Diversity and Abundance of Sulfur-Oxidizing Bacteria in Wadden
Sea Sediments
Revealed by DsrAB Phylogeny and dsrAB-Targeted
Real-Time PCR
Master Thesis
to obtain the academic degree Master of Science (M.Sc.)
of the International Max Planck Research School for Marine Microbiology
and the University of Bremen
Submitted by
SABINE LENK
First Reviewer: Prof. Dr. Rudolf Amann
Second Reviewer: Dr. Marc Mußmann
Submitted at: 17.03.2006
Day of defence: 31.03.2006
1
The paper at hand has been produced as a Master Thesis at the MPI for Marine
Microbiology, Department of Molecular Ecology of Prof. Dr. R. Amann during the
period of September 2005 to March 2006.
Bremen, 17.03.2006
2
Table of Contents
Table of Contents
(A) Abbreviations ............................................................................................................... 3
(B) Summary....................................................................................................................... 4
1. Introduction..................................................................................................................... 5
2. Material and Methods ................................................................................................... 14
2.1 Material ................................................................................................................... 14
2.2 Chemicals................................................................................................................ 15
2.3 Sampling ................................................................................................................. 17
2.4 Primer design .......................................................................................................... 18
2.5 Amplification of dsrAB from Wadden Sea sediments............................................ 19
2.5.1 Establishment of dsrAB clone libraries ........................................................... 19
2.5.2 Sequencing....................................................................................................... 26
2.6 Amplification of dsrAB from cultivated SOP......................................................... 28
2.7 Specificity test of dsrAB primers dsrA 240 F and dsrA 240 F* ........................... 30
2.8 Determination of dsrB copy number in Wadden Sea sediments ........................ 30
2.8.1 Primer specificity check................................................................................... 30
2.8.2 Standard Generation......................................................................................... 31
2.8.3 Quantitative Real-time PCR ............................................................................ 32
3. Results........................................................................................................................... 34
3.1 Primer optimization ................................................................................................ 34
3.2 Amplification of dsrAB from Wadden Sea sediment ............................................. 36
3.3 Phylogeny and diversity of the DsrAB from SOP .................................................. 37
3.3.1 Overall DsrAB diversity .................................................................................. 37
3.3.2 Comparative analysis of DsrAB consensus tree and 16s rRNA gene tree ...... 41
3.3.3 Comparative analysis of insertions and deletion patterns................................ 43
3.3.4 Vertical DsrAB diversity ................................................................................. 44
3.3.5 Rarefaction analysis ......................................................................................... 44
3.3.6 Comparison 16S rRNA gene identity and DsrAB identity.............................. 45
3.4 Amplification of dsrAB from cultivated SOP......................................................... 46
3.5 Primer selectivity test.............................................................................................. 46
3.6 Real-time Quantitative PCR ................................................................................... 47
4. Discussion ..................................................................................................................... 50
4.1 Primer evaluation and methodological aspects....................................................... 50
4.1.1 Evaluation of dsrAB primers for diversity studies and qRT-PCR ................. 50
4.1.2 Amplification of DsrAB gene from cultivated SOP ........................................ 51
4.2 DsrAB and 16S rRNA phylogeny of cultured SOP and evidence for LGT ........... 52
4.3 Diversity of SOP in WS sediment .......................................................................... 54
4.3.1 Overall SOP diversity ...................................................................................... 54
4.3.2 Vertical SOP diversity ..................................................................................... 56
4.4 Quantitative Real-Time PCR .................................................................................. 57
4.5 General value and limitations of DsrAB gene as molecular marker for SOP
diversity studies and qRT-PCR..................................................................................... 58
4.6 Outlook ................................................................................................................... 59
(C) Literature .................................................................................................................... 61
(D) Appendix .................................................................................................................... 66
(E) Acknowledgement ...................................................................................................... 70
(F) Statement..................................................................................................................... 71
Abbreviations
(A) Abbreviations
ARB
APS
ATP
BSA
CARD
DNA
dNTP
Dsr
dsrAB
DsrAB
et al.
F
FISH
IPTG
JSS
LB
PBS
PCR
MQ
NAD(P)
r
R
RNA
RT
SOP
S
SDS
SOP
Sox
DQR
sp.
Table
TAE
Taq
U
UK
USA
UV
V
X-Gal
Arbor
adenosine phophosulfate
adenosine triphosphate
bovine serum albuminw
Catalyzed Reporter Deposition
deoxyribonucleic acid
Desoxyribonucleosidtriphosphat
dissimilatory (bi)sulfite reductase
nucleotide sequence (gene) of the Dsr
amino acid sequence of the Dsr
et alii (und andere)
forward
Fluoreszenz in situ Hybridisierung
Isopropyl-ß-D-thiogalactosid
Janssand
Luria-Bertani
phosphate buffered saline
Polymerase Chain Reaction
MilliQ demineralized water in Millipore Quality
nicotineamid adenosine dinucleotide phosphate
ribosomal
reverse
ribonucleic acid
real-time
sulfur oxidizing prokaryotes
Svedberg
sodiumdodecyl sulfate
sulfur-oxidizing prokaryotes
sulfur oxidation
sulfide:quinone oxidoreductase
species
Table
Tris-Acetat-EDTA
Thermus aquaticus
Units
United Kingdom (Großbritannien)
United States of America (Vereinigte Staaten von Amerika)
Ultraviolett
Volt
5-Brom-4-chlor-3-indoxyl-ß-D-galactosi
3
4
Summary
(B) Summary
Microbial sulfur oxidation is an ancient metabolic process where reduced sulfur
compounds serve as electron donors for phototrophic and chemotrophic metabolism. The
role of sulfur-oxidizing prokaryotes (SOP) and the applied sulfur oxidation pathways in
marine sediments are poorly understood. Since SOP are phylogenetically diverse and are
often closely related to non-SOP, a solely 16S rRNA gene-based analysis is inadequate
for identification of SOP.
In this study the dissimilatory sulfite reductase (DsrAB), a key enzyme in both
SRP and also some SOP, was used as a phylogenetic marker gene for uncultured SOP.
For this purpose novel primer sets were developed and tested for applicability. In the
backbarrier tidal area (Janssand) of Spiekeroog Island in the German Wadden Sea sulfur
is extensively cycled between sulfate reduction and sulfur oxidation. From the respective
sediments three depth-specific clone libraries were generated. Phylogenetic analysis of 64
fully sequenced clones suggests five distinct sequence clusters of which 46 clones
affiliated with Gammaproteobacteria. In addition, one cluster of 18 clones displays a yet
unclear phylogenetic position but exhibits some similarities to Alphaproteobacteria. Most
sequences are only distantly related to known SOP indicating a large hidden diversity of
sulfur oxidizers in marine sediments. Comparative analysis of DsrAB and 16S rRNA
phylogeny of cultured SOP revealed a polyphyletic origin of the DsrAB sequences of the
alphaproteobacterial Magnetococcus sp. and Magnetospirillum spp.. These findings
strongly indicate the involvement of lateral gene transfer in the evolution of SOP.
Moreover, different treeing methods indicate a close evolutionary linkage between the
DsrAB of the bacterial sulfur oxidizing Chlorobium spp. and the archaeal sulfate reducer
Archaeoglobus fulgidus.
In addition, a novel quantitative real-time PCR assay using SYBR Green was
developed to quantify the dsrB copy number in Janssand sediments. Up to 1.85×107
copies/ml sediment were determined reaching a similar order of magnitude as previous
FISH counts of Gammaproteobacteria. The data imply that the “reverse” Dsr pathway
besides other pathways may play a major role in sulfur oxidation in Janssand sediments.
However, methodological improvements are necessary to enhance reproducibility of the
assay. Overall, the obtained results strongly support the relevance of
Gammaproteobacteria in sulfur oxidation in Janssand sediments.
Introduction
5
1. Introduction
Microbial sulfur oxidation is a key process for the biogeochemical sulfur cycle in
marine sediments and closely linked to the cycling of other elements like oxygen,
nitrogen, and carbon. The process is one of the oldest types of biological energy
conservation (Wachtershauser, 1990). Sulfur compound oxidizing prokaryotes are
phylogenetically (Figure 1) and metabolically (Table1) highly diverse (Lane et al., 1992;
Brock et al., 2006).
Figure 1: 16S rRNA tree of sulfur-oxidizing prokaryotes
Many bacteria and some archaea can carry out dissimilatory sulfur oxidation of
reduced inorganic sulfur compounds, e.g. sulfide, thiosulfate, polythionates, elemental
sulfur and sulfite (Brüser et al., 2000). The latter are employed as donors for electron
transport coupled to ATP synthesis or reduction of NAD(P)+ and other electron carriers.
Phototrophic and chemotrophic organisms exploit reduced sulfur compounds as source of
reducing equivalents (e-, H+) for assimilatory processes. In addition, chemolithotrophic
sulfur oxidizers gain energy from sulfur compound oxidation by coupling it to respiratory
electron transport.
Introduction
6
Sulfur chemolithotrophs grow primarily aerobic, i.e. using molecular oxygen as
terminal electron acceptor. However, some species (Beggiatoa sp., Thioploca sp.,
Thiobacillus denitrificans, Thiomicrospira denitrificans) oxidize H2S anaerobically
coupling it to nitrate reduction (Brock et al., 2006). SOP which exploit alternative
electron acceptors like iron or manganese have not been identified yet (Schippers and
Jorgensen, 2001, 2002). Further physiological differences can be found in the ability to
use various sulfur compounds. Consequently the biochemical sulfur oxidation pathways
are found to be variable.
Table 1: Metabolic lifestyles of sulfur oxidizers1
1
Term
Energy source
e- Donor
C source
organism
Chemolithoautotroph
chemical
H2S…
CO2
Paracoccus, Starkeya
Beggiatoa, Thiobacillus, Thiomargaritha,
Thioploca, Thiomicrospira,
Sulfolobus, Acidianus
Chemolithoheterotroph
(Mixotrophy)
chemical
H2S…
org. C
Beggiatoa, Thiothrix, Leucothrix
Photolithoautotroph
light
H2S…
CO2
Chlorobium, Chromatium, Allocromatium,
Thiocapsa Halochromatium, Rubrivivax,
Rhodobacter, Chloroflexus
Data collected from (Brüser et al., 2000; Brock et al., 2006)
The oxidation of sulfide, sulfur and polythionates is linked to thiosulfate oxidation
and can therefore easily be included into the existing thiosulfate oxidation pathways
(Brüser et al., 2000). So far several biochemical models for thiosulfate oxidation have
been proposed: the tetrathionate pathway (e.g. Thiobacillus thioparus, Acidithiobacillus
sp. and Acidiphilum sp.), the multi-enzyme-complex or SOX pathway (e.g. Paracoccus
denitrifcans, Starkeya novella) and the branched thiosulfate oxidation pathway (e.g.
Allochromatium vinosum, Thiobacillus denitrificans, Thiocapsa roseopercicina) (Brüser
et al., 2000).
The best studied archaeal sulfur oxidizer is the aerobic Acidianus ambivalens,
which has been shown to possess a sulfur oxygenase:reductase (SOR) and
thiosulfate:quinone oxidoreductase (TQO) (Sun et al., 2003; Muller et al., 2004; Urich,
2005). These enzymes catalyze the conversion of sulfur to sulfite, thiosulfate or hydrogen
sulfide and the oxidation of thiosulfate to tetrathionate, respectively (Figure 2).
Introduction
7
Figure 2: Model for sulfur oxidation in
Acidianus ambivalens. Elemental sulfur is
oxidized by a cytoplasmatic sulfur
oxygenase:reductase (SOR) yielding
thisosulfate, sulfite and hydrogen sulfide. The
latter are substrates for thiosulfate:quinone
oxidoreductase (TQO) and sulfite:acceptor
(SAOR) which are coupling the substrate
oxidation to energy conservation.
Adenylsulfate (APS) reductase and
adenylylsulfate: phosphate adenylyltransferase
(APAT), enzymes involved in the generation
of ATP from sulfite by substrate level
phosphorylation, have been identified.
Sulfide:quinone oxidoreductases (SQR), which
catalyzes the oxidation of sulfide activity has
so far not been shown, although homologues
to sqr genes can be found in the genome. CQ
Caldariella quinone (after Kletzin et al., 2004)
In the tetrathionate pathway of Acidithiobacillus and Acidiphilum spp. thiosulfate
is oxidized to the stable intermediate tetrathionate prior to cleavage by a hydrolase
(Brüser et al., 2000). For the oxidation of elemental sulfur and sulfide a different model
not involving tetrathionate has been developed (Figure 3) (Rohwerder and Sand, 2003).
Figure 3: Model for sulfur and sulfide oxidation in
Acidithiobacillus and Acidiphilum spp.. Extracellular
elemental sulfur (S8) is mobilized as persulfide sulfan
sulfur by outer membrane proteins (OMP) and
oxidized by a periplasmatic sulfur dioxygenase
(SDO). The resulting sulfite is further oxidized to
sulfate by sulfite:acceptor oxidoreductase (SOR).
Free sulfide is oxized to elemental sulfur by a
separate
sulfide:quinon
oxidoreductase.
Cyt
Cytochromes, Q Quinonens (after Rohwerder and
Sand, 2003)
Among the sulfur chemotrophs members of the genera Thiobacillus and
Paracoccus are best studied, whereas Allochromatium vinosum is the best studied
phototrophic sulfur oxidizer.
The “sulfur oxidation (SOX) pathway” or “Paracoccus sulfur oxidation (PSO)
pathway” (Kelly et al., 1997) is widespread among Alphaproteobacteria. The latter
harbors a complete thiosulfate-oxidizing multi-enzyme system (Kelly et al., 1997). The
encoding sox gene cluster (Friedrich et al., 2001) comprises 15 genes, seven of which
8
Introduction
(soxXYZABCD) encoding four essential proteins, and was first described for Paracoccus
panthotropus (Friedrich et al., 2005). Catalytic activities of the Sox proteins involve the
oxidation of sulfite, thiosulfate, sulfur and hydrogen sulfide coupled to cytochrome C
reduction without the formation of any free intermediate (Figure 4).
Figure 4: Model of thiosulfate oxidation in
Paracoccus panthotropus (Friedrich et al., 2001).
SoxAX catalyzes the oxidative and covalent transfer
of thiosulfate to the substrate carrier protein SoxYZ.
The SO3- is cleaved off by the hydrolase SoxB,
yielding sulfate and subsequently the S0 group is
oxidized to SO3- by SoxCD and again hydrolyzed by
SoxB (after Urich, 2005).
Sulfur-oxidizing members of the Beta- and Gammaproteobacteria as well as the
Chlorobiaceae (e.g Thiobacillus denitrificans, Allochromatium vinosum and Chlorobium
tepidum) possess an incomplete sox gene cluster and lack the sulfur dehydrogenase
(Friedrich et al., 2005). This points at a different pathway for oxidation of sulfur to
sulfate (Figure 5).
Figure 5: Schematic overview of key genes/enzymes
putatively associated with sulfur coumpound oxidation
in Thiobacillus denitrificans. The presence of sox and
dsr genes suggestes that different pathways enable the
oxidation of thiosulfate and sulfide to sulfate. Moreover
different enzymes may be involved in sulfur oxidation
under aerobic versus anaerobic conditions. (after Beller
et al., 2006)
Introduction
9
For phototrophs of the gamma-subclass and obligate chemotrophs of the betasubclass of Proteobacteria the branched thiosulfate oxidation pathway is established
(Figure 6). Dahl and Trüper (1994) proposed a general scheme for dissimilatory sulfur
metabolism in anaerobic phototrophic bacteria, which includes three essential steps: (1)
oxidation of sulfide or thiosulfate resulting in the formation of elemental sulfur, (2)
oxidation of sulfide or elemental sulfur to sulfite and (3) formation of sulfate as the final
product. Moreover, pioneering work on Allochromatium vinosum identified dissimilatory
sulfite reductase (DsrAB) to be involved in the anaerobic oxidation of sulfide and sulfur
(Dahl and Trüper, 1994; Pott and Dahl, 1998; Dahl et al., 2005). The respective enzyme
is well known from sulfate-reducing prokaryotes (SRP), mediating the reduction of
sulfite to sulfide (Hatchikian and Zeikus, 1983; Wolfe et al., 1994; Molitor et al., 1998).
The entire SOP dsr operon contains 15 genes, with dsrAB encoding an α2β2-structured
“reverse” operating DsrAB that catalyzes the oxidation of sulfide to sulfite (Figure 7).
Figure 6: Model of the branched thiosulfate oxidation pathway. SCS “sulfide carrier system” (after Brüser
et al., 2000)
10
Introduction
Figure 7: Two models for the interaction of Dsr proteins (Dahl et al., 2005) The reverse ctyoplasmatic
“DsrAB” oxidizes sulfide to sulfite. So far, unresolved remains how the substrate HS- is made available
from extracytoplasmatically stored sulfur and how electrons resulting from the sulfite reductase reaction are
fed into the electron transport chain. Model (1) Stored sulfur is reduced to the level of sufide ansd diffuses
as H2S or is transported in an unknown form and by an unknown mechanism over the cytoplasmatic membrane. DsrK and DsrM accept electrons from the Dsr feeding it into the photosynthetic electron flow. A
previously discovered potential NADP:acceptor oxidoreductase DsrL (not shown) could be involved in the
reductive release of sulfide from an organic perthiol carrier molecule. Model (2) DsrK is involved in the
reduction of the perthiol and obtains the required electrons from quinol via DsrM, releasing HS-. The model
can not explain the presence of the previously discovered membrane bound DsrJOP (not shown) and how
electrons from the sulfite reductase reaction are fed into the photosynthetic electron chain. (after Pott and
Dahl, 1998)
Besides the various enzymes and pathways for sulfur oxidation and its ecological
importance, so far, very little is known about the diversity and abundance of key
organisms driving sulfur oxidation in marine sediments. The polyphyly of SOP
complicates 16S rRNA based approaches. Comparative 16S rRNA sequence analysis
does not allow a conclusion on metabolic properties of the respective organisms. Hence,
only potential sulfur oxidizers can be revealed. The use of functional marker genes, i.e.
genes encoding proteins essential to a specific metabolic process, can overcome these
problems since it enables to focus on a defined physiological group. Phylogenies of key
enzymes involved in sulfur oxidation already exist for SoxB; (Petri et al., 2001),
sulfide:quinone oxidoreductases (SQR) (Theissen et al., 2003) and adenine
phosphosulfate (APS) reductase (Kuever, J., unpublished data; Rühland, C. unpublished
data).
Here, DsrAB was applied as functional marker for sulfur-oxidizing bacteria,
employing the “reverse” Dsr pathway (Schedel and Truper, 1979; Schedel et al., 1979).
The homologous protein of SRP is frequently used in environmental studies (Wagner et
al., 2005). So far an universal phylogenetic marker gene for all sulfur oxidizers is lacking.
However, the DsrAB is presumed to be the environmentally prevalent enzyme involved
Introduction
11
in sulfur compound oxidition (personal communication C. Dahl). Recently, the relevance
of DsrAB in unculturred SOP has been shown in Riftia pachyptila endosymbionts
(Markert et al., 2005), Olavius algarvensis gammaproteobacterial endosymbionts (Woyke
et al., unpublished data) and uncultured Beggiatoa sp. (Mußmann et al., unpublished
data).
Lateral gene transfer is regarded as a driving force in the evolution of metabolic
pathways among prokaryotes (Doolittle, 1999; Gogarten et al., 2002; Boucher et al.,
2003). It has frequently been reported for genes involved in sulfate reduction, eg. dsrAB
and apsA of APS reductase (Klein et al., 2001; Friedrich, 2002) and sulfur oxidation, e.g.
soxB, SQR genes and dsrAB (Petri et al., 2001; Theissen et al., 2003; Sabehi et al., 2005).
