Environmental stress response in filamentous fungi [Elektronische Ressource] : the impact of ion homeostasis on gene regulation / vorgelegt von Anja Spielvogel

De
Environmental stress response in filamentous fungi: the impact of ion homeostasis on gene regulation vorgelegt von Diplom-Ingenieurin Anja Spielvogel Von der Fakultät für Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Ingenieurwissenschaften -Dr.-Ing.- genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Roland Lauster Berichter: Prof. Dipl.- Ing. Dr. Ulf Stahl rof. Dr. Johannes Wöstemeyer Tag der wissenschaftlichen Aussprache: 08.02.2008 Berlin 2008 D83 Meiner Familie gewidmet. Ich widme diese Arbeit besonders meinen Großeltern Herbert und Gerda Grothe. Ihre Liebe wird mich ein Leben lang begleiten. Danksagung Die vorliegende Arbeit wurde in dem Zeitraum von 2003 bis 2007 im Fachgebiet Mikrobiologie und Genetik des Institutes für Biotechnologie der TU Berlin erstellt. Mein besonderer Dank gilt Herrn Prof. Dr. Ulf Stahl für die wissenschaftliche Betreuung, die stete Bereitschaft zu konstruktiven Diskussionen und seine herzliche und motivierende Unterstützung. Prof. Dr.Wöstemeyer danke ich sehr für die Übernahme des Gutachtens dieser Arbeit. Ganz besonderer Dank gilt Frau Dr. Vera Meyer, in deren Arbeitsgruppe die Arbeit angefertigt wurde.
Publié le : mardi 1 janvier 2008
Lecture(s) : 35
Tags :
Source : D-NB.INFO/1009686550/34
Nombre de pages : 145
Voir plus Voir moins


Environmental stress response in filamentous fungi: the
impact of ion homeostasis on gene regulation






vorgelegt von
Diplom-Ingenieurin
Anja Spielvogel


Von der Fakultät für Prozesswissenschaften der
Technischen Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Ingenieurwissenschaften
-Dr.-Ing.-



genehmigte Dissertation



Promotionsausschuss:
Vorsitzender: Prof. Dr. Roland Lauster
Berichter: Prof. Dipl.- Ing. Dr. Ulf Stahl rof. Dr. Johannes Wöstemeyer

Tag der wissenschaftlichen Aussprache:
08.02.2008


Berlin 2008
D83
















Meiner Familie gewidmet.
Ich widme diese Arbeit besonders meinen Großeltern Herbert und
Gerda Grothe. Ihre Liebe wird mich ein Leben lang begleiten.















