Dissimilatory (bi-)sulfite reductase as a marker for phylogenetic and ecological studies of sulfate-reducing prokaryotes [Elektronische Ressource] / Michael Dieter Klein

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 123
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Lehrstuhl für Mikrobiologie
der Technischen Universität München



Dissimilatory (bi-) sulfite reductase as a marker for phylogenetic and ecological
studies of sulfate-reducing prokaryotes



Michael Dieter Klein



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
(Dr. rer. nat.)

genehmigten Dissertation.











Vorsitzende r: Univ.- Prof. Dr. Erwin Grill

Prüfer der Dissertation: 1. Univ.- Prof. Dr. Michael Wagner
Universität Wien / Österreich

2. Univ.- Prof. Dr. Karl-Heinz Schleifer




Die Dissertation wurde am 21.10.2004 bei der Technischen Universität München eingereicht und
durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am
17.12.2004 angenommen.

Parts of this work have been published in advance:


Dubilier, N., C. Mulders, T. Ferdelman, D. de Beer, A. Pernthaler, M. Klein, M.
Wagner, C. Erseus, F. Thiermann, J. Krieger, O. Giere and R. Amann (2001).
“Endosymbiotic sulphate-reducing and sulphide-oxidizing bacteria in an
oligochaete worm.” Nature 411(6835): 298-302.

Klein, M., M. Friedrich, A. J. Roger, P. Hugenholtz, S. Fishbain, H. Abicht, L. L.
Blackall, D. A. Stahl and M. Wagner (2001). “Multiple lateral transfer events of
dissimilatory sulfite reductase genes between major lineages of sulfate-reducing
prokaryotes.” Journal of Bacteriology 183(20): 6028-6035.

Schmid, M., U. Twachtmann, M. Klein, M. Strous, S. Juretschko, M. Jetten, J. W.
Metzger, K. H. Schleifer and M. Wagner (2000). “Molecular evidence for genus
level diversity of bacteria capable of catalyzing anaerobic ammonium oxidation.”
Syst Appl Microbiol 23(1): 93-106.




In preparation:

Zverlov, V., Klein, M., Lücker, S., Friedrich, M.W., Kellermann, J., Stahl, D.A., Loy,
A., and M. Wagner (2004). „Lateral Gene Transfer of Dissimilatory (Bi)Sulfite
Reductase Revisited“, submitted to J. Bacteriol.

Abbreviations:

AMP Adenosine-mono-phosphate
APS Adenosine – 5´phosphosulfate
ATP Adenosinetriphosphate
bp base pairs
DNA Deoxyribonucleic acid
dsr Gene coding for the dissimilatory sulfite reductase
Dsr Dissimilatory sulfite reductase, the protein
dsrA/ DsrA Partial sequence of dsr / Dsr alpha subunit as sequenced by the primers presented
dsrAB approximately 1.9 kb long fragment of the dsr operon, encompassing most of the alpha and
beta subunit of the dsr
DsrAB Amino acid sequence inferred form dsrAB
dsrB /DsrB Partial sequence of dsr / Dsr beta subunit as sequenced by the primers presented
et al. et alteri und andere
g Gravity
GC or GC% Mol % Guanine + Cytosine
h Hour
+
H Proton
H Hydrogen 2
Indel Insertion / deletion events of certain sequences
IS Insertion sequences
kb Kilo base, 1000 nucleotide bases
kDa Kilo Dalton
min Minute
ml milliliter
mM milli molar
nm Nanometer
PAGE Polyacrylamidegelelectrophoresis
PAPS Phospho-adenosine – 5´phosphosulfate
PCR polymerase chain reaction
rRNA Ribosomal ribonucleic acid
-1S Svedberg (s )
s Second
2-
S or H S Sulfide or hydrogen sulfide 2
SDS Sodium- dodecyl- sulfate
2-
SO Sulfite 3
2-SO Sulfate 4
SRB Sulfate reducing bacteria/bacterium
SRP Sulfate reducing prokaryotes/prokaryote
UTP Uraciltriphosphate



A INTRODUCTION.........................................................................................................1
A.1 The global sulfur cycle.............................................1
A.2 Physiological traits of sulfate reducing prokaryotes........................2
A.3 Habitats of sulfate reducing prokaryotes............................................................................................................4
A.4 Phylogeny of sulfate reducing prokaryotes........5
A.5 Detection of SRP using 16S rRNA as marker ...................................................................................................7
A.6 The dissimilatory sulfite reductase – an alternative marker molecule for sulfate reducing
prokaryotes .........................................................................................................................................................................9
A.7 Extension of the DsrAB data base ......................................................................................................................11
A.8 The aims of this Ph.D. thesis.................................12
B MATERIAL AND METHODS..................................................................................13
B.1 Reference strains, and clone maintenance........................................13
B.2 DNA extraction from pure cultures and environmental samples ..............................................................15
B.3 Polymerase chain reaction amplification of dsrAB genes .............................................................................16
B.4 Molecular cloning of dsrAB genes into pCRTM2.1 or pCR-XL-TOPO vectors and identification of
dsrAB carrying clones....................................................................................................................................................16
B.5 Sequencing of cloned dsrAB gene fragments....................................................................................................17
B.6 Gelretardation ..........................................................................................17
B.7 Phylogenetic Analysis .............................................................................................................................................17
B.7.1 Pure culture databank....................................................................................................... 17
B.7.2 Environmental databank.. 18
B.7.3 Alignment and phylogenetic analysis of 16rRNA gene data..... 18
B.7.4 Alignment and phylogenetic analysis of DsrAB data (amino acid based analysis).............................. 19
B.7.5 Calculation of Distance matrices.................................................................................................................... 19
B.7.6 Analysis of short environmental DsrAB sequence fragments................................... 19
B.7.7 Alignment of DsrA to DsrB and phylogenetic analysis of this databank................ 20
B.7.8 Phylogenetic analysis of dsrAB gene sequences (nucleic acid based analysis)..... 21
B.8 Preparation of tissue of the oligochaet Olavius algarvensis for fluorescence in situ hybridization
(FISH) .................................................................................................................................................................................21
B.9 Fluorescence in situ hybridization......................21
C RESULTS AND DISCUSSION...............................................................................23
C.1 Establishment of reference strain DsrAB databank ......................................................23
C.1.1 Reevaluation of PCR primers for amplification of dsrAB gene fragments............................................. 23
C.1.2 Inconsistencies between Edman degradation/ inference from nucleotide sequencing.......................... 26
C.1.3 DsrAB sequence alignment and characteristic motives ............................................. 27
C.1.4 Conservation profile of DsrAB....................................................................................... 28
C.2 Comparison of 16S rRNA and DsrAB-based phylogeny for SRP reference strains.............................29



C.2.1 Comparative analysis of 16S rRNA gene and DsrAB sequence based phylogeny............................... 29
C.2.2 Further analysis on dsrAB originating from Desulfobacula toluolica ..................................................... 35
C.2.3 Comparison of 16S rRNA gene sequence similarity and DsrAB identity values between SRP ......... 41
C.2.4 Further reflections on lateral gene transfer................................................................................................... 45
C.2.4.1 Comparison of G+C% content of dsrA and dsrB............... 45
C.2.4.2 Comparative analysis of genomic G+C% content and dsrAB G+C% content.............................. 46
C.2.4.3 Consequences of lateral gene transfer... 47
C.3 Analysis of environmental DsrAB sequences...................................................................................................49
C.3.1 Global traits in DsrAB based environmental surveys................. 49
C.3.2 Consensus tree containing available pure culture and environmental DsrAB sequences of good
sequence quality.....................................................................................................................................................