Thus detailed comparison of functional gene and 16S rRNA trees is necessary to track
putative events of horizontal transmission.
Since no fluorescence in situ hybridization probes are available that target all SOP
the determination of the in situ abundance of SOP is as yet limited to specific
phylogenetic groups such as Beggiatoa.(Mussmann et al., 2003), Thioploca (Schulz et
al., 1999), Arcobacter spp. (Llobet-Brossa et al., 1998). Moreover, available probes, e.g.
GAM660 and GSO477 target only subgroups of organisms affiliating with specific
clusters of endosymbiotic and free living hydrothermal vent associated
Gammaproteobacteria. Cultivation dependent most probable-number-counts (MPN)
revealed 0.93 to 9.30 × 105 cells/ml of chemolithoautotrophic sulfur-oxidizing bacteria in
Wadden Sea sediment. Thereof 0.93 to 9.30 × 103 cell/ml belonged to the genus
Thiomicrospira sp. (Brinkhoff et al., 1998). This numbers are probably underestimated
due to the selectivity of the cultivation approach. Thus, cultivation- as well as phylumindependent approaches to quantify SOP are essential.
Quantitative PCR (qPCR) represents a promising option. Here the copy number of
a certain marker gene is determined and thereof in situ cell number can be deduced. The
approach has already been applied for quantification of microorganisms in environmental
samples, targeting either 16S rRNA or functional marker genes (Suzuki et al., 2000;
Hermansson and Lindgren, 2001; Stubner, 2002; Kolb et al., 2003). Of course
methodological aspects like lyses efficiency in DNA extraction, PCR biases and
amplification efficiency, in addition to copy number of the respective target gene per cell
have to be considered. Consequently, qPCR is a semiquantitative method for the
assessment of bacterial abundances.
Marine sediments represent an ecosystem where extensive sulfur
transformations, including a large sulfur cycle of sulfate reduction and sulfide oxidation,
occur. The intertidal sand flats (Figure 8) at the backbarrier tidal area of Spiekeroog
Island (Janssand region, German Wadden Sea) offer an excellent sampling site for the
investigation of sedimentary SOP.
Moreover, intertidal sulfur pools (Figure 8) occur where elemental sulfur
granules accumulate in the residual water over time. This hints at a significant oxidation
of sulfide which outgases from the underlying sediment and might be catalyzed by SOP.
12
Introduction
Figure 8: Janssand sediments and sulfur pool, German Wadden Sea (M. Mußmann 2003/2005)
Previous 16S rRNA based community studies already reported sulfate reducing
Deltaproteobacteria, e.g. Desulfosarcinales and Desulfobulbaceae, to be abundant in
Wadden Sea sediments (Llobet-Brossa et al., 1998; Llobet-Brossa et al., 2002; Ishii et al.,
2004; Mussmann et al., 2005). Using quantitative CARD-FISH Ishii, Mussmann et al.
(2004) identified Gammaproteobacteria as the most abundant group in janssand
sediments. Furthermore, Arnds (2006) suggested a high abundance of potentially sulfuroxidizing Gammaproteobacteria and Roseobacter at the same site and a minor
significance of typical sulfur oxidizers such as Beggiatoa and Thiomicrospira spp..
Introduction
13
The aim of this study was to investigate the phylogeny and diversity of sulfuroxidizing bacteria employing the dissimilatory sulfite reductase (DsrAB) in Wadden Sea
sediments. For this purpose novel PCR primers specific for dsrAB of SOP were
developed. The applied primers were used to establish clone libraries from different
sediments depths and to amplify dsrAB from cultured sulfur oxidizers. Since the only
available phylogeny of DsrAB of SOP (Sabehi et al., 2005) comprises only sequences to
date, one goal was to generate a more comprehensive phylogeny by using a larger
sequence set. Moreover a phylogenetic comparison with DsrAB of SRP should be
facilitated.
In addition, a quantitative real-time PCR assay for the DsrB subunit was
developed to quantify the dsrB copy number per ml of sediment over the vertical profile.
This approach was supposed to give a first impression of the relevance of the DsrAB in
sulfur oxidation.
In detail this worked focused on the following questions:
1. What is the diversity of DsrAB of SOP in Janssand sediments?
2. What are the main groups of sulfur oxidizers?
3. Can novel groups of SOP be detected?
4. Is there a vertical stratification of certain phylotypes?
5. To what extent is the DsrAB diversity of SOP reflected on 16S rRNA level?
6. Has LGT also affected the evolutionary path of microbial sulfur oxidation?
7. How abundant are SOP employing the “reverse” Dsr pathway?
8. Is there a variation of dsrB copy number and hence cell number of SOP over the
vertical sediment profile?
9. Who are the putative key players mediating sulfur oxidation in Janssand
sediments?
Material and Methods
14
2. Material and Methods
In the following section the methodological procedure including all used material
and methods is described.
2.1 Material
Instruments:
•
Agilent 2100 Bioanalyzer
•
Autoclave Lam-201 (SANOclav, Bad Überkingen-Hausen)
•
Incubator Modell 800 (Memmert, Schwabach)
•
CYBERTECH CS1 Photodocumentation System (Cybertech, Berlin)
•
Demineralisation system MembraPure (MembraPure, Bodenheim)
•
Electrophoresis chamber (Horizon 58, GIBCO BRL, Life Technologies,
Gaithersburg, MD USA)
Kits:
•
TOPO TA Cloning Kit (Invitrogen, Karlsruhe)
•
QIAquick PCR purification kit (Fa. Quiagen, Hilden)
•
MinElute 96 UF PCR Purification Kit (Quiagen)
•
Plasmid MiniPrep96 Kit (Montage)
•
Plasmid MiniPrep96 Kit (Quiagen)
•
Power Soil DNA Kit (MO Bio Laboratories, Inc.,CA, USA)
PC-Software:
•
Agilent 2100 Bioanalyzer
•
ARB 2.4
•
BioEdit
•
Canvas 8.0.4(Microsoft)
•
ClustalW 1.8.1
•
Endnote 7
•
Excel (Microsoft)
•
Photoshop (Microsoft)
•
Sequencher 4.5.1 (GeneCodes)
•
Sequencing Analysis 5.2 (Applied Biosystems)
•
Word (Microsoft)
Material and Methods
15
•
Plates
•
OmniTray Singlewell 86 x 128 mm (Nalge Nunc International, New York, USA)
for preparation of agar plates for 96 clones
•
96-well Standard Microtiterplate (ABgene House, Surrey, UK) for long term
storage of 96 clones at 24 °C
•
Thermo-Fast® 96 Skirted mit Domed Cap Strips (ABgene House, Surrey, UK)
•
Mikroplatte 96 V (Roth, Karlsruhe)for PCR with fluorescence dye SybrGreen
•
MultiScreen®-HV (Millipore, Molsheim, Frankreich) for parallel purification of
96 Cyle Sequencing PCR products
•
Shaker KS 250 basic (IKA Labortechnik, Wilmington, USA)
•
Sequencer 3130xl Genetic Analyzer (Applied Biosystems, Foster City, USA)
•
Sequencer ABI Big Dye ABI PRISM 3100 genetic analyzer (Applied Biosystems,
Foster City, California, USA)
•
Thermocycler for PCR and Cycle Sequencing PCR
•
Mastercycler (Eppendorf, Hamburg)
•
Mastercycler gradient (Eppendorf, Hamburg)
•
Mastercycler personal (Eppendorf, Hamburg)
•
Vortexer MS1 Minishaker (IKA Labortechnik, Wilmington, USA)
•
Watherbaths
•
Typ 1086 (Memmert, Schwabach)
•
Typ 1002 (Memmert, Schwabach)
•
Centrifuges
•
Centrifuge 5810R (Eppendorf, Hamburg)
•
Centrifuge 5415R (Eppendorf, Hamburg)
•
UV-Transilluminator (Vilber Lourmat, Marne la Vallée, Frankreich)
2.2 Chemicals
•
Acetic acid (glacial) (Fluka, Buchs, Schweiz)
•
Agar-Agar (Difco, Heidelberg)
•
Agarose (Seakem, LE, Cambrex Bio Science Rockland, Inc. Rockland, ME USA)
•
Agarose, low melting (Metaphor, Southborough, USA)
Material and Methods
16
•
Ampicillin Sodiumsalt (Roth, Karlsruhe)
•
5-Brom-4-chlor-3-indolyl-ß-D-galactosid [X-Gal] (Roth, Karlsruhe)
•
Bromphenol-Blue
•
BSA solution (bovine serum albumine, Sigma, Taufkirchen)
3mg/ml in PCR water
•
Casein (Sigma-Aldrich, Steinheim)
•
3.8-Diamino-5-ethyl-6-phenylphenanthridiniumbromid
[Ethidiumbromid] (Sigma-Aldrich, Steinheim)
•
dNTP solution (Roche, Mannheim)
•
Dinatriumhydrogenphosphat Heptahydrat (Fluka, Buchs, Schweiz)
•
DNA molecular weight markers
•
96% Ethanol (Merck, Darmstadt)
•
ExoSap-IT enzymes (Bioscience, Amersham)
•
Glycerol (Fluka, Buchs, Schweiz)
•
Isopropyl-ß-D-thiogalactosid [IPTG] (Promega, Mannheim)
•
Low DNA mass ladder (Invitrogen, Breda, Netherlands)
•
Primer 50 pmol/µl (Biomerse, Ulm)
•
LiChrosolv Chromatographie Wasser (Merck, Darmstadt)
•
Na2EDTA
•
PCR Water (Sigma-Aldrich, Steinheim)
•
SephadexTM G-50 Superfine (Amersham Bioscience, Freiburg)
•
Sucrose
•
Tris (Biomol, Hamburg)
•
Taq Polymerase 5U/µl (Eppendorf, Hamburg)
•
Taq reaction buffer (10x) (Eppendorf, Hamburg)
•
Tris (Biomol, Hamburg)
•
Xylene Cyanol
•
Yeast extract (Fluka, Buchs, Schweiz)
Material and Methods
17
2.3 Sampling
An intertidal sandflat in the backbarrier tidal area of Spiekeroog Island (German
Wadden Sea, Janssand region 53°43`45``North and 7°41`80``East) served as sampling
site (Figure 9). The respective area of investigation was already described in detail in a
previous study, for comprehensive information it is referred to Arnds (2006).
North Sea
Janssand
Spiekeroog
Germany
Figure 9: Satellite image of the sampling site Janssand in the backbarrier tidal area of Spiekeroog Island
Sediment cores for the SOP diversity study were taken in Novemer 2004 and
April 2005 and DNA was extracted by M. Mußmann from three distinct depths. In
addition, an intertidal pond with precipitated sulfur was sampled in April 2005 and DNA
was extracted from the pond water. A clone library was constructed from the surface
layer 0 – 3 cm (sediment core 02/04/2005) as well as from the deeper layers 8 – 19 cm
and > 19 cm (sediment core 02/11/2004). In addition a sediment core was taken for the
quantitative survey of SOP in November 2005 frozen and cut into slices. Sediment
samples were taken in triplets from 7 distinct sediment depths, including oxic and anoxic
sediment layers and the oxic-anoxic interface as indicated by the sediment colour (Figure
10) To allow calculation of copy number per ml sediment, each sediment sample was
weighed and the mass of 0.5 ml reference sediment was determined for each depth. DNA
was extracted using the Power Soil DNA Kit (MO Bio Laboratories, Inc.,CA, USA)
according to the manufactoresrs protocol.
Material and Methods
18
LAYER
SAMPLED DEPTH [cm]
OXIC (BROWHNISH)
0.0 – 0.5
1.0 – 1.5
3.0 – 3.5
3.5 – 4.0
OXIC - ANOXIC
ANOXIC (GREYISH)
6.0 – 6.5
11.0 – 11.5
Figure 10: Sediment core for the quantitative survey of SOP taken in November 2005
2.4 Primer design
Design of appropriate primers was the precondition to facilitate the amplification
of dsrAB and dsrB from SOP. Therefore, published (www.ncbi.nlm.nih.gov/Genbank)
and unpublished dsrAB sequences (Table 2) of exclusively bacterial origin were aligned
in BioEdit using ClustalW algorithm. No archaeal dsrAB sequences were available.
Based on the sequence alignment (see appendix Figure A1 and A2) new, degenerated
primers (Table 3).were manually designed for target sites of high sequence conservation
(M. Mußmann, unpublished).
Table 2: Overview of dsrAB sequences used for primer design
Organism
Source
Allochromatium vinosum
GenBank
Alkalilimnicola ehrlichei
GenBank
Chlorobium limicola
GenBank
Chlorobium phaeobacteroides BS1
GenBank
Chlorobium phaeobacteroides DSM
GenBank
Chlorobium tepidum
GenBank
Magnetococcus sp.
GenBank
Magnetospirillum magneticum
GenBank
Magnetospirillum magnetotacticum
GenBank
Pelodictyon phaeoclathratiforme
GenBank
Prosthecochloris aestuarii
GenBank
Prosthecochloris vibrioformi
GenBank
Thiobacillus denitrificans
GenBank
Thiobacillus denitrificans
GenBank
Thiobacillus denitrificans
GenBank
Sargasso Sea uncultured bacterium
GenBank
uncultured bacterium MED1313K9
GenBank
uncultured bacterium Fosmid194A5
Dr. M. Mußmann
Olavius algarvensis gamma1 symbiont
Dr. N. Dubilier
Olavius algarvensis gamma 2 symbinot
Dr. N. Dubilier
Accession number
AAC35394/AAC35395
EAP35273/EAP35272
EAM42077/EAM42078
EAM62784/EAM62785
EAM34529/EAM34528
AAM72088/AAM72089
EAN29021/EAN29022
BAE52171/BAE52172
ZP_00053119/ZP_00053120
EAN24264/EAN24263
EAN22044/EAN22045
EAO15265/EAO15264
AAZ98438
AAZ97262
AAZ97322
EAJ64896/EAJ64897
AAY89969/AAY89968
unpublished
unpublished
unpublished
Material and Methods
19
Table 3: Overview of primers designed for dsrAB of SOP
Binding site
(Chlorobium limicola)
Primer1
Target
Sequence2 (5´
dsrA 116F
Bacteria
115 - 131
GGNCCNTGGCCBAGYTT
dsrA 240F
Bacteria
226 - 242
GGNTAYTGGAARGGYGG
dsrA 240F*
Bacteria
226 - 242
GGNTAYTGGAARGGNGG
dsrB 403F
Bacteria
385 - 401
CAYACNCARGGNTGGY
dsrB 403R
Bacteria
385 - 401
ARCCANCCYTGNGTRTG
dsrB 808R
Bacteria
781 - 797
CCDCCNACCCADATNGC
3´)
1
Reference Mußmann, unpublished; Names according to alignment positions of Chlorobium limicola.
Ambiguity base codes: R = A, G; Y = C, T; B = C, G, T; N = A, T, C, G
F – Forward primer (binds to 5´ end of the target region)
R – Reverse primer (binds to 3´ end of the target region)
2
2.5 Amplification of dsrAB from Wadden Sea sediments
2.5.1 Establishment of dsrAB clone libraries
Polymerase chain reaction
Polymerase chain reaction after (Saiki et al., 1988) was used for in vitro selective
amplification of the dsrAB of sulfide-oxidizing bacteria. This method is based on the use
of two flanking, single stranded oligonucleotid primers (~20 nt) which bind specific to
their target sequences at both ends of the amplifiable region and a thermostable Taqpolymerase. The principle of repetitive cycles, where newly synthesized strands serve as
template during the following cycles, already allows the amplification of minimal
amounts of DNA.
Material and chemicals
•
Taq reaction buffer (10x) (Eppendorf, Hamburg)
•
dNTP solution (Roche, Mannheim)
o 2.5 MM dATP
o 2.5 MM dCTP
o 2.5 MM dGTP
o 2.5 MM dTTP
•
BSA solution (bovine serum albumine, Sigma, Taufkirchen)
Material and Methods
20
o 3mg/ml in PCR water
•
Taq Polymerase 5U/µl (Eppendorf, Hamburg)
•
Primer 50 pmol/µl (Biomerse, Ulm)
•
PCR water (Sigma, Taufkirchen)
Procedure
Standard reaction of 25 µl volume for amplification of dsrAB
•
Taq buffer (10x)
2.5 µl
•
BSA (3mg/ml)
2.5 µl
•
Dntps (10mM)
2.5 µl
•
Primer F (50µM)
1.0 µl
•
Primer R (50µM)
1.0 µl
•
Taq Polymerase (5U/µl)
0.1 µl
•
Template
1 µl
•
PCR-H2O
ad 25 µl
Table 4: Program for amplification of dsrAB from environmental samples
Phase of reaction
Duration of phase [min]
Temperature [°C]
Number of cycles
Initial denaturation
4
95
1
Cycle denaturation
0.5
95
30
Annealing
0.5
55.5
30
Elongation
2
72
30
Final elongation
10
70
1
Primer test by Temperature gradient PCR
To find the optimal annealing temperature of the new, degenerated primer pair
dsrA 240F and dsrB 808R a temperature gradient PCR was performed. Positive dsrAB
PCR products from environmental DNA JSS 0-3cm and resuspended cells from
Thiobacillus denitrificans (Betaproteobacteria) served as template. PCR reactions were
prepared according to the PCR standard reaction protocol containing 0.1 µl of template
(dsrAB PCR product from environmental DNA JSS 0-3cm) and 0.3 µl of template (dsrAB
PCR product from Thiobacillus denitrificans, 1:10 dilution).
Material and Methods
21
Amplification of dsrAB from environmental DNA
The dsrAB fragment was amplified using the degenerated primer pair dsrA 240F
and dsrB 808R specific for dsrAB of sulfide-oxidizing bacteria.
Sediment as well as sulfur pond samples were amplified in five replicate reactions
to minimize stochastic PCR bias (Cavanaugh et al., 1998). Each 25 µl reaction was
prepared according to the PCR protocol (see Table ) containing 2 µl of template DNA for
the surface sediment sample JSS 0-3cm and accordingly 1µl of template for the two
deeper sediment samples JSS 8-19cm and JSS > 19cm. Amplification was carried out in
an Eppendorf Mastercycler using the conditions shown in Table 4. After completion
samples were cooled down to 12 ° C and stored at 4°C if not immediately processed
futher. The replicates of the PCR products were combined and 40 µl of each amplificate
were applied to an 1.3% agarose gel for excision of band.
Agarose gel electrophoresis
Material and chemicals
TAE buffer (50x) stock solution
•
Tris
2.0 M
•
Acetic acid (glacial)
0.95 M
•
Na2EDTA (pH 8.0)
0.5 M
The TAE buffer (1x) working solution has to be diluted 10 fold with Milli-Q.
DNA molecular weight marker
•
Low DNA mass ladder (Invitrogen, Breda, Netherlands)
Agarose gel (1.3%)
•
1.3 gram Agarose per 100ml TAE (1x) working solution
Loading buffer (6x)
•
Bromphenol-Blue (0.25%)
2.5 g/l
•
Xylene Cyanol (0.25%)
2.5 g/l
•
Sucrose (40%)
400 g/l
Ethidiumbromid staining bath
•
Ethidiumbromid
0.4 µg×ml-1 in Milli-Q
Material and Methods
22
Procedure
The gel electrophoresis was used for analysis of the fragment size. Electrophoretic
separation of nucleic acids was performed according to Sambrook et al. (2001). Samples
were prepared with > 0.2 vol.-% loading buffer and applied to an 1.3% agarose gel
covered with TAE (1x) in a horizontal electrophoresis chamber (Horizon 58, GIBCO
BRL, Life Technologies, Gaithersburg, MD USA). In addition, 2µl of DNA molecular
weight markers mixed with µl loading buffer were applied for size determination and
quantification of the fragments. For spatial separation an electrical field was applied,
according to a voltage power of 4 V/cm distance anode – cathode, on average 100 – 200
mV, for 10 to 20 min. Afterwards the agarose gel was stained for 10 to 20 min in an
Ethidiumbromid staining bath. Detection of the nucleic acids was carried out via a UVTransilluminator (Vilber Lourmat, Marne la Vallée, Frankreich). Stained agarose gels
were documented with the CYBERTECH CS1 Photodocumentation System (Cybertech,
Berlin).
