Characterisation of the lectin microvirin from Microcystis aeruginosa PCC 7806 and new insights into the role of microcystin [Elektronische Ressource] / von Jan-Christoph Kehr

Jan-Christoph Kehr

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2009
• Nombre de lectures: 48
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

Characterisation of the lectin microvirin from
Microcystis aeruginosa PCC 7806 and new
insights into the role of microcystin

Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
im Fach Biologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von
Jan-Christoph Kehr
geboren am 21.02.1977 in Osnabrück
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Christoph Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Lutz-Helmut Schön
Gutachter: 1. Prof. Dr. Elke Dittmann
2. Dr. Nicole Tandeau de Marsac
3. PD Dr. Hans von Döhren
Tag der mündlichen Prüfung: 11.06.2009 Contents
CONTENTS
CONTENTS........................................................................................................... 2
ZUSAMMENFASSUNG............................................................................................ 5
ABSTRACT ........................................................................................................... 6
1 INTRODUCTION ............................................................................................... 7
1.1 CYANOBACTERIA ..................................................................................................................................7
1.1.1 Microcystis aeruginosa..............................................7
1.1.2 Microcystin11
1.2 MICROVIRIN .......................................................................................................15
1.3 MRPC...............................................17
1.4 LECTINS.............18
1.4.1 Mechanism of carbohydrate binding.......................................................19
1.4.2 Functions of lectins ..................................................................................20
1.4.3 Animal lectins...........................20
1.4.4 Plant lectins ..............................................................................................................................21
1.4.5 Bacterial lectins........................22
1.4.6 Cyanobacterial lectins..............................................................................................................23
1.4.7 Microcystis lectins....................23
1.4.8 Cyanovirin-N.............................................................................................................................24
1.5 AIM OF THIS WORK26
2 M ATERIALS AND METHODS .............................................................................28
2.1 MATERIALS ........................................................................................................................................28
2.1.1 Bacterial strains........................................................................................................................28
2.1.2 Kits...........30
2.1.3 Chemicals.................................................................................................................................30
2.1.4 Enzymes...................................32
2.1.5 Antibodies................................32
2.1.6 Nucleic acids............................................................................................................................33
2.1.7 Filters and Membranes ............................................................................................................34
2.1.8 List of Manufacturers...............34
2.2 METHODS..........................................36
2.2.1 Microbiological methods..........................................................................................................36
2.2.2 Molecular biological methods..36
2.2.3 Protein biochemical methods..40
2 Contents
3 RESU LTS........................................................................................................50
3.1 CHARACTERISATION OF MICROVIRIN.....................................................................................................51
3.1.1 Characterisation of heterologously expressed His-MVN........................51
3.1.2 Mass spectrometric analysis of His-Mvn.................................................................................52
3.1.3 Assigment of disulphide bonds in His-Mvn.............52
3.1.4 Size determination of native Mvn from M. aeruginosa............................................................54
3.1.5 Carbohydrate specificity of Mvn ..............................................................55
3.1.6 Anti-HIV activity of Mvn............................................................................58
3.1.7 Detection of mannan moieties on the M. aeruginosa cell surface..........59
3.1.8 In situ detection of Mvn in M. aeruginosa PCC 7806 .............................................................63
3.1.9 Microvirin binds to LPS............................................................................................................65
3.1.10 The loss of Mvn leads to a reduced production of microcystin............67
3.2 DIRECT INTERACTION OF MICROCYSTIN AND MICROVIRIN........69
3.2.1 Binding of microcystin to Mvn .................................................................................................69
3.2.2 Microcystin binding can be suppressed by blocking of thiol groups......70
3.2.3 Microcystin binds to cysteines via N-methyl-dehydroalanine.................................................72
3.2.4 Influence of microcystin binding on the oligomerisation of Mvn.............74
3.3 INFLUENCE OF OXIDATIVE STRESS CONDITIONS ON MICROCYSTIN-PROTEIN INTERACTIONS ........................77
3.4 OCCURENCE AND EVOLUTION OF MVN.................................................................................................81
3.4.1 Distribution of mvn among different Microcystis species........................................................81
3.4.2 Evidence for the loss of Mvn in M. aeruginosa NIES 843.......................83
3.4.3 Comparison of Mvn sequences from various M. aeruginosa strains......................................84
3.4.4 Phylogeny of CV-N domains....................................................................85
3.5 FIELD STUDIES....................................89
3.5.1 Morphotype diversity and sample quality................................................................................89
3.5.2 Immunofluorescence detection of Mvn...................94
3.5.3 LBA on field samples ...............................................96
3.5.4 Immunofluorescence detection of MrpC.................................................................................98
3.5.5 The MrpC antibody recognises accompanying bacteria......................100
4 DISCUSSION................................................................................................. 102
4.1 LIMITATIONS.....................................................................................................................................102
4.2 GENERAL CHARACTERISATION OF MICROVIRIN.....................................................................................103
4.3 MICROCYSTIN BINDS TO PROTEINS.....104
4.3.1 Microcystin binds covalently to cysteine SH-groups of MVN...............104
4.3.2 General considerations on in vivo microcystin binding .........................................................105
4.4 IMPACT OF MICROCYSTIN ON MICROVIRIN ...........................................................109
4.4.1 Microcystin could affect the carbohydrate binding properties of Mvn in multiple ways.......109
3 Contents
4.4.2 Influence of microcystin-binding on disulphide bond formation ...........................................110
4.5 IMPLICATIONS ON MICROCYSTIN DETECTION AND RISK ASSESSMENT.....................111
4.6 IMPLICATIONS FROM FIELD STUDIES....................................................................112
4.7 HYPOTHESIS FOR THE FUNCTION OF MICROVIRIN.................................................114
4.7.1 Mvn in intraspecies interactions and morphotype determination.........114
4.7.2 Mvn and buoyancy regulation ...............................................................................................116
4.7.3 Microcystin and Mvn in stress adaptation.............................................116
4.8 PHYLOGENETIC ASPECTS OF MVN.....117
4.9 CONCLUDING REMARKS AND OUTLOOK ..............................................................................................120
REFERENCES .................................................................................................... 122
SUPPLEMENT .................................................................................................... 142
ABBREVI ATIONS ................................................................................................ 144
PUBLICATIONS .................................................................................................. 147
SELBSTÄNDIGKEITSERKLÄRUNG ........................................................................ 149

4 Zusammenfassung
ZUSAMMENFASSUNG
Sowohl in Süßwasserseen als auch in marinen Gewässern kommt es in den Sommermonaten
immer wieder zu Massenentwicklungen von Cyanobakterien, sogenannten “Blüten”. In Seen
werden diese oftma