Diversity, Biomineralization and Rock Magnetism of Magnetotactic Bacteria

Springer Theses
Recognizing Outstanding Ph.D. Research
Wei Lin
Diversity,
Biomineralization
and Rock Magnetism
of Magnetotactic
Bacteria
Springer Theses
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Wei Lin
Diversity, Biomineralization
and Rock Magnetism of
Magnetotactic Bacteria
Doctoral Thesis accepted by
Chinese Academy of Sciences, China
123
Author Supervisors
Dr. Wei Lin Prof. Dr. Yongxin Pan
Institute of Geology and Geophysics Institute of Geology and Geophysics
Chinese Academy of Sciences Chinese Academy of Sciences
Beijing Beijing
People’s Republic of China People’s Republic of China
Prof. Dr. Dirk Schüler

Bereich Mikrobiologie/Department
Biologie I
Ludwig-Maximilians-Universität
München
Munich
Germany
ISSN 2190-5053 ISSN 2190-5061 (electronic)

ISBN 978-3-642-38261-1 ISBN 978-3-642-38262-8 (eBook)
DOI 10.1007/978-3-642-38262-8
Springer Heidelberg New York Dordrecht London
Library of Congress Control Number: 2013939046
Springer-Verlag Berlin Heidelberg 2013

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Parts of this thesis have been published in the following journal articles:
Wei Lin, Christian Jogler, Dirk Schüler and Yongxin Pan (2011) Metagenomic
analysis reveals unexpected subgenomic diversity of magnetotactic bacteria within
the Phylum Nitrospirae. Applied and Environmental Microbiology, 77(1):323–
326.
Wei Lin and Yongxin Pan (2010) Temporal variation of magnetotactic bacterial
communities in two freshwater sediment microcosms. FEMS Microbiology
Letters, 302(1):85–92.
Wei Lin and Yongxin Pan (2009) Uncultivated magnetotactic cocci from Yuandadu
park in Beijing, China. Applied and Environmental Microbiology, 75(12):4046–
4052.
Wei Lin and Yongxin Pan (2009) Specific primers for the detection of freshwater
alphaproteobacterial magnetotactic cocci. International Microbiology, 12(4):237–
242.
Wei Lin, Jinhua Li, Dirk Schüler, Christian Jogler and Yongxin Pan (2009)
Diversity analysis of magnetotactic bacteria in Lake Miyun, northern China, by
restriction fragment length polymorphism. Systematic and Applied Microbiology,
32(5):342–350.
Dedicated to my family
Supervisors’ Foreword
Research in biomineralization investigates the mechanisms and properties of

minerals deposited by organisms. Biogenic minerals have been discovered in a
number of organisms, including microorganisms, insects, mollusks, fish, birds, and
human beings. Several dozens of biominerals (metals, oxides, carbonates, phos-
phates, sulfates, etc.) have been identified, which are of great importance in
aspects of life and earth sciences.
Magnetotactic bacteria (MTB) are one of the most fascinating examples of
biologically controlled mineralization, which synthesize intracellular magnetic
minerals of magnetite (Fe3O4) and/or greigite (Fe3S4) magnetosomes. Since the
pioneering work by Salvatore Bellini and Richard Blakemore, study of MTB has
become a hot interdisciplinary research topic. Biomineralization in MTB is a key
issue for understanding processes of biogenic minerals assembly in eukaryotes.
Therefore, MTB are attractive model systems for investigating fundamental bio-
mineralization mechanisms. Because MTB are able to accumulate large amounts
of iron in cells, they may play functionally important roles in ecosystems, espe-
cially in global biogeochemical cycling of iron. Moreover, the geological signif-
icance of fossil magnetosomes preserved in sediments is also important, such as
potential contributions to the bulk magnetization of sediments and serve as
archives of ancient environmental changes.
Like most microorganisms, the cultivated species of MTB only represent a
minor fraction of environmental MTB community, and our knowledge of uncul-
tivated MTB is rudimentary due to the lack of efficient approaches and techniques.
During Wei Lin’s Ph.D. studies he hunted MTB in field sites both in China and
Germany. In Wei Lin’s thesis, novel MTB enriching and detecting methods have
been developed which allow quick and efficient collection, purification and
characterization of uncultivated MTB from environmental samples, circumventing
the need to cultivate these bacteria. The diversity, distribution, genomic, and
magnetic properties of uncultured MTB communities around Beijing, China have
been investigated through a combined approach of molecular biology, electron
microscopy, and magnetic measurements. The results of the thesis lead to a better
understanding of the diversity, ecophysiology, and biogeography of natural pop-
ulations of MTB in nature, and improve our knowledge of the interaction of MTB
communities with key environmental factors. Magnetic properties of MTB
ix
x Supervisors’ Foreword
magnetosomes are important for decoding the paleomagnetic signals of sediments

containing fossil magnetosomes. We hope that this thesis will be useful to
researchers and graduate students who are interested in biology and magnetism of
magnetotactic bacteria, the process of magnetic nanoparticle biomineralization,
and its potential biotechnological applications.
Beijing Yongxin Pan

Munich Dirk Schüler
Acknowledgments
Sincere thanks are given to my supervisors Yongxin Pan for his support to my
Ph.D. project and guidance throughout the research, and Dirk Schüler for giving
me the opportunity to study in his lab and providing advice with regard to the
molecular techniques. I could not complete this work without their guidance and
support.
I would like to thank many people in the Paleomagnetism and Geochronology
Laboratory at Institute of Geology and Geophysics, Chinese Academy of Sciences
who helped me during my Ph.D. project: Rixiang Zhu, Chenglong Deng, Qingsong
Liu, Baochun Huang, Fei Wang, Huaiyu He, and others. I am also grateful to
Nikolai Petersen in the Department of Earth and Environmental Sciences at
Ludwig-Maximilians-Universität München who taught me a lot about how to
detect and collect magnetotactic bacteria.
Further, I would like to express my gratitude to my colleagues in the Bio-
geomagnetism Group at Institute of Geology and Geophysics, Chinese Academy
of Sciences for their helpful comments and discussions: Lanxiang Tian, Jinhua Li,
Bi Li, Changqian Cao, Xinlin Ji, Lei Wang, Wenfang Wu, Yinzhao Wang, and
Haitao Cheng. In addition, I would like to thank my colleagues in Schüler’s Lab at
Ludwig-Maximilians-Universität München: Claus Lang, Katja Junge, Rene Uebe,
Emanuel Katzmann, Susanne Ullrich, Damien Faivre, and Zuzana Safrankova. In
particular, humble thanks go to Christian Jogler for his advice and help during my
study in Munich.
Finally, I would like to thank my parents and my wife for their support and
encouragement.
The financial supports from the Chinese Academy of Sciences, the National
Natural Science Foundation of China, and the Max Planck Society are kindly
acknowledged.
Beijing, China Wei Lin
xi
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 General Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.1 Diversity of MTB . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.1.2 Distribution of MTB . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.1.3 Molecular Mechanism of MTB Biomineralization. . . . . . 5
1.1.4 Magnetic Properties of MTB and Magnetosomes . . . . . . 6
1.1.5 Fossil Magnetosomes and Paleoenvironmental
Reconstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2 Aims and Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3 Outline of this Thesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2 Enrichment, Purification and Characterization of Environmental

Magnetotactic Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2 Novel Apparatus and Strategy for MTB Enrichment . . . . . . . . . 16
2.3 Specific Primers for Freshwater Alphaproteobacteria
Magnetotactic Cocci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.4 In silico Evaluation of Restriction Endonucleases for RFLP
Analysis of MTB. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3 Diversity of Magnetotactic Bacteria and Its Environmental

Implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.2.1 Sampling Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.2.2 Physical–Chemical Analyses . . . . . . . . . . . . . . . . . . . . 32
3.2.3 Magnetic Collection of MTB . . . . . . . . . . . . . . . . . . . . 34
3.2.4 Transmission Electron Microscope Observation . . . . . . . 34
3.2.5 PCR, Cloning and DNA Sequencing . . . . . . . . . . . . . . . 34
xiii
xiv Contents
3.2.6 Phylogenetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 35

3.2.7 Fluorescence In Situ Hybridization . . . . . . . . . . . . . . . . 35
3.2.8 Statistical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.3.1 MTB Cell Morphologies and Magnetosomes . . . . . . . . . 36
3.3.2 Phylogenetic Diversity of MTB . . . . . . . . . . . . . . . . . . 37
3.3.3 FISH Analysis of MTB Population from
Yuandadu Park . . . . . . . . . . . . . . . . . . . . . . . . ...... 38
3.3.4 Spatiotemporal Variation of MTB Communities
from Lake Miyun . . . . . . . . . . . . . . . . . . . . . . ...... 40
3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 44
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...... 47
4 Metagenomic Analysis of Uncultivated Magnetotactic Bacteria