Gel electrophoresis yielded gene products of different sizes including an
approximately 2kb DNA fragment for the three distinct sediment samples. The latter
band was excised and DNA was purified from the gel using the QIAquick PCR
purification kit (Fa. Quiagen, Hilden) according to the instructions of the manufacturer.
Amplification of dsrAB from the sulfur pond yielded no visible band in the agarose gel.
Cloning
Material and chemicals
TOPO-TA Cloning Kit (Invitrogen, Karlsruhe) containing
•
pCR®4-TOPO® Vector (10 ng µl-1) 3956 bp
o Triton X-100
0.1%
o Glycerol
50%
o Tris-HCl (pH 7.4)
50 mM
o EDTA
1 mM
o DTT
2 mM
o BSA
100 µg ml-1
o Phenol red
•
•
salt solution
o Sodium chloride
1.20 M
o Magnesium chloride
0.06 M
strain Escherichia coli TOP10
genotyp: lacZΔM15 hsdR lacX74 recA endA tonA
Material and Methods
•
23
SOC-medium (pH 7.3)
o Trypton
2.0%
o Yeast extract
0.5%
o Sodium chloride
10 mM
o Potassium chloride
2.5 mM
o Magnesium chloride
10 mM
o Magnesium sulfate
10 mM
o Glucose
20 mM
TOPO-TA Cloning ligation reaction
•
Salt
1µl
•
PCR product
2µl
•
sterile
3µl
•
PCR 4 – TOPO vector
1µl
Procedure
Ligation and transformation
PCR products of each depth were cloned separately using the TOPO TA Cloning
Kit (Invitrogen, Breda, Netherlands) according to the manufacturer´s protocol. In brief,
two microliters of the purified PCR product were ligated into pCR 4 – TOPO vector
incubated at room temperature for 30 min. Afterwards 2 µl of the ligation reaction were
added into one vial of chemically competent Escherichia coli cells. Incubation time on
ice was prolonged to 30 min to ensure a sufficient attachment of vector onto the cells.
Transformation was achieved via heat shock. Finally 40 and 80 µl of each transformation
reaction were spread on selective LB plates and incubated overnight at 37°C.
Selection of recombinant clones
•
LB/AMP/IPTG/x-Gal selective agar plates
•
LB medium liquid
•
LB medium liquid + 10 vol.-% glycerol
•
Ampicillin stock solution
100 µg µl-1 Ampicillin (Roth, Karlsruhe) were dissolved in MQ - water gelöst,
sterile filtered (pore size 0.2 µm) and stored at -20°C -20°C
Material and Methods
24
•
0.5 M IPTG stock solution
Isopropyl-ß-D-thiogalactosid [IPTG] (Promega, Mannheim) were dissolved in
50% ethanol, sterile filtered (pore size 0.2 µm) and stored at -20°C
•
X-Gal stock solution 100 mg ml-1 5-Chlor-4-brom-3-indol-ß-D-galactosid [X-Gal]
(Roth, Karlsruhe) were disolved in N, N-dimethyl-formamid and stored at -20°C
•
LB-Medium (pH 7.3-7.5)
o 1.0%
Casein
o 0.5%
Yeast extract
o 0.5%
Sodiumchlorid
o 1.5%
Bacto-Agar (for solid LB)
All reagents were dissolved in MQ-water and autoclaved for 45 min. Solid LB-medium
was taken out at a temperature of 80 °C to keep the solution liquid for pouring. For
plating of clones 100 µg ml-1 ampicillin, 20 µg ml-1 IPTG und 20 µg ml-1 X-Gal were
added to the LB-medium. Solutions were stored autoclaved and at 4 °C.
Cells containing plasmid inserts were selected using the blue white screening
method (Sambrook et al., 2001). After final incubation at 4°C white colonies were
transferred on new selective LB agar plates and simultaneously into 96 well-format plates
(NunclonTM, Nunc, Wiesbaden) containing 150 µl liquid LB medium. Both were
incubated overnight at 37°C, the liquid cultures on a rotary shaker at 200 rpm. For
permanent storage clones from liquid culture were transferred into each 150 µl liquid LB
containing glycerol, incubated overnight on a rotary shaker at 200 rpm and afterwards
frozen at -20°C.
Screening of clones
Material and chemicals
PCR reagents see point 2.5.1
Procedure
Screening for the dsrAB fragment was carried out by PCR using the M13 forward
and reverse vector primers (see Table ). 20 clones of each depth were randomly picked
and controlled for the correct insert size (~2 kb). Each 25 µl reaction was prepared
according to the PCR protocol (see Table ) containing 0.3 µl of primer mix and 1 µl of
liquid LB culture as template. Afterwards gel electrophoresis through a 1.3% agarose gel
was performed.
Material and Methods
25
Table 5: PCR Primers used for amplification of the dsr inserts in E. coli
Primer
M13F
M13R
dsrB 403R
Target/
Binding
site
Vector
355 - 378
Vector
199 - 222
Bacteria
403 - 419
Sequence (5´
3´)
Referenz
ACGACGTTGTAAAACGACGGCCAG
TTCACACAGGAAACAGCTATGACC
Yanisch-Perron
et al. (1983)
Mußmann,
unpublished
ARCCANCCYTGNGTRTG
F – Forward primer (binds to 5´ end of the target region)
R – Reverse primer (binds to 3´ end of the target region)
Table 6: Program for amplification of insert from pCR 4 – TOPO vector
Phase of reaction
Duration of phase [min]
Temperature [°C]
Number of cycles
Initial denaturation
4
95
1
Cycle denaturation
0.5
95
30
Annealing
30
50
30
Elongation
2
72
30
Final elongation
10
70
1
Purification of PCR products
Material and chemicals
•
•
ExoSap-IT enzymes (Bioscience, Amersham)
MinElute 96 UF PCR Purification Kit (Quiagen)
Procedure
PCR products of correct size were purified using either the MinElute 96 UF PCR
Purification Kit (Quiagen) according to he manufacturers instructions or ExoSap
purification protocol. Therefore 0.25 of ExoSap-IT enzymes and 3.25 PCR water were
combined with 1.5 µl of PCR reaction and incubated in an Eppendorf Mastercycler at
first for 30 minutes at 37°C and afterwards for 15 minutes at 80°C. 3 µl of ExoSap
reaction were used as template for sequencing.
Since neither of the PCR purification methods yielded a product from which
sequences could be obtained plasmids were extracted for sequencing of dsrAB.
Material and Methods
26
Plasmid preparation
Material and chemicals
•
Plasmid MiniPrep96 Kit (Montage)
•
Plasmid MiniPrep Kit (Quiagen)
•
2x LB medium liquid
Procedure
15 µl of liquid LB culture were transferred into 1 ml of liquid 2x LB and
incubated overnight at 37°C on a rotary shaker at 200 rpm. To obtain a compact pellet,
cells were centrifuged at 1500 g for ten minutes. LB medium was completely removed
and cells were processed further according to the Plasmid MiniPrep96 protocol (Montage)
for clones from environmental sample JSS 8-19 cm and JSS >19 cm or Plasmid MiniPrep
Kit (Quiagen) for clones from environmental sample JSS 0-3 cm.
2.5.2 Sequencing
Material and chemicals
PCR reagents see point 2.5.1
Procedure
Standard sequencing reaction of 5 µl volume for dsrAB
•
Big Dye
1.0 µl
•
Buffer
1.0 µl
•
Primer F (50µM)
0.1 µl
•
Primer R (50µM)
0.1 µl
•
Template
2.5 µl
•
PCR-H2O
ad 5 µl
Material and Methods
27
Table 7: Program for sequencing
Phase of reaction
Duration of phase
Temperature [°C]
Number of cycles
Initial denaturation
20 sec
96
1
Cycle denaturation
10 sec
5 sec
30sec
4 min
96
> 50
≤ 50
60
60
60
30 sec
21
1
Annealing
Elongation
60
Sequencing of the complete dsrAB inserts was achieved by using the M13 F and
M13R vector primers and in addition, the internal new, degenerated dsrB 403R primer.
For the latter the optimal annealing temperature was detected by performing a
temperature gradient PCR of a dsrAB insert containing clone. Vector primed sequencing
with the M13F and M13R was performed at an annealing temperature of 48 °C and 57°C
respectively for plasmids from sediment samples JSS 8-19 as well as JSS >19 and JSS
0-3 cm respectively. Sequencing with the dsrB 403R primer was performed at an
annealing temperature of 57 °C for all samples.
2.5.3. Phylogenetic analysis
The dsrAB sequences were checked against sequences in the Genbank database
using BLAST for similarity searches (Altschul et al., 1990). Partial sequences were
analyzed for sequence quality using the Sequencing Analysis software package (Applied
Biosystems). Full length sequences were generated by assembling partial sequences with
the Sequencer Version 4.5.1 software package. The dsrA and dsrB clone sequences were
aligned separately together with the respective published sequences using the ClustalX
and Bioedit software and subsequently translated into proteins. Further phylogenetic
inference of the DsrAB sequence data was performed with the ARB software package
(www.arb-home.de).
102 full length sequences from the environmental clone libraries (64 different
sequences), cultivated (14 sequences) and uncultivated (4 sequences) bacteria as well as
cultivated representatives of SRP (20 sequences (Klein et al., 2001)) and 3 partial length
sequences of uncultured SOP were entered into the dsrAB ARB database, translated into
amino acids and computational and manually aligned. In brief, first SOP DsrAB
sequences were roughly aligned against the existing SRP alignment. Afterwards the
resulting SOP DsrAB alignment was corrected on the basis of the Thiobacillus
denitrificans sequences. Finally, 20 representatives of SRP were aligned against the
refined SOP DsrAB alignement. A total number of 532 amino acid positions (alpha
subunit 318; beta subunit 214) were included in DsrAB analysis (excluding insertions and
deletions). Phylogenetic trees were calculated by performing Neighbour Joining,
Maximum Parsimony, Maximum Likelihood and Tree Puzzle analysis. For tree
reconstruction only full length sequences were used. Partial length DsrAB sequences of
Beggiatoa, Riftia pachyptila sulfur oxidizing endosymbiont and Olavius algarvensis
sulfur oxidizing gamma 1 symbiont were added to the final trees using the ARB
Parsimony Qick Add tool. To root the tree the 20 representatives of cultivated SRP (Klein
Material and Methods
28
et al., 2001) served as outgroup. In addition to the inference methods mentioned above,
we employed MrBayes phylogenetic analysis starting from the Neighbour joining tree.
Calculation parameters included 30000 generations, a print frequency of 100 and a
sample frequency of 10 resulting in a total number of 3000 trees. For final tree
construction the first 2000 trees were discarded.
2.6 Amplification of dsrAB from cultivated SOP
For completion of the phylogenetic tree dsrAB fragments should be amplified
from sulfur-oxidizing reference strains (Table 8)
Table 8: Sulfur oxidizing bacteria chosen for amplification of dsrAB
Species
Strain no/Referenz
Beggiatoa sp.
Susanne Hink
Halochromatium salexigens
DSM4395
Halothiobacillus neapolitanus
DSM581
Leucothrix mucor
Thomas Holler
Marichromatium gracile
DSM203
Magnetospirillum gryphiswaldense
PD Dr.Dirk Schüler
Rhodobacter capsulatus
DSM1710
Rubrivivax gelatinosus
DSM6859
Starkeya novella
DSM506
Thiobacillus thioparus
DSM505
Thiobaca trueperi
DSM13587
Thiocapsa sp.
DSM5653
Thiocococcus pfennigii
DSM226
Thiomicrospira psychrophila
Dr. Katrin Knittel
Thiomicrospira arctica
Dr. Katrin Knittel
Thiomicrospira kuenenii
DSM12350
Thiomicrospira frisia
DSM12351
Thiomicrospira pelophila
DSM1534
Phylum
Gamma-Proteobacteria
Gamma-Proteobacteria (PSB)
Gamma-Proteobacteria
Gamma-Proteobacteria
Gamma-Proteobacteria
Alpha-Proteobacteria
Alpha -Proteobacteria (PNSB)
Beta-Proteobacteria (PNSB)
Beta-Proteobacteria
Beta-Proteobacteria
Gamma-Proteobacteria
Gamma-Proteobacteria (PSB)
Gamma-Proteobacteria (PSB)
Gamma-Proteobacteria
~
~
~
~
Primer test by Temperature gradient PCR
Standard reaction of 25 µl volume according to point 2.5.1
•
Primer F (50µM) 16s/dsr
0.25/ 1.0 µl
•
Primer R (50µM) 16s/dsr
0.25/ 1.0 µl
Table 9: PCR Primers used for amplification of dsrAB from cultured SOP
Primer
Target
Binding site Sequence (5´
dsrA 240F*
Bacteria
240 - 256
dsrB 808R
Bacteria
808 - 824
3´)
Referenz
GGNTAYTGGAARGGNGG Mußmann,
unpublished
CCDCCNACCCADATNGC
F – Forward primer (binds to 5´ end of the target region)
R – Reverse primer (binds to 3´ end of the target region)
* modified dsrA 240 F primer containing an additional wobble at position 15
Material and Methods
29
For amplification of the dsrAB fragment from cultured SOP the new degenerated
dsrA 240F* primer was designed on the basis of an additional dsrAB sequence from
fosmid clone 194A5 (unpublished, M. Mußmann). Here, an additional degeneracy was
introduced in the previously designed primer dsrA 240F, making it fully complementary
to the respective target site. To find the optimal annealing temperature of the new dsrA
240F* and dsrB 808R primer pair an annealing temperature dependent amplification of
the dsrAB gene fragment from Magnetospirillum gryphiswaldense was performed. Cells
from pure culture were frozen and thawn and served as template for PCR using the PCR
standard reaction protocol and conditions under 2.5.1 without a final elongation.
Amplification of 16s rRNA gene and dsrAB gene fragment from reference strains
Cell pellets of the freeze-dried reference strains (obtained from DSMZ ) were
directly resuspended in 30 – 60 µl PCR water (Sigma, Taufkirchen). Living cultures were
centrifuged (Eppendorf Centrifuge 5810) at 1500 g for 10 minutes to gain a sufficient cell
pellet. Amplification of dsrAB gene fragment from the reference strains was performed
according to 2.5.1 using an annealing temperature of 57.5 and 51.3 °C respectively.
Magnetospirillum gryphiswaldense served as positive control.
To ensure the right DNA amount for potentially successful amplification of DNA
from target cells and to proof the correct template concentration, the 16s rRNA gene was
amplified simultaneously from all reference strains. Besides using the 16s rDNA primers
(Table 10), a primer specific annealing temperature (Table 11), the standard reaction
protocol (see above) and PCR conditions were analogue to dsr amplification.
Table 10: Primers used for amplification of the 16s rRNA gene
Primer
Target
Binding site Sequence (5´
3´)
8F
16s rDNA
8-24
AGAGTTTGATCMTGGC
1492 R
16s rDNA
1492-1507
TACCTTGTTACGACTT
Referenz
Muyzer et al.
1995
F – Forward primer (binds to 5´ end of the target region)
R – Reverse primer (binds to 3´ end of the target region)
Table 11: Program for amplification of dsrAB from cultured SOP
Phase of reaction
Duration of phase [min]
Temperature [°C]
Number of cycles
Initial denaturation
4
95
1
Cycle denaturation
0.5
95
35
Annealing 16s/dsr
0.5
48.0/ 57.5; 51.3
35
Elongation
2
72
35
Final elongation
10
70
1
30
Material and Methods
2.7 Specificity test of dsrAB primers dsrA 240 F and dsrA
240 F*
To test the specificity of the dsrA 240 F and dsrA 240 F* primer with regard to
the additional degeneracy PCR was performed on the Fosmid194A5 containing the dsrAB
gene fragment according to the protocol under point 2.5.1. using 0.5 µl of fosmids as
template and 35 PCR cycles. A dsrAB containing plasmid served as positive control.
2.8 Determination of dsrB copy number in Wadden Sea
sediments
To quantify the copy number of dsrAB from SOP over the vertical sediment
profile qRT -PCR based on a SybrGreen assay was employed. This method allows for
simultaneous amplification and quantification of a target template by detecting the
amplification associated fluorescence. Using PCR for quantification seems contradictory
at the first glance since this method was originally developed for massive amplification of
smallest DNA amounts, i.e. the pure qualitative detection. Nevertheless, it is based on a
exponential amplification which follows mathematical rules. Thus a quantification is
principle possible (Mülhardt, 1999).
The applied SYBR Green assay is generally based on the detection and
quantification of the respective fluorescence reporter. SYBR Green is a fluorescence dye
which intercalates with double stranded DNA. The fluorescence of SYBR Green
increases when bound to accumulating amplification product. Consequently, a higher
fluorescence signal will be detected during each subsequent PCR cycle. Since the
reaction is monitored continuously an increase in fluorescence signal can be viewed at
real-time. The parameter used for quantification, is the threshold cycle, or Ct-value,
namely, the cycle at which a significant increase in ∆Rn fluorescence/PCR product can
be observed. (Dorak). In general, homogeneous amplification of the target gene is a
precondition for reliable quantification. Since the amplification efficiency is higher for
shorter templates (Mülhardt, 1999), we amplified a 400 bp sized gene fragment of the
DrsB subunit. An absolute quantification assay, i.e. copy number was quantified by
interpolation from a standard curve, was employed for determination of the dsrAB copy
number in the environmental samples.
2.8.1 Primer specificity check
As a consequence of the unspecific binding of SybrGreen to dsDNA the measured
fluorescence reports only an increase in DNA but not necessarily an increase in specific
product. Hence specific amplification of the target gene has to be ensured. To identify
possible contaminations gel electrophoresis, sequencing and melting curve analysis were
employed.
Material and Methods
31
2.8.2 Standard Generation
For generation of an appropriate standard a dsrB clone library was established.
Therefore a dsrB gene fragment of approximately 400bp was already amplified from
environmental DNA JSS 0-3 cm using the degenerated primer pair dsrB 403F and dsrB
808R by Marc Mußmann. The PCR was performed according to the protocol under 2.5.1
using an annealing temperature of 52.9°C. Prior to further processing the stored product
was incubated under the conditions shown in Table 12 to restore sequence quality at the
end of the sequences. The PCR product was stored at -18°C.
Table 12: PCR products Cycle Program for incubation of stored
Phase of reaction
Duration of phase [min]
Temperature [°C]
Denaturation
4
95
Elongation
10
72
Final elongation
60
60
For further library construction steps listed under 2.5.1 were performed. Since
only a single band was observed the PCR product was cloned directly without excision
from the agarose gel. Vector primed sequencing of 16 clones, containing the correct size
insert, was performed using the M13 F primer according to 2.5.2.
Obtained sequences were checked against sequences in the Genbank database
(www.ncbi.nlm.nih.gov/blast/) using BLAST (Altschul et al., 1990) for similarity
searches.
