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          Institute: MPI für Evolutionsbiologie     Collection: Ecophysiology     Display Documents



ID: 319760.0, MPI für Evolutionsbiologie / Ecophysiology
Diversity of functional genes in the aquatic nitrogen cycle
Advisors:Imhoff, Johannes F.
Authors:Kim, Ok-Sun
Referees:Schönheit, Peter
Language:English
Date of Approval (YYYY-MM-DD):2007-07-06
Name of University:Christian-Albrechts-Universität
Place of University:Kiel
Physical Description
(e.g. Total Number of Pages):
158,XIV S.
Audience:Not Specified
Table of Contents:Summary.......................................................................................................................1
Zusammenfassung........................................................................................................3
1. INTRODUCTION....................................................................................................5
1.1 Nitrogen cycle......................................................................................................5
1.2 Microorganisms and enzymes related to nitrification.........................................6
1.3 Microorganisms and enzymes related to denitrification....................................10
1.4 Molecular-based analysis of microbial diversity...............................................13
1.5 Purpose of the thesis..........................................................................................14
1.6 Thesis outline.....................................................................................................15
2. MATERIALS AND METHODS..........................................................................17
2.1 Sampling sites....................................................................................................17
2.2 Sample collection..............................................................................................18
2.3 Strains................................................................................................................18
2.4 DNA extraction and PCR..................................................................................18
2.4.1 16S rDNA of ammonia-oxidizing bacteria............................................19
2.4.2 Amplification of amo genes...................................................................19
2.4.3 amoA and pmoA genes...........................................................................20
2.4.4 nirK and nirS genes................................................................................20
2.5 Fingerprinting techniques..................................................................................20
2.5.1 Denaturing gradient gel electrophoresis (DGGE)..................................20
2.5.2 Terminal restriction fragment length polymorphism (T-RFLP)............20
2.6 Cloning and sequencing....................................................................................21
2.7 Diversity estimation of clone libraries..............................................................21
2.8 Phylogenetic analysis........................................................................................21
2.9 Nucleotide sequence accession numbers..........................................................22
2.10 Bioinformatic analyses for chapter I...............................................................22
2.10.1 Sequences and alignments...................................................................22
2.10.2 Primer analyses....................................................................................23
CONTENTS
3. RESULTS...............................................................................................................25
I Comparative analysis and description of PCR primers for ammoniamonooxygenase
genes of ammonia-oxidizing bacteria...................................25
II Application of novel PCR strategies and sequence analysis to study the
amoCAB operon in beta ammonia-oxidizing bacteria.....................................41
III Comparative analysis of ammonia-oxidizing bacterial communities in two
lakes in North Germany and the Baltic Sea.....................................................53
IV Diversity of ammonia monooxygenase (amoA) genes in the water column and
sediment of two lakes and the Baltic Sea.........................................................67
V Genetic diversity of the evolutionarily related enzymes ammonia
monooxygenase (AMO) and particulate methane monooxygenase (pMMO) in
two lakes and the Baltic Sea.............................................................................85
VI Distribution of nitrite reductase (nirK and nirS) genes in the water column and
sediment-water interface of two lakes and the Baltic Sea..............................105
4. DISCUSSION.......................................................................................................127
4.1 Diversity of ammonia-oxidizing bacteria (AOB).............................................127
4.1.1 Strategy to study AOB in environments................................................127
4.1.2 Community comparison between 16S rDNA and amoA.......................128
4.1.3 Comparison of amoA and pmoA diversity.............................................129
4.2 Diversity of denitrifying bacteria.....................................................................130
4.2.1 Strategy to study denitrifying bacteria in environments......................130
4.1.2 Comparison of the diversity of nirK and nirS.......................................130
4.3 How many species are there?...........................................................................131
4.4 Selection pressure.............................................................................................132
4.4 Gene expression...............................................................................................132
REFERENCES.........................................................................................................135
ACKNOWLEDGEMENTS.....................................................................................153
CURRICULUM VITAE..........................................................................................155
ERKLÄRUNG..........................................................................................................157
Appendix A....................................................................................................................i
Appendix B.................................................................................................................vii
Abstract / Description:In this PhD thesis the diversity of functional bacterial genes in the nitrogen
cycle was investigated with molecular methods in the lakes Plußsee and Schöhsee,
and the Baltic Sea. The diversity of ammonia-oxidizing bacteria (AOB) was studied
by diversity of specific 16S rDNA and amoA genes. Dominant sequences from Baltic
Sea water column and Schöhsee 1 m were related to different Nitrosospira clusters.
Sequences from Plußsee 7 m, Schöhsee 12 m and sediment of both lakes were
clustered into a purely environmental cluster with no cultivated representatives. Two
groups of amoA sequences from Baltic Sea sediment were related to environmental
clusters from brackish and marine habitats.
The diversity of the evolutionarily related genes for ammonia monooxygenase
(AMO) and particulate methane monooxygenase (pMMO) was analyzed. A higher
frequency of pmoA sequences, mainly belonging to methane oxidizing bacteria of the
gamma subgroup of proteobacteria (γ-MOB), was detected. Dominant amoA
sequences were related to ammonia oxidizing bacteria of the beta subgroup of
proteobacteria (β-AOB), no sequences related to amoA of the γ-AOB were detected.
The deduced amino acid sequences of some clones from lake sediments were distantly
related to PmoA from Crenothrix polyspora, a filamentous methane oxidizer with an
unusual methane monooxygenase.
The distribution of denitrifying bacteria was studied by the nitrite reductase
genes nirK and nirS. The dominant sequences of nirK from all clone libraries
belonged to two distinct phylogenetic clusters, while nirS sequences from both lakes
were scattered over several clusters throughout the complete phylogenetic tree, and
only few sequences from Baltic Sea overlapped. In the Baltic Sea, nirK-denitrifiers
were diverse throughout the water column, while nirS-denitrifiers were dominant in
the sediment and almost absent in the water column. In Plußsee the community
composition was inverted: nirK-denitrifiers were more diverse in the water column
and nirS-denitrifiers in the sediment. In Schöhsee nirS-denitrifiers were highly diverse
in water and sediment samples.
For nirK and nirS the sequences of the protein were less conserved than those
of the gene while the amoA and pmoA protein were conserved, which might be an
indication of a differential selection pressure.
Document Type:PhD-Thesis
Affiliations:MPI für Limnologie/Abt. Ökophysiologie
Identifiers:LOCALID:Diss/11482 [Signatur/Zugangsnummer]
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