Within the Phylum Nitrospirae . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
4.2.1 Sampling and Collection of MTB . . . . . . . . . . . . . . . . . 52
4.2.2 Preparation of High Molecular DNA for Library
Construction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 53
4.2.3 Fosmid Library Construction, Screening
and Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . ... 53
4.2.4 Fosmids Annotation and Phylogenetic Analyses . . . . ... 54
4.2.5 Oligonucleotide Probes and Fluorescence
In Situ Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.2.6 Transmission Electron Microscope (TEM) . . . . . . . . . . . 55
4.2.7 Nucleotide Sequence Accession Numbers . . . . . . . . . . . 55
4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.3.1 Library Construction and Screening . . . . . . . . . . . . . . . 56
4.3.2 Analyses of Five Fosmid Clones. . . . . . . . . . . . . . . . . . 56
4.3.3 In Situ Characterization of ‘‘Ca. Magnetobacterium
bavaricum’’-Like Bacteria. . . . . . . . . . . . . . . . . . . . . . . 59
4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5 Magnetic Properties of Uncultivated Magnetotactic Bacteria . . . . . 67

5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
5.2.1 Sample Sites and MTB Collection . . . . . . . . . . . . . . . . 68
5.2.2 Transmission Electron Microscopy Observation . . . . . . . 68
5.2.3 Magnetic Measurements of MTB . . . . . . . . . . . . . . . . . 68
Contents xv
5.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
5.3.1 Room-Temperature Properties . . . . . . . . . . . . . . . . . . . 69
5.3.2 Low-Temperature Properties. . . . . . . . . . . . . . . . . . . . . 71
5.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Chapter 1
Introduction
Iron is the fourth most common element in the Earth’s crust. It is a crucial nutrient
for almost all organisms and plays an important role in the oceanic primary pro-
duction process (Martin and Fitzwater 1988). Plenty of organisms in nature are
found to form iron minerals, either extracellularly or intracellularly, which may
play significant roles in local or global iron cycles (Kirschvink 1989; Winklhofer
2010). One of the most interesting examples is found in magnetotactic bacteria
(MTB), a physiologically and phylogenetically diverse group of microorganisms
that are able to mineralize intracellular membrane-enveloped iron minerals of
magnetite (Fe3O4) and/or greigite (Fe3S4) (Schüler and Frankel 1999; Amann et al.
2007; Jogler and Schüler 2009). These magnetic particles (known as magneto-
somes) generally range from 30 to 120 nm, and fall into a range of stable single
domain (Bazylinski and Frankel 2004; Pan et al. 2004). As a consequence of
evolution, magnetosomes are normally organized into one or multiple chain
structures in order to optimize the magnetic moment, which enable MTB cell to
swim along the geomagnetic field lines (Fig. 1.1). It is widely accepted that, under
the influence of the Earth’s magnetic field, magnetotaxis facilitates the navigation
of MTB to their preferred low-oxygen microenvironments in chemically stratified
environments (Bazylinski and Frankel 2004).
In the 1960s–1970s, MTB were independently discovered by Salvatore Bellini
(2009) and Richard Blakemore (1975). After more than four decades of research,
the knowledge on MTB and magnetosomes has been greatly improved. The
members of MTB are found to globally distribute, and so far they have been
discovered in diverse aquatic and/or sedimentary environments, e.g., freshwater
lakes, salt ponds, estuaries, mangrove swamps, intertidal zones, deep-sea sedi-
ments, and even extreme environments (Amann et al. 2007; Faivre and Schüler
2008; Lefèvre et al. 2010; Lin et al. 2012b). Considering their high intracellular
iron content, MTB have been attributed as an important role in global iron cycling
and bulk magnetization of sediments (Pan et al. 2004; Faivre and Schüler 2008).
Furthermore, fossil magnetosomes that preserved in sediments could serve as a
potential proxy for paleoenvironmental reconstructions (Kopp and Kirschvink
2008). Magnetosomes formed by MTB as a unique biomaterials have potential
W. Lin, Diversity, Biomineralization and Rock Magnetism 1

of Magnetotactic Bacteria, Springer Theses, DOI: 10.1007/978-3-642-38262-8_1,
Ó Springer-Verlag Berlin Heidelberg 2013
2 1 Introduction
Fig. 1.1 Transmission

electron micrograph of a
magnetotactic bacterium. The
black arrow indicates
magnetosomes that organized
into a chain structure.
Bar = 500 nm
applications in various fields, such as magnetic separation, diagnostics, and

detection of analytes (Lang and Schüler 2006; Matsunaga et al. 2007).
1.1 General Information
1.1.1 Diversity of MTB
The majority of MTB populations in nature are so far uncultivable. However,

MTB can be enriched from environmental samples simply by taking advantage of
their active magnetotactic behavior. MTB exhibit diverse morphologies, such as
coccoid, spirillar, rod-shaped and multicellular cells with one, two, or more chains
of magnetosomes. The morphology of magnetosome crystals is different in distinct
MTB populations, such as cuboidal, elongated prismatic, bullet-shaped, and
anomalous magnetosomes (Fig. 1.2). MTB are ubiquitous in aquatic microbial
communities, where they sometimes can be detected in high abundance (Spring
et al. 1993; Simmons et al. 2007). Based on 16S rRNA gene analysis, to date all
discovered MTB are affiliated with the Alphaproteobacteria, Deltaproteobacteria,
Gammaproteobacteria, phylum Nitrospirae, and the candidate division OP3
(Amann et al. 2007; Lefèvre et al. 2011b; Kolinko et al. 2012; Kolinko et al. 2013).
In most environments in nature, bacteria in the Alphaproteobacteria are the
dominant populations of MTB (Fig. 1.3). Spring et al. (1992, 1994) first utilized
16S rRNA gene-based molecular approach to characterize the diversity of
uncultivated MTB. They have found several coccoid MTB populations dominant
in the upper sediment layers of Lake Chiemsee in Germany, which phylogeneti-
cally belonged to the Alphaproteobacteria. Since these studies, Alphaproteobac-
teria MTB are found to ubiquitously exist in both freshwater and saline
environments, including the Itaipu lagoon in Rio de Janeiro, Brazil (Spring et al.
1998), the McFarland pond in Ames, USA (Schüler et al. 1999), Baldwin Lake in
Los Angeles, USA (Cox et al. 2002), several sites in Germany and Sweden
1.1 General Information 3
Fig. 1.2 Representative shapes of magnetosome crystals. Bars = 100 nm
(Flies et al. 2005), and the Huiquan Bay in Qingdao, China (Pan et al. 2008; Xing
et al. 2008). The majority of cultivated representatives of MTB also affiliate with
this class, such as Magnetospirillum magnetotacticum strain MS-1 (Blakemore
et al. 1979), Magnetospirillum magneticum strain AMB-1 (Kawaguchi et al. 1992),
Magnetospirillum gryphiswaldense strain MSR-1 (Schleifer et al. 1991; Schüler
and Kohler 1992), Magnetococcus marinus MC-1 (Bazylinski et al. 2012), mag-
neto-ovoid strain MO-1 (Lefèvre et al. 2009), magnetotactic spirillum strain QH-2
(Zhu et al. 2010), and Magnetospira thiophila MMS-1 (Williams et al. 2012).
Besides Alphaproteobacteria, molecular analyses have revealed a great deal of
diversity in the Deltaproteobacteria MTB (Fig. 1.3). One of intriguing examples is
found in multicellular magnetotactic prokaryotes (MMP), which consist of mul-
tiple (normally 10–40) individual cells (Keim et al. 2007). MMP were first dis-
covered by Farina and de Barros (1983). So far these special MTB have been
found in various saline environments and represent an unexpected phylogenetic
diversity (DeLong et al. 1993; Simmons and Edwards 2007; Wenter et al. 2009;
Zhou et al. 2012). Besides MMP, other MTB, including the cultivated Desulf-
ovibrio magneticus strain RS-1 (Kawaguchi et al. 1995), an uncultivated barbell-
shaped population (Simmons et al. 2006), and a group of sulfate-reducing bacteria
that could form magnetite and/or greigite (Lefèvre et al. 2011b), were also dis-
covered to affiliate with the Deltaproteobacteria.
The finding of a greigite-producing magnetotactic rod provides the first infor-
mation for the existence of MTB in the Gammaproteobacteria (Simmons et al.
2004). This bacterium was discovered in a stratified coastal salt pond in Massa-
chusetts, USA, and was found in the hypolimnion in chemically stratified
4 1 Introduction
Fig. 1.3 Phylogenetic tree of representative magnetotactic bacterial strains
environment. Recently, two novel magnetotactic rods were isolated and identified to
affiliate with the Gammaproteobacteria. Unlike bacteria from Salt Pond, these two
novel populations produce magnetite magnetosomes (Fig. 1.3) (Lefèvre et al. 2012).
Besides Proteobacteria, MTB populations affiliated with the phylum Nitros-
pirae and the candidate division OP3 are detected as well (Fig. 1.3). MTB in the
phylum Nitrospirae was first reported by Spring et al. (1993). It is a large rod-
shaped bacterium (named ‘‘Candidatus Magnetobacterium bavaricum’’) that forms
hundreds of bullet-shaped magnetite magnetosomes in a single cell. In recent
studies, several MTB strains from various ecosystems with distinct morphologies
were also indentified belonging to this phylum (Flies et al. 2005; Lefèvre et al.
2010; Lefèvre et al. 2011a; Lin et al. 2012a). In addition, Kolinko et al. (2012)
have found a novel MTB which is morphologically similar to a population in the
phylum Nitrospirae. However, using a single cell-based approach and fluorescence
in situ hybridization, the authors identified that this bacterium belongs to the
candidate division OP3. This study has suggested that magnetotactic bacteria are
not restricted to the phyla Proteobacteria and Nitrospirae. The ability of forming
magnetosomes may occur widely in other bacterial taxa.
1.1.2 Distribution of MTB
Through comprehensive studies of MTB communities across different ecosystems,

