A multi-gene approach in the systematics of the phototrophic purple sulfur bacteria genera Marichromatium and Allochromatium with description of the new species Allochromatium humboldtianum and the new biotype Marichromatium gracile biotype thermosulfidiphilum [Elektronische Ressource] / Wilbert Serrano

Wserrano

Sujets

• Biologie

Informations

• Publié par: universitat_bremen
• Publié le: 01 janvier 2009
• Nombre de lectures: 101
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

A multi-gene approach in the systematics of the
phototrophic purple sulfur bacteria genera
Marichromatium and Allochromatium with description of
the new species Allochromatium humboldtianum and the
new biotype Marichromatium gracile biotype
thermosulfidiphilum.



Dissertation
zur
Erlangung des Grades eines
Doktors der Naturwissenschaften
-Dr. rer. nat.-






dem Fachbereich Biologie/Chemie der
Universität Bremen vorgelegt von






Wilbert Serrano
aus
Lima-Peru






Bremen 2009















































Die Untersuchungen zur vorlegelegten Doktorarbeit wurden in der Zeit von Juli 2005
bis Juni 2009 am UFT der Universität Bremen und am Max Planck Institut für Marine
Mikrobiologie in Bremen durchgeführt.


























1. Gutachter: Prof. Dr. Ulrich Fischer
2. Gutachter: Prof. Dr. Rudolf Amann

Tag des Promotionskolloquiums: 23. Juli 2009.




















Für meine Eltern






















Table of contents
Table of contents …………………………….. .....................................................3
List of abbreviations………………………………………………………………… ..5
Summary.……………………………………………………………………………….7
Zusammenfassung .............................................................................................9

Chapter 1
Introduction .......................................................................................................11
1. A general overview of bacterial sytematics ..................................................11
2. A multi-gene taxonomy in prokaryotes ........................................................17
3. Photosynthetic bacteria …………………………………………………….….20
3.1. Antenna complex………..…………………………………..………….….24
3.2. Reaction center ……………….……………………………………….….25
4. The family Chromatiaceae………………………………………………….….26
4.1. Environmental factors …………….………………………………….….27
4.2. Importance………………….……………..………………………………28
4.3. Carbon fixation and sulfur oxidation by purple sulfur bacteria……..…29
5. Material and methods…………………………………………………...............30
5.1. Source of microorganisms… …….………………………………….….30 5.2. Media and cultivation conditions………………………………….….32
5.3. Enrichment and isolation……….…………………………………….….34 5.4. Genomic DNA isolation and molecular analysis……………….….35
5.4.1. Genomic DNA "fishing" extraction protocol…………………….….35 5.4.2. G+C mol% determination..……………..……………………….….37
5.4.3. DNA-DNA reassociation..……………..……………………….….38 5.4.4. Protein-coding gene analysis…………..……………………….….39
5.4.5. Population genetic analysis…………..……………………….….40
6. Aim of this study… .......................................................................................41


- 3 - 3
7. Results and discussion..................................................................................42
7.1. Characterization of a new purple sulfur bacterium, Marichromatium
gracile biotype thermosulfidiphilum (Chapter 2). ……………………42
7.2. A multi-gene phylogeny of the genus Marichromatium (Chapter 3).44 7.3. A multi-glogeny of the genus Allochromatium (Chapter 4)….47
7.4. Description of a novel Allochromatium species; A. humboldtianum sp.
. nov. (Chapter 5)………….……………..…………………………..…….49
8. This work (List of publications)……………….………………………………..50
9. References ...................................................................................................52

Chapter 2
A new moderately thermophilic and high sulfide tolerant biotype of
Marichromatium gracile, isolated from tidal sediments of the German Wadden
Sea: Marichromatium gracile biotype thermosulfidiphilum……………………… 63

Chapter 3
Evaluation of the use of Multilocus Sequence Analysis (MLSA) to resolve
taxonomic conflicts within the genus Marichromatium. ….................................79

Chapter 4
The Genus Allochromatium (Chromatiales, Chromatiaceae) revisited: a study of
its intragenic structure based on multilocus sequence analysis (MLSA) versus
DNA-DNA Hybridization (DDH)…………….………………..…….……………...117

Chapter 5
Allochromatium humboldtianum sp. nov., a novel Allochromatium species
isolated from soft marine sediments from Peruvian coast ..............................137

Appendices .....................................................................................................149
Acknowledgements .........................................................................................156


- 4 -
List of abbreviations

AAI Amino acid identity
Bchl Bacteriochlorophyll

BOX A1R Highly conserved repeated sequence, based on BOX
repetitive elements present in the chromosome of
Streptococcus pneumoniae
DDH DNA-DNA hybridization
DIG DNA Digoxigenin-dUTP labeling DNA probe
DSMZ Deutsche Sammlung von Mikroorganismen und
Zellkulturen
dN/dS Ratio of the rate of non synonymous substitutions (dN) to
the rate of synonymous substitutions (dS)
ERIC Enterobacterial repetitive intergenic consensus sequences
H gene diversity
HGT Horizontalgene transfer
ITS Internal transcriber spacer

LH Photosynthetic light harvesting complex

MLEE Multilocus enzyme electrophoresis
MLST Multilocus sequence typing
MLSA us sequence analysis
Mol% G+C Percentage of guanine-cytosine content in the genome
M13-Core Bacteriophage M13 core repeated sequence

nucleotide diversity

PhyML Phylogenies by maximum likelihood

RAPD Ramdom amplified polymorphic DNA
RC Photosynthetic reaction center complex
SG Segregation site
TD-PCR Touch-down PCR
T’D Tajima test of neutrality







- 5 -





























- 6 -
Summary
In the present thesis two genera of the photosynthetic Purple Sulfur Bacteria (PSB)
of the family Chromatiaceae are revised by molecular techniques which include
extensive DNA-DNA hybridization (DDH) studies and a multi-gene analysis.
Traditionally, species of PSB have been described based on some easily
recognizable phenotypic properties. Separation of the PSB in two groups was
possible by a diagnostical key property, e.g. internal or external storage of sulfur
globules. This separation of PSB in the two families Chromatiaceae and
Ectothiorhosdopiraceae was confirmed by comparing their 16S rRNA gene
sequences (Imhoff et al., 1998, Int. J. Syst. Bacteriol. 48, 1129-1143).
Within the Chromatiaceae the genus Chromatium was also separated in several
genera being the most important the genera Marichromatium and Allochromatium.
However, until now only a low number of species have been reported within each
genus. Type species from both genera do not exhibit remarkable phenotypic
differences and they seem to be closely related phylogenetically (16S rRNA-based)
and genomically (Serrano et al., 2009, Syst. Appl. Microbiol. 32, 1-7).
Type species and strains of both genera examined in the present study were
obtained from culture collections as well as isolated from geographically distant
locations. A multi-gene analysis was performed based on PCR amplification and
sequencing of a set of protein coding-genes such as pufM, soxB, recA, fusA, dnaK,
and gyrB for Marichromatium and additionally cbbL-1, mfd, and groES genes for
Allochromatium. In addition, extensive cross-DDH comparison among all the
available strains within each genus were also made.
Strains within the genera Marichromatium and Allochromatium showed a high 16S
rRNA gene sequence similarity ranging from 98 to 100% (mean 99.3%) and 97.0 to
100% (mean 98.0%) respectively. However, cross-DDH comparison, a standard tool
for the definition of bacteria species, showed ambiguous results. On one hand they
confirmed the 16S rRNA-based phylogenetic relationship, on the other hand they
were contradicting, thus making species definition within both genera difficult.
However, these phylo-genomic incongr