Determination of copy number in standard
For generating a standard curve the respective ~ 400bp sized dsrB fragment was
amplified from a clone according to 2.5.1. The resulting PCR product of approximately
570bp was purified using the MinElute 96 UF PCR Purification Kit (Quiagen) and served
as template for serial dilutions. For the quantification of copy number in the standard the
fragment size [bp] and the DNA amount [ng/µl] was determined via the 2100 Bio
analyzer and Bio Sizing software Version A.02.12 (Agilent Technologies, USA).
Table 13: Determination of copy number in standard
Standard
Fragment size
[bp]
Concentration
[ng/µl]
Copy number
[mol of dsDNA/µl]
Copy number
per µl
sample 1
569
10.7
2,85052E-14
1,72E+10
sample 2
574
10.8
2,84895E-14
1,72E+10
Conversion of µg to pmol:
pmol of dsDNA = μg (of dsDNA) × 1515
fragment size in bp
(1)
Material and Methods
32
Conversion of pmol to absolute copy number:
mol DNA × 6, 02214 × 10 23
(2)
A two fold dilution series, comprising 11 dilution steps, was prepared starting
from a 1000fold diluted purified PCR product.
2.8.3 Quantitative Real-time PCR
Material and chemicals
•
iCycler iQTM5 Multicolour Real-Time PCR Detection systems (Bio-Rad, CA,
USA)
•
Platiunum SYBR Green qPCR Super Mix-UDG Kit (Invitrogen, Kalsruhe)
containing:
•
Platinum® SYBR® Green qPCR SuperMix-UDG
•
50 mM Magnesium Chloride (MgCl2) 1 ml 2 ×
•
ROX Reference Dye
•
20X Bovine Serum Albumin (ultrapure, non-acetylated) (1 mg/ml) 300 µl
Platiunum
•
SYBR Green qPCR Super Mix-UDG composition
•
SYBR Green I, 60 U/ml Platinum® Taq DNA polymerase, 40 mM Tris-HCl (pH
8.4), 100 mM KCl, 6 mM MgCl2, 400 µM dGTP, 400 µM dATP,
•
400 µM dCTP, 400 µM dUTP, 40 U/ml UDG, and stabilizers
Procedure
Standard reaction of 25 µl volume for Real-time Quantitative PCR of dsrAB
•
Platinum SYBR Green qPCR Super Mix-UDG
15.6 µl
•
Primer F (10µM)
1.0 µl
•
Primer R (10µM)
1.0 µl
•
Template
1 µl
•
PCR-H2O
ad 25 µl
Material and Methods
33
Table 14: Program for amplification of dsrB from standard and environmental samples
Cycle Repeats
1
2
3
4
1
1
45
81
Time Temperature
[min]
[°C]
Step
1 UDG Incubation
1 Initial denaturation
1 Cycle denaturation
2 Annealing
3 Elongation
1 Final Denaturation
2.0
2.0
0.5
0.5
0.5
0.5
50
95
95
54
72
55
PCR/Melt
DataAcquisition
Real time1
Melt curve2
1
Real time – Step at which real time data were collected
Melt curve – Step at which melt curve data were collected
2
Standards and environmental samples were analyzed in triplets (technical triplets)
to allow discrimination of technical measurement inaccuracies. To minimize pipetting
errors 3 µl of template were added to 75 µl of Mastermix (see standard reaction) and
subsequently separated into triplets using 23 µl of reaction volume. RT-qPCR was
performed according to the protocol (Table 14) The amplification associated fluorescence
was detected at each cycle during PCR. Melting curve analysis was performed after PCR
was completed over a temperature gradient of 0.5 °C temperature change starting from a
temperature of 55 °C to a final temperature of 95 °C.
Determination of dsrB copy number in Wadden Sea sediment
Data analysis and standard curve generation was performed using the Bio-RadiQ 5 software. Copy number was determined per µl of sample and manually converted
to copy number per µl of sediment. Average values of the copy numbers of the technical
triplets [copy number/µl of applied DNA] were multiplied with the dilution factor of 20
to obtain copy numbers per volume of extracted DNA [copy number/mg]. Therefore, a
DNA extraction efficiency of 100% was assumed. Some samples were not optimally
extracted and were excluded from final data set. Samples with non-optimal DNA
extraction were excluded from the final data set. Based on calculated volume of extracted
sediment [ml] values were finally converted to copy number per ml of sediment.
TM
(3)
0.5 ml
volume of extr. sediment [ml ] =
× weight of extr. sed iment [g ]
weight of 0.5 ml sed iment [g ]
copy number per ml sediment =
copy number per extr. sediment
× 1ml
volume of extr. sediment [ml ]
(4)
Data conversion and generation of the diagrams was performed using the Microsoft Excel
(Windows 2000) software.
Results
34
3. Results
3.1 Primer optimization
Prior to amplification of the dsrAB fragment from the environmental samples as
well as the cultivated SOP a temperature gradient-PCR was conducted to identify the
optimal annealing temperature of the newly developed primers. Primer pair 240 F and
dsrB 808 R were used for amplification of dsrAB from the environmental samples at an
annealing temperature of 55.5 °C (Figure 11).
environmental DNA JSS 0-3cm
Thiobacillus denitrificans
1200 bp
2000 bp
63.5
60.8
58.1
55.5
53.2
51.4
63.5
60.8
58.1
55.5
53.2
51.4
Figure 11: Temperature gradient PCR for amplification of dsrAB from Thiobacillus denitrificans and
environmental DNA JSS 0-3cm using the dsrA 240 F and dsrB 808 R primer. For primer optimisation
amplification was carried out at different annealing temperatures (T in °C).
2000 bp
NC
PC
1
2
3
4
5
Figure 12: Amplification of dsrAB from environmental DNA JSS 0-3cm. Amplification was carried out in
5 replicate reactions (number 1 – 5) at an annealing temperature of 55.5 °C. PC positive control, NC;
negative control
The dsrAB of Magnetospirillum gryphiswaldense was successfully amplified over
a wide range of annealing temperatures (Figure 13). Moreover, the 16S rRNA gene
fragment was amplified successfully from all reference strains (Figure 14 and Figure 15)
Thus the right DNA template concentration was provided. The DsrAB gene fragment
could not be amplified from the majority of the respective organisms at an annealing
temperature of 57.5°C (Figure ) A decrease in annealing temperature to 51.3° in the
following PCR also yielded no products (data not shown). Besides Magnetospirillum
gryphiswaldense successful amplification of the dsrAB fragment was achieved for
Thiobacillus denitrificans and Thiobacillus thioparus (data not shown), using the forward
primer dsrA 240F and an annealing temperature of 55.5°C.
2000 bp
51.3
52.2
53.2
54.3
55.4
56.5
57.3
58.4
NC
Figure 13: Temperature gradient PCR for amplification of dsrAB from Magnetospirillum gryphiswaldense
using the dsrA 240 F* and dsrB 808 R primer. For primer optimisation amplification was carried out at
different annealing temperatures (T in °C). The negative control was amplified at a temperature of 55.4 °C.
5
Results
35
1200 bp
2000 bp
DsrAB gene
16s rRNA gene
1200 bp
2000 bp
1
3
2
4
5
6
7
8
9
10
NC
12
Figure 14: Amplification of DsrAB gene fragment and 16s rRNA gene from cultured reference strains.
The 16s rRNA gene could be amplified from all reference strains except Rhodobacter capsulatus (3).
Amplification of the DsrAB gene fragment at an annealing temperature of 57.5 °C was only succesful for
Magnetospirillum gryphiswaldense (8). Number 1 – 10 Marichromatium gracile, Starkeya novella,
Rhodobacter capsulatus, Thiocapsa sp., Thiobacter truepii, Thiocococcus pfennigii, Thiomicrospira
pelophila, Magnetospirillum gryphiswaldense Leucothrix mucor, uncultured Beggiatoa; NC negative
control
1200 bp
2000 bp
1
2
3
4
5
6
7
8
9
10
NC
NC
Figure 15: Amplification 16s rRNA gene from cultured reference strains. The 16s rRNA gene could be
amplified from all reference strains except Thiomicrospira arctica (1). Thiomicrospira kuenenii (3) and
Starkeya novella (8). The 16s rRNA gene could be amplified from the former organisms in previous a PCR
(data not shown). Simultaneous amplification of the DsrAB gene fragment at an annealing temperature of
51.3 degree did no yield any visible band for any of the respective organisms. Number 1 – 10
Thiomicrospira arctica, Thiomicrospira psychrophila, Thiomicrospira kuenenii, Halothiobacillus
neapolitanus, Thiobacillus thioparus, Halochromatium salexigens, Rubrivivax gelatinosus, Starkeya
novella, Rhodobacter capsulatus, Magnetospirillum gryphiswaldense; NC negative control
Newly designed primers were tested for selectivity in order to detect whether the
covered sequence diversity was possibly less than the actual existing. The design of
primers on the basis of multiple sequence led to the integration of numerous
degeneracies. Due to up to 5 wobble positions per primer many variants are possible.
Hence, it was necessary to evaluate whether the primer variability was also reflected in
sequence diversity or whether certain sequences where amplified selectively.
Environmental clone dsrAB sequences were screened for the nucleotide composition at
the primer sites dsrA 240F and dsrB 808R to detect a possible selectivity of certain
primers variants. All respective nucleotides present at the wobble position of the primers
were represented in the sequences of the primer binding site. Moreover, the majority of
nucleotide combinations could be detected.
36
Results
3.2 Amplification of dsrAB from Wadden Sea sediment
A DNA fragment of ca. 2 kb size, encompassing most of the alpha and beta
subunit genes of the dissimilatory sulfite reductase, could be amplified from three distinct
sediment depths of the Wadden Sea, but not from the intertidal sulfur pond. Although
amplification of the 16s rRNA gene from the respective pond was successful for a
already low number of 20 cycles, dsrAB amplification with 35 cycles did not yield any
visible product in the electrophoresis gel. Partial and full sequences were obtained from
the respective PCR products. BLAST analysis of overall 182 dsrA partial sequences
against the GenBank database yielded 57 non DsrA sequences (Table 15), all of which
did not encode for similar protein functions as Dsr. This high fraction can be mainly
ascribed to direct sequencing without size specific prescreening of inserts. However,
some screening PCR products had the same size like the dsrA fragment and thus could
not be discriminated. Furthermore, the application of degenerated primers was reflected
in target sequence diversity, i.e. wobble position were highly divers. Hence a broad
spectrum of dsrAB sequences could be covered.
Table 15: Overview of non DsrAB BLAST hits of environmental sequences amplified with primer pair
dsrA 240 F and dsrB 808 R
Target sequence
cytochrome c5530 family protein [Methylococcus capsulatus str. Bath]
TPR repeat protein [Nitrosococcus oceani ATCC 19707]
TPR repeat [Nitrosomonas europaea ATCC 19718]
ccdB fusion protein [Cloning vector pTarg2]
Acyl-CoA dehydrogenase-like [Geobacter metallireducens GS-15]
hypothetical protein RferDRAFT_3095 [Rhodoferax ferrireducens DSM 15236]
hypothetical protein CA2559_10383 [Croceibacter atlanticus HTCC2559]
conserved hypothetical protein [Alkalilimnicola ehrlichei MLHE-1]
TonB-dependent receptor [Rhodospirillum rubrum ATCC 11170]
TonB-dependent copper receptor [Magnetococcus sp. MC-1]
LacOPZ-alpha peptide from pUC9 [Cryptosporidium hominis]
Predicted periplasmic solute-binding protein [Microbulbifer degradans 2-40]
conserved hypothetical protein [Parachlamydia sp. UWE25]
hypothetical protein AcidDRAFT_4187 [Solibacter usitatus Ellin6076]
ketol-acid reductoisomerase [Pelobacter carbinolicus DSM 2380
alpha-galactosidase [Bacillus clausii KSM-K16
sialic acid-specific 9-O-acetylesterase [Bacteroides thetaiotaomicron
GCN5-related N-acetyltransferase [Anabaena variabilis ATCC 29413]
putative integrase [Nitrosococcus oceani ATCC 19707]
O-acetylserine sulfhydrylase [Methanosarcina thermophila]
hypothetical protein PatlDRAFT_2122 [Pseudoalteromonas atlantica
urf53 [Chlorella vulgaris]
conserved hypothetical protein [Xanthomonas campestris pv. campestris str. 8004]
putative succinate dehydrogenase flavoprotein subunit [Nocardia farcinica]
probable serine/threonine protein kinase [Rhodopirellula baltica SH 1]
hypothetical protein BURPS1710b_A1869 [Burkholderia pseudomallei 1710b]
hypothetical protein SfumDRAFT_2942 [Syntrophobacter fumaroxidans MPOB]
IstB-like ATP-binding protein [Solibacter usitatus Ellin6076]
hypothetical protein BbacK_01001192 [Bartonella bacilliformis KC583]
Oxidoreductase, N-terminal [Solibacter usitatus Ellin6076]
TPR repeat [Chlorobium phaeobacteroides DSM 266]
antigen, putative [Treponema denticola ATCC 35405]
ribonuclease G [Xanthomonas axonopodis pv. citri str. 306]
Acyl-CoA dehydrogenase-like protein [Burkholderia sp. 383]
Histidine kinase A, N-terminal [Methanococcoides burtonii DSM 6242]
CoA-binding [Chloroflexus aurantiacus J-10-fl]
PREDICTED: hypothetical protein XP_896092 [Mus musculus]
hypothetical protein Npun02005532 [Nostoc punctiforme PCC 73102]
E value
3e-33
1e-86
9e-47
2e-33
e-17
1e-12
8e-36
6e-15
2e-42
6e-36
3e-12
4e-39
1e-55
7e-61
4e-125
7e-111
1e-36
4e-19
6e-79
1e-88
6e-15
7e-24
1e-24
2e-46
2e-52
0.055
0.48
2e-16
0.010
1e-40
3e-12
1e-17
4e-69
0.011
1e-23
2e-57
0.86
7e-28
Identity
107/278 (38%)
184/319 (57%)
106/223 (47%)
78/116 (67%)
52/158 (32%)
67/222 (30%)
70/108 (64%)
40/67 (59%)
121/317 (38%)
105/277 (37%)
38/40 (95%)
83/133 (62%)
110/252 (43%)
133/321 (41%)
221/269 (82%)
190/308 (61%)
105/253 (41%)
62/173 (35%)
149/226 (65%)
180/288 (62%)
55/147 (37%)
56/123 (45%)
73/199 (36%)
111/263 (42%)
21/286 (42%)
45/164 (27%)
70/259 (27%)
2/75 (56%)
18/51 (35%)
88/226 (38%)
41/120 (34%)
43/99 (43%)
135/249 (54%)
40/97 (41%)
53/107 (49%)
126/277 (45%)
20/68 (29%)
69/179 (38%)
Accession
AAU90385
ABA57571
CAD85209
CAD27778
ABB32438
EAO41905
EAP86435
EAP35670
ABC24246.1
EAN28739.1
EAL34605.1
ZP_00314664.1
CAF23477.1
EAM58117.1
ABA89756
BAD62954.1
AAO79196.1
ABA23672.1
ABA57146.1
AAG01804.1
EAO67128.1
BAA99566.1
AAY48221.1
BAD55792.1
CAD77107.1
ABA51353.1
EAO22105.1
EAM60184.1
ZP_00947034.1
EAM54819.1
EAM34521.1
AAS13216.1
AAM37615.1
ABB05792.1
EAM99616.1
EAO57496.1
XP_901185.1
ZP_00106364.2
Results
37
3.3 Phylogeny and diversity of the DsrAB from SOP
Full sequences were loaded into the ARB database and comparative sequence
analysis was performed based on a combined DsrAB dataset, comprising a maximum
number of 532 comparable amino acid positions. Overall 102 DsrAB full length
sequences from the environmental clone library (64 different sequences), cultivated (14
sequences) and uncultivated (4 sequences) bacteria as well as selected, cultured
representatives of SRP (20 sequences (Klein et al., 2001)) and additionally 3 partial
length sequences from uncultivated SOP served for reconstruction of the phylogeny of
the DsrAB.
3.3.1 Overall DsrAB diversity
DsrAB Consensus tree
For reconstruction of DsrAB phylogeny different inference methods were applied,
including Neighbor Joining, Maximum Parsimony, Maximum Likelihood and Tree
Puzzle, to achieve optimal resolution of evolutionary relationships. The obtained tree
topologies showed no major discrepancies Differences occurred in the phylogenetic
positioning of Thiobacillus denitrificans as well as the Wadden Sea clone clusters C, B
and E. Based on this results a consensus tree (Figure 16) was constructed representing the
phylogenetic relatedness as best as possible.
The DsrAB clone library contained 64 distinct sequences and was dominated by
clones which affiliated with Gammaproteobacteria (46 different sequences) constituting
72%. Eighteen of 64 clones (28%) grouped together in the monophyletic cluster E with
probable alphaproteobacterial affiliation. The majority of gammaproteobacterial
associated sequences (57%) grouped together in the monophyletic Cluster A (16 different
sequences) and B (10 different sequences) with a sequence identity between 81.9 and
99.8% and 92.3 and 99.8% respectively. Clones JSS d1_F3 and JSS d3_G8 were related
to an uncultured bacterium from the German Wadden Sea (Fosmid 194A5, Janssand)
displaying 95.5 and 94.2% identity to the fosmid sequence. Clone JSS d1_F6 clustered
with the sulfur-oxidizing gamma 1 symbiont of Olavius algarvensis (90.9% sequence
identity). Clones JSS d1_A2, JSS 2_B5 and d2_A9 JSS (96.6 – 98.4% sequence identity)
of the monophyletic cluster C were more distantly related to that sequence (84.6 – 85.4%
seqeunce identity). A fourth group of sequences formed the monophyletic Cluster D (92.5
–99.8% sequence identity) and affiliated with the sulfur oxidizing gamma 2 symbiont of
Olavius algarvensis (90.2 – 96.2% sequence identity). In addition, clone JSS d2_dB1
showed a close relationship to the gamma 2 symbiont (96.0% sequence identity). Clone
JSS d2_D7 took an intermediary position displaying 82.6% sequence identity to the
gamma 2 symbiont of Olavius algarvensis and 82.6% sequence identity to uncultured
bacterium from the Mediterranean Sea (BAC13K9). The phototrophic sulfur oxidizer
Allochromatium vinosum was found to be the closest cultured relative of the majority of
the Gammaproteobacteria-related clones. Only one sequence (JSS d2_D9) grouped
closer with Alkalilimnicola ehrlichei (73.3% sequence identity).
38
Results
The monophyletic Cluster E comprised nearly one third of all clones, including 18
distinct sequences (79.3 – 99.7% sequence identity) and hence constituted the second
largest fraction in the DsrAB clone library, accounting for 28% of all clones. Maximum
Likelihood and Neighbour Joining methods indicated an alphaproteobacterial affiliation
of this cluster. However, Maximum Parsimony yielded contradictory results. Moreover,
sequence identity between the clones and Alkalilimnicola ehrlichei was on average 1.3
higher compared to the Magnetospirillum sequences (71.2% compared to 72.5%).
Interestingly, neither of the clones affiliated with the sulfur oxidizing
Betaproteobacterium Thiobacillus denitrificans or the phototrophic chlorobi . The latter
phylum branched deepest within the SOP. Moreover, the DsrAB of two Chlorobium
phaeobacteroides strains, DSM266 and BS1, was represented within the phylogenetic
tree and branched of separately within the chlorobial cluster.
Interestingly, the closest relatives among the sulfate-reducing prokaryotes were
members of the deep-branching SRP phyla, i.e. Euryarchaeota and Firmicutes.
Archaeoglobus fulgidus affiliated closest displaying 49.4% sequence identity to
Chlorobium phaeobacteroides. Furthermore, the DsrAB sequence of the uncultered
sulfate reducing bacterium (Wadden Sea Fosmid 39f7) showed a comparable high
percentage of sequence identity (49.7%). However this does not necessarily result from
close evolutionary relatedness as reflected in the tree topology.