growing evidence has suggested that the composition of MTB communities rep-
resents a biogeographic distribution across heterogeneous environments, which are
regulated by various environmental factors. In single locations, the vertical dis-
tribution of MTB is found to correlate with geochemical gradients. For most MTB
populations, their preferential locations are near the oxic-anoxic transition zones
(OATZ) of sediments. For example, in the deep-sea sediments, the majority of
MTB cells concentrated near the anaerobic zones, while only a small portion of
MTB occurred in the aerobic layers (Petermann and Bleil 1993). The vertical
distribution of these MTB is found to correlate with nitrate and oxygen concen-
trations (Petermann and Bleil 1993). In addition, a marked stratification of dif-
ferent MTB populations is observed in a stratified salt pond, where magnetite-
producing MTB were found at the top of the oxycline, while greigite-producing
MTB were observed at the base of the oxycline (Simmons et al. 2004). For MTB
across multiple locations, recent studies have revealed an intra- and inter-lake
variability of MTB communities. For instance, both salinity and the ratio of iron
and bioavailable sulfur concentrations are identified to regulate the population
density of ‘‘Candidatus Magnetoglobus multicellularis’’ (Martins et al. 2009;
Sobrinho et al. 2011). In the Gulf of Fos in French, the cell abundance of MTB is
found to correlate with the redox state of sulfur compounds as revealed by a
microcosm-based analysis (Postec et al. 2012).
1.1.3 Molecular Mechanism of MTB Biomineralization
Biomineralization of magnetosomes in MTB is a key issue for understanding

processes of biogenic minerals assembly in eukaryotes. Since their discovery,
MTB have become an attractive model system for investigating the fundamental
biomineralization mechanisms. Over the past decade, the molecular mechanism of
magnetosome chain formation is becoming clear for a few strains completely or
partially sequenced. A plausible model for magnetosome chain formation for
genus Magnetospirillum is: (1) magnetosome membrane biogenesis, (2) targeting
of magnetosome proteins to the magnetosome membrane, (3) magnetosome
membrane chain arrangement, and (4) magnetite biomineralization (Komeili 2007;
6 1 Introduction
Faivre and Schüler 2008; Schüler 2008; Komeili 2012). The entire process of
magnetosome formation in genus Magnetospirillum is strictly controlled by a
group of genes that are organized into a coherent genomic fragment described as
magnetosome island or MAI. Recent studies have revealed that MAI structure is
ubiquitous in genomes of both isolated and uncultivated MTB species (Jogler et al.
2009; Nakazawa et al. 2009; Abreu et al. 2011; Jogler et al. 2011; Lefèvre et al.
2011b). The existence of MAI across several magnetotactic bacterial species has
indicated the hypothesis that MAI may be horizontally transferred (Jogler et al.
2009). However, divergence in sequences and gene content of MAIs from different
MTB groups was also noticed, which likely accounts for the diversity in mag-
netosomes observed in MTB from various environments (Jogler and Schüler
2009).
Since most MTB are still unculturable, our knowledge of the mineralization in
uncultivated MTB with different morphologies and phylotypes remains very
limited. Indeed those uncultivated bacteria are normally the dominant MTB
populations in nature and therefore may play more important environmental roles
than their cultured counterparts. The characteristics and alignments of magneto-
somes in uncultivated MTB [e.g. ‘‘Candidatus Magnetobacterium bavaricum’’ in
the phylum Nitrospirae (Jogler et al. 2011)] are not found in cultivated bacteria.
Therefore, cultivation-independent approaches, such as metagenomics and single-
cell analysis, are useful methods to acquire the genomic information of unculti-
vated MTB, which may lead to ultimately answer the key question whether MAI is
horizontally transferred among all MTB populations. Moreover, by comparative
genomics, it is also possible to identify the core magnetosome genes that con-
served in most if not all MTB genomes or metagenomes. These core magnetosome
genes may potentially useful for the biosynthesis of magnetic minerals.
1.1.4 Magnetic Properties of MTB and Magnetosomes
Magnetite is one of the principal recorders of paleomagnetic information on Earth.

It is also an important material that has been widely applied in various fields.
Understanding its physical properties is thus of great importance. Although studied
for several decades, the magnetic properties of magnetite remain somewhat
enigmatic. Magnetite magnetosomes formed by MTB display narrow size distri-
butions and uniform morphologies, which is not easily accessible by chemical
synthesis. These magnetosomes provide an ideal material for investigating fun-
damental properties of magnetite. Moreover, investigation of magnetic properties
of magnetosomes may also help us better understand the mechanisms of mag-
netosome biomineralization processes in MTB cells.
The first magnetic measurements of pure MTB was conducted by Denham et al.
(1980), who have compared magnetic properties of whole M. magnetotacticum
strain MS-1 cells, whole cells without magnetosomes, and isolated magnetosomes.
The authors have revealed that magnetic properties of isolated magnetosomes are
different from that of whole MS-1 cells due to the stronger magnetic interaction.
Following this study, Moskowitz and colleagues have compared both room- and
low-temperature magnetic properties of several cultivated MTB strains under
different conditions (Moskowitz 1988; Moskowitz et al. 1989, 1993). They sug-
gested a combination of low-temperature magnetic measurements and remanence
coercivity analyses to identify MTB magnetite magnetosomes in the sediments
(Moskowitz et al. 1993). In these magnetic criteria, the most widely used criterion
is the low-temperature field cooling (FC) and zero field cooling (ZFC) method,
known as ‘‘Moskowitz test’’, i.e. dFC =dZFC [ 2ðd ¼ ðJ80K J150K Þ=J80K ;
J80K and J150K represent remanence at 80 and 150 K, respectively). In recent
years, detailed magnetic measurements were conducted for various cultivated
MTB strains, which have a single magnetosome chain structure per cell (Weiss
et al. 2004a; Hergta et al. 2005; Kopp et al. 2006; Pósfai et al. 2006; Chen et al.
2007; Prozorov et al. 2007; Alphandéry et al. 2008; Fischer et al. 2008; Carvallo
et al. 2009; Li et al. 2009) (Table 1.1). While due to requirement of large amount
of cells, magnetic measurements on environmental uncultivated MTB samples
were only occasionally performed (e.g., Pan et al. 2005b; Li et al. 2010). There-
fore, our knowledge of uncultivated MTB, especially those with multiple chains of
magnetosomes, remains very limited.
Table 1.1 Magnetic properties of various MTB samples

Sample Bc Bcr Bcr/ Mr/ Tv (K)d d Reference
(mT)a (mT)b Bc Mcs ratioe
Cultivated MS-1 26.7 37.8 1.42 0.53 100–110 [2 (Moskowitz 1988;
MTB Moskowitz et al. 1989,
strains 1993)
AMB- 18.1 23.3 1.29 0.45 106 2 (Li et al. 2009)
1
MSR-1 16.27 20.34 1.25 0.44 *100 1.16 (Fischer et al. 2008)
MC-1 NMf NM NM NM 102 NM (Prozorov et al. 2007)
MV-1 NM NM 1.1 0.49 117 [2 (Moskowitz et al. 1989,
1993; Prozorov et al.
2007)
RS-1 NM 46.7 NM 0.44 86 5.54 (Pósfai et al. 2006)
Uncultivated P2 26.7 40 1.5 0.47 *110 2.3 (Pan et al. 2005a, b)
MTB P3 33.4 45.5 1.36 0.51 *110 3.7 (Pan et al. 2005a, b)
samples
a
coercivity
b
coercivity of remanence
c
Ms: saturation magnetization, Mrs: saturation remanence
d
Verwey transition temperature
e
dFC/dZFC
f
not measured
8 1 Introduction
1.1.5 Fossil Magnetosomes and Paleoenvironmental

Reconstruction
Magnetosomes formed by MTB may be preserved in sediments as fossil records.