Danksagung
Die vorliegende Arbeit wurde in dem Zeitraum von 2003 bis 2007 im Fachgebiet
Mikrobiologie und Genetik des Institutes für Biotechnologie der TU Berlin erstellt.
Mein besonderer Dank gilt Herrn Prof. Dr. Ulf Stahl für die wissenschaftliche Betreuung, die
stete Bereitschaft zu konstruktiven Diskussionen und seine herzliche und motivierende
Unterstützung.
Prof. Dr.Wöstemeyer danke ich sehr für die Übernahme des Gutachtens dieser Arbeit.
Ganz besonderer Dank gilt Frau Dr. Vera Meyer, in deren Arbeitsgruppe die Arbeit
angefertigt wurde. Sie stand mir als direkte Ansprechpartnerin immer hilfreich und
freundschaftlich zur Seite, viele Diskussionen und Anregungen haben sehr zum Gelingen
dieser Arbeit beigetragen.
Sehr dankbar bin ich Herrn Dr. Eduardo A. Espeso für die Begleitung der Arbeit seit 2005.
Die Möglichkeit der Forschungsaufenthalte in Madrid am CSIC, die stetige
Diskussionsbereitschaft, die Weitergabe seiner Erfahrungen und Methoden sowie die
liebenswerte Atmosphäre in der spanischen Arbeitsgruppe zusammen mit Lidia, America,
Olga, Antonio und Elena haben einen großen Anteil am Gelingen dieser Arbeit.
Ebenso möchte ich mich bei Prof. Herb Arst und Frau Helen Findon (Imperial College,
London) für die Erstellung und Überlassung der A. nidulans Stämme HHF17a, HHF17d -
HHF17f bedanken.
Mein besonderer Dank gilt Frau Susanne Engelhardt dafür, dass sie mit ihrer exzellenten
technischen Unterstützung sehr zum Gelingen der Arbeit beigetragen hat. Ihr großartiges
Engagement, auch in den Durststrecken, hat letztendlich zum Erfolg geführt.
Bei Frau Barbara Walewska bedanke ich mich sehr herzlich für die tatkräftige Unterstützung
vor allem bei der Messung der Reporteraktivitäten in dieser Arbeit.
Ein großes „Danke“ an Herrn Jochen Schmid für die vielen kleinen und großen
Aufmunterungen und dafür, dass er immer für mich da war.
Herrn Dr. Udo Schmidt, Herrn Dr. Dirk Müller-Hagen, Frau Dr. Silke Hagen, Frau Cornelia
Luban, Herrn Dr. Falk Matthäus, Herrn Dr. Thomas Lautz, Frau Eva Graf und Frau Birgit
Baumann danke ich für viele anregende Gespräche und Diskussionen und für die freundliche
Arbeitsatmosphäre.
Frau Dr. Vera Meyer, Herr Dr. Espeso, Frau Dr. Silke Hagen, Herrn Tom Spielvogel und
Frau Roslin Bensman danke ich für die kritische Durchsicht der Arbeit, sowie für die
Korrektur der englischen Sprache.
Allen weiteren Mitarbeiterinnen und Mitarbeitern des Fachgebietes Mikrobiologie und
Genetik danke ich für die nette und kooperative Zusammenarbeit, insbesondere Frau Rita
Waggad, Frau Roslin Bensmann und Frau Sonja Leberecht.
Danke auch an meine Volleyballmannschaft des VSV Havel Oranienburg. Sie hat für den
besten Ausgleich gesorgt, den man sich für diese Arbeit vorstellen kann.
Abschließender und überaus herzlicher Dank gebührt meinen Freunden und meiner Familie,
insbesondere meiner Mutter Margrit und meinem Bruder Tom Spielvogel. Hier habe ich
immer Liebe und Verständnis gefunden und auf ihre Unterstützung konnte ich mich immer
verlassen.
Anja Spielvogel

