Alexander Loy ProkaryotesLehrstuhl für Mikrobiologie
der Technischen Universität München

DNA Microarray Technology
for Biodiversity Inventories
of Sulfate-Reducing Prokaryotes

Alexander Loy

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

genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. Gert Forkmann
Prüfer der Dissertation: 1. Univ.-Prof. Dr. Michael Wagner, Universität Wien/Österreich
2. Univ.-Prof. Dr. Karl-Heinz Schleifer
3. Univ.-Prof. Dr. Rudi F. Vogel

Die Dissertation wurde am 13.03.2003 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 02.06.2003 angenommen. ABBREVIATIONS
apsA gene encoding alpha subunit of adenosine-5`-phosphosulfate reductase
ApsA alpha subunit of adenosine-5`-phosphosulfate reductase
BLAST Basic Local Alignment Search Tool
bp base pairs
Cy5 5,5’-disulfo-1,1’-di(X-carbopentynyl)-3,3,3’,3’-tetramethyindole-Cy5.18-
derivative, N-hydroxysuccimidester
Cy5-dCTP 5-amino-propargyl-2'-deoxycytidine 5'-triphosphate coupled to Cy5 fluorescent
cDNA complementary deoxyribonucleic acid
DGGE denaturing gradient gel electrophoresis
DNA deoxyribonucleic acid
dsrAB genes encoding alpha and beta subunit of dissimilatory (bi)sulfite reductase
DsrAB alpha and beta subunits of dissimilatory (bi)sulfite reductase
dCTP deoxycytidine 5'-triphosphate
dNTPs deoxynucleotide triphosphates
DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
(German Collection of Microorganisms and Cell Cultures)
EDTA ethylenediamine tetraacetic acid
FISH fluorescence in situ hybridization
kb kilobases
LSU large-subunit
mg milligram
ml milliliter
ng nanogram
OPD Oligonucleotide Probe Database
PBS phosphate-buffered saline
PCR polymerase chain reaction
pmol picomole
RDP Ribosomal Database Project
rRNA ribosomal ribonucleic acid
RSGP reverse sample genome probing
SDS sodium dodecyl sulfate
SSU small-subunit
SRAs sulfate-reducing archaea
SRBs sulfate-reducing bacteria
SRPs sulfate-reducing prokaryotes
SSC standard sodium citrate
TMAC tetramethylammonium chloride
µg microgram
µl microliterCONTENTS
Introduction 1
I. Classification and phylogeny of sulfate-reducing prokaryotes 3
II. Habitats and general ecological aspects of SRPs 6
III. Current molecular approaches to determine SRP community structure 9
IV. DNA microarrays for determinative studies in microbiology 14
V. Scope of this thesis 16
VI. References 18
Materials and methods 29
Results and discussion 43
I. Phylogeny of hitherto recognized SRPs based on
comparative 16S rRNA sequence analyses 45
II. In silico design and evaluation of SRP-specific, 16S rRNA-targeted
oligonucleotide probes suitable for DNA microarray application 57
III. Electronic forum for the maintenance and evaluation of rRNA-targeted
oligonucleotide probes (http://www.probeBase.net) 63
IV. Specificity and sensitivity of a 16S rRNA-based oligonucleotide
microarray for detection of all recognized SRPs (SRP-PhyloChip)
as evaluated with pure cultures 67
V. Application of the SRP-PhyloChip for analysis of
complex environmental and medical samples 73
VI. Conclusions and perspectives: the PhyloChip approach 84
VII. References 86
Summary/Zusammenfassung 93
Appendices I/II/III 97
List of publications/oral presentations/poster presentations 141INTRODUCTION