These fossil magnetosomes or magnetofossils have been found in various aquatic
environments, such as marine (Petersen et al. 1986; Chang and Kirschvink 1989;
Hesse 1994; Roberts et al. 2011; Larrasoaña et al. 2012) and freshwater lake
sediments (Vali et al. 1987; Snowball 1994; Snowball et al. 2002; Pan et al.
2005a). So far, the robust identification of the oldest magnetite magnetofossils
traces back to the Cretaceous (Kopp and Kirschvink 2008), while the oldest gre-
igite magnetofossils are found back to Pliocene (Vasiliev et al. 2008).
Due to their stable single domain behavior, magnetofossils may act as stable
carriers of natural remanent magnetization and play important roles in the bulk
magnetization in particular sediment layers (Pan et al. 2005a; Moskowitz et al.
2008; Kopp et al. 2009). In addition, magnetofossils preserved in sediments are
potential archives of paleoenvironmental information. For example, the variation
of magnetosomes in marine sediments is found to correlate with glacial and
interglacial periods (Hesse 1994). Snowball et al. (2002) have revealed that the
amount of magnetofossils in Swedish varved lake-sediments is consistent with the
total organic carbon content, which suggested that magnetofossils might provide
information on variation of lake productivity. In a recent study, Roberts and
colleagues have found that iron and organic carbon may control the abundance of
MTB in deep-sea sediments (Roberts et al. 2011).
In 1996, McKay and colleagues have identified putative magnetite magneto-
fossils and carbonate globules in a Martian meteorite ALH84001, which suggests
that this Martian meteorite may harbor relics of early biological activity (McKay
et al. 1996). ALH84001 is a member of the Shergottites–Nakhlites–Chassigny
group of meteorites that crystallized at *4.5 Ga (Weiss et al. 2002). Since this
report, various different approaches have been performed to characterize this
meteorite, with the key question that is magnetite inside of chemical or biogenic
origin, or a mixture of both (Scott 1999; Buseck et al. 2001; Thomas-Keprta et al.
2001; Weiss et al. 2004b; Thomas-Keprta et al. 2009). Although several criteria
have been developed to identify the origin of magnetite (Thomas-Keprta et al.
2001; 2002; Kopp and Kirschvink 2008), the debate over the origin of magnetite in
meteorite ALH84001 is far from being over (Nealson and Cox 2002; Thomas-
Keprta et al. 2009; Martel et al. 2012).
1.2 Aims and Motivation
The general aims of this thesis are:

1. To develop effective approaches for collecting, purifying and characterizing
environmental MTB.
1.2 Aims and Motivation 9
2. To investigate the diversity and distribution of MTB communities across var-

ious environments.
3. To characterize the genomic information and potential ecological functions of
MTB in nature.
4. To characterize the magnetic properties of environmental MTB and their
potential contributions to the bulk magnetism of sediments.
Results of this thesis may lead to better understanding of diversity, biogeog-
raphy and biomineralization mechanisms of MTB in nature, and improve our
knowledge on the contributions of MTB to biogeochemical cycles of elements and
sedimentary magnetism.
1.3 Outline of this Thesis
Chapter 1 contains a short introduction of MTB. Chapter 2 is devoted to develop

new approaches and techniques for MTB enrichment and diversity analysis.
Chapter 3 describes the diversity of MTB communities from two freshwater
locations in Beijing, China through a combined cultivation-independent approa-
ches, and further compares spatiotemporal variation of MTB communities from
Lake Miyun over a 3-month period. Chapter 4 has combined the metagenomic
analysis and fluorescence in situ hybridization to explore the phylogenetic diver-
sity and genomic information of environmental Nitrospirae MTB. Chapter 5
characterizes the room- and low-temperature magnetic properties of two natural
MTB samples. Chapter 6 is devoted to conclude the whole thesis, and Chapter 7 is
the Appendix.
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Chapter 2
Enrichment, Purification
and Characterization of Environmental
2.1 Introduction
Although magnetotactic bacteria (MTB) are ubiquitous in various aquatic or

sedimentary environments, the majority of them are still unculturable. One of the
most intriguing properties of MTB, in contrast to other bacteria, is their ability to
swim along the magnetic field lines, which provides an opportunity to enrich and
purify uncultivated MTB cells directly from environmental samples. The principal
steps of the proposed procedure for MTB diversity analysis are (1) magnetic
enrichment and purification of MTB cells from samples, (2) PCR amplification of
16S rRNA genes or other functional marker genes, (3) construction of clone
libraries, (4) sequences sequencing, (5) phylogenetic analysis, and (6) other cul-
tivation-independent analyses, such as fluorescence in situ hybridization or single-
cell analysis.
For step (1), i.e. magnetic enrichment and purification of MTB, ‘capillary
racetrack’ method (Wolfe et al. 1987), which uses a capillary tube and magnet to
enrich MTB cells from sediments, has been successfully applied in a number of
previous studies (e.g. Flies et al. 2005a). Although ‘capillary racetrack’ is an
efficient approach, it has the following two drawbacks. First, only a small number
of MTB cells (normally 103–105 cells, occasionally up to 107 cells) could be
enriched, which is not enough for further metagenomic characterizations and
magnetic measurements. Second, this approach only enriches MTB cells swim-
ming in one direction, but cannot simultaneously recover both north- and south-
seeking subpopulations. To overcome the first drawback, Lins et al. (2003) have
developed a homemade large-volume apparatus for harvesting much more MTB
cells than using the ‘capillary racetrack’ method. However, this apparatus only has
one end and thus cannot simultaneously recover both north- and south-seeking
cells either.
Another way of investigating MTB diversity is to directly amplify 16S rRNA
genes of MTB cells from environmental metagenomic DNA without requirement
of magnetic collection. In this way, PCR primers that specifically target particular
MTB populations are necessary. Since MTB are polyphyletic, it is difficult to

16 2 Enrichment, Purification and Characterization
design one set of primers to target all MTB communities scattered among the
Proteobacteria, Nitrospirae, and the candidate division OP3. So far only one set of
specific primers (MCF and MCR) for freshwater Alphaproteobacteria magneto-
tactic cocci has been reported (Thornhill et al. 1995). These primers amplify a very
short fragment (*266 bp) and fail to target newly detected MTB populations in
this group.
Restriction fragment length polymorphism (RFLP) analysis is a popular method
used to survey the phylogenetic diversity of microorganisms (Nocker et al. 2007).
This approach is performed to investigate MTB diversity in several studies (e.g.
Flies et al. 2005a; Pan et al. 2008). Restriction endonucleases are used in this
approach to produce different RFLP patterns, which are defined as operational
taxonomic units (OTUs) inferred as single populations within a community.
Clearly, selection of restriction endonucleases plays a critical role in RFLP
analysis results. Therefore, it is necessary to evaluate the power of different sets of
restriction endonucleases in RFLP for identifying the actual populations of MTB.
The aim of this chapter is to develop efficient approaches for MTB enrichment,
purification, and community characterization. A double-ended open magnetic
separation apparatus and a two-step magnetic enrichment strategy are developed,
which allow mass collection of pure environmental MTB for further molecular,
genomic and magnetic analyses (Jogler et al. 2009). In addition, a new set of
specific primers for freshwater Alphaproteobacteria magnetotactic cocci is
designed, which could amplify longer product (about 864 bp) and target more
populations compared to the old primers (Lin and Pan 2009a). Finally, the
resolving powers of different restriction endonucleases are assessed on selected
MTB 16S rRNA gene sequences by in silico experiments (Lin et al. 2009).
2.2 Novel Apparatus and Strategy for MTB Enrichment
For magnetic enrichment of large amount of both north- and south-seeking MTB
from environment, a specifically double-ended open separation apparatus (named
as MTB trap) is designed (Fig. 2.1) (Jogler et al. 2009). This glass device consists
of a reservoir with two opposite funnels connecting with 1.5-ml tubes through
rubber stoppers. A small piece of sterilized cotton is loosely packed into the
funnel, which will yield much more purified MTB samples. A homemade
Helmholtz coil was designed to generate homogenous magnetic field with the
strength of about 5–8 times greater than the Earth’s magnetic field (approximately
0.25–0.4 mT), by which both north- and south-seeking MTB in the trap can
simultaneously swim to the collecting tubes.
Sediments that contain MTB were divided into 600-ml plastic flasks (micro-
cosms), which were covered with about 100 ml of water. A two-step magnetic
enrichment strategy is developed for the MTB trap to harvest large amount of pure
MTB cells from microcosms (Fig. 2.2). Step 1 magnetic enrichment: MTB cells
were magnetically collected by situating one south magnetic pole and one north
2.2 Novel Apparatus and Strategy for MTB Enrichment 17
Fig. 2.1 a The photo and b The schematic diagram of the double-ended open separation
apparatus ‘MTB trap’
magnetic pole of two ferrite magnets, respectively, on opposite sides of the wall of
a microcosm, just above the sediment-water interface. Step 2 magnetic concen-
tration and purification: after 1–2 h of collection, 5 ml of water with accumulated
MTB cells near each magnets were removed by a pipette and transferred to the
MTB trap. In this step, 20–50 microcosms could be simultaneously collected and
pooled. MTB cells in the trap swim along the applied homogeneous magnetic field
to the northern or southern collecting tubes. After 2–5 h collection, visible pellets
of accumulated pure MTB could be seen by the naked eye at the tips of both
collecting tubes (Fig. 2.2).
After MTB collection, carefully disassemble both collecting tubes and centri-
fuge the tubes in a micro centrifuge at the maximum speed (14,500 rpm) for 3 min
Fig. 2.2 Procedures of magnetic collection and purification of environmental MTB cells from
sediment samples
to pellet MTB cells. Carefully remove the supernatant, add 100 ll of sterilized
double-distilled water, gently resuspend the MTB pellet, and mix samples from
two tubes. The enriched MTB cells were then observed under light and trans-
mission electron microscope (TEM). A drop of enrichment (20 ll) was placed on a
glass coverslip to check the presence of MTB using the hanging-drop method
(Greenberg et al. 2005) under a Olympus BX51 research light microscope
(Olympus Optical, Tokyo, Japan) at magnifications of 9400 and 91,000
(Fig. 2.3). For TEM observation, a 20-ll drop of magnetic enrichment was
deposited on a formvar-carbon coated grid and allowed to air dry. The grid was
Fig. 2.3 Schematic diagram of the hanging-drop method