Figure 16: DsrAB concensus tree for the known SOP and SRP as well as environmental clone sequences
based on Neighbor Joining, Maximum Parsimony, Maximum Likelihood and Tree Puzzle phylogenetic
inference methods. Multifurcations were introduced when branching orders were not supported by all
phylogenetic inference methods used. Environmental clone sequences are colour coded according to the
depth they originated from. Phylogenetic sequence clusters are indicated by brackets.
Figure 17: DsrAB tree for the known SOP and SRP as well as environmental clone sequences as calculated
with MrBayes phylogenetic inference method. Environmental clone sequences are color coded according to
the depth they originated from. Phylogenetic sequence clusters are indicated by brackets.
(see following pages)
Results
39
40
Results
Results
41
DsrAB MrBayes tree
We applied the MrBayes inference method for phylogeny of DsrAB (Figure 17)
to check our results from the consensus tree. Overall, both trees displayed congruent tree
topologies. Slight differences occurred in the phylogenetic positioning of the sulfuroxidizing gamma 1 symbiont of Olavius algarvensis branching of between
Allochromatium vinosum (92.02% sequence identity) and the uncultured bacterium
(fosmid 194A5) from the German Wadden Sea (91.73% sequence identity). The Wadden
Sea clone Cluster B showed monophyletic affiliation with the Allochromatium vinosum
associated sequences, as before revealed by Maximum Likelihood analysis. Consistently
with results from Maximum Parsimony analyis clone d2_D7 affiliated with cluster C
(80.7 – 98.4% sequence identity), which likewise branched of differently. Furthermore,
the inferred phylogeny strongly supported the affiliation of the cluster D with the sulfur
oxidizing gamma 2 symbiont of Olavius algarvensis. Additionally, a clustering of
sequences into surface and deeper sediment layers associated subclusters became
evidently. Moreover, the topology of the MrBayes tree strongly supported the relatedness
of clone cluster E to the alphaproteobacterial Magnetospirillum spp. sequences.
Accordant to the consensus tree, SRP of the Euryarchaeota and Firmicutes affiliated
closest with the chlorobi.
3.3.2 Comparative analysis of DsrAB consensus tree and 16s
rRNA gene tree
The comparison of the tree topologies of DsrAB and 16s rRNA gene (Figure 18)
revealed one putative major LTG event within the SOP. The chlorobial affiliation of the
alphaproteobacterial Magnetococcus sp. points at horizontal transmission of the dsrAB.
Analysis of the insertion and deletion patterns yielded additional evidence.
Figure 18: Comparison of the DsrAB consensus and 16S rRNA trees. Known SOP are colour coded
according to their taxonomic classification. (see next page)
42
Results
Results
43
3.3.3 Comparative analysis of insertions and deletion patterns
As additional indications of the deduced phylogenetic relationships, particularly
with respect to LGT, insertion and deletion patterns within the DsrAB amino acid
sequences of SOP and SRP were examined (Figure 19).
Within the DsrAB sequences of the analyzed SOP Thiobacillus denitrificans
harbored three unique insertions within the alpha subunit at alignment positions 33 to 37,
68 and 252 to 267. The first insertion was also found in the DsrAB sequence of
Alkalilimnicola ehrlichei and Wadden Sea clone JSS d2_D7. One unique insertion at
alignment position 211 to 212 (DsrA) was found for Magnetococcus sp.. In addition, the
latter shared a unique deletion in the alpha subunit present in all members of the chlorobi
at alignment position 268, supporting the suggested LGT event. In contrast, the missing
amino acid was highly conserved in all other DsrAB sequences of SOP and SRP.
Interestingly, all SOP shared an insertion at alignment position 332 to 333 with
Desulforhopalus vacuolatus, Desulfobacter latus and Desulfitobacterium hafniense.
SOP associated deletions at alignment positions 211 to 226 (DsrA) and 620 to 626
(DsrB) are also present within the DsrAB sequences of Desulfotomaculum alkaliphilum,
Thermodesulfovibrio islandicus, Desulfitobacterium hafniense, Archaeoglobs fulgidus
and fosmid clone 39F2, with Desulfoarculus baarsii and Desulfobacca acetoxidans
displaying the deletion within the beta subunit as well. Overall, one deletion at alignment
positions 702 to 703 (DsrB) was found to be characteristic for the DsrAB sequences of
all analyzed SOP, in contrast, all analyzed SRP possessed a unique insertion at this
position.
D. vacuolatus
D. baarsii
D. latus
D. acetoxidans
D. variabilis
D. vulgaris
D. kuznetsovii
D. tiedjei
D. alakiliphilum
T. norvegica
T. commune
T. islandicus
D. hafniense
Fosmid clone 39F7
A. fulgidus
T. denitrificans
A. ehrlicheii
A. vinosum
Magnetococcus sp.
C. tepidum
C. limicola
M. magnetotacticum
M. magneticum
M. gryphiswaldense
30
40
....|....|....|......
PKRFPGV-----EHFHTMRAN
PNLFPGV-----EHFHTVRVN
PNLFPGV-----EHFHTMRVN
PNEFPGL-----EHFHTMRIN
PETFPGV-----AHFHTMRIN
PEKFPGV-----AHFHTVRVA
PEKFPGI-----AHFHTIRVN
PQQFPGV-----AHFHTVRVN
PEQFPNV-----AEFHTVRLN
PDLFPNV-----AHFHTMRVN
QERFPAI-----WAFHTIRVH
ADQFPGV-----AHFHTVRVN
PEDYPNL-----AAFHTVRIN
PDKWPGI-----AHFHTMRIN
GEQIPEV-----EHFHTMRIN
KDENHQPLFPEAAEFHTLRIQ
KDENGKAKFPDAAEFHTMRVM
GKVFPSSK-----EFHTVRVQ
PELFPEAK-----EFHTMRIQ
KDKFPEAA-----EFHTMRIQ
KDKIPEAA-----EFHTMRIQ
AAQFPESA-----EFHTLRIM
AAQFPESS-----EFHTLRIM
ADKHPESS-----EFHTLRIM
200
210
220
230
240
.|....|....|....|....|....|....|....|....|..
QAAVKEYIAGNYPGNGGAHSGRDWG---KFDIQKEVIDLCPTEC
QAAVAAYAGEXIPPRAAAHGARGV----KLDVQKEVXKLCPTGC
QDAVNKYVEQDPAYPANAGAHKGKDWG-PFDIEKEVINLCPTGC
QSAVKAYAGGELKPRGGGSPYA------KLDIQADVVELCPTGC
QEAVASYVGGEFAPNGGAHSGRNWG---AFDIQKEVIDLCPTRC
AEAVKAYVAGEFKPNAGAHSGRDWG---KFDIEAEVVNRCPSKC
QEAVKAYIAGDIVPRGGSHKGRDWG---KFDIQKEVINLCPSQC
QEAVKAYVGGELKPNAGAHSSRDWG---KFDIIAEVINLCPTNC
QEAVREYVNNGFD------------------IQELVIDRCPTGC
QEAVRAYVAGELVPNGGAHGFEKRP----LDIQAEVIDKCPTRC
QEAVRAYVRGELKPNAGAFSDRDWGP---FDIKKEVIDLCPTKC
QDKVKDYAKKGMN------------------IQEEIVNFCPGKC
QEAVKAYVAEGLN------------------IQAVVCDRCPTKC
KAAVTEY--AKTG----------------MDIKADVVDNCPAKC
QEAVKEYASWMDI-------------------ENEVVKLCPTGA
EEQVQAYYKAHGM----------------NDLVNDVISKCPTKA
QEEVKAFVARKGR----------------KYVVDNVISRCPTNA
QAEVKHYIADKGR----------------QYYIDNVITRCPTKA
EAEVAKFIETRGVAY--------------LTNNVITRCPTNAIV
HDEVKAWIAEKGV----------------DALVNNVINRCPTKA
RDEVTAWVEKKGM----------------DALVNQVINLCPTRA
QKEVQAKVKQLGR----------------KEFINQVIRMCPTQA
QDEVKAKVKQLGR----------------KEFINQVIRMCPTQA
QDEVKAKVAQLGR----------------KEFINQVIRMCPSQA
D. vacuolatus
D. baarsii
D. latus
D. acetoxidans
D. variabilis
D. vulgaris
D. kuznetsovii
D. tiedjei
D. alakiliphilum
T. norvegica
T. commune
T. islandicus
D. hafniense
Fosmid clone 39F7
A. fulgidus
T. denitrificans
A. ehrlicheii
A. vinosum
Magnetococcus sp.
C. tepidum
C. limicola
M. magnetotacticum
M. magneticum
M. gryphiswaldense
250
260
270
..|....|....|....|....|....|....|...
MWMEDGE-LK----------------I-DNSECTRC
MSYEGGK-LA----------------I-NNRECTRC
MKFENKE-LT----------------I-NDEHCTRC
MSFDGS-TLK----------------I-DNRNCNHC
MKYEGGK-LA----------------I-NTKECTRC
MKWDGSK-LS----------------I-DNKECVRC
MWMEDGK-LV----------------I-DNKECVRC
MCWDGSK-LE----------------I-NNAECVRC
LEWDGNE-LK----------------L-DAPECVRC
MEWDGKN-LK----------------I-WDEDCVRC
MYWDENEQKLX---------------I-NNSECNRC
ITWDGNN-LT----------------I-NNSDCLHC
LKFDAETQELS---------------V-IAEECTRC
MDWDGKE-IH----------------I-ENSYCRKC
IKWDGKE-LT----------------I-DNRECVRC
ITLVASDKFAPSETVSAANLGDGNTLCIDNKNCVRC
LSLNDDDTLD----------------V-DNGSCVRC
LSLNDDDTLD----------------V-NNRDCVRC
MNEDGKSITI------------------NNSDCVRC
IRLQDGDIDI------------------STRDCVRC
ISLKEGEMHI------------------ETRDCVRC
LGLNDDDTLD----------------V-DNKSCVRC
LGLNDDDTLD----------------V-DNKSCVRC
LALNDDDTLD----------------I-DNKSCVRC
330
340
....|....|....|....|....
FMKIEKENDYQ-ELIDLIEKIWDW
FIKMEEPYDEF---KEFVANTWDW
FVEVNADNDYK-EITEIIENIWDW
FMEMEPPYDNL---KEIAGNIMEW
FVKVEEPYDGI---KEVIESIWNW
FVAAEEPFDEI---KEVVEKIWDW
FMKAEPPYDNV---KEVIEKIWEF
FMKMEPPFDEL---KELIEKVWDW
FMKMEAPYQEV---KDLLEKIWEW
FMKMEPPYEEF---HEFVEKMWDW
FMKVEPPYDEL---KDFIYKAWDY
FMGMKPPYQEI---KDLIKNMWDW
FMKMEVEDDFQ-EFKDMIERIWEW
FYKMEPPYTEL---KDIMERIWDL
FVEVEKPYDEI---KEILEAIWDW
FMKLESEEDFE-KLNDLARNIIDF
FLPLNTEEDYD-RVVEIAEEIIDF
FKKLETEEDYE-SLVELAETIIDF
FMKMEDEDDIE-AFIDMIDSMIDY
FMKMETDEDRE-AFIELIEEIIDW
FMKMESDEDVE-TFIEQIETIIEW
FMKLETEEDFE-KLVELAHNMLDF
FMKLETEEDFA-GLVELAHNMLDF
FMKLETDEDYE-RLLEMAHNMLDF
Results
44
D. vacuolatus
D. baarsii
D. latus
D. acetoxidans
D. variabilis
D. vulgaris
D. kuznetsovii
D. tiedjei
D. alakiliphilum
T. norvegica
T. commune
T. islandicus
D. hafniense
Fosmid clone 39F7
A. fulgidus
T. denitrificans
A. ehrlicheii
A. vinosum
Magnetococcus sp.
C. tepidum
C. limicola
M. magnetotacticum
M. magneticum
M. gryphiswaldense
620
630
640
....|..|....|....|....|....|....|....
LLDTLASYGN-----SYPVGGT------GAGVTNIVH
LKKPCRAG-------NMPIGGT------GHSVTNIVH
LKNDLASRKFAGGSQKFPIGGT------GAGVTNIVH
LKAELAQR-------GIPKGGT------GHSITNIVH
LIKDLESRKFDGGSFKFPIGGT------GAGVTNIIH
LKEDLASRKFDGGSLKFPIGGT------GAGVSNIVH
LKKDLWSRKFVSGSYKFPIGGT------GASITNIVH
LIDDLKGRKFGAGSYKFPIGGT------GSGITNIVH
LIAEVKAK-------GLPVGGT------GNSITNMVH
LIDDLKSRGN-----WFPIGGT------GASVTNIVH
LIEDLKNRKHPNGSYKFSIGGT------GAGISNIVH
LLDELKAK-------KYMIGGI------GSRVSNIVH
LIAALGAK-------GLPVGGT------GASVSNIVH
LIKALKDI-------GHPVGGI------GNAISSIVH
LINEVQERV------GFPCGGTWDAVKGEYGLSNIVH
LIKALSDK-------GYPIGGT------ANSVSMIAH
LIDKLRAE-------GFPVGGT------GPSVAMISH
LIDALEGA-------GFVVGGT------QNSVAMISH
LIKALEAA-------GHPVGGT------GNSITSMAH
MIAELESL-------GFPVGGT------GMCVSAVSH
MIQELESL-------GFPVGGT------GMCVSSVSH
LVKRLEAE-------GFPVGGT------GNSVGFISH
LIERLEKD-------GFPVGGT------GNSVGFISH
LIDRLEAA-------GFPVGGT------GNSVGFISH
700
710
..|....|....|..
MCGAVHASDTAILGI
MCGAVHCSDIAILGV
MCGAVHCSDIAILGY
MCGAVHCSDIAILGV
MCGAVHCSDIAILGY
MCGAVHCSDIGVVGI
MCGAVHCSDIALLGI
MCGAVHCSDIAILGI
MCGAVHCSDIAILGI
MCGAVHCSDIAILGV
MCGAVHCSDLALVGI
MCGAVHCSDIAFVGI
MCGAAHCSDIAIVGV
MCGAVHCSDIAILGI
MCGAVHASDIAIVGI
NCGGQ--ADIAIIVQ
NCGGQ--GDIAINIQ
NCGGQ--GDIAINIQ
NCGGQ--ADIAIVVQ
NCGGQ--ADIAVVVK
NCGGQ--ADIAIVVK
NCGGQ--GDIAINVQ
NCGGQ--GDIAINVQ
NCGGQ--GDIAINVQ
Figure 19: Insertion and deletions patterns of DsrAB of selected SOP and SRP
3.3.4 Vertical DsrAB diversity
Clone libraries of dsrAB from different sediment layers were established to
elucidate vertical distribution of SOP. Overall 28 different full sequences were obtained
from the surface sample (JSS 0 – 3 cm), 21 (JSS 8 – 19cm) and 15 (JSS > 19cm) different
full sequences, respectively, from the deeper sediment layers.
Clones from all depths seemed to be randomly distributed over the both major
clone groups, the gammaproteobacterial branch as well as the Wadden Sea clone cluster
E. Nevertheless, a clustering of sequences of different depths could be observed. Within
cluster A clones JSS d2_B2 – d3_B3 (93.43 –99.8% sequence identity) of the deeper
sediment layers and clones JSS d1_G4 – d1_H5 (83.7 –99.2% sequence identity) of the
surface layer formed two distinct subclusters. A similar pattern could be found in Cluster
D were clones JSS d3_B4 – d3_F9 (89.9 – 99.7% sequence identity) and clones JSS
d1_B1 – d1_D4 (95.3 – 99.8% sequences identity) clustered together to two distinct
subgroups from the depth and surface sample, respectively. Sequences of cluster B
originated almost exclusively from the surface sediment (92.3 – 99.8% sequence
identity). As already mentioned above, clones from all sediment depths were also present
within the cluster E, in which again clones JSS d1_D1 – JSS d1_E6 (88.9 –89.0%
sequence identity) from the upper sediment layer clustered together in a distinct
subgroup.
Some clones within the clusters A, B, D and E displayed very high amino acid
sequence identity, e.g. d2_D2 and D2_G9 (99.8%), d1_C6, d1_A1 and d1_A3 (99.8%),
d1_C2 and d3_F12 (99.7%) as well as d3_E11 and d2_C6 (94.7%), which can also be
found on the nucleotide basis. These findings point at the the existence of identical clones
3.3.5 Rarefaction analysis
To estimate overall diversity of SOP in the Wadden Sea sediment a DsrAB clone
library was established from three distinct sediment depths and a total number of 112
were screened. In order to proof whether the number of retrieved sequences efficiently
covered the sequence diversity in our clone library we performed a rarefaction analysis
(Heck et al., 1975) on a combined dsrA dataset including sequences from all depths
(Figure 20). Based on sequence comparison of the dsrA fragment (~ 600 bp) 46 distinct
Results
45
sequence types were identified among 112 clone sequences. DsrAB diversity was higher
than expected as represented by the continous slope. However the rarefaction curve
slightly turned into the saturation limit. Thus we could assume coverage of the major
dsrAB sequence types by numer of anaylzed clones but further screening of the clone
library was predicted to yield additional novel dsrAB sequence types.
50
Number of sequence types
45
40
35
30
25
20
15
10
5
0
0
10
20
30
40
50
60
70
80
90
100
110
120
Number of screened clones
Figure 20: Rarefaction curve for the different dsrA sequence types. The number of sequence types is plotted versus the
number of dsrA clones. The dotted lines represent 95% confidence intervals.
3.3.6 Comparison 16S rRNA gene identity and DsrAB
identity
To evaluate the degree of sequence conservation of DrsAB and to compare
sequences identity on the basis of the functional gene with the 16S rRNA gene, identity
matrices (see appendix Table A1 and A2) were calculated for the respective nucleotide
and aminoacid sequences of representative organisms using the ARB Similarity Matrix
calculation editor. Comparative sequence analysis reavaled a high degree of sequence
conservation of the the DsrAB, with an average sequence identity of 64. 01% the
organisms analyzed. Absolute values varied between 54.2% (Thiobacillus denitrificans Chlorobium tepidum and Magnetococcus sp., respectively) and 96.41%
(Magnetospirillum magnetotacticum - Magnetospirillum magneticum). Overall, the
degree of sequence conservation of the 16s rRNA gene (82.0% average sequence
identity) exceeded the one of DsrAB. In general, aminoacid sequences were highly
conserved for species closely related on 16S rRNA gene level. For example, members of
the chlorobi, displaying a 16S rRNA gene sequence identity between 91.8% and 99. 2%
(average value 96.4%), showed comparable high values for the DsrAB, i.e. between
73.7% and 90.5% sequence identity (average value 83.9%). Similarly, the
alphaproteobacterial Magnetospirillum sp. sequences (95.8 – 99.4% 16 s rRNA gene
sequence identity, average value 97.1%) exhibited a considerable high DsrAB sequence
identity (87.5 – 96.4%, average value 90.9% ). Highest conservation of DsrAB was found
46
Results
between the closely related species Magnetospirillum magnetotacticum and
Magnetospirillum magneticum (99.4% 16s rRNA gene sequences identity) displaying
96.4% aminoacid and 92.8% nucleotide sequence identity. Although the dsrAB
nucleotide sequence in general was less conserved, displaying 41.92% average sequence
identity, a high degree of sequence conservation on the aminoacid level could also be
detected on nucleotide basis (53.4 – 81.1% sequence identity for the chlorobi, average
value 70.5%; 84.2 – 92.8% sequence identity for the Magnetospirillum sp. sequences,
average value 87.7%). Interestingly, the DsrAB of the two Chlorobium phaeobacteroides
species (95% 16 s rRNA gene sequence identity) was less conserved, displaying 74.8%
aminoacid sequence identity. However, in comparison, the nucleotide sequence identity
of 73.2% was high. Moreover the DsrAB of the Chlorobium sp. sequences (95.6%
average sequence identity 16s rRNA gene to Chlorobium phaeobacteroides) showed a
much higher sequence identity to Chlorobium phaeobacteroides DSM 266 (88.7%
average) than to Chlorobium phaeobacteroides BS 1 (74.9%). Interestingly, within the
gammaproteobacterial cluster Allochromatium vinosum displayed a lower DsrAB
sequence identity to Alkalilimnicola ehrlichei (76.1%) than to the sulfur oxidizing gamma
2 endosymbiont of Olavius algarvensis (80.6%). In contrast, the respective 16 s rRNA
gene sequence identity was higher (90.1% compared to 86.7%).
