A proteomics view of quorum-sensing regulated and surface induced genes in representative Pseudomonas and Burkholderia species [Elektronische Ressource] / Catalina Arévalo-Ferro

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• Biologie

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 68
• Poids de l'ouvrage: 47 Mo

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Lehrstuhl für Mikrobiologie
der Technischen Universität München






A proteomics view of quorum-sensing regulated
and surface induced genes in representative
Pseudomonas and Burkholderia species






Catalina Arévalo-Ferro






Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.









Vorsitzender: Univ.-Prof. Dr. W. Höll

Prüfer der Dissertation: 1. Univ.-Prof. Dr. K.-H. Schleifer
2. apl.-Prof. Dr. A. Görg








Die Dissertation wurde am 19. 04. 2004 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 28. 05. 2004 angenommen. Table of contents
1 Introduction ----------------------------------------------------------------------------------- 1
1.1 Biofilms and quorum-sensing: rather a social than an isolated life. ---------------------- 1
1.2 Quorum-sensing regulated phenotypes: two sides of one coin ---------------------------- 3
1.2.1 Pseudomonas aeruginosa---------------------------------------------------------------- 4
1.2.2 Burkholderia cepacia complex-------------------------- 6
1.2.3 Pseudomonas putida----- 8
1.3 Quorum-sensing: a bacterial double-edged sword---------10
1.4 Comparative proteome analysis: an essential tool to study biological systems----------11
1.5 Aims of this work-----------------------------------------------------------------------------14
2 Materials and methods-------15
2.1 Bacterial strains--------------15
2.2 Cultivation of organisms: Planktonically and biofilm -------------------------------------16
2.2.1 Pseudomonas aeruginosa---------------------------------------------------------------16
2.2.2 Burkholderia cenocepacia------------------------------17
2.2.3 Pseudomonas putida----17
2.2.4 Biofilm models---------------------------------------------------------------------------17
2.2.5 Haem acquisition-------20
2.3 Fractionation of cellular proteins-----------------------------------------------------------20
2.3.1 Sample preparation from planktonically grown cells. -------------------------------20
2.3.2 Phenol extraction---------------------------------------21
2.3.3 Preparation of biofilm cells----------------------------22
2.4 Protein biochemical methods--------------------------------22
2.4.1 SDS- Polyacrylamide gel electrophoresis (SDS-PAGE)22
2.4.2 Western Blot---------------------------------------------22
2.4.3 Quantification of protein concentration--------------23
2.4.4 Quantification of enzymatic activities and exoproducts. ----------------------------23
2.5 Two-dimensional electrophoresis (2-DE)-------------------24
2.5.1 First dimension: isoelectric focussing (IEF)--------------------------------------------25
2.5.2 Equilibration of the IPG stripes------------------------25
2.5.3 Preparation and casting of SDS gels for second dimension. -------------------------26
2.5.4 Second dimension: SDS-PAGE ----------------------------------------------------------27
2.5.5 Fixing ----------------------------------------------------27
2.5.6 Visualization--------------------------------------------27
2.5.7 Image capturing and gel analysis.---------------------28
2.5.8 Protein identification -----------------------------------28
3 Results and discussion--------29
3.1 Part I- Global analyses of quorum-sensing regulated genes of Pseudomonas aeruginosa-
-----------------------------------------------------------------29
3.1.1 General mapping of quorum-sensing regulated proteins----------------------------29
i 3.1.2 Impact of the superregulators Gac and Vfr on the QS regulon of P. aeruginosa.--36
3.1.3 Quorum-sensing and pathogenesis----------------------------------------------------42
3.2 Part II- Quorum-sensing in Burkholderia cenocepacia H111, a global regulatory system
-----------------------------------------------------------------44
3.3 Part III-Identification of quorum-sensing and surface induced genes in Pseudomonas
putida.-----------------------------------------------------------45
3.3.1 Establishing of a biofilm growth model for 2-DE analyses --------------------------45
3.3.2 Differentially expressed proteins between the biofilm mode of growth and
planktonic cultures of P. putida-----------------------48
3.3.3 Quorum-sensing regulated genes in P. putida Iso F----------------------------------50
3.3.4 Impact of quorum-sensing on biofilm development--55
4 Summary --------------------------------------------------------------------------------------56
5 References---------------------60
6 Appendix70
6.1 Identification of quorum-sensing regulated proteins in the opportunistic pathogen
Pseudomonas aeruginosa by proteomics -----------------------------------------------------71
6.2 Proteome analysis of intraclonal diversity of two Pseudomonas aeruginosa TB clone
isolates ----------------------------------------------------------92
6.3 Analysis of the quorum-sensing regulon of the opportunistic pathogen Burkholderia
cepacia H111 by proteomics -------------------------------------------------------------------98
6.4 Protein spots induced or repressed in the PAO1 gacA mutant and PAO1 mvrf mutant. 110
6.5 Identification and spot signal intensities of induced or repressed proteins in the biofilm
growth on glass wool of P. putida IsoF ----------------------------------------------------- 112
6.6 Orthologous proteins between P. aeruginosa and P. putida. --------------------------- 115
6.7 Identification of induced or repressed proteins in the biofilm growth on silicone tubes of
P. putida------------------------------------------------------- 117
6.8 Protein spots induced or repressed in the biofilm growth on silicone tubes of P. putida
IsoF.----------------------------------------------------------- 119
6.9 Identification of quorum-sensing regulated proteins of P. putida IsoF. --------------- 122
6.10 Protein spots induced or repressed by the quorum-sensing system of P. putida IsoF. 124
6.11 Publications list----------------------------------------------------------------------------- 126