2.2 Novel Apparatus and Strategy for MTB Enrichment 19
Fig. 2.4 Representative transmission electron micrographs of MTB cells in Beijing, China.
Bars = 500 nm
rinsed with sterilized double-distilled water twice and then observed on a JEM-
2100 transmission electron microscope with an accelerating voltage of 200 kV.
Enrichment of enough pure cells is a prerequisite for further diversity, phylo-
genetic, genomic and magnetic analyses of uncultivated MTB. The MTB trap
apparatus and the two-step collection method have the advantages that not only
enrich large amount of MTB cells but also collect both north- and south-seeking
MTB at the same time. Hanging drop observation and TEM analysis have revealed
that contaminant nonmagnetic organisms were virtually absent from the enrich-
ment(\1 %) (Fig. 2.4a–i). The apparatus and the approach developed here are
efficient and easy to manipulate, and thus can be used for various MTB studies.
The MTB trap is also applicable for collecting and purifying uncultivated mag-
netotactic protists (e.g., Bazylinski et al. 2012).
2.3 Specific Primers for Freshwater Alphaproteobacteria

Magnetotactic Cocci
In the present study, we focused on the Alphaproteobacteria magnetotactic cocci

in freshwater environments that form a coherent phylogenetic lineage. These
bacteria are the most common MTB group encountered in nature (Moench and
Konetzka 1978; Spring et al. 1992; Spring et al. 1994; Thornhill et al. 1995; Spring
et al. 1998; Cox et al. 2002; Simmons et al. 2004; Flies et al. 2005b; Flies et al.
2005a; Simmons et al. 2007; Lin et al. 2008; Pan et al. 2008; Lin et al. 2009; Lin
and Pan 2009b). To develop a set of specific primers, nineteen16S rRNA gene
sequences longer than 1,200 bp were compared (Table 2.1). The specific primers
were designed using the Primrose v2.17 program (available at http://www.
bioinformatics-toolkit.org/). The forward primer, FMTCf (50 -TAAAGCCCTTTY
AGTGGGAA-30 ), corresponds to positions 431-450 of Escherichia coli, and the
reverse primer, FMTCr (50 -ACTKCAATCYGAACTGAGACGAGY-30 ), corre-
sponds to positions 1360–1383 of E. coli. The primer FMTCf was a modification
of the primer MCF (Thornhill et al. 1995), which substituted the C at position 12
for Y, thus facilitating targeting more sequences of freshwater magnetotactic cocci.
This primer set could amplify PCR product of approximately 864 bp.
Theoretical evaluation of these primers was tested using the PROBE_MATCH
program of the Ribosomal Database Project II (RDP-II, Release 10, Update 11)
with default parameters (Cole et al. 2003). FMTCf matched with 47 hits, 44 of
which belonged to magnetotactic cocci, the rest were clustered into the Gamma-
proteobacteria. While, FMTCr matched 58 hits, 36 of which were from mag-
netotactic cocci. The others belonged to non-MTB bacteria, such as phyla
Firmicrtes, Acidobacteria, Bacteroidetes, BRC and unclassified Bacteria.
Although each of primers potentially binds to bacteria other than MTB, the
combined use of both primers exclusively targets the freshwater magnetotactic
cocci.
16S rRNA gene of uncultured freshwater Magnetococcus sp. clone 17 (OTU A)
(Lin et al. 2009) was used as template to test the applicability of primers FMTCf
and FMTCr, and to find their optimal PCR condition. PCR reactions in a final
volume of 20 ll were carried out, using 0.2 mM of each of the four dNTPs, 2 mM
MaCl2, 1.25 U Taq DNA polymerase (TaKaRa Bio., Shiga, Japan) with 1 9 Taq
buffer, 8 pmol of each primer and 0.5 ll of template DNA. The PCR processes
were optimized using a T-Gradient thermocycler (Whatman-Biometra, Göttingen,
Germany) using the following conditions: 5 min of initial denaturation at 95 °C,
followed by 30 cycles of 30 s at 94 °C, 30 s at 40–60 °C, and 1 min at 72 °C.
Final extension was carried out at 72 °C for 10 min. All amplification products
were analyzed by gel electrophoresis (0.8 %, w/v). Single bands with expected
size (*864 bp) were successfully amplified at annealing temperatures from 40 to
60 °C (Fig. 2.5a), suggesting that the specificity of primers FMTCf and FMTCr
was not affected in this temperature range. However, the concentration of the PCR
products declined below 48.7 °C (lane 6 in Fig. 2.5a) and above 59.4 °C (lane 11
2.3 Specific Primers for Freshwater Alphaproteobacteria Magnetotactic Cocci 21
Table 2.1 In silico analysis of specific primers compared to published 16S rRNA gene
sequences of freshwater Alphaproteobacteria magnetotactic cocci larger than 1,200 bp
Sequence GenBank Sequence In silico test of primera Reference
name accession length (bp) b
MCF ? MCR FMTCf ? FMTCr
number
CS103 X61605 1,495 + + (Spring et al. 1992)
CS308 X61607 1,494 + +
CS92 X81182 1,498 - + (Spring et al. 1994)
CS81 X81184 1,497 - +
M. coccus X80996 1,496 + +
TB12 X81183 1,493 + +
TB24 X81185 1,494 - -
CF2 AJ863135 1,427 + + (Flies et al. 2005b)
MYG-4 EF370484 1,462 + + (Lin et al. 2008)
MYG-5 EF370485 1,462 + +
MYG-22 EF370486 1,462 + +
MYG-38 EF370487 1,462 + +
YDC-1a FJ667777 1,464 - - (Lin and Pan 2009b)
YDC-1b FJ667778 1,464 - -
Clone 7 EU780674 1,466 - + (Lin et al. 2009)
Clone 10 EU780675 1,462 - +
Clone 17 EU780677 1,462 + +
Clone 29 EU780680 1,464 - -
Clone 37 EU780681 1,466 - +
Reprinted with the permission from Lin and Pan (2009a). Copyright 2009 by Spanish Society for
Microbiology
a
In silico test of primer set: + indicates matches to the sequence in target regions; - indicates
mismatches to the sequence in target regions
b
Primers according to reference (Thornhill et al. 1995)
in Fig. 2.5a), providing empirical annealing limits for these primers. We therefore
chose 50 °C as the annealing temperature for primers FMTCf and FMTCr in the
following experiments.
We further examined the robustness of primers FMTCf and FMTCr using
environmental genomic DNA. Surface sediments (5–10 cm) were collected from
Lake Miyun (40°270 46.0200 N, 116°560 5.8200 E) and Lake Kunming (39°590 38.5200 N,
116°150 49.1400 E) in Beijing, China. Genomic DNA was extracted from 0.5 g (wet
weight) of each sediment using the UltraClean Soil DNA Isolation Kit (MoBio
Laboratories Inc., Carlsbad, USA) according to the manufacturer’s instruction.
Although some nonspecific faint bands of various sizes were noted, PCR products
of expected bands of about 864 bp were obtained from both tested sediment
samples (Fig. 2.5b). The target bands were then cloned and transformed using a
commercially available TOPO TA cloning kit (Invitrogen, San Diego, USA). In
total 29 clones were randomly selected and sequenced using an ABI 3730 genetic
analyzer (Beijing Genomics Institute, Beijing, China). The resulting sequences
were checked for chimera formation with the CHECK_CHIMERA software of the
Fig. 2.5 a PCR amplification at increasing annealing temperatures using primers FMTCf and
FMTCr. The template used in these reactions was the 16S rRNA gene of uncultured freshwater
Magnetococcus sp. clone 17 (OTU A, EU780677). Lane M, size marker; lane 1, annealing
temperature of 40 °C; lane 2, 40.6 °C; lane 3, 41.9 °C; lane 4, 44 °C; lane 5, 46.4 °C; lane 6,
48.7 °C; lane 7, 51.3 °C; lane 8, 53.6 °C; lane 9, 56 °C; lane 10, 58.1 °C; lane 11, 59.4 °C; and
lane 12, 60 °C. b Gel electrophoresis of PCR products from environmental metagenomic DNA
with primers FMTCf and FMTCr. Lane M, size marker; lane 1, Lake Kunming (Beijing, China);
lane 2, Lake Miyun (Beijing, China); and lane 3, PCR negative control. Reprinted with the
permission from Lin and Pan (2009a). Copyright 2009 by Spanish Society for Microbiology
RDP-II (Cole et al. 2003) and the Bellerophon server (Huber et al. 2004). Cloned
sequences were then compared with existing 16S rRNA genes using the GenBank
and RDP-II. The sequences retrieved in this study were deposited in the GenBank
database under accession numbers GQ338449 to GQ338468. It is apparent that the
development of primers FMTCf and FMTCr is successful since 100 % of retrieved
sequences were most related to known freshwater magnetotactic cocci at [95 %
similarity (Fig. 2.6). The sequences and their close neighbors were subsequently
aligned with ClustalW (Thompson et al. 1994), and the phylogenetic tree was
constructed by MEGA 4.0 using a neighbor-joining method with 100 bootstrap-
ping (Tamura et al. 2007). All resulting sequences affiliated with the Alphapro-
teobacteria and the overall similarities of them ranged from 90 to 100 %,
indicating that the primers FMTCf and FMTCr are able to amplify a broader
coverage of freshwater magnetotactic cocci (Fig. 2.6).
The novel specific primers FMTCf and FMTCr developed here provide a
cultivation-independent, reliable and efficient tool to screen environmental sam-
ples for the presence of freshwater magnetotactic cocci. Combined with other
approaches (e.g., magnetic enrichment, transmission electron microscope, fluo-
rescence in situ hybridization, or single-cell analysis), this new primer set will shed
new light on the diversity, distribution, and ecological function of freshwater
magnetotactic cocci. More sequences retrieved by these primers in future may also
be helpful in directing cultivation or identification of novel magnetotactic cocci
from freshwater ecosystems. Furthermore, this new primer set could be used to
detect fossil 16S rRNA gene sequences of freshwater magnetotactic cocci and
therefore may help us better understand paleoenvironmental changes.
2.3 Specific Primers for Freshwater Alphaproteobacteria Magnetotactic Cocci 23
Fig. 2.6 Bootstrapped neighbor-joining phylogenetic tree showing the relationships of the16S
rRNA genes of freshwater Alphaproteobacteria magnetotactic cocci retrieved in this study and
their close relatives. Modified after the permission from Lin and Pan (2009a). Copyright 2009 by
Spanish Society for Microbiology
2.4 In silico Evaluation of Restriction Endonucleases