Contents
Contents...................................................................................................................................................................I
List of Figures and Tables ................................................................................................................................. III
List of Abbreviations: ................................................................................................................................... V
1 Calcium signalling in eukaryotic organisms ..................................................................1
2+1.1 Ca – a divalent cation with a special task ..................................................................................... 1
1.2 Calcium homeostasis and signalling................................................................................................ 5
2+1.3 Ca - signalling related proteins in fungi, plants, and animals ....................................................... 6
1.4 Calcium mediated control of transcription .................................................................................... 13
1.5 Cellular events dependent on calcium signalling........................................................................... 17
1.6 Concluding remarks and future directions 23
2 Subject description.......................................................................................................24
2.1 The antifungal protein AFP and its application ............................................................................. 24
2.2 Transcriptional regulation of the afp gene of Aspergillus giganteus ............................................. 25
2.3 Aim of the thesis............................................................................................................................ 27
3 Materials and Methods .................................................................................................29
3.1 Equipment...................................................................................................................................... 29
3.2 Enzymes, chemicals and kits ......................................................................................................... 29
3.3 Strains............................................................................................................................................ 30
3.4 Plasmids......................................................................................................................................... 30
3.5 Cloning strategy for newly generated plasmids............................................................................. 31
3.6 Oligonucleotides............................................................................................................................ 32
3.7 Culture media ................................................................................................................................ 33
3.8 Buffers, reagents, and solutions..................................................................................................... 35
3.9 Cultivation conditions for bacteria, yeast and filamentous fungi .................................................. 35
3.10 Methods for DNA and RNA analysis and modification................................................................ 36
3.11 Methods for protein isolation, purification and enzyme activity test............................................. 39
3.12 Transformation methods................................................................................................................ 39
4 Results..........................................................................................................................41
4.1 Transcriptional regulation of the afp promoter.............................................................................. 41
4.2 CrzA, the Crz1p orthologue in Aspergillus nidulans..................................................................... 44
4.3 Generation and characterisation of a crzA deletion strain.............................................................. 49
4.4 CrzA directly influences expression of the afp gene 56
4.5 Expression analysis of putative CrzA target genes........................................................................ 66
4.6 Characterisation of CrzA binding activity and specificity............................................................. 69
4.7 the DNA binding motif of CrzA 71
4.8 In addition to CrzA, SltA is involved in Aspergillus salt stress response...................................... 75
4.9 Characterisation of SltA in A. nidulans ......................................................................................... 82
5 Discussion.....................................................................................................................90
5.1 Filamentous fungi possess a transcription factor that is homologous to yeast Crz1p.................... 90
5.2 The role of CrzA in environmental stress tolerance of Aspergillus............................................... 94
5.3 The role of SltA in environmenolerance in Aspergillus 102
5.4 The activity of both CrzA and SltA is necessary for sustained calcium homeostasis.................. 104
5.5 Regulation of gene expression by CrzA and SltA in A. nidulans – a summary........................... 108
5.6 The interplay of SltA, CrzA and other transcription factors in afp expression............................ 109
5.7 Regulation of afp gene expression – a summary ......................................................................... 113
5.8 Conclusion and future prospects.................................................................................................. 114
6 Summary.....................................................................................................................116
7 Appendix:.....................................................................................................................IV
Reference list ...............................................................................................................................................IV
CrzA promoter region................................................................................................................................ XV
SltA promoter region...................... XV
I
PacC promoter region ...............................................................................................................................XVI
ChsB promoter regionXVI
EnaA promoter region..............................................................................................................................XVII
AN 7250 promoter region:..................................................................................................................... XVIII
VcxA promoter regionXIX
Curriculum vitae......................................................................................................................................XXII


