Quorum sensing by the bacterial signal autoinducer-2 [Elektronische Ressource] : phylogenetic distribution of the synthesis gene LuxS, and its role in Shewanella oneidensis / von Ágnes Mária Bodor

Agb

Sujets

• Biologie

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2008
• Nombre de lectures: 57
• Langue: Deutsch
• Poids de l'ouvrage: 25 Mo

Extrait

Quorum Sensing by the Bacterial Signal Autoinducer-2:
Phylogenetic Distribution of the Synthesis Gene LuxS, and
its Role in Shewanella oneidensis


Von der Fakultät für Lebenswissenschaften

der Technischen Universität Carolo-Wilhelmina

zu Braunschweig

zur Erlangung des Grades einer

Doktorin der Naturwissenschaften

(Dr. rer. nat.)

genehmigte

D i s s e r t a t i o n










von Ágnes Mária Bodor
aus Kecskemét, Ungarn















































1. Referentin: Professor Dr. Irene Wagner-Döbler
2. Referent: Professor Dr. Dieter Jahn
eingereicht am: 04.06.2008
mündliche Prüfung (Disputation) am: 18.07.2008

Druckjahr 2008







Vorveröffentlichungen der Dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für
Lebenswissenschaften, vertreten durch die Mentorin/den Mentor der Arbeit, in
folgenden Beiträgen vorab veröffentlicht:



Publikationen

Bodor, A., Elxnat, B., Thiel, V., Schulz, S. & Wagner-Dobler, I. (2008). Potential
for luxS related signalling in marine bacteria and production of autoinducer-2 in the
genus Shewanella. BMC Microbiol 8, 13.



Tagungsbeiträge

Bodor, A., Schulz, S. & Wagner-Dobler, I.: Phylogenetic distribution of the luxS and
sahH genes in marine bacteria and production of autoinducer-2 in the genus
Shewanella. (Poster) 336. 11th International Symposium on Microbial Ecology,
August 20-25, Vienna, Austria (2006).







CONTENT
Contents
Index of tables ...................................................................................................................... V
Index of figures....................................................................................................................VI
Abbreviations ................................................................................................................... VIII
List of chemicals..................................................................................................................IX
1 Introduction .............................................................................................. 1
1.1 Quorum sensing................................................................................................... 1
1.1.1 Alternative interpretations........................................................................ 1
1.1.2 Regulated phenotypes .............................................................................. 2
1.1.3 Autoinducer structures ............................................................................. 5
1.2 Autoinducer-2...................................................................................................... 6
1.2.1 History 6
1.2.2 Synthesis 7
1.2.2.1 Synthesis via the Pfs/LuxS enzymatic pathway ....................... 7
1.2.2.2 AI-2 forms: epimeric DPD derivatives..................................... 9
1.2.3 Signal response....................................................................................... 10
1.2.3.1 Vibrio harveyi......................................................................... 10
1.2.3.2 Salmonella typhimurium and Escherichia coli....................... 12
1.2.3.3 Other species: phenotypes of luxS mutants and their
complementation ................................................................................... 13
1.2.4 Pseudomonas aeruginosa....................................................................... 16
1.2.5 Phylogenetic distribution of the Pfs/LuxS and SAH pathway ............... 18
1.2.6 AI-2 production and AI-2 activity.......................................................... 20
1.2.7 The genus Shewanella............................................................................ 20
1.2.8 Shewanella oneidensis 21
2 Aim of the work ......................................................................................22
3 Materials and Methods ..........................................................................23
3.1 Strains ................................................................................................................ 23
3.2 Plasmids............................................................................................................. 26
I
CONTENT
3.3 Primers............................................................................................................... 27
3.4 Bacterial cultivation........................................................................................... 29
3.4.1 Media...................................................................................................... 29
3.4.1.1 Luria-Bertani Medium............................................................ 29
3.4.1.2 Marine broth (MB) ................................................................. 29
3.4.1.3 LB sea salt broth (LBSS)........................................................ 29
3.4.1.4 Shewanella marine medium (SM) .......................................... 29
3.4.1.5 Shewanella Defined Minimal medium (SDM)....................... 30
3.4.1.6 Autoinducer bioassay medium (AB) ...................................... 31
3.4.1.7 SOC medium .......................................................................... 32
3.4.2 Antibiotics .............................................................................................. 32
3.4.3 Cultivation conditions ............................................................................ 32
3.4.4 Glycerol stock ........................................................................................ 33
3.5 Design of degenerated primers 33
3.6 Bioassays ........................................................................................................... 34
3.6.1 The Vibrio harveyi bioassay................................................................... 34
3.6.2 Bioassays with Pseudomonas aeruginosa strains .................................. 36
3.7 Standard DNA techniques ................................................................................. 36
3.7.1 Isolation of genomic and plasmid DNA................................................. 37
3.7.2 PCR ........................................................................................................ 37
3.7.2.1 Standard PCR ......................................................................... 37
3.7.2.2 High-fidelity PCR................................................................... 38
3.7.3 Restriction digestion............................................................................... 38
3.7.4 DNA blunting......................................................................................... 39
3.7.5 DNA dephosphorylation and phosphorylation....................................... 39
3.7.6 Ligation .................................................................................................. 39
3.7.7 Sequencing ............................................................................................. 40
3.8 DNA introduction into bacterial cells................................................................ 40
3.8.1 Transformation of Escherichia coli by electroporation ......................... 40
3.8.2 Shewanella oneidensis conjugation........................................................ 41
3.8.2.1 Biparental mating ................................................................... 41
3.8.2.2 Triparental mating .................................................................. 42
II
CONTENT
3.8.3 Transformation of Pseudomonas aeruginosa ........................................ 42
3.9 The sacB counter selection................................................................................ 43
3.10 Biofilm cultivation............................................................................................. 43
3.10.1 Biofilm formation assay......................................................................... 45
3.11 Secretome analysis ............................................................................................ 46
3.12 Siderophore analysis.......................................................................................... 47
4 Results......................................................................................................48
4.1 Response of P. aeruginosa virulence genes to autoinducer-2........................... 48
4.2 Phylogenetic distribution of the Pfs/LuxS and SahH pathways........................ 52
4.2.1 Alphaproteobacteria and Bacteroidetes................................................. 53
4.2.2 Gammaproteobacteria ........................................................................... 54
4.3 AI-2 production of Shewanella sp