for RFLP Analysis of MTB
RFLP analysis enables a rapid investigation of microorganism community diver-

sity. The selected restriction endonucleases play a critical role for the analysis
results. Moyer et al. (1996) first systematically evaluated the rationale for selecting
restriction endonucleases and found that the choice of different restriction enzymes
could enhance the information gained from the same bacterial community. In this
study, we focused on the evaluation of appropriate restriction endonucleases that
are efficient in detecting different MTB populations. Twenty-three nearly complete
16S rRNA gene sequences of MTB were selected for in silico analysis, which were
from different types of MTB, including cocci, spirilla, vibrioid and rod-shaped
bacteria (Table 2.2). Multiple sequence alignments were performed by using
Table 2.2 Similarity values of selected MTB 16S rRNA sequences from the Genbank database. Two sequences with the similarity above 98 % belong to
24
the same OTU

OTU Strain Accession no. 1 2 3 4 5 6 7 8 9 10 11
I 1. CF2 AJ863135 100
II 2. CS103 X61605 94.8 100
III 3. CS308 X61607 96.7 94.9 100
IV 4. MRT-81 EF371482 90.6 91.7 90.1 100
IV 5. MRT-82 EF371483 90.7 91.9 90.3 99.9 100
V 6. M-52 EF371485 90.5 90.4 90.7 91.9 92.0 100
VI 7. M-40 EF371486 90.5 90.4 90.4 92.2 92.3 97.9 100
VII 8. M-67 EF371491 91.2 92.2 91.3 90.6 90.8 90.9 91.1 100
VIII 9. MRT-97 EF371493 90.6 91.0 91.0 91.1 91.2 91.0 91.2 91.5 100
IX 10. Mabrj12 Y13215 91.7 92.1 91.5 90.6 90.8 90.9 90.9 96.8 91.5 100
X 11. Mabrj58 Y13211 90.3 90.5 90.8 91.7 91.9 96.3 96.0 91.0 91.6 89.9 100
XI 12. MC-1 NC008576 89.2 89.4 90.0 89.3 89.4 89.7 89.3 90.5 91.2 90.3 89.6
XII 13. MYG-22 EF370486 94.1 96.9 92.9 91.7 91.8 90.0 90.0 92.0 90.8 91.8 90.5
XII 14. MYG-38 EF370487 93.9 96.7 92.7 91.4 91.6 89.8 89.7 91.7 90.6 91.6 90.2
XIII 15. MV-1 L06455 83.5 84.6 84.1 83.4 83.6 82.7 83.3 83.8 83.8 84.2 83.0
XIV 16. RS-1 D43944 82.6 83.0 82.7 82.3 82.4 81.8 81.5 82.3 84.0 82.0 81.3
XV 17. AMB-1 D17514 84.5 84.8 83.8 84.5 84.6 83.5 83.9 83.4 84.6 83.6 83.5
XV 18. CC-26 AB033746 84.7 84.5 83.9 84.3 84.5 83.3 83.7 83.3 84.7 83.6 83.5
XV 19. MGT-1 D17515 84.1 84.4 83.4 84.1 84.3 83.1 83.6 83.0 84.2 83.3 83.1
XVI 20. MS-1 M58171 84.1 84.1 83.5 84.0 84.1 82.8 83.2 82.9 84.0 83.1 82.9
(continued)
2 Enrichment, Purification and Characterization
Table 2.2 (continued)
OTU Strain Accession no. 1 2 3 4 5 6 7 8 9 10 11
XV 21. MSM-6 Y17391 84.8 85.1 84.2 84.8 84.9 83.5 83.9 83.7 84.9 83.9 83.5
XVII 22. MSR-1 Y10109 84.9 85.5 84.2 84.9 85.0 83.9 84.5 84.3 84.6 84.4 83.9
XVIII 23. WM-1 DQ899734 82.8 83.1 82.3 82.8 83.0 81.8 82.2 81.9 82.9 82.2 81.9
OTU Strain Accession no. 12 13 14 15 16 17 18 19 20 21 22 23
I 1. CF2 AJ863135
II 2. CS103 X61605
III 3. CS308 X61607
IV 4. MRT-81 EF371482
IV 5. MRT-82 EF371483
V 6. M-52 EF371485
VI 7. M-40 EF371486
VII 8. M-67 EF371491
VIII 9. MRT-97 EF371493
IX 10. Mabrj12 Y13215
X 11. Mabrj58 Y13211
XI 12. MC-1 NC008576 100
XII 13. MYG-22 EF370486 89.7 100
XII 14. MYG-38 EF370487 89.5 99.8 100
XIII 15. MV-1 L06455 83.1 84.2 84.1 100
XIV 16. RS-1 D43944 82.2 82.5 82.5 80.5 100
XV 17. AMB-1 D17514 82.9 84.8 84.6 87.3 80.1 100
XV 18. CC-26 AB033746 82.8 84.6 84.4 87.3 80.3 98.7 100
XV 19. MGT-1 D17515 82.5 84.4 84.2 86.9 79.9 99.6 98.4 100
XVI 20. MS-1 M58171 82.5 84.1 84.0 87.2 80.0 97.7 97.7 97.4 100
2.4 In silico Evaluation of Restriction Endonucleases for RFLP Analysis of MTB
XV 21. MSM-6 Y17391 83.3 85.0 84.8 87.2 80.5 99.3 99.0 99.0 98.0 100
XVII 22. MSR-1 Y10109 83.2 85.3 85.1 87.5 80.9 95.7 95.2 95.3 94.7 95.8 100
XVIII 23. WM-1 DQ899734 81.3 83.1 82.8 86.0 78.8 97.6 97.2 97.3 96.2 98.0 94.3 100
25
Vector NTI Advance 10.1.1 (Invitrogen, Carlsbad, USA) with default parameters.
The exterior ends of sequences were manually trimmed, and the lengths of size
were *1,350 bp. Since the sequence similarity between two well-studied model
organisms, Magnetospirillum magnetotacticum strain MS-1 and M. magneticum
strain AMB-1, was 97.7 %, here we arbitrarily defined that the identities between
two populations[98 % belonged to the same OTU (Sait et al. 2002). According to
this criterion, 23 selected MTB were affiliated with 18 OTUs (Table 2.2).
Twelve commonly used sets of restriction endonucleases were evaluated here:
RsaI; MspI; AluI plus RsaI; MspI plus HaeIII; MspI plus RsaI; HhaI plus HaeIII;
HinP1I plus MspI; MspI plus HhaI; RsaI plus HindIII; HhaI, RsaI plus HaeIII;
HhaI, RsaI plus BstUI; and AluI, DdeI plus MspI. The restriction site determina-
tion, the size of RFLP fragments and the expected results of analysis via gel
electrophoresis were simulated in silico by using Vector NTI Advance 10.1.1
(Invitrogen, Carlsbad, USA). Restriction fragments shorter than 99 bp were not
considered and fragments that differ about 7 bp or less in sizes were treated as
identical here (Urakawa et al. 1999; Stakenborg et al. 2005). Two parameters were
defined in this study: OTUmin, representing the minimal identity of nucleic acid
sequences between two strains in the same OUT, and OTUmax, reflecting the
maximal identity of nucleic acid sequences between two strains in different OTUs.
Both parameters were utilized to evaluate the resolution of the selected restriction
endonucleases. The ideal values of OTUmin and OTUmax should be identical
with the similarity threshold for different MTB species (i.e. 98 % in this study).
For all selected MTB sequences, the theoretical restriction patterns of different
sets of restriction endonucleases were calculated and performed in silico. Initial
observation revealed that different restriction endonucleases had different power in
the ability to differentiate OTUs of MTB (Table 2.3). For example, MspI plus
