Molecular characterization of a novel quorum sensing system of the diazotrophic grass endophyte Azoarcus sp. strain BH72 [Elektronische Ressource] / Lena Hauberg

Lena Eyl

Sujets

• Biologie

Informations

• Publié par: universitat_bremen
• Publié le: 01 janvier 2010
• Nombre de lectures: 42
• Langue: English
• Poids de l'ouvrage: 20 Mo

Extrait

Molecular characterization of a novel quorum
sensing system of the diazotrophic grass
endophyte Azoarcus sp. strain BH72
Lena Hauberg
Dissertation submitted in partial fulfilment
of the requirements for the degree
Dr. rer. nat.
Bremen, May 2010The experiments of the presented study have been carried out from December 2006 until
February 2010 under the guidance of Prof. Dr. Barbara Reinhold-Hurek in the Laboratory of
General Microbiology at the Department 2 Biology/Chemistry of Bremen University,
Germany.
First reviewer: Prof. Dr. Barbara Reinhold-Hurek
Second reviewer: Prof. Dr. Uwe Nehls
Date of thesis defence: 01. July 2010 Publications
Parts of the presented results have been published:
Lena Hauberg, Frank Schmidt, Christian Scharf, Juliane Dörr, Uwe Völker and
Barbara Reinhold-Hurek: Proteomic characterization of a pilR regulatory mutant of
Azoarcus sp. strain BH72 with the aid of gel-based and gel-free approaches. Proteomics.
2010; 10(3):458-69.
Manuscripts in preparation:
Melanie Böhm, Frauke Friedrich, Lena Hauberg, Juliane Dörr, Jörg Plessl and Barbara
Reinhold-Hurek: Regulation of the type IV pilin gene expression in the grass endophyte
Azoarcus sp. strain BH72 is affected by carbon starvation and cell density. Submitted to
Journal of Bacteriology.
Lena Hauberg, Christian Scharf, Uwe Völker, Anke Becker and Barbara Reinhold-
Hurek: Characterization of quorum-sensing regulated gene and protein expression of the
grass endophyte Azoarcus sp. BH72. In preparation.
Angela Sessitsch, Pablo Hardoim, Jessica Döring, Alexandra Weilharter, Andrea
Krause, Tanja Woyke, Birgit Mitter, Lena Hauberg, Frauke Friedrich, Monali
Rahalkar, Thomas Hurek, Abhijit Sarkar, Levente Bodrossy, Leo van Overbeek,
Darshan Brar, Jan Dirk van Elsas and Barbara Reinhold-Hurek: Metagenome analysis
of endophytes in rice roots reveals a highly adapted community affecting plant productivity.
In preparation. Table of contents
TABLE OF CONTENTS
Abbreviations ......................................................................................................................... I
Summary...............................................................................................................................II
German summary – Zusammenfassung.................................................................................III
I Introduction....................................................................................................................1
I 1 Quorum sensing in the microbial world...................................................................1
I 1.1 Quorum sensing signal molecules....................................................................2
I 1.2 The complex quorum sensing hierarchy of Pseudomonas ................................4
I 1.3 Quorum sensing is integrated in different regulatory networks.........................6
I 2 The grass endophyte Azoarcus sp. BH72...............................................................10
I 2.1 Type IV pili and colonization ........................................................................11
I 2.2 Azoarcus sp. BH72 communicates via a novel quorum sensing system..........12
I 3 Objectives.............................................................................................................13
II Material & Methods......................................................................................................15
II 1 Chemicals and Buffers ..........................................................................................15
II 2 Oligonucleotides ...................................................................................................15
II 3 Strains and Plasmids .............................................................................................16
II 4 Molecular weight marker and DNA size marker....................................................17
II 5 Culture media and cultivation of microorganisms..................................................17
II 5.1 Culture media................................................................................................18
II 5.2 Antibiotics and other supplements .................................................................19
II 5.3 Cultivation of microorganisms.......................................................................19
II 6 Determination of β-glucuronidase activity.............................................................21
II 7 Determination of fluorescence of the green fluorescent protein .............................21
II 8 Heterogeneity test and fluorescence microscopy....................................................22
II 9 DNA techniques....................................................................................................22
II 9.1 Isolation of E. coli Plasmid DNA...................................................................22
II 9.2 Isolation of Azoarcus sp. chromosomal DNA ................................................23
II 9.3 Estimation of DNA concentration..................................................................23
II 9.4 Cleavage of DNA with restriction endonucleases ..........................................24
II 9.5 Agarose gel electrophoresis to separate DNA24
II 9.6 Cloning procedures........................................................................................24
II 9.7 DNA hybridization to test the correct plasmid integration..............................26
II 10 Proteome studies ...................................................................................................27 Table of contents
II 10.1 Two-dimensional gel electrophoresis.............................................................27
II 10.2 Analyses of protein gels with Image Master 2D.............................................29
II 10.3 Protein identification by mass spectrometry...................................................29
II 11 Transcriptome studies ...........................................................................................33
II 11.1 RNA isolation (Hot phenol procedure) ..........................................................33
II 11.2 DNase I Treatment ........................................................................................34
II 11.3 Determination of RNA concentration ............................................................34
II 11.4 Reverse transcription of total RNA to yield aminoallyl-labelled first strand
cDNA for microarray experiments ................................................................................34
II 11.5 Coupling of fluorescent dyes to aminoallyl-labelled first strand cDNA..........35
II 11.6 Oligonucleotide Microarray...........................................................................36
II 11.7 Analyses of microarray data ..........................................................................38
II 11.8 Real-time PCR experiments39
II 12 Computational based analyses...............................................................................41
II 12.1 Analyses of quorum sensing regulated genes and different protein parameters
41
II 12.2 Analyses of microbiome samples and endophytic genomes ...........................41
III Results..........................................................................................................................44
III 1 Quorum sensing systems in microbiome samples ..................................................44
III 2 Quorum sensing systems in several endophytes.....................................................48
III 3 Quorum sensing bioassays and gene expression studies.........................................52
III 3.1 The genes pilAB are quorum sensing targets in Azoarcus sp. .........................52
III 3.2 The gene azo3874 is a newly discovered quorum sensing target ....................55
III 3.3 Influence of growth medium on quorum sensing target gene expression ........56
III 3.4 Possible distribution of the novel quorum sensing signal molecule ................60
III 4 Heterogeneity in Azoarcus sp. cultures..................................................................63
III 5 The proteome of Azoarcus sp. BH72 under aerobic standard growth conditions ....65
III 5.1 Protein identification with gel-based and gel-free approaches........................65
III 5.2 Most abundant proteins of Azoarcus sp. BH72...............................................67
III 5.3 Characterization of Azoarcus sp. BH72 protein parameters............................69
III 6 The Azoarcus sp. BH72 proteome under quorum sensing conditions .....................74
III 7 The Azoarcus sp. BH72 transcriptome under quorum sensing conditions...............77
III 7.1 Genome-wide expression profile of Azoarcus sp. BH72 ................................79
III 7.2 Monitoring differential gene expression of Azoarcus sp. BH72 grown under
quorum sensing conditions............................................................................................80
III 7.3 Validation of microarray results with real-time PCR......................................82 Table of contents
III 8 Gene expression and protein synthesis of Azoarcus sp. BH72 are altered under
quorum sensing conditions................................................................................................84
III