Proteomics and kinetic modeling analysis of a 4-chlorosalicylate degrading bacterial community [Elektronische Ressource] / von Roberto Andrés Bobadilla Fazzini

Sujets

• Biologie

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2006
• Nombre de lectures: 30
• Poids de l'ouvrage: 4 Mo

Extrait

PROTEOMICS AND KINETIC MODELING ANALYSIS OF A 4-
CHLOROSALICYLATE DEGRADING BACTERIAL
COMMUNITY
Von der Fakultät für Lebenswissenschaften
der Technischen Universität Carolo-Wilhelmina
zu Braunschweig
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)
genehmigte
D i s s e r t a t i o n


von Roberto Andrés Bobadilla Fazzini
aus Santiago de Chile, Chile


1. Referent: Prof. Dr. Kenneth N. Timmis
2. Referent: Prof. Dr. Dieter Jahn
eingereicht am: 25.09.2006
mundliche Prufung (Disputation) am: 07.11.2006
Druckjahr 2006

ii
AKNOWLEDGEMENTS ............................................................................................................. V
ABSTRACT ............................................................................................................................... VI
I. INTRODUCTION.................................................................................................................1
II. PROJECT RATIONALE.......................................................................................................3
III. LITERATURE REVIEW........................................................................................................7
3.1 BACTERIAL COMMUNITIES ..............................................................................................7
3.1.1 Characterization of bacterial communities ................................................................8
3.1.2 Bacterial communities and communication.............................................................11
3.1.3 Bacterial Communities and Biodegradation............................................................12
3.2 PROTEOMICS...............................................................................................................16
3.2.1 Protein identification techniques.............................................................................16
3.2.2 Protein separation techniques................................................................................17
3.2.3 Proteomics and stress response ............................................................................19
3.2.4 Proteomics and Bacterial Communities ..................................................................22
3.3 METABOLIC MODELLING ...............................................................................................24
IV. MATERIALS AND METHODS........................................................................................33
4.1 STRAINS......................................................................................................................33
4.2 CHEMICALS .................................................................................................................33
4.3ULTURE CONDITIONS .................................................................................................33
4.4 DYNAMIC STATE: SUBSTRATE SHOCK LOAD36
4.5 ENUMERATION OF BACTERIA AND QUANTIFICATION OF BIOMASS ...................................36
4.6 METABOLIC PROFILE: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY..........................36
4.7 FLOW CYTOMETRY ANALYSIS .......................................................................................37
4.7.1 Cell viability determination......................................................................................37
4.7.2 Fluorescence in situ hybridization (FISH) ...............................................................38
4.8 PROTEOMICS...............................................................................................................39
4.8.1 Cell collection and Protein extraction......................................................................39
4.8.2 First dimension: isoelectric focusing .......................................................................39
4.8.3 Second dimension: Equilibration and SDS-PAGE ..................................................40
4.8.4 Protein Identification...............................................................................................41
4.8.5 Protein differential expresison analysis...................................................................41
V. RESULTS AND DISCUSSION...........................................................................................43
5.1 STEADY STATE CULTURES43
5.1.1 Pseudomonas sp. MT1 steady state continuous cultures .......................................43
5.1.1.1 Low dilution rate steady state continuous cultures of Pseudomonas sp. MT1.52
5.1.1.2 High dilution rate steady state contures of onas sp. MT1 58
5.1.2 Pseudomonas sp. MT1 and Achromobacter xylosoxidans strain MT3 steady state
cultures..............................................................................................................................60
5.1.2.1 Low dilution rate steady state continuous community cultures of Pseudomonas
sp. MT1 and Achromobacter xylosoxidans strain MT3 ...................................................61
5.1.3 Comparison of steady state pure cultures of Pseudomonas sp. MT1 and community
culture of Pseudomonas sp MT1 and Achromobacter xylosoxidans MT3 at the low dilution
-1rate of 0.1 d ......................................................................................................................65
5.1.4 Comparison of steady state pure cultures of Pseudomonas sp MT1 and mixed
culture of Pseudomonas sp MT1 and Achromobacter xylosoxidans MT3 at reference
-1dilution rate of 0.2 d . ........................................................................................................67
ii i5.1.5 Discussion overview of steady state cultures..........................................................72
5.2 DYNAMIC STATE CULTURES..........................................................................................74
5.2.1 Metabolic profile of Pseudomonas sp. MT1 dynamic state cultures........................74
5.2.2 Pseudomonas sp. MT1 shock load stress dynamic state proteomics .....................77
5.2.3 onas sp. MT1 and Achromobacter xylosoxidans MT3 community shock
load stress dynamic state proteomics ................................................................................83
5.2.4 Discussion overview of dynamic state cultures.......................................................88
5.2.5 Kinetic metabolic modeling of dynamic states.........................................................92
5.2.5.1 Kinetic Modeling of Pseudomonas sp. MT1 dynamic states............................92
5.2.5.1.1 Pseudomonas sp. MT1 kinetic metabolic mathematical statements and
model structure ..........................................................................................................94
5.2.5.1.2 Experimental determination of initial parameter values for Pseudomonas sp.
MT1 kinetic model ......................................................................................................98
5.2.5.1.3 Parameter sensitivity analysis of Pseudomonas sp. MT1 kinetic model ....103
5.2.5.1.4 Pseudomonas sp. MT1 kinetic model validation........................................108
5.2.5.2 Kinetic Modeling of Pseudomonas sp. MT1 and A. xylosoxidans MT3
community dynamic states...........................................................................................110
5.2.5.2.1 Pseudomonas sp. MT1 and A. xlosoxidans MT3 community kinetic metabolic
mathematical statements and model structure .........................................................110
5.2.5.2.2 Parameter estimation and sensitivity analysis of Pseudomonas sp. MT1 and
A. xylosoxidans MT3 community kinetic model114
5.2.5.2.3 Community model validation.....................................................................116
5.2.6 Discussion overview of kinetic modeling in dynamic states...................................117
VI. CONCLUSIONS...........................................................................................................120
VII. OUTLOOK...................................................................................................................124
VIII. REFERENCES............................................................................................................129
IX. APPENDIX141
iv A mi esposa Alejandra
A mis hijos Emilia, Andrés y Benjamín
iv AKNOWLEDGEMENTS
During the development of my work there are several persons who collaborated in one
way or another to accomplished it. Special thanks to my direct supervisor Dr. Dipl-Ing.
Vitor Martins dos Santos who provide guidance and gave me the chance to perform this
study and to PD Dr. Dietmar Pieper, Prof. Dr. Burkhard Tümmler, Dr. Volker Hecht and Dr.
Max Schobert for fruitful discussions.
Thanks to all the Environmental Microbiology Department leaded by Prof. Dr. Kenneth N.
Timmis and most specially to my group mates, Amit, Filip, Jacek, Massimo, Miguel and
Piotr.
AGRADECIMIENTOS
No quisiera dejar pasar la oportunidad de agradecer a los amigos que han generado un
ambiente grato y de mucho compañerismo, haciendo mas fáciles aquellos momentos de
nostalgia y soledad en tierras tan lejanas. Agata, Alexandre, Andrew, Bea, Christiane,
Faiza, Felipe, Gonçalo, Howard, Magally, Marcelo, Mariela, Melissa, Nacho, Pablo, Peter,
Popi, Rosalila, Silvana, Tom, u Pedro y Victoria