HaeIII, AluI, DdeI plus MspI, and MspI plus RsaI were able to detect a large
number of different OTUs (18, 17 and 16 OTUs, respectively), while RsaI, RsaI
plus HindIII, and HhaI, RsaI plus BstUI could only distinguish 12 OTUs. The
average number of restriction fragments per strain for each set of restriction
endonucleases was estimated (Table 2.3). The results showed a range of 4–6
fragments per strain. Additionally, the percentages of successfully identified OTUs
divided by the real number of OTUs (18 OTUs) were presented (Table 2.3). Of 12
sets of restriction endonucleases, AluI, DdeI plus MspI, MspI plus RsaI and MspI
plus HaeIII were most efficacious for identifying corrected OTUs (Table 2.3).
We further compared the identities of nucleic acid sequences among same and
different OTU(s) acquired by the three sets of restriction endonucleases mentioned
above (Table 2.4). The higher OTUmax of MspI plus HaeIII and AluI, DdeI plus
MspI, 99.6 and 99.8 % respectively, meant that using these two sets of restriction
endonucleases might overestimate the number of OTUs, i.e., the retrieved number
of OTUs by these enzymes was more than the real number of OTUs for some
MTB. Correspondingly, lower OTUmin of MspI plus HaeIII (89.4 %) showed that
different strains of MTB might share the same RFLP patterns, which lead to
underestimate of community diversity. However, MspI plus RsaI presented
appropriate values of OTUmin and OTUmax (96.7 and 97.7 %, respectively,
2.4 In silico Evaluation of Restriction Endonucleases for RFLP Analysis of MTB 27
Table 2.3 Summary of in silico RFLP analysis of 23 selected MTB 16S rRNA gene sequences
Restriction Number Mean %Successful OTUs which cannot be
endonuclease of number of affiliationsa differentiated by this set of
detected restriction enzymesb
OTUs fragments
per strain
MspI plus 18 5 77.8 OTU II, XI and XII
HaeIII
AluI, DdeI 17 5 83.3 OTU II and XII; OTU V and VI
plus MspI
MspI plus 16 5 77.8 OTU II and XII; OTU V and VI
RsaI
AluI plus 15 6 66.7 OTU II and XII; OTU VII, IX and X; OTU V
RsaI and VI
RsaI, HhaI 15 5 66.7 OTU VII and IX; OTU V and X; OTU XV
plus and XVIII
HaeIII
MspI 15 4 66.7 OTU II and XII; OTU V and VI; OTU X and
XI
Hinp1I plus 14 4 66.7 OTU II and XII; OTU V, VI and VIII
MspI
MspI plus 14 4 66.7 OTU II and XII; OTU V, VI and VIII
HhaI
HaeIII plus 13 6 55.6 OTU I and III; OTU V, VII, IX and X; OTU
HhaI XV and XVIII
RsaI 12 4 50.0 OTU II, V, VI, X and XII; OTU VII and IX;
RsaI Plus 12 4 50.0 OTU II, V, VI, X and XII; OTU VII and IX;
HindIII OTU XV and XVI
HhaI, RsaI 12 5 38.9 OTU II and XII; OTU VII and IX; OTU V,
plus VI and X; OTU VIII and XI; OTU XV
BstUI and XVI
a
Percentage of successfully identified OTUs divided by the real number of OTUs (18 OTUs,
Table 2.2)
b
Detailed information of OTUs are shown in Table 2.2
Reprinted by permission from Elsevier Science Ltd: Ref. (Lin et al. 2009), copyright 2009
Table 2.4), which were close to the ideal value of 98 %. Thus, MspI plus RsaI
outperformed the others and could better reflect the diversity of selected MTB
sequences.
These results clearly indicate that utilization of more restriction endonucleases
does not increase the selective power for differentiating MTB. For example, MspI
alone is able to detect more OTUs than several sets of two or three restriction
endonucleases (Table 2.3). Although, set of MspI plus HaeIII and set of AluI, DdeI
plus MspI detect more OTUs in this study, partial of them are false OTUs because
the OTUmax of them are very high (99.60 and 99.80 %, Table 2.4). Furthermore,
89.4 % of OTUmin with set of MspI plus HaeIII suggests that this set of enzymes
cannot effectively differentiate selected sequences. Our findings demonstrate that
Table 2.4 Comparison of OTUmin and OTUmax among three sets of restriction endonucleases
Restriction endonucleases OTUmina (%) OTUmaxb (%)
MspI plus HaeIII 89.40 99.60
AluI, DdeI plus MspI 96.90 99.80
MspI plus RsaI 96.70 97.70
a
Minimal identity of nucleic acid sequences between two strains in the same OTU
b
Maximal identity of nucleic acid sequences between two strains in different OTUs
Reprinted by permission from Elsevier Science Ltd: Ref. (Lin et al. 2009), copyright 2009
exclusive use of RFLP method alone to analyze the community structure of MTB
should be treated with care, since this method might bias the diversity of com-
munities by using inappropriate set of enzymes. RsaI plus HindIII and MspI were
previously used to investigate the diversity of MTB in microcosms and marine
samples (Flies et al. 2005a; Pan et al. 2008). However, both of them only detect 50
and 66.7 % corrected OTUs (Table 2.3), which suggests that these two sets of
restriction endonucleases are not appropriate to unravel the real community
structure of uncultivated MTB. According to our in silico analysis, the MspI plus
RsaI RFLP analysis should have a better resolution power to screen the diversity of
MTB. It should be noted that given the faster advances and cheaper price in DNA
sequencing technology, MTB clone libraries might be screened by direct
sequencing in future rather than using traditionally fingerprint methods, such as
RFLP. However, compare to SSU rRNA gene sequencing RFLP could be directly
applied with rRNA PCR products to characterize the changes in MTB commu-
nities across time and space (so-called community RFLP), and further link these
changes with environmental factors.
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Chapter 3
Diversity of Magnetotactic Bacteria
and Its Environmental Implications
3.1 Introduction
Magnetotactic bacteria (MTB) are ubiquitous in aquatic and sedimentary envi-