Surprisingly, the Alphaproteobacterium Magnetococcus sp. displayed a
significantly lower degree of sequence conservation on the DsrAB level (58.1% average
sequence identity) to the alfaproteobacterial sequences than to the chlorobial sequences
(65.2% average sequence identity).
3.4 Amplification of dsrAB from cultivated SOP
In addition to amplification from environmental DNA dsrAB should also be
amplified from cultivated SOP to better resolve phylogenetc relationsships. Temperature
gradient PCR yielded a dsrAB product over a wide range of annealing temperatures from
Magnetospirillum gryphiswaldense. Subsequently, primer pair 240F* and dsrB 808R
were used for amplification of dsrAB from cultivated SOP at an annealing temperature of
57.5 (Figure 14) and 51.3°C (Figure 15), respectively. Nevertheless, amplification of
dsrAB from sulfur-oxidizing reference strains was not successful in the majority of cases.
Finally, amplification of the dsrAB could only be achieved for Thiobacillus thioparus,
displaying a weak band of approximately 2000bp in the agarose gel (data not shown).
Repetition of the PCR is necessary to approve this result.
3.5 Primer selectivity test
Primer dsrAB 240F was tested for selectivity in order to detect wether the covered
sequence diversity is possibly less than the actual existing and in order to reveal a
selective amplification of dsrAB from environmental samples. Amplification of the dsrAB
from the fosmid failed with both primer combinations. Besides the positive control no
visible PCR product of dsrAB from fosmid clone 194A5 was detected in the gel for
neither of the forward primers, dsrA 240F and dsrA 240F*. To test wether primer 240F is
capable of amplifying the dsrAB fosmid target sequence and to reveal a possible
selectivity in the amplification of dsrAB from the environmental samples a repetition of
the PCR is necessary.
Results
47
3.6 Real-time Quantitative PCR
Primer specificity
In order to ensure a specific amplification of the target gene we employed gel
elctrophoresis, sequencing and melting curve analysis. Gel elctrophoresis exclusively
yielded a visible band for the respective 400bp sized dsrB fragment. Furthermore a
similarity based BLAST search (Altschul et al., 1990) of the obtained clone seqeuences
against the Genbank database (www.ncbi.nlm.nih.gov/blast) did always hit the dsrB
subunit as closest relative for all analyzed sequnces. In addition, results from melting
curve analysis (Figure 21) implied a contamination free amplification of the dsrB target
fragment. All standards as well as all samples showed an identical melting melting
temperature (Tm) of approximately 86.5°C. Neither primer dimers, nor multiple products
could be detected.
Figure 21: Melting curve for the dsrB fragment amplified from the standard (blue) and the environmental
DNA (red). The first derivative of the fluorescence plotted against temperature (-Δ(RFU)/ ΔT) is plotted
against temperature resulting in a peak at the specific melting temperature (Tm).
An approximately 600bp-sized DNA fragment amplified from Wadden Sea clone
E2 containing the respective 400bp sized dsrB fragment served as template for the
generation of an appropriate standard curve. The applied dilution series sufficiently
covered the range of the environmental samples. Triplets of the high diluted standards
showed significant variations in the treshold cycle (Figure 22).
48
Results
Figure 22: Standard curve for amplification of dsrB from the standard with primers dsrA 403F and dsrB
808R for concentration from 1.7 ×107 copies × µl-1 to 1.66 ×104 copies × µl-1
Data analysis revealed remarkable variation of the copy numbers of dsrB per ml
of extracted DNA for the technical as well as the biological triplets of the respective
depth intervals (Figure 23). Final calculated copy numbers of the dsrB fragment ranged
from 106 to 107 copies per ml sediment over the vertical profile and displayed relevant
variations between each sediment depth (Figure ) Standard deviations were considerable
high, displaying an order of magnitudes of 106 copies per ml sediment. Highest copy
numbers of 1.52 to 1.82×107 were reached in the zone of the oxic-anoxic interface in 3 to
4 cm depth and in the anoxic zone of the deepest sediment layer in 16.5 to 17cm depth
(1.85×107 copies). Close to the surface of the sediment significantly lower copy numbers
of 1.11 to 1.13×107 numbers were detected.
Results
49
copy number per µl extracted DNA
5,00E+04
5×104
1,00E+05
1×105
1,50E+05
1.5×105
2,00E+05
2×105
2,50E+05
2.5×105
3.5. - 4.0
16.5 - 17.0
depth interval [cm]
1.0 - 1.5
0,00E+00
0
Figure 23: Copy number of dsrB per µl of extraced DNA for the three replicates of a certain sediment
depth (Note, the standard deviation for the technical triplets as well as the considerable variation of copy
numbers of the biological triplets for the same sediment depth.)
copy number per ml sediment
7 1,50E+0
7 2,00E+0
7 2,50E+0
7 3,00E
0,00E+0
5,00E+06 1,00E+0
0
5×10
1×10 1.5×10
2×10
2.5×10
0
6
7
7
7
7
7
-2
0
2
depth [cm]
4
6
8
10
12
14
16
18
Figure 24: Copy number of the dsrB per ml sediment over the vertical sediment profile
Literature
50
4. Discussion
Aim of the present study was to reveal the diversity of SOP in Wadden Sea
sediments based on the establishment of a phylogeny of a functional gene essential for
sulfur oxidation, namely the DsrAB. Due to the polyphyly of SOP, this gene of the
“reverse” Dsr sulfur oxidation pathway offers an adequate tool for environmental analysis
of the SOP. Moreover, the abundance of the respective organisms should be assessed by a
newly developed dsrB targeted quantitative real- time PCR approach. The results of the
completed work will be discussed as follows:
At first methods and primer issues will be evaluated. Afterwards the DsrAB and
16S rRNA phylogeny of cultured SOP will be introduced and compared with regard to
putative events of lateral gene transfer. This is followed by a discussion of the main focus
of this study, the diversity of environmental SOP in the Janssand sediment. Subsequently
the SybrGreen based quantitative real-time PCR assay for the assessment of the dsrB
copy number is debated. Finally, a synopsis in combination with an outlook will provide
a critical view of the whole dsrAB approach and moreover propose molecular
experiments for future ecological studies.
4.1 Primer evaluation and methodological aspects
4.1.1 Evaluation of dsrAB primers for diversity studies and
qRT-PCR
In the course of the study several primers were tested to optimize amplification of
dsrAB of sulfur oxidizing bacteria. Previously, only primers were available to specifically
amplify dsrAB from SRP (Wagner et al., 1998; Klein et al., 2001) but not from SOP.
Hence, there was a clear lack to apply this functional marker molecule for diversity
studies of SOP employing the “reverse” Dsr pathway and to elucidate the evolution of
SOP and SRP. The primers dsrA 240F and dsrB 808R were chosen for the DsrAB
diversity survey since best results in amplification of environmental dsrAB were
obtained. Initially conducted test PCRs yielded a lower product with the primer
combination dsrA 116F and dsrB 808R (pers. communication M. Mußmann 2005). The
primer 240F was improved after the additional dsrAB sequence of the fosmid clone
194A5 became available. The phenomenon of numerous “non-dsrAB” amplificates is
probably associated with the use of degenerated primers and has already been reported by
Klein et al. (2001) for the dsrAB of SRP. The latter is homolog to the covered dsrAB
fragment of SOP in this study. The used primers target all published sequences of
cultured and uncultured organisms. Just a very few sequences are not fully matched.
Primer dsrB 403F was designed for quantitative real-time PCR to allow the amplification
of a rather short dsr fragment. The screening of the nucleotide composition of the
environmental clone dsrAB and dsrB sequences at the primer sites dsrA 240F, dsrB 403F
and dsrB 808R indicated no selectivity of certain primer variants. Virtually most primer
sequence variants are represented in the clone libraries indicating little bias in primer
binding. Analysis of the environmental dsrAB sequences additionally revealed that the
employed primers are potentially able to cover a wide range of sequences.
Literature
51
However, unexpectedly dsrAB of some strains such as Thiocapsa marina and
Marichromatium gracile was not amplified , although the latter organism and Thiocapsa
roseopersicina were shown to contain Dsr genes by Southern hybridization (Dahl et al.,
1999). Furthermore, dsrAB from the fosmid clone 194A5 was not targeted at all by
completely matching primers. Unfortunately the selectivity test of Primer dsrA 240F
failed. The lack of amplification of dsrAB from the fosmid 194A5 by both primers, dsrA
240F and 240F*, does not allow for a conclusion on the selectivity of primer dsrA 240F.
Primer dsrA 240F* should have amplified the respective gene fragment. The lack of
product could be due to suboptimal PCR conditions, e.g. template concentration or
annealing temperature. Overall, these results indicate that primers and PCR conditions
need to be optimized further in order to cover a maximal diversity of SOP.
4.1.2 Amplification of DsrAB gene from cultivated SOP
For comprehensive reconstruction of the DsrAB phylogeny and a detailed
resolution of the affiliations of the environmental sequences dsrAB should be amplified
from a set of reference species, encompassing several lineages of SOP. Moreover, this
refined framework should enable us to clarify whether Dsr genes, in addition to
undergoing vertical transfer, have also undergone lateral transmission.
The unsuccessful amplification from most of the reference strains might result
from non-optimal PCR conditions despite a sufficient template concentration. The chosen
annealing temperature of 57.1°C appeared optimal since dsrAB was amplified
successfully from the Magnetospirillum gryphiswaldense pure culture. Although
temperature gradient PCR indicated a broad range of annealing temperature a decrease
was not suitable for amplification. Besides PCR conditions, the overall absence of DsrAB
from the respective reference strains might be the reason for amplification failure. During
this study it became obvious, that Rhodobacter sphaeroides does not possess a DsrAB
(www.img.jgi.doe.gov/cgi-bin/pub/main.cgi?page=taxonDetail&taxon_oid=400140000).
This is also likely to be the case for the tested strain Rhodobacter capsulatus. Similarly,
the genome of Thiomicrospira crunogena does not contain Dsr genes
(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=genome
and
http://www.genome.jp/kegg/pathway.html).
Although the reference strains were selected on the basis of the capability to
oxidize sulfur compounds of biochemical pathways mediating the respective process
without employing DsrAB could be prevalent. For example, the examined reference
organisms Halobacillus neapolitanus, Starkeya novella, Thiobacillus thioparus and
Thiomicrospira crunogena possess the soxB gene (Petri et al., 2001), which might
involve the absence of a DsrAB and thus explain the negative PCR results. Likewise, the
whole Thiomicrospira cluster may employ a DsrAB independent sulfur oxidation
pathway. In contrast, Chlorobium limicola, Chlorobium tepidum and Pelodictyon
phaeoclathratiforme have been shown to harbour both genes (Petri et al., 2001) (Eisen et
al., 2002) and the sulfur-oxidizing gammaproteobacterium Leucothrix mucor most likely
possesses an APS reductase (Grabovich et al., 1999) (Grabovich et al., 2002) suggesting a
possible presence of DsrAB.
52
Literature
4.2 DsrAB and 16S rRNA phylogeny of cultured SOP and
evidence for LGT
All previously available DsrAB sequences of cultured and uncultured organisms
served for the built up of the phylogenetic DsrAB backbone. The addition of
environmental clone sequences stabilized the constructed evolutionary tree.
Overall, the detailed phylogenetic relationships of DsrAB among the alpha-, beta- and
gammaproteobacterial subdivions remain unresolved as indicated by the multifurcations
in the phylogenetic trees. The unique DsrAB sequence of Thiobacillus denitrificans as the
only representative of Betaproteobacteria and to the conspicuous insertion/deletion
pattern, points at a phylogenetic distinct position. Only the insertion shared with
Alkalilimnicola ehrlichei gives evidence for a gammaproteobacterial affiliation. This is
consistent with 16S rRNA gene phylogeny (Woese, 1987) which declares a concomitant
origin of these two subdivisions.
Independent of the treeing method the chlorobi along with Magnetococcus sp.
were found to be the deepest branching SOP. Assuming that the highly conserved 16S
rRNA genes have undergone no lateral transfer and thus the 16S rRNA gene based
phylogeny reflects the true organismal phylogeny (Woese, 1987) events of lateral gene
transfer can be deduced from major inconsistencies between the DsrAB and 16S rRNA
gene tree. Based on this assumption Magnetococcus sp. possess non-orthologous DsrAB
genes since its DsrAB sequence differs from the Magnetospirillum-like sequences that
group with other Proteobacteria. Consistently the putative xenologous DsrAB sequence
of Magnetococcus sp. shares a unique deletion in the alpha subunit typical for all
members of the chlorobi, which can be considered as additional signpost for the separated
phylogenetic position. Thus the phylogenetic affiliation of the non-orthologous
Magnetococcus sp. DsrAB with the presumably orthologous DsrAB of the chlorobi most
likely reflects lateral gene transfer. Analysis of the genomic GC content with regard to
variability towards the dsrAB GC content (Lawrence and Ochman, 1997) could proof this
hypothesis.
The presence of DsrAB in both sulfate-reducing and sulfur-oxidizing organisms
raises the question of its evolutionary origin and the progenitor of Dsr involved in
catalysis of sulphur-oxidation or sulfate reduction. The DsrAB harbouring ancestor of
SOP and SRP was consistently placed between the chlorobi and the deep-branching
sulfate reducers, such as Archaeoglobus fulgidus, Thermodesulfovibrio islandicus and
members of the Firmicutes. Insertion/deletion-patterns shared between all SOP and some
deep-branching SRP (here Thermodesulfovibrio islandicus, Desulfitobacterium
hafniense, Archaeoglobus fulgidus and environmental fosmid clone 39F2) give additional
evidence for the deduced phylogenetic relationship. Moreover, a synopsis of different
treeing methods suggested Archaeoglobus sp. and the chlorobi as the closest relatives of
both functional groups of prokaryotes. With regard to 16S rRNA gene phylogeny
(Woese, 1987) these findings might hint at a lateral transfer of DsrAB genes from
Archaeoglobus fulgidus to the ancestor of the chlorobi since Chlorobium tepidum is not
deeply branching within the bacterial 16S rRNA tree.
To date, the sulfate reducing archaeon Archaeoglobus fulgidus is the oldest
known organism possessing a Dsr but is supposed to have acquired its Dsr genes laterally
from a bacterial donor (Klein et al., 2001). However, the postulated bacterial origin can
not be taken for granted since the employed paralogous rooting method is controversial
Literature
53
and additional evidence from the genome of Archaeoglobus fulgidus identifying the Dsr
genes as clear xenologs is lacking. Here, a paralogous rooting approach of the DsrA and
B subunit of SOP would be worthwhile to approve the deep branching relationships of
SOP. Eisen et al. (2002) postulated lateral gene transfer of archaeal genes to the
thermophilic Chlorobium tepidum based on the findings that many genes resembled those
of the also thermophilic Archaeoglobus fulgidus. Interestingly, the sulfur oxidation genes
of Chlorobium tepidum are more similar to homologous genes in Archaeoglobus fulgidus
than to genes in the sulfur-oxidizing archaea Sulfolobus sp.. In addition, a recent analysis
of the phylogeny and alignment patterns of membrane proteins mediating Dsr dependent
sulfate reduction suggest an affiliation with both Chlorobium tepidum as well as
Archaeoglobus fulgidus (Mussmann et al., 2005). A thermophilic lifestyle of organisms
involved in LGT has already been reported from other studies (Friedrich, 2002). Further
tree topology suggests, after a putative later gene transfer event from Archaeoglobus
fulgidus to the chlorobi, a subsequent transfer of DsrAB to the proteobacterial lineages
involving vertical as well as horizontal tansmission. Indeed, Sabehi et al. (2005) recently
found an uncultured proteorhodopsin-containing bacterium (BAC) to possess a
gammaproteobacterial reverse sulfite reductase operon. In contrast, the proteorhodopsin
sequence and data from other genes supported an alphaproteobacterial affiliation. Hence,
these findings suggest an additional lateral transfer of DsrAB between the alpha- and
gammaproteobacterial subdivisions. However, such putative events within the
Proteobacteria were not detected in our study. This can probably be addressed to the
limited availability of reference sequences.
Overall, our findings yield additional evidence for the close relatedness of the
DsrAB genes involved in either sulfate reduction or sulfur oxidation and their putative
lateral transfer between these functional groups.
S0
H2S/FeS2
SO42-
Figure 25: Limited microbial sulfur cycle before 2.45 billion years (Ga). Partial sulfide oxidation and
sulfur reduction, which are commonly considered to be ancient metabolic processes were present in
addition to localized sulfate reduction. Processes that were possible after oxygen became available are
indicated by red arrows. (modified after Blank, 2004)
One key for understanding the evolutionary origin of Dsr is the early sulfur cycle
(Figure 25). Anaerobic sulfur oxidation is thought to be one of the earliest metabolic
pathways used for energy conservation by ancient prokaryotes. The assumed ancient
chloroflexi have not been shown to possess a DsrAB. In contrast, all recent anoxygenic
phototrophic SOP containing DsrAB group within evolutionary younger phyla.
Regardless of the 16S rRNA gene-based phylogeny comparative sequence
analysis of photosynthesis genes gave evidence for Chlorobium tepidum and
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Chloroflexus aurantiacus to be each others closest relatives (Xiong et al., 2000). Thus,
Chlorobium may be more ancient than previously thought and might be a potential
candidate for an ancestral DsrAB. However, a lateral gene transfer of photosynthesis
genes was suggested for this conclusion and may weaken the argumentation (Xiong et al.,
2000; Igarashi et al., 2001). The ‘Wächtershäuser hypothesis’ postulates the formation of
pyrite and hydrogen from ferrous iron sulfide and hydrogen sulfide as ancient energy
yielding reaction for chemolithoautotroph microbial metabolism (Wachtershauser, 1990;
Brock et al., 2006). Moreover sulfur oxidation dependent reduction of ferric iron might
be a primordial, energy yielding process. However, microbial iron sulfide and pyrite
oxidation coupled to electron acceptors other than O2 or NO3-, like Mn(IV) or Fe(III)
could not been detected (Schippers and Jorgensen, 2001, 2002; Blank, 2004) so far. To
trace back the evolutionary pathway of DsrAB the reconstruction of the SOP and SRP
phylogeny should bee seen in context with geological record. Records from sulfur
isotopic fractionation indicate that large scale bacterial sulfate reduction began in marine
environment not until 2.35 billion years ago (Ga) (Cameron, 1982) followed by or nearly
concurrent with rapid oxygenation of the atmosphere about 2.2 Ga (Feng et al., 2000).
Consistent with the availability of oxygen in the biosphere was the diversification of
aerobic sulfide and pyrite oxidizing organism (Blank, 2004). Thus it seems likely that the
Dsr possessing progenitor has to be further searched among either anaerobic sulfide
oxidizers or very early sulfate reducers.
