japan (Kinoshita et al., 1957). It is an aerobic, non-sporulating, and immobile
bacterium marked by a high G+C content of the DNA. The characteristic rod-shape (koryne,
Greek word for rod) is eponymous for corynebacteria. Another feature of this genus is the
so-called snapping cell division, during which cells are laterally connected prior to the actual
division (figure 2.1). Due to the complex composition of the cell wall, corynebacteria are
referred to as mycolic acids-containing actinomycetes (Stackebrandt et al., 1997). Besides
nocardia, well-known human pathogens such as Corynebacterium diphtheriae,
Mycobacterium leprae, and Mycobacterium tuberculosis are members of this group
(Pascual et al., 1995). However, C. glutamicum is non-pathogenic and therefore save to
handle. Because of these properties and the availability of a broad range of experimental
techniques, C. glutamicum is suitable as model organism for its pathogenic relatives.
The identification of C. glutamicum as a
glutamate-excreting bacterium by Kinoshita
and coworkers (1957) laid the foundation for
an extended biotechnological application of
this organism. Under conditions of biotin
limitation, treatment with antibiotics or certain
surfactants, C. glutamicum is able to
accumulate high amounts of glutamate in the
surrounding medium (Gutmann et al., 1992).
Fig. 2.1: Scanning-electromicrogram of
During the last decades, improved C. glutamicum cells. Snapping cell division
as well as the characteristic V-shape of the production strains were created by random
cells is visible (Forschungszentrum Jülich).
mutagenesis programs, and within the last
years rational design approaches were applied (Sahm et al., 1996; Ohnishi et al., 2002).
Upscaling and optimization of cultivation conditions as well as the use of heat-tolerant
species such as Corynebacterium efficiens contributed to an increase in yield combined
with a reduction of production costs (Fudou et al., 2002; Hermann, 2003). So, today
C. glutamicum is one of the most important organisms used in biotechnological production
processes. This biotechnological relevance of the organism is reflected in recent numbers
on amino acid production by various C. glutamicum strains. Of the flavor enhancer L-Introduction 4
glutamate 1.5 million tons per year and of the feed additive L-lysine 650,000 tons per year
are produced. In addition to that, other amino acids such as L- alanine, L-isoleucine, and L-
proline as well as vitamins and nucleotides are yielded by fermentation with C. glutamicum
strains (Leuchtenberger et al., 2005).
This rapid increase in industrial importance led to an intense study of this organism.
Whereas first approaches mainly focused on the respective pathways of amino acid
biosynthesis as well as on import and export systems, today more emphasis is put on
global approaches. While the central metabolism has also been studied in detail already,
the publication of the full genome sequence by two different industry-funded groups (Ikeda
& Nakagawa, 2003; Kalinowski et al., 2003) enables a closer look on interacting metabolic
pathways and connected regulatory mechanisms. Besides the characterization of enzymes
on the biochemical and genetic level, currently techniques are available that allow global
investigation of transcriptome (Wendisch, 2003), proteome (Schaffer and Burkovski, 2005),
and metabolome (Strelkov et al., 2004). Within the last decade especially nitrogen
metabolism has been well investigated, leading to new insights into regulatory processes
and signal transduction in response to the environment (for overview, Burkovski, 2003a;
2003b; 2005; 2007). This approach has recently even been extended to related
actinomycetes with published genome sequence, namely, C. diphtheriae, the causative
agent of diphtheria (Cerdeno-Tarraga et al., 2003), Corynebacterium jeikeium (Tauch et al.,
2005) a pathogen found in the human skin flora, and the heat-tolerant amino acid producer
C. efficiens (Fudou et al., 2002). Currently, nitrogen metabolism is in the focus of research
in connection with finding a common theme among related organisms (Walter et al., 2007).


2.2 Uptake and assimilation of nitrogen sources
Nitrogen is an essential macro-element, which is part of important components of the cell
such as nucleotides and amino acids or amino sugars within the bacterial murein sacculus.
In general, bacteria can use a variety of different organic and inorganic nitrogen sources
depending on their genetic repertoire. As a first step of utilization, compounds need to be
taken up by the cell. This can either occur via passive diffusion along a gradient of
concentration or by active transport processes. In C. glutamicum, transport systems and
respective assimilatory enzymes have been characterized biochemically and on the genetic
level for ammonium, creatinine, glutamate, and urea (Börmann et al., 1992; Kronemeyer et
al., 1995; Siewe et al., 1996; Jakoby et al., 1997; Nolden et al., 2000; Beckers et al., 2001;
Meier-Wagner et al., 2001; Nolden et al., 2001a; Schulz et al., 2001; Beckers et al., 2004;

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