II
List of Figures and Tables

2+ 2+Figure 1: Ribbon drawing of Ca binding protein motifs and EF-hand coordination of Ca ............... 3
2+Figure 2: Schematic illustration of Ca transport and binding proteins (Niki et al., 1996) ................... 6
Figure 3: Calcium dependent protein kinases in mammals and plants and the structural analogue in
fungi ........................................................................................................................................................ 8
Figure 4: Schematic presentation of classical calpain domain structure in animals, plant and fungi ... 11
Figure 5: Calcium and calcium binding proteins in signal transduction............................................... 13
Figure 6: Schematic representation of the action of RIC3 and RIC4 in plant polarised growth........... 20
Figure 7: Changes in morphology and a putative model for axon specification in neuronal growth
(Arimura and Kaibuchi, 2007) .............................................................................................................. 21
Figure 8: Cell cycle of eukaryotic cells................................................................................................. 22
Figure 9: Environmental conditions that influence afp expression and putative cognate regulatory
elements within the afp promoter (adapted from Meyer et al., 2002)................................................... 25
Figure 10: Calcineurin-dependent gene regulation in S. cerevisiae in response to different stress
conditions, Figure adapted from Stathopoulos and Cyert, 1997 ........................................................... 27
Figure 11: afp Expression is induced upon CR and NaCl treatment..................................................... 43
Figure 12: afp expression is induced upon calcium treatment ............................................................. 44
Figure 13: Amino acid sequence of the CrzA coding region of A. nidulans (AN 5726) ...................... 46
Figure 14: Alignment of the zinc-finger region of annotated and putative Crz1p-like proteins ........... 47
Figure 15: Identification of a crzA homologue in A. giganteus ............................................................ 48
Figure 16: Strategy of the crzA replacement......................................................................................... 49
Figure 17: Growth behaviour of isogenic wt and ∆crzA strains 51
Figure 18: Inhibitory effects of calcium ions on crzA deletion strains.................................................. 52
2+Figure 19: Ca sensitivity of ∆crzA is partially rescued by elevated magnesium concentrations........ 52
Figure 20: AFP susceptibility of A. nidulans wild-type (MAD1425) and crzA deletion strain (BER02).
............................................................................................................................................................... 54
Figure 21: The phenotype of the crzA deletion strain ........................................................................... 55
Figure 22: Reporter measurements of afp expression levels in the crzA deletion strain....................... 58
Figure 23: Five putative CDREs have been identified within the afp promoter ................................... 58
Figure 24: Purification of GST::CrzA123 zinc-finger fusion protein................................................... 60
Figure 25: Binding specificity of GST::CrzA123 to CDRE 2/3 ........................................................... 60
Figure 26: Gel retardation assay using the putative CDRE-5 element.................................................. 62
Figure 27: EMSA with A. nidulans protein extracts at different conditions......................................... 63
Figure 28: Competition EMSA with protein extracts of A. nidulans to identify CrzA specific
complexes.............................................................................................................................................. 64
Figure 29: Comparison of DNA-protein complex pattern in the wild-type and in the ∆crzA strain..... 65
Figure 30: Expression analysis of putative CrzA targets genes ............................................................ 68
Figure 31: C H Zinc-finger structure ................................................................................................... 70 2 2
Figure 32: Protein – DNA binding analysis of GST::CrzA12 to CDRE-5 ........................................... 70
Figure 33: Schematic representation of C H of zinc-finger II and atypical zinc-finger III ................. 71 2 2
Figure 34: The third finger of CrzA is formed by an atypical C HC structure ..................................... 71 2
Figure 35: Point mutation within the GGC core prevents binding to GST::CrzA123 .......................... 72
Figure 36: Alignment of proteins that contain a Trp in adjacent zinc-fingers ...................................... 74
Figure 37: The significance of zinc-finger III....................................................................................... 74
Figure 38: The Cys knuckle Trp in finger III plays an essential role in DNA binding......................... 75
Figure 39: Identification of a SltA homologue in A. giganteus ............................................................ 76
Figure 40: Alignment of the zinc-finger region of SltA and CrzA ....................................................... 78
Figure 41: Expression of GST::SltA in E.coli....................................................................................... 78
Figure 42: SltA from A. nidulans recognises putative SDEs within the afp promoter.......................... 79
Figure 43: Binding affinity of CrzA and SltA is interchangeable......................................................... 81
Figure 44: Reporter activity depending on CrzA and SltA ................................................................... 82
Figure 45: Identification of sltA homologues in filamentous fungi ...................................................... 83
Figure 46 : The phenotype of ∆sltA and a double deletion strain of A. nidulans.................................. 84
III
Figure 47: Phenotypes of ∆crzA, ∆sltA and double deletion on different media .................................. 87
2+ +Figure 48: Expression analysis of the putative SltA target – the Ca / H exchanger.......................... 88
+Figure 49 Expression analysis of the putative SltA target –ATPase Na pump.................................... 89
Figure 50: Domain structure of the Crz1p and its homologue in A. nidulans CrzA ............................. 92
Figure 51: Schematic representation of the PHO pathway in S. cerevisiae ........................................ 101
2+Figure 52: The inositol phosphate cycle and IP3 mediated Ca release via IP3 receptor channels in
animal cells, adapted from Balla et al. (2006)..................................................................................... 107
Figure 53: Schematic model for CrzA, SltA and putatively PacC target genes in A. nidulans........... 109
Figure 54: The cell wall integrity pathway in yeast and in silico reconstruction in Aspergillus species
............................................................................................................................................................. 112
Figure 55: Transcriptional regulation of afp expression ..................................................................... 114