4.3 Diversity of SOP in WS sediment
4.3.1 Overall SOP diversity
One major aim of the study was to assess the diversity of SOP in the Wadden Sea
sediment based on comparative sequence analysis of a key enzyme involved in
biochemical sulfur-oxidation, namely the DsrAB.
Overall, the presented phylogenetic reconstruction of the DsrAB was based on
numerous almost full-length environmental sequences and only on a few sequences of
cultured SOP. The resulting low resolution of the DsrAB tree in comparison to the 16S
rRNA phylogeny limits the conclusiveness of our diversity analysis. For example, the
phototrophic sulfur oxidizer Allochromatium vinosum was found to be the closest
cultured relative of the vast majority of gammaproteobacterial-related sequences.
Furthermore, the putative alphaproteobacterial Wadden Sea clone cluster E affiliated with
Magnetospirillum species, which are currently the only representatives of the
Alphaproteobacteria. This phenomenon can probably be addressed to a lack of DsrAB
sequence data, rather than to close phylogenetic relatedness.
Rarefaction analysis of 112 clones suggests that the actual diversity in our clone
libraries was not completely covered. However the major groups discovered were
identical with those detected in 16S rRNA gene libraries by screening of 543 clones
(Arnds, 2006). Moreover, the analysis was based on the identification of dsrA nucleotide
sequences. Since the genetic code is degenerated unique sequence types on DNA level do
not necessarily result in distinct amino acid sequences. In addition, the high degree of
sequence identity within the clone clusters A, B, D and E suggests the existence of
identical clones which could not be discriminated due to slight sequencing errors. The
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55
close relatedness of DsrAB within each Wadden Sea cluster along with the high sequence
identity suggests more limited species diversity.
Comparative amino acid sequence analysis of the DsrAB revealed a dominance of
gammaproteobacterial sequences among all three clone libraries. In addition, sequences
were identified that can not clearly be assigned to a specific proteobacterial group using
Neighbor Joining, Maximum Parsimony and Maximum Likelihood but the Bayesian
inference method supported an alphaproteobacterial affiliation. These findings are in
good accordance with previous results. 16S rRNA gene based studies (Arnds, 2006) from
the same site already reported the presence of Gammaproteobacteria which affiliated
predominantly with sulfur-oxidizing endosymbionts and some free-living SOP. Arnds
(2006) found numerous 16S rRNA sequences that clustered with the sulfur-oxidizing
gamma 1 (93 – 96% identity) and gamma 2 (96 – 98% identity) symbiont of Olavius
algarvensis. Environmental sequences clustering with the sulfur oxidizing gamma
symbionts of Olavius algarvensis were also detected in the dsrAB clone libraries.
Respectively one clone closely affiliated with the gamma 1 and gamma 2 symbiont.
Additionally, 11 sequences, forming the Wadden sea clone cluster D, grouped with the
gamma 2 symbiont. However, the detected organisms more likely represent permanent
free-living Gammaproteobacteria forming a distinct phylogenetic subgroup together with
the endosymbionts. Unfortunately, the phylogenetic position of the Wadden Sea clone
clusters A and B is of only minor resolution. Here, the phototrophic Allochromatium
vinosum represents the closest cultured relative. Since the prevalence of a
chemolithotrophic lifestyle among sedimentary sulfide oxidizers is more likely than a
phototrophic one, further analysis of pure cultures is necessary to uncover the identity of
the respective organisms.
As supported by tree topology the putative alphaproteobacterial sequences
distantly affiliated with Magnetospirillum sp.. Magnetotactic bacteria are ubiquitous in
marine sediments and their occurrence appears to be dependent on the presence of
opposing gradients of oxidized and reduced compounds, such as oxygen and reduced
sulfur species (Spring and Bazylinsky, 1999-2006). However, members of the genus
Magnetospirillum have only been isolated from freshwater habitats so far (Spring and
Bazylinsky, 1999-2006). In addition, Arnds (2006) did not detect Magnetosprillum-like
sequences. The respective alphaproteobacterial sequences instead clustered with
Roseobacter species and were also frequently detected by FISH. Indeed, members of the
Rosebacter clade harbour the ability to drive on the oxidation of inorganic sulfur
compounds (Sorokin, 1995; Buchan et al., 2005). It can be speculated whether
alphaproteobacterial sequences found in the dsrAB clone libraries belong to Rosebacterlike organisms.
The absence of Betaproteobacteria- and chlorobi-related sequences again is
consistent with findings from 16S rRNA data from the same sampling site (Arnds, 2006).
Similarly, Llobet-Brossa et al. (1998) and Ishii et. al.(2004) reported a minor contribution
of Betaproteobacteria to the bacterial community of the Wadden Sea sediments.
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4.3.2 Vertical SOP diversity
In order to reveal potential stratifications of the SOP community over the vertical
sediment profile three clone libraries from different depths were established. Generally, it
is not possible to draw any conclusion about relative in situ abundance from clone
frequencies due to e.g. PCR bias. However, the sequence diversity of DsrAB from
Janssand sediments points at some differences in the vertical distribution of certain
phylotypes. Particularly in the Wadden Sea clone clusters A and B and less pronounced
in the cluster C some sequence subclusters mainly originate either from the oxic/suboxic
surface layers or from anoxic layers below 8 cm depths. The clustering of sequence types
obtained from the surface samples (within Wadden Sea clone clusters A, D, and E) points
at organisms which are capable to use oxygen or alternatively even nitrate as electron
acceptor in sulfur oxidation.
The affiliation of phylotypes from the deeper anoxic sediment layers with each
other (clusters A and D) may indicate sulfur-oxidizers exhibiting an anaerobic life style
such as fermentation or iron or manganese dependent sulfide oxidation. Hence, our
DsrAB based diversity study may reflect the prevalence of different lifestyles as a
consequence of adaptation to different environmental conditions and subsequent niche
differentiation. Nevertheless, the detection of DsrAB sequences in all depths does not
necessarily mean that biological sulfur oxidation proceeds throughout the sediment.
Further analysis has to prove the actual activity of the respective organisms.
The occurrence of Alpha- and Gammaproteobacteria-related sequences
throughout the vertical sediment profile is consistent with previous findings of the same
site based on FISH. Ishii et al. (2004) observed Gammaproteobacteria throughout a depth
of 40 cm. In contrast, Alphaproteobacteria were only found in the upper 10 cm. Arnds
(2006) detected numerous Alpha- and Gammaproteobateria, including potential sulfuroxidizing, up to a sampled depth of 5.5 cm.
In the intertidal sulfur pools the “reverse” Dsr pathway apparently contributes less
to sulfur oxidation, since dsrAB could not be amplified in contrast to 16S rDNA (Arnds,
2006). The minor significance of biological sulfur oxidation by Gammaproteobacteriarelated organisms in these pools is corroborated by the low abundances of
Gammaproteobacteria and lacking 16S rDNA sequences related to known SOP (Arnds,
2006). In contrast, Roseobacter accounted for 3% of all cells in these pools. Accordingly,
amplification of dsrAB should have yielded a product from the sulfur pool if the putative
alphaproteobacterial sequences of our clone libraries indeed represent Roseobacter
relatives. Overall, sulfur oxidation in the intertidal sulfur pool may proceed
predominantly chemically rather than biologically.
In conclusion, our findings are consistent with previous studies based on
comparative 16S rRNA analysis indicating that potential sulfur-oxidizing
Gammaproteobacteria are frequent in marine sediments. It can be proposed that these
organisms are mainly responsible for sulfur oxidation at Janssand site. Especially the
organisms previously targeted by the FISH probe Gam660 (Arnds, 2006) are candidates
for carriers of DsrAB. However this needs to be proven further in future research
projects.
Since it could be demonstrated that LGT is involved in the evolution of SOP the
phylogenetic inferences in the present environmental diversity study may be interpreted
cautiously.
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4.4 Quantitative Real-Time PCR
The community ecology of sulfur oxidizers is important for understanding the
relevance of biological sulfur oxidation in the natural habitat. Due to the high
physiological and phylogenetic diversity approaches based on 16S rRNA such as FISH
does only allow the quantification of distinct organisms potentially oxidizing sulfur
compounds. In order to fill this gap a quantitative real-time PCR assay for the dsrB from
SOP was developed to get a first impression of the abundance of microorganism applying
the “reverse” Dsr pathway over the vertical sediment profile. Quantitative real-time PCR
is a semiquantitaive method since organismal abundances, in terms of total cell numbers
are not directly measured by cell counts like in microscopy based methods, e.g. FISH.
Cell abundances are rather deduced from gene copy number. This, of course, is sensitive
for biases, leading to a possible overestimation of cell number. An increase in dsrB copy
number in relation to cell number could result from growing cells, possessing more than
one copy in their genome or multiple replication of the respective gene. Indeed, a
possible replication of the Dsr genes within the genomes of certain SOP could complicate
the interpretation of the qRT-PCR data. Genomic analysis revealed that SRP (Klein et al.,
2001) and the majority of here examined SOP (www.img.jgi.doe.gov/cgibin/pub/main.cgi?page=findGenomes) harbors only a single copy of the dsrAB genes
apart from Chlorobium tepidum possesing two identical dsrAB copies (Eisen et al., 2002).
A particular position takes Thiobacillus denitrificans, harboring three putative copies of
the dsrA (Beller et al., 2006) of which probably one is functional. Hence, besides method
intrinsic biases, the deduction of cell numbers from copy number of dsrA remain critical.
Nevertheless, numerous examples have shown the value of qPCR in environmental
microbiology (Suzuki et al., 2000; Hermansson and Lindgren, 2001; Stubner, 2002; Kolb
et al., 2003).
The established qRT-PCR assay was checked for replicability and specificity.
Sequencing and further PCR based tests verified that fluorescence increase was due to
specific PCR products. However, despite the indicated high specificity of the applied
qPCR primers it should be considered that the general primer bias could result in an
underestimation of copy number. Since amplification proceeded equally for standards and
environmental DNA, as indicated by a similar slope of amplification curves, a high
efficiency of the PCR is assumed. Remarkable are the high standard deviations which
most likely result from the low amount of applied DNA. An increased amount of DNA as
PCR template could minimize pipetting errors and further decrease the threshold cycle
which will probably ensure higher detection sensitivity. For this at least the ten-fold
amount of applied sediment should be used. However, higher amounts of sediment for
DNA extraction bear the risk to introduce PCR inhibitors, e.g. salts, polysaccharides,
humic acids, which will influence the amplification efficiency.
Copy numbers of dsrB determined by qRT-PCR analysis were in the lower range
of FISH detection limit for cell counts of potential sulfur oxidizing Alpha- and
Gammaproteobacteria. Arnds (2006) reported cell numbers of 107 to 108ml-1 for the
respective organisms. Hence, the obtained maximum dsrB copy numbers are in the same
order of magnitude. Theoretically dsrB copy numbers should be as high as cell numbers
since the majority of SOP possess one copy per cell. However, the low DNA template
volumes in combination with a lyses efficiency probably below 100% and general primer
bias might be an explanation for a potential underestimation.
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Besides overall coverage of the dsrB copy number, one aim was the assessment of
its distribution over the vertical sediment profile. The high standard deviation does not
allow a conclusion on the vertical stratification of SOP. However, highest copy numbers
were detected in the oxic – anoxic transition zone. These findings are in good match with
previous studies. Arnds (2006) also found maxima for Gammaproteobacteria (9.6 × 108
cells ml-1) and Roseobacter (2.1× 108ml-1) in the sub-oxic transition zone of sulfide and
oxygen. Maximum total cell numbers and microbial sulfate reduction rates in this zone
have already been reported by (Böttcher et al., 2000).
Thus, aerobic SOP extensively consuming available sulfide and oxygen seem to
be abundant. Moreover, the detection of dsrB sequences in anoxic sediment layers
suggests the presence of so far unidentified organisms capable to drive on sulfur
compound oxidation under anaerobic conditions. Based on comparative analysis of
DsrAB or 16S rRNA (Arnds, 2006) none of the clones affiliated with the known
denitrifying species Beggiatoa sp., Thiobacillus denitrificans or Thiomicrospira
dentrificans. SOP using manganese or possibly iron as potential electron acceptors have
not been identified yet (Schippers and Jorgensen, 2001, 2002). Unfortunately, the dsrB
gene based approach does not give any information whether the respective organisms
actively catalyze sulfur oxidation thus further analysis has to reveal this.
In summary, as a first attempt to use dsrB as a quantitative marker for qRT-PCR
the obtained results are of value. To date there is no publication attempting to quantify
sulfur oxidizers independently of their phylogeny. The data suggest that the the “reverse”
Dsr pathway may be very important for biochemical sulfur oxidation in Janssand
sediments.
4.5 General value and limitations of DsrAB gene as
molecular marker for SOP diversity studies and qRTPCR
In the context of this work the value of DsrAB as molecular marker for
environmental diversity studies is limited by the scarce number of sequences from
cultured representatives of SOP. The resulting limited overall resolution of the DsrAB
phylogeny allows only constricted information about the detailed evolutionary
relationships and the environmental diversity of SOP. Thus, additional DsrAB sequences
from known sulfur oxidizers have to be added to the phylogenetic DsrAB backbone to
better resolve the evolutionary relationships in future diversity studies. Complementation
of the phylogenetic framework of the DsrAB gene marker will allow the to link
environmental sequences to recognized lineages of SOP.
If there indeed exists a great variety of metabolic pathways facilitating biological
sulfur oxidation without relying on DsrAB, then comparative sequence analysis of the
respective enzyme will cover only part of the natural microbial diversity. Since there are
obviously several biochemical pathways of sulfur oxidation the DsrAB possessing
organisms only represent a fraction of sulfur oxidizers in a given habitat. However, the
“reverse” Dsr pathway is probably more prevalent in the environment than the sox
pathway (pers. communication C. Dahl 2005) and thus further investment is worthwhile.
Of course, alternative pathways also need to be studied to reveal which one is most
important in marine sediments.
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Nevertheless, the problem of the applicability of a functional gene as molecular
marker also holds for other enzymes involved in dissimilatory sulfur metabolism. For
example the flavoprotein sulfide:quinone oxidoreductase (SQR), catalyzing the oxidation
of sulfide to sulfur, is widely distributed among bacteria (Theissen et al., 2003).
However, the poor sequence conservation (average amino acid sequence identity 20%)
and proposed lateral transfer of SQR genes among prokaryotes here again pose a
challenge to the use as molecular marker in phylogenetic analysis. In contrast, the level of
sequence conservation of the soxB gene, present in Alpha-, Beta- and
Gammaproteobacteria as well as Green sulfur bacteria, seems sufficient (amino acid
sequence identity 41 – 98%) but the putative involvement of LGT also holds for this
functional gene (Petri et al., 2001).
In view of the variation of biochemical sulfur oxidation pathways and the wide
distribution of different sulfur oxidizing enzymes throughout the microbial world
(Brune, 1995) it is not likely that an universal molecular marker will be available for all
sulfur oxidizing prokaryotes. Moreover the coexistence of different functional genes in
several SOP complicates a combination of approaches aimed at different molecular
markers.
4.6 Outlook
Overall, the congruence of the present results with 16S rRNA data encourages to
further use dsrAB as molecular marker in environmental diversity studies of SOP.
Therefore, the phylogenetic backbone of DsrAB has to be complemented. Primer
optimization might be necessary for the retrieval of additional DsrAB sequences data
from cultured sulfur oxidizers. In the future, additional genome sequences may be
available. Alternatively, southern blotting of the dsrAB fragment (Dahl et al., 1999)
combined with subcloning and sequencing could yield the respective sequences from
selected reference strains. Based on both enhanced primers can be designed.
Since the comparative sequences analysis of DsrAB gives no information about
the identity of uncultured environmental SOP, the direct linkage of a functional gene to a
16S rRNA phylotype is one of the main future goals. Here, application of fluorescence
associated cell sorting (FACS) (Sekar et al., 2004) of certain subgroups, e.g. hybridized
with 16S rRNA FISH probes GAM 660 or ROS 537, and subsequent amplification of
dsrAB from the sorted cell fractions is a promising approach. Moreover, screening of
dsrAB in metagenome libraries combined with screening for 16S rRNA genes and
subsequent analysis of tetranucleotide patterns (Teeling et al., 2004)could unravel the
identity of uncultured environmental sulfur oxidizers. Finally, the design of group
specific probes could elucidate the community structure of sulfur oxidizers and
furthermore allow an evaluation of the role of specific groups by quantitative FISH
(Moraru, 2006).
In future studies the expression of dsrAB may be tracked by determination of dsrB
mRNA copy number under distinct environmental conditions. Therefore, the application
of reverse transcription PCR prior to quantitative-real time PCR constitutes a promising
method (Neretin et al., 2003). In addition, immunocytochemistry using antibodies raised
against DsrAB could overcome the expression problem of DNA and mRNA targeted
assays (Pernthaler and Amann, 2004) and detect the presence of DsrAB in situ.
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The application of the newly developed dsrB targeted qRT-PCR assay for the first time
enabled quantitative assessment of a specific physiological group of SOP. The partial
congruence with CARD-FISH and clone library data (Arnds, 2006) encourages for
further surveys. Since the relatively lower sensitivity of the SybrGreen compared to the
TaqMan assay has been reported (Stubner, 2002) the application of a probe-based
approach should be considered.
To confirm the relevance of the “reverse” Dsr pathway in Wadden Sea sediments,
the role of competing pathways such as the “sox PSO pathway” has to be explored by e.g.
soxB targeted qRT-PCR. Overall, the improvement of the current protocol is a promising
approach for the quantitative detection of SOP in sediments of the Wadden Sea and other
environments.
