A unique way of energy conservation in glutamate fermenting clostridia [Elektronische Ressource] / vorgelegt von Elamparithi Jayamani

Manfred Irmler

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

A unique way of energy conservation in glutamate fermenting
clostridia




Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)


dem
Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von



Elamparithi Jayamani
aus Chennai, Indien






Marburg/Lahn 2008
Die Untersuchungen zur vorliegenden Arbeit wurden von April 2004 bis Dezember 2008 am
Fachbereich Biologie der Philipps-Universität Marburg unter der Leitung von Herrn Prof. Dr.
W. Buckel durchgeführt.






Vom Fachbereich Biologie
der Philipps-Universität Marburg als Dissertation am _______________ angenommen.








Erstgutachter: Prof. Dr. Wolfgang Buckel
Zweitgutachter: Prof. Dr. Rudolf K. Thauer



Tag der mündlichen Prüfung: _______________








2
Die im zeitlichen Rahmen dieser Dissertation erzielten Ergebnisse sind in folgenden
Publikationen veröffentlicht:

Boiangiu, C. D., Jayamani, E., Brügel, D., Herrmann, G., Kim, J., Forzi, L., Hedderich,
R., Vgenopoulou, I., Pierik, A. J., Steuber, J. & Buckel, W. (2005) Sodium ion pumps
and hydrogen production in glutamate fermenting anaerobic bacteria. J. Mol. Microbiol.
Biotechnol. 10, 105-119.

Herrmann, G., Jayamani, E., Mai, G. & Buckel, W. (2008) Energy conservation via
electron transferring flavoprotein (ETF) in anaerobic bacteria. J. Bacteriol. 190, 784-
791.

Frank, I., Biegel, E., Jayamani, E., Buckel, W., Müller, V. (2007) Dissection of the
caffeate respiratory chain in the acetogen Acetobacterium woodii: identification of an
Rnf-type NADH dehydrogenase as a potential coupling site. J. Bacteriol. 189, 8145-
8153.

Jayamani, E*., Boiangiu, C. D*., Dietmar, L., and Buckel, W. Rnf-related ferredoxin-
+NAD reductases from Clostridium tetanomorphum. (Manuscript in preparation).
*equally contributed to this work.



















3
CONTENTS
CONTENTS .................................................................................................................... 3
ABBREVI ATIONS: ........................................................................................................ 8
ZUSAMMENFASSUNG ................................................................................................ 9
SUMMARY ................................................................................................................... 11
1. INTRODUCTION .................................................................................................... 13
1.1 A brief overview on anaerobic metabolism .......................................................... 13
1.2 Glutamate fermentation by clostridia ................................................................... 15
1.2.1 The methylaspartate pathway ............................................................................ 16
1.2.2 Rnf complex ....................................................................................................... 18
1.2.3 Putative role of the Rnf system in Clostridium tetani ....................................... 19
1.2.4 The hydroxyglutarate pathway .......................................................................... 21
1.3 4-Hydroxybutyryl-CoA pathway ........................................................................... 23
1.4. Bacteria ................................................................................................................... 23
1.4.1. Clostridium tetanomorphum: Cluster I ............................................................. 23
1.4.2 Clostridium pascui: Cluster I ............................................................................. 24
1.4.3 Clostridium pasteurianum: Cluster I ................................................................. 24
1.4.4 Clostridium aminobutyricum: Cluster X ............................................................ 24
1.4.5 Clostridium symbiosum: Cluster XIVa .............................................................. 24
1.4.6 Clostridium propionicum: Cluster XIVb ........................................................... 25
1.4.7 Eubacterium (Clostridium) barkeri: Cluster XV ............................................... 25
1.5. Goals of my work ................................................................................................... 27
2. Materials and Methods ............................................................................................. 28
2.1. Instruments ............................................................................................................. 28
2.2. Columns .................................................................................................................. 28
2.3. Radioisotopes .......................................................................................................... 28
2.4. Anaerobic setup ...................................................................................................... 28
2.5. Clostridium tetanomorphum .................................................................................. 29
2.6. Clostridium pascui .................................................................................................. 30
2.7. Clostridium aminobutyricum.................................................................................. 30
2.8. Clostridium symbiosum .......................................................................................... 31 4
2.9. Preparation of membranes extracts ..................................................................... 31
2.10. Purification of Rnf complex from C. tetanomorphum ...................................... 31
2.11. Ferredoxin purification from C. tetanomorphum .............................................. 32
2.12. Partial purification of hydrogenase from Clostridium pasteurianum .............. 33
2.13. Purification Bcd-Etf complex from C. pascui .................................................... 33
2.14. Purification of BCD-Etf complex from C. tetanomorphum .............................. 34
2.15. Assays .................................................................................................................... 34
2.15.1. Rnf with Ferricyanide ..................................................................................... 34
2.15.2. Rnf with ferredoxin and Ti(III)Citrate ............................................................ 35
2.15.3. Hydrogenase to generate reduced ferredoxin ................................................. 35
2.15.4. Butyryl-CoA dehydrogenase with ferricenium hexafluorophosphate (FcPF6)
.................................................................................................................................... 35
2.15.5. Bcd/Etf with NADH and crotonyl-CoA ......................................................... 36
2.15.6. Citramalate lyase ............................................................................................. 36
2.15.7. 2-hydroxyglutarate dehydrogenase and Glutamate dehydrogenase ............... 37
2.15.8. NADH oxidation assay ................................................................................... 37
2.16. Preparation of inverted vesicles .......................................................................... 37
2.17. Reconstitution Methods ....................................................................................... 38
2.18. Preparation of liposomes ..................................................................................... 38
+2.19. Na translocation assay 39
2.20. Determination of protein concentration ............................................................ 39
2.21. SDS-PAGE ............................................................................................................ 40
2.22. Amino acid sequence analysis ............................................................................. 41
2.23. Spectral analysis ................................................................................................... 41
2. 24. Method 1 .............................................................................................................. 41
2.25. Method 2 ............................................................................................................... 42
2.26. Flavin quantification by HPLC .......................................................................... 42
2.27. Non-heme iron determination ............................................................................. 42
2.28. CoA thioester synthesis ....................................................................................... 43
2.29. Genomic DNA isolation 44
2.30. Determination of DNA concentration ................................................................ 44 5
2.31. DNA Agarose Gel Electrophoresis preparation ................................................ 44
2.32. DNA Extraction from Gel .............................................................................