Table 1: Properties of calcium versus magnesium ions .......................................................................... 2
2+Table 2: Ca concentration in cellular compartments ............................................................................ 4
2+Table 3: Overview of selected calcium regulated transcription factors regulated by Ca / calmodulin................................ 17
Table 4: Putative regulatory elements within the afp promoter (adapted from Meyer et al., 2002) ..... 26
Table 5: Oligonucleotides used for cloning strategies and PCR probe generation ............................... 32
Table 6: Comparison of regulatory elements of 79 co-regulated genes upon cell wall stress in S.
cerevisiae............................................................................................................................................... 41
Table 7: Identification of sublethal concentration that exert cell wall stress on A. giganteus .............. 42
Table 8: Multiway protein alignment (BLOSUM 62)........................................................................... 45
Table 9: Genotypes of selected ∆crzA reporter strains.......................................................................... 56
Table 10: Comparison of CDRE oligonucleotides used in gel retardation assays. ............................... 62
Table 11: Putative CrzA-dependent genes selected for expressional analysis...................................... 67
Table 12: Comparison of the affinities of CrzA to CDREs of the afp promoter................................... 73
Table 13: Comparison of Crz1p/CrzA and ACEI/SltA DNA binding site ........................................... 77
Table 14: Comparison of the affinities of CrzA and SltA to CDREs and SDEs of the afp promoter.. 80
Table 15: Binding affinities of peptides taken from yeast calcineurin targets 92
Table 16: Comparison of calcineurin and Crz1p homologues deletion phenotype of different fungi .. 93

IV
List of Abbreviations:

dCTP 2’-Deoxycytosine 5’-triphosphate kb Kilobase
kDa Kilodalton Kd Dissociation constant
aa Amino acid MIC Minimal inhibitory concentration
AFP Antifungal protein mRNA Messanger RNA
APS Ammonium- peroxidsulfate MW Molecular weight
BAPTA 1,2-bis(o-aminophenoxy)ethane-OD optical density
N,N,N',N'-tetraacetic acid
bp Basepair ORF Open reading frame
CDRE Calcineurin dependent responsive PAA Polyacrylamide
element
CHS Chitin synthase PAGE Polyacrylamide gel electrophoresis
dATP 2’-Deoxyadenosine 5’-triphosphate PBS Phosphate buffer saline
cADP PCR Polymerase chain reaction
DBD DNA binding domain PEG Polyethylene glycol
dH2O Deionised water PMSF Phenylmethylsulfonyl
DNA Deoxyribonucleic acid RNA Ribonucleic acid
dNTP 2’- Desoxynucleosid 5’- rpm Rotations per minute
triphosphate
e.g. for example RT Room temperature
EDTA Ethylenediamine-tetra-acetic acid SDE Salt dependent element
ER Endoplasmatic reticulum SDS Sodium Dodecyl Sulfate
Fig. Figure SDS- PAGE Sodium dodecyl sulphate
polyacrylamid gel electrophoresis
GST Glutathion-S-Transferase TEMED N,N,N',N'-
Tetramethylethylenediamine
GTP Guanosine triphosphate Tris Tris(hydroxymethyl)aminomethane
HEPES 4-(2-hydroxyethyl)-1- X-Gal 5-Bromo-4-chloro-3-indolyl- beta-D-
Piperazineethanesulfonic acid galactopyranoside
IPTG Beta-galacotosidase Isopropyl β-D-1- β-gal
thiogalactopyranoside

Nucleobases

A Adenine R A oder G
C Cytosine Y C oder T
G Guanine S G oder
T Thymine W A ode

Amino acids

A Ala Alanine L Leu Leucine
R Arg Arginine K Lys Lysine
N Asn AsparagiM Met Methioni
D Asp Aspartic acid FPhePhenylalanine
C Cys Cysteine P Pro Proline
Q Gln Glutamine S Ser Serine
E Glu ic acid T Thr Threonine
G Gly Glycine W Trp Tryptophan
H His Histidine Y Tyr Tyrosine
I Ile Isoleucine V Val Valine

Standard SI units are used throughout.
V

List of genes:

Gene Function Organism
CCH1 Calcium channel S. cerevisiae
Crz1 Calcium related zinc finger protein transcription factor S. ce
CrzA Calcium related zinc finger protein transcription factor A. nidulans
MID1 Calcium channel S. cerevisiae
PacC Ambient pH transcription factor A. nidulans
PalB Calpain-like protease A. nidulans
TPC1 Mitochondrial membrane transporter S. cerevisiae
VCX1 Vacuolar calcium exchanger S. ce


List of abbreviated proteins, enzymes and cell lines:

α-CREM cAMP response element modulator
bHLH Basic helix-loop-helix
bZIP Basic leucine zipper
CaMK Calmodulin dependent kinases
CaMKK CaMK kinase
CaM-like Calmodulin-like
CAMTA Calmodulin-binding transcriptional activator
2+CCaMK Ca /calmodulin dependent kinases
CCAT Calcium channel associated transcriptional regulator
CDPK Calcium dependent protein kinase
CNGC cyclic nucleotide-gated channel
COS CV-1 (simian) in Origin, and carrying the SV40 (virus) genetic material
CREB cAMP response element binding protein
CRK Calmodulin related kinase
DAG Diacylglycerol
DRE Downstream regulatory element
DREAM Downstream regulatory element antagonist modulator
ER Endoplasmatic reticulum
HEK Human Embryonic Kidney cells
iGlu Ionotrophic glutamate
IP3 Inositol 1,4,5 phosphat
MADS The MADS box is a highly conserved sequence motif found in a family of transcription factors.
The conserved domain was recognized after the first four members of the family, which were
MCM1, AGAMOUS, DEFICIENS and SRF (serum response factor). The name MADS was
constructed form the "initials" of these four "founders".
MEF-2 Myocyte-specific enhancer factor 2
MYB myeloblastosis
NAADP nicotinic acid adenine dinucleotide phosphate
NCS Neuronal calcium sensor
NF-AT Nulcear factor of activated T-cells
PIP2 Phosphatidylinositol -4,5-biphosphate
PKC Protein kinase C
PLC Phospholipase C
PLD Phospholipase D
PS Phophatidylserin
RCN Regulator ofcalcineurin
TF-1 Activating transcription factor 1
TM Transmembrane
TRP Transient receptor potential
VDCC Voltage dependent calcium channel

VI
Calcium signalling in eukaryotic organisms

1 Calcium signalling in eukaryotic organisms

Calcium has been selected by nature to be an essential messenger that transduces signals
throughout the entire lifespan of a cell. The following section gives an introduction into the
2+vast field of Ca signalling in mammalian, plant and fungal cells. Many signal transduction
pathways are conserved within these kingdoms. However, some regulatory circuits have
evolved in one kingdom only. Moreover, one signalling task can be fulfilled by different
protein families. It is beyond the scope of the next chapter to give a detailed description of all
2+cellular events that are related to Ca signalling. Focus will be drawn to the basics such as
calcium homeostasis and how this is achieved, as well as calcium mediated transcriptional
2+ response. Furthermore, cellular events that depend on Ca are exemplified by polarised
growth, cell cycle and apoptosis.
2+1.1 Ca – a divalent cation with a special task

2+Ca ions exhibit unique features that are necessary for the cell to differentiate between
2+ 2+another very abundant divalent cation: Mg ions (Table 1). Targets of Ca must respond in a
2+100 – 10.000 fold excess of Mg . Binding flexibility, geometry as well as charge density
2+ 2+different to Mg make reversible Ca binding to biomolecules principally easy (Malmendal
2+ 2+et al., 1998). In contrast to Mg , Ca is able to bind sites of irregular geometry (Table 1).
Nevertheless, proteins that are specifically regulated by binding calcium ions can complex
2+magnesium ions in the absence of calcium ions. Furthermore, Mg has been shown to
2+stabilise calcium binding proteins in their Ca free state (Mukherjee et al., 2007).
Subsequently, fine tuning between these two ions is very important for cellular signalling and
the activity of proteins.

- 1 -

Soyez le premier à déposer un commentaire !

17/1000 caractères maximum.