ii List of Tables
Table 1 Bacterial strains--------------------------------------------------------------------------------15
Table 2 Isoelectric focusing running conditions----------------------25
Table 3 Standard volumes for casting 12 2-DE gels------------------26
Table 4 Novel QS regulated proteins-------------------------------------------------------------------30
Table 5 Proteins affected by the quorum-sensing inhibitor C-30---35
Table 6 Identification of differential expressed proteins between wild type and superregulator
mutants, gacA and mvfr. Regulation refers to each one of the indicated mutants. Protein
were identified by MALDI-TOF MS peptide mass mapping -------------------------------------37
Table 7 Global comparison of differentially expressed proteins between GacA, MvfR and lasI rhlI
mutants.--------------------------------------------------------------------------------------------41
Table 8 Identification and functional cathegory of genes that are differentially expressed in P.
putida after 18 h of attachment to glass wool.-----------------47
Table 9 Identification and functional category of the proteins, which are regulated by the ppuI
sytem of P.putida IsoF and/or differentially expressed between cells grown as biofilm in
silicone tubes and planktonic cultures. The regulation in the quorum-sensing comparison is
referring to the wild type and the surface-induced genes are referred to the biofilm growth.
------------------------------------------------------------------------------------------------------53

iii List of Figures
Figure 1 Schematic drawings of the quorum-sensing circuits of a) P. aeruginosa (las/rhl system),
b) B. cenocepacia (cep system), c) P. putida (ppu system).---------------------------------------- 7
Figure 2 Schematic drawing of the silicone tube biofilm growth model system.-------------------19
Figure 3 Percentage of quorum-sensing regulated proteins in the P. aeruginosa PAO1 wild type.
----------------------------------------------------------------------32
Figure 4 Western blot analysis of FliD expression.----------------------------------------------------32
Figure 5 Biofilm formation under anaerobiosis in minimal medium with arginine as sole carbon
source.------------------------------34
Figure 6 Growth of the PAO1 wild type and the lasI rhlI double mutant in medium containing
haemoglobin as the sole iron source.-------------------------------------------------------------34
Figure 7 Comparative two-dimensional gel electrophoresis (2-DE) of P. aeruginosa PAO1wild type
and superregulator mutants.-------------------------------------39
Figure 8 Percentage of differentially expressed protein spots between P. aeruginosa