ronments, where they sometimes can be detected in high abundance (Spring et al.
2000; Amann et al. 2007; Edwards and Bazylinski 2008). MTB play pivotal roles
in biogeochemical cycle of iron and perhaps other nutrients such as sulfur and
nitrogen (Simmons and Edwards 2007a; Faivre and Schüler 2008). It is important
to understand MTB diversity in various environments not only for basic scientific
research, but also the prerequisite for addressing how changes in MTB community
influence functions of ecosystems. In addition, studies of MTB diversity and
phylogenetic analysis may shed light on the origin and evolution of magnetotactic
behavior and microbial biomineralization.
Cultivation-based analysis is a powerful approach to characterize the phylo-
genetic composition and structure of MTB community in nature. Several MTB
strains in the Alphaproteobacteria, Deltaproteobacteria and Gammaproteobacte-
ria have been successfully isolated (Schüler et al. 1999; Amann et al. 2007; Zhu
et al. 2010; Lefèvre et al. 2011b, 2012a, b). However, the majority of MTB
populations in nature are so far uncultivable, with distinct morphologies and
phylogenetic positions that are not represented by their cultivated counterparts.
Thus culture-dependent method alone may bias our view of the true diversity of
MTB. The use of 16S rRNA gene-based molecular approach could improve our
understanding of MTB diversity.
Although studied for several decades, it appears that a significant component of
MTB communities still remain to be described. This is suggested by a number of
newly described populations and the discovery of the unexpected depth of the
molecular phylogeny (Amann et al. 2007; Simmons and Edwards 2007b; Lefèvre
et al. 2011b; Kolinko et al. 2012, 2013). In this chapter, we focused on the MTB
communities in freshwater sites around Beijing, China. The structures of MTB
communities in Lake Miyun and Pond Xiaoyue are investigated by various
approaches, including light and transmission electron microscope observation, 16S
rRNA gene-based clone library, restriction fragment length polymorphism

32 3 Diversity of Magnetotactic Bacteria
(RFLP), fluorescence in situ hybridization (FISH), and phylogenetic analysis. The

spatiotemporal variation of MTB communities from two sampling sites in Lake
Miyun was additionally characterized and compared over a 3-month period. These
results will provide new insights into the diversity and ecology of MTB.
3.2 Materials and Methods
3.2.1 Sampling Sites
Sediment samples were collected from two freshwater sites, Lake Miyun (Lin et al.
2009) and Pond Xiaoyue (Lin and Pan 2009) in Beijing, China. At each sampling
site, sediments from the top 5–20 cm were collected and aliquoted into 4–14
plastic bottles (600 ml) covered with about 100 ml of in situ water. The climate in
Beijing is temperate, with an average annual temperature of 10–12 °C and annual
rainfall values of 600 mm. Lake Miyun is located approximately 80 km northeast
of Beijing, which has a water body exceeding 1 billion m3 and a maximum water
depth of 40 m. The pH of water from the sampling sites in Lake Miyun is
determined to be 7.4–7.6. Pond Xiaoyue is located in the Yuan Dynasty Capital
City Wall Relics (Yuandadu) Park. The pH and temperature of sediments were 7.7
and 16.8 °C during the sampling time, respectively. The salinities of these two
locations are all below 0.5 parts per thousands (ppt), indicating that they are
freshwater.
To investigate the spatiotemporal variation of MTB communities, two micro-
cosms (MY8 and MY11) collected from two separate sites in the southern margin
of Lake Miyun were analyzed (Fig. 3.1). Approximately 10 L of surface sediments
from each site were collected and transferred to 20-L aquaria and overlaid with
lake water (microcosms) in lab. The microcosms were loosely covered and stored
in dim light at room temperature without disturbance. For both microcosms, MTB
were separately collected in 2009 at February (MY8a and MY11a), March (MY8b
and MY11b) and April (MY8c and MY11c).
3.2.2 Physical–Chemical Analyses
The oxygen concentrations of surface sediment were determined using an HQ40d

Oxygen Meter (HACH, Loveland, Colorado, USA). The pore water was separated
from the sediments by centrifugation at 1,0009g for 20 min and was further
filtered through 0.45 lm membrane filters. The pH and salinity of pore water were
measured using a Mettler Toledo Delta 320 pH meter (Mettler-Toledo, Greifensee,
Switzerland) and a Salinity Meter AZ-8371 (Instrument Corp., China), respec-
tively. Physical–chemical analyses of various anions and major cautions in Lake
3.2 Materials and Methods 33
Fig. 3.1 Locations of two sampling sites (MY8 and MY11) in the Lake Miyun near Beijing,
China
Miyun were conducted at the Analytical Laboratory Beijing Research Institute of

Uranium Geology, using a Dionex-500 chromatograph (BioPortfolio) and 785
DMP Titrino (Metrohm AG, Herisau, Switzerland). The concentrations of total
iron of pore water were measured using HR-ICP-MS (Finnigan Mat, San Jose,
USA).
3.2.3 Magnetic Collection of MTB
The existence of MTB in all sediment samples was checked through the ‘‘hanging-
drop’’ method (see Chap. 2). MTB in the sediment were magnetically enriched
using the MTB trap and the two-step magnetic enrichment strategy as described in
Chap. 2. Briefly, both north- and south-seeking MTB cells were first magnetically
collected from sediments in each microcosm by using magnets. Collected MTB
were then purified from nonmagnetic contaminations by using the MTB trap. MTB
in the trap were collected and purified under a homogenous magnetic field (5 times
greater than the Earth’s magnetic field).
3.2.4 Transmission Electron Microscope Observation
A drop of magnetically enriched MTB (10–20 ll) was deposited onto a copper
grid covered by a carbon-coated forever film. The cells were allowed to settle on
the grid for 1 h. Excess liquid was removed with a filter paper. All grids were
rinsed at least twice with distilled water before TEM observation. In case of
negative staining, wet grids were treated with a drop of negative staining solution
(1.5 % uranyl acetate) for 1 min. Examinations were performed with a FEI Tecnai
20 transmission electron microscope with an accelerating voltage of 120 kV, or a
JEM-2100 transmission electron microscope with an accelerating voltage of
200 kV.
3.2.5 PCR, Cloning and DNA Sequencing
Nearly complete 16S rRNA genes were amplified directly from magnetically
enriched MTB cells by using the universal bacterial primers 27F (50 -AGA-
GTTTGATCCTGGCTCAG-30 ) and 1492R (50 -GGTTACCTTGTTACGACTT-30 )
(Lane 1991). PCR was performed using a T-Gradient thermocycler (Whatman
Biometra, Göttingen, Germany). The PCR amplification program consisted of
5 min at 95 °C; 30 cycles of 1.5 min at 92 °C, 1 min at 50 °C, and 2 min at 72 °C;
and a final extension at 72 °C for 10 min. The PCR products were purified by
0.8 % (wt/vol) agarose gel electrophoresis. The purified PCR products were cloned
into the pMD19-T vector and chemically competent DH5a cells (both from Ta-
KaRa, Dalian, China) by following the manufacturer’s instructions. The trans-
formed cells were incubated overnight on Luria–Bertani agar plates with
ampicillin. Clones were randomly selected and were either directly sequenced
using an ABI 3730 genetic analyzer (Beijing Genomics Institute, Beijing, China),
or screened by RFLP before complete sequencing as described in Chap. 2. For
RFLP analysis, transformed clones were screened for 16S rRNA gene inserts and
amplified with the primers specific for the pMD19-T vector. Ten microliters of
PCR products were digested with 0.1 U of MspI plus 0.1 U of RsaI (MBI Fer-
mentas, Vilnius, Lithuania) overnight at 37 °C. The resulting fragments were
separated by gel electrophoresis in 3 % (wt/vol) agarose in 1 9 TAE buffer. RFLP
patterns were compared and different operational taxonomic units (OTUs) were
defined.
3.2.6 Phylogenetic Analysis
Sequences retrieved in this chapter were first checked for chimera formation using
the CHECK_CHIMERA software of the Ribosomal Database Project II (RDP-II)
(Cole et al. 2009) or the Bellerophon server (DeSantis et al. 2006) before being
compared with the GenBank database (Benson et al. 2005) by using the BLAST
program to search for related sequences with high similarities. These sequences
and their close relatives were then aligned using CLUSTALW software
(Thompson et al. 1994) and corrected by manual inspection. Phylogenetic trees
were subsequently constructed with MEGA version 4.0 by using the neighbor-
joining method (Tamura et al. 2007). Bootstrap analysis for 100 replicates was
performed for the estimation of reproducibility of tree topologies.
3.