Literature
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Appendix
66
(D) Appendix
Allochromatium vinosum
Fosmid 194A5
Alkalilimnicola ehrlichei
Environmental sequence IBEA_CTG_1982486
Proteorhodopsin clone MED13K9
Magnetospirillum magnetotacticum
Magnetospirillum magneticum
Magnetococcus sp. MC-1
Chlorobium limicola
Prosthecochloris aestuarii
Prosthecochloris vibrioformis
Chlorobium tepidum
Chlorobium phaeobacteroides EAM34529
Chlorobium phaeobacteroides EAM62784
Pelodictyon phaeoclathratiforme
Thiobacillus denitrificans AAZ98438
Thiobacillus denitrificans AAZ97262
Thiobacillus denitrificans AAZ97322
Olavius algarvensis Gamma2 symbiont
Olavius algarvensis Gamma1 symbiont
116f
Allochromatium vinosum
Fosmid 194A5
Alkalilimnicola ehrlichei
Environmental sequence IBEA_CTG_1982486
Proteorhodopsin clone MED13K9
Magnetospirillum magnetotacticum
Magnetospirillum magneticum
Magnetococcus sp. MC-1
Chlorobium limicola
Prosthecochloris aestuarii
Prosthecochloris vibrioformis
Chlorobium tepidum
Chlorobium phaeobacteroides EAM34529
Chlorobium phaeobacteroides EAM62784
Pelodictyon phaeoclathratiforme
Thiobacillus denitrificans AAZ98438
Thiobacillus denitrificans AAZ97262
Thiobacillus denitrificans AAZ97322
Olavius algarvensis Gamma2 symbiont
Olavius algarvensis Gamma1 symbiont
240
CTCGAGACCGGTCCCTGGCCCAGCTTCATCTCCGGCATCAAGCGTCTGCG
CTGGAAGAAGGCCCCTGGCCGAGCTTCATCAGCGGTATCAAGCGTCTGCG
CTGGAAAGCGGCCCCTGGCCCAGTTTTGTCACCGGCCTGAAGCACCTGGC
CTAGAAACTGGTCCATGGCCTAGTTTCATTAGCGGAATTAAAAAACTCAG
CTTGAGAATGGCCCATGGCCAAGCTTCATAAGCGGCATTAAAAGACTCAG
TTGGAAAGCGGTCCCTGGCCCAGCTTCGTGACCGGCTTGAAGCGTCTGGC
CTGGAAAGCGGTCCCTGGCCCAGCTTCGTGACGGGCCTGAAGCGTCTGGC
CTCGAAGAGGGACCCTGGCCCAGTTTCATCTCCGGGTTCAAAGAACTCTA
CTGGAAAGCGGCCCCTGGCCAAGCTTCGTTTCCGGCTTCAAGGAACTTGC
CTTGAAAGCGGCCCCTGGCCGAGCTTCGTATCCGGTTTCAAGGAGATGGC
CTGGAAAAAGGCCCCTGGCCAAGTTTTGTCTCTGGCTTCAAGACTCTTGC
CTTGAAAGCGGTCCGTGGCCCAGCTTCATTTCGGGCTTCAAGGCGCTTGC
CTGGAAAAAGGCCCCTGGCCGAGTTTTATTTCCGGTTTCAAAGAGCTTGC
CTTGAAAGCGGCCCCTGGCCGAGTTTTGTGACCGGTCTGAAGGACCTTGC
TTGGAAAAAGGTCCTTGGCCGAGTTTTATCAGCGGTTTCAAGGAACTTGC
CTCGAGCGCGGACCCTGGCCCAGCTTCGTCACCGGGCTCAAGCGTCTGGC
CTCGAGAAAGGCCCGTGGCCCAGCTTCGTGACCGGGCTGAAGCGTTTGGC
CTCGAGAAAGGGCCTTGGCCCAGCTTCGTGACCGGCCTCAAGCGCCTCGC
CTCGAGAACGGTCCCTGGCCAAGCTTTATTTCAGGGATCAAGCGTTTACG
TTGGAAACCGGCCCCTGGCCGAGTTTCATTTCCGGTATCAAGCGTCTGCG
GGNCCNTGGCCBAGYTT-----------------------AGACCCGCAAGGGCTACTGGAAGGGCGGTACCGTGTCGGTGTT
AGACCCGCAAGGGTTACTGGAAAGGAGGCACCGTCTCTGTCTA
AGGACCGCAAGGGTTACTGGAAGGGCGGCACCGTCAGTGTCTA
AAACCCGCAAAGGATATTGGAAGGGTGGCACTGTCAGTGTTTA
AAACACGTAAAGGGTATTGGAAAGGCGGCACAGTGAGTGTATA
AGACCCGCAAGGGCTATTGGAAGGGCGGCACGGTGGGCGTGTT
AGACCCGCAAGGGCTATTGGAAGGGCGGCACGGTGGGCGTGTT
AAACCCGCATGGGTTACTGGAAAGGTGGCGTGGTAGGTGTACA
ACACGAAAATGGGGTACTGGAAGGGCGGTCTTGTTACGGTTGA
ACACCAAAATGGGCTACTGGAAAGGCGGTCTGGTGACTGTTGA
ATACCAAGATGGGTTACTGGAAGGGTGGGCTTGTGACGGTTGA
AGACCAAGATGGGCTACTGGAAAGGCGGTCTGGTCACGGTAGA
ATACCAAGATGGGGTATTGGAAGGGCGGTCTGGTAACGGTGGA
AGACAAGGATGGGGTATTGGAAGGGCGGTCTTGTTACCGTGAA
ACACCAAGATGGGCTACTGGAAAGGCGGTCTTGTTACTGTTGA
AGACCAAGACCGGCTACTGGAAAGGCGGCACGGTCGGGGTGTT
AGACCAAGTTCGGCTACTGGAAGGGCGGCACGGTCGGCGTGTT
AGACCAAGAAAGGCTATTGGAAGGGCGGCACGGTCGGCGTGTT
AGACCCGTAAGGGTTACTGGAAAGGCGGCACCGTCAGCGTCTA
AGACCCGCAAGGGGTATTGGAAGGGTGGCACCGTTTCGGTGTA
GGNTAYTGGAARGGYGG---------------
Figure A1: Sequence alignments of primer binding sites for dsrA
Appendix
Allochromatium vinosum
Fosmid 194A5
Alkalilimnicola ehrlichei
Environmental sequence IBEA_ CTG_2027414
Environmental sequence IBEA_CTG_1982486
Environmental sequence IBEA_CTG_2018072
Proteorhodopsin clone MED13K9
Magnetospirillum magnetotacticum
Magnetospirillum magneticum
Magnetococcus sp. MC-1
Chlorobium limicola
Prosthecochloris aestuarii
Prosthecochloris vibrioformis
Chlorobium phaeobacteroides
Chlorobium tepidum
Chlorobium phaeobacteroides
Pelodictyon phaeoclathratiforme
Thiobacillus denitrificans
Olavius algarvensis Gamma2 symbiont
Olavius algarvensis Gamma1 symbiont
404_for
Allochromatium vinosum
Fosmid 194A5
Alkalilimnicola ehrlichei
Environmental sequence IBEA_ CTG_2027414
Environmental sequence IBEA_CTG_1982486
Environmental sequence IBEA_CTG_2018072
Proteorhodopsin clone MED13K9
Magnetospirillum magnetotacticum
Magnetospirillum magneticum
Magnetococcus sp. MC-1
Chlorobium limicola
Prosthecochloris aestuarii
Prosthecochloris vibrioformis
Chlorobium phaeobacteroides
Chlorobium tepidum
Chlorobium phaeobacteroides
Pelodictyon phaeoclathratiforme
Thiobacillus denitrificans
Olavius algarvensis Gamma2 symbiont
Olavius algarvensis Gamma1 symbiont
dsr_808_rev (Target sequence)
CATGATCTCGCACACCCAGGGCTGGCTGCACTGCGACATCCCG
CATGATCTCGCACACCCAGGGCTGGCTGCATTGCGACATCCCC
CATGATCAGCCACACCCAGGGCTGGCTGCACTGTGACATCCCA
TATGATTTCTCATACACAGGGCTGGTTACACTGTGATATACCA
AATGATTTCACATACTCAAGGTTGGTTACATTGTGATATACCC
TATGATTTCTCACACCCAAGGTTGGCTGCATTGTGATATTCCT
AATGATTTCACATACACAAGGATGGTTACATTGTGATATCCCA
CTTCATCAGCCATACCCAGGGCTGGCTGCACTGCGACATCCCC
CTTCATCAGCCATACCCAGGGCTGGCTGCACTGCGACATCCCC
CTCCATGGCCCACACCCAGGGCTGGTTGCACTGTGACATTCCT
TTCGGTATCGCACACGCAGGGATGGCTGCACTGCGATATTCCC
CTCGGTCTCCCATACCCAGGGTTGGCTCCATTGCGATATTCCT
GTCGGTATCGCACACCCAGGGGTGGCTCCATTGCGACATTCCC
ACCGATAGCGCATACGCAGGGATGGCTCCACTGCGATATTCCG
AGCCGTTTCGCACACCCAGGGCTGGCTGCACTGCGACATTCCG
CTCGGTGTCGCATACGCAGGGTTGGCTTCATTGCGATATTCCT
TTCTGTTTCTCATACACAAGGTTGGCTTCATTGCGATATTCCG
GATGATCGCCCACACCCAGGGCTGGCTGCACTGCGACATCCCC
GATGATATCGCACACCCAGGGCTGGCTGCACTGCGACATTCCG
CATGATCTCCCATACCCAGGGCTGGTTGCACTGCGACATCCCG
CAYACNCARGGNTGGYT---------------CGCCGA-TGCAGATCAACGACGCCGAGCACTCCAAGCTGGCCATCTGGGTCGGCGGCAACCACTCCAACGCGCGCGGCACGCCGA-TGCAGATCAACGACGCCGAACACACCAAGCTCGCCATCTGGGTGGGCGGTAACCACTCCAATGCACGTGGCACCCCCA-TGGAGATCAACGACCCGGAGCACTCCAAACTGGCGATCTGGATCGGGGGCAACCACTCCAACGCCCGTGGCGCTCCAA-TGCAAATCAATGACCCTGAGCACAGTAAACTTGCTATTTGGGTTGGTGGTAATCACTCTAATGCAAGAGGCACTCCTA-TGCAGATCAATGACCCCGAACATAGTAAATTAGCTATATGGGTTGGCGGCAACCACTCGAATGCGAGAAGCACTCCAA-TGCAAATCAATGATGCCGAACATACAAAACTTTCAATCTGGGTCGGTGGTAATCATTCAAATGCGAGAGGAACACCTA-TGCAAATTAATGATCCAGAGCATAGTAAGCTTGCAATATGGGTAGGTGGAAACCACTCAAATGCAAGGAGTACGCCCA-TGCAGATCAACGATCCCATCCATTCCAAGATCGCCATCTGGGTGGGCGGCAAGCATTCCTCTACCCGGTCCACGCCCA-TGCAGATCAACGACCCGGTCCATTCCAAGATCGCCATCTGGGTGGGCGGCAAGCACTCCTCCACCCGTTCCACTGCGA-TGGAGATCAACCATCCTGAGTACTCCAAACTGGCTATCTGGATCGGTGGTAAGAACGCCAACACCCGTTCCCCCGCCA-TGGAGATCAACCATCCCGAGCACTCCAAGTTTGCGATCTGGGTCGGCGGCAAGAACAGCAACGCCCGTTCCACTGCAA-TGGAGATCAACCATCCCGAGCATTCAAAATTCGCTATCTGGGTAGGAGGCAAGAACAGTAATGCCCGATCCACGGCAA-TGGAAATAAATCATCCCGAGCACTCCAAGTTTGCAATCTGGGTTGGCGGAAAGAACAGCAACGCCCGCTCAACTTCGA-TGGAGATCAACCATCCTGAATACTCCAGGTTTGCTATCTGGGTTGCCGGCAAGAACAGTAATGCCCGTTCAACGGCCA-TGGAGATCAACCACCCCGAGCACTCCAAGTTTGCCGTCTGGGTCGGCGGCAAAAACAGCAACGCCCGCTCGACTGCCA-TGGAGATCAACCATCCGGAACACTCAAAGTTTGCCATATGGGTAGGCGGCAAAAACAGCAATGCCCGTTCAACTTCCA-TGGAGATCAACCACCCGGAACATTCCAAATTTGCAATCTGGGTCGGCGGGAAAAACAGCAATGCCCGATCAACGCCGA-TGCAGATCAACGATCCCGAGCACTCGAAGATCGCGATCTGGGTTGGCGGCAAA----AACTCGAA-------CGCCGA-TGCAAATCAACGACCCCGAACATACCAAGCTAGCTATCTGGGTCGGCGGTAACCACTCCAACGCGCGCGGCACGCCCA-TGCAGATCAACGATGCCGAGCATTCCAAGCTGGCCATCTGGGTCGGCGGTAACCACTCCAACGCCCGCGGCA------------------------------------GCNATHTGGGTNGGHGG
Figure A2: Sequence alignments of of primer binding sites for dsrB
67
Appendix
68
Table A1: Sequence similarities1 of amino acid (lower triangle) and nucleotide sequences (upper triangle) of the DsrAB
Reference species
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
1
Beggiatoa (uncultured)
M. magnetotacticum
M. magneticum
T. denitrificans
O. algarvensis gam 2
O. algarvensis gam 1
A. vinosum
A. ehrlichei
C. phaeobacteroides DSM266
C. limicola
P. aestuarii
P. vibrioformis
P. phaeoclathratiforme
C. phaeobacteroides BS1
C. tepidum
M. spec.
M. gryphiswaldense
R. pachyptila symbiont
Sequence similarity [ % ]
1
2
3
4
25.3
24.6
23.9
68.4
92.8
32.3
67.4
96.4
33.1
56.8
64.5
64.0
77.9
66.0
65.7
63.4
68.8
69.3
67.2
79.8
67.1
68.0
65.4
74.7
69.0
69.6
66.9
50.0
56.7
57.1
55.2
45.7
56.4
56.4
54.5
52.1
57.1
57.2
55.3
50.0
58.1
57.7
54.7
48.9
57.1
57.1
54.8
52.1
55.6
56.3
55.1
54.3
55.4
55.5
54.2
39.8
57.7
58.3
54.2
65.3
87.5
88.7
63.9
85.0
63.4
63.8
55.6
5
23.9
50.0
51.1
32.2
84.6
80.6
73.3
56.2
55.3
57.8
56.3
56.6
55.6
57.5
54.5
66.4
63.6
6
23.9
43.4
45.1
36.2
48.9
91.7
74.7
60.3
60.0
61.1
59.9
60.8
58.8
59.9
64.2
69.4
50.4
7
22.5
71.7
72.9
33.2
56.8
52.9
76.1
59.1
57.8
59.4
57.8
58.7
56.5
59.4
57.6
68.6
63.0
8
26.3
31.8
33.1
35.6
32.2
35.1
32.9
58.4
57.4
58.0
58.5
58.7
57.7
57.7
56.1
70.4
68.5
9
25.9
38.6
39.3
28.1
38.9
41.4
39.5
29.5
90.5
90.0
86.5
88.9
74.8
87.6
66.3
58.5
47.8
10
28.0
40.4
41.6
30.3
39.3
42.6
41.1
30.6
81.1
90.4
86.3
86.5
73.8
87.0
64.4
57.5
45.9
11
27.6
40.3
40.9
29.6
40.5
41.9
41.7
30.4
80.1
80.9
86.5
86.5
76.0
89.8
66.8
58.5
47.8
12
28.3
39.6
40.8
31.1
39.3
43.3
41.2
33.0
55.0
56.7
56.9
85.6
75.3
85.3
65.9
57.5
46.1
13
27.3
37.7
38.7
27.3
37.7
40.1
38.9
28.5
79.3
77.6
76.8
54.2
74.5
84.3
64.1
57.6
47.0
Similarity values were calculated using the Similarity Matrix calculation editor from the ARB software package.
14
29.7
38.3
39.3
28.3
39.4
39.1
39.9
28.9
73.2
72.2
73.1
53.4
72.2
74.6
63.3
56.5
47.0
15
24.9
42.4
43.8
32.3
42.1
45.6
44.2
34.3
77.4
79.7
79.4
57.2
73.4
70.8
65.4
57.5
49.1
16
27.3
38.9
39.7
30.4
38.7
40.1
39.7
30.2
39.0
40.0
41.1
41.4
39.3
39.0
41.4
58.4
45.2
17
27.3
84.1
86.1
32.8
50.3
44.5
72.7
32.6
39.2
40.7
41.0
39.4
37.6
39.0
43.7
39.7
61.4
18
26.3
28.7
29.0
29.4
27.6
27.8
28.6
27.1
24.5
26.8
26.1
24.8
24.5
24.4
27.5
27.5
28.0
Appendix
69
Table A2: Sequence similarities1 of DsrAB amino acid (lower triangle) and 16S rRNA gene nucleotide (upper triangle) sequences.
Reference species
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
1
Beggiatoa (uncultured)
M. magnetotacticum
M. magneticum
T. denitrificans
O. algarvensis gam 2
O. algarvensis gam 1
A. vinosum
A. ehrlichei
C. phaeobacteroides DSM266
C. limicola
P. aestuarii
P. vibrioformis
P. phaeoclathratiforme
C. phaeobacteroides BS1
C. tepidum
M. spec.
M. gryphiswaldense
R. pachyptila symbiont
Sequence similarity [ % ]
1
2
3
4
82.3
82.3
82.0
68.4
99.4
81.4
67.4
96.4
80.7
56.8
64.5
64.0
77.9
66.0
65.7
63.4
68.8
69.3
67.2
79.8
67.1
68.0
65.4
74.7
69.0
69.6
66.9
50.0
56.7
57.1
55.2
45.7
56.4
56.4
54.5
52.1
57.1
57.2
55.3
50.0
58.1
57.7
54.7
48.9
57.1
57.1
54.8
52.1
55.6
56.3
55.1
54.3
55.4
55.5
54.2
39.8
57.7
58.3
54.2
65.3
87.5
88.7
63.9
85.0
63.4
63.8
55.6
5
85.6
82.2
82.0
82.6
84.6
80.6
73.3
56.2
55.3
57.8
56.3
56.6
55.6
57.5
54.5
66.4
63.6
6
87.0
83.5
83.4
82.5
88.0
91.7
74.7
60.3
60.0
61.1
59.9
60.8
58.8
59.9
64.2
69.4
50.4
7
86.3
82.7
82.5
83.1
86.7
91.9
76.1
59.1
57.8
59.4
57.8
58.7
56.5
59.4
57.6
68.6
63.0
8
87.6
84.3
84.2
82.6
88.4
91.0
90.1
58.4
57.4
58.0
58.5
58.7
57.7
57.7
56.1
70.4
68.5
9
76.5
77.2
77.0
73.2
74.5
75.7
75.7
76.7
90.5
90.0
86.5
88.9
74.8
87.6
66.3
58.5
47.8
10
76.9
76.7
76.8
73.9
76.4
76.9
76.3
78.0
96.4
90.4
86.3
86.5
73.8
87.0
64.4
57.5
45.9
11
76.1
76.8
76.8
74.2
75.3
75.9
76.1
76.6
92.8
92.3
86.5
86.5
76.0
89.8
66.8
58.5
47.8
12
76.4
77.0
77.0
73.4
75.6
76.6
76.0
77.1
93.4
94.6
93.5
85.6
75.3
85.3
65.9
57.5
46.1
13
76.3
77.8
77.8
73.2
75.2
76.5
76.7
77.7
96.3
96.1
91.8
93.4
74.5
84.3
64.1
57.6
47.0
Similarity values were calculated using the Similarity Matrix calculation editor from the ARB software package.
14
76.7
76.8
76.7
73.3
75.3
76.3
76.4
76.9
92.6
93.0
95.5
92.4
92.3
74.6
63.3
56.5
47.0
15
76.7
76.8
76.8
72.5
75.7
76.3
75.8
77.0
92.5
94.8
93.2
95.5
92.8
92.1
65.4
57.5
49.1
16
82.1
83.2
82.8
80.3
81.4
83.5
82.1
83.8
76.4
77.5
76.7
77.6
76.6
76.8
77.6
58.4
45.2
17
82.5
96.0
95.8
81.4
82.4
83.3
83.3
84.7
77.6
77.0
76.8
77.7
77.9
76.9
77.9
83.5
61.4
18
86.2
82.4
82.2
83.6
91.6
89.7
89.0
89.8
75.3
77.7
76.7
76.3
76.8
75.9
76.3
82.7
82.5
70
(E) Acknowledgement
I would like to thank my thesis supervisor Dr. Marc Mußmann for providing
such an exciting research topic and for his encouragement. His fascination for sulfur
oxidizers greatly supported this work. He always found time for helpful discussions.
Many thanks go to Prof. Dr. Rudolf Amann for setting up an excellent research
framework and for taking the part of the first reviewer.
Dr. Jens Harder is acknowledged for suggesting me for the marmic program and
thus offering me the possibility to participate in this outstanding Master degree of the
International Max Planck Research School for Marine Microbiology.
Moreover, Dr. Christiane Glöckner is very much acknowledged for caring
management of the entire marmic class.
Finally, I want to thank all members of the Molecular Ecology group for the
support and kind working atmosphere.
This work was financially supported by an educational grant of the Max Planck
Society.
Allen voran besonderen Dank meiner Familie und Falk☺ für die Unterstützung
und tatkräftiges zur Seite stehen in den gesamten Studienjahren.
71
(F) Statement
I herewith confirm that I have written this thesis unaided and that I used no other
resources than those mentioned.
Bremen 17.03.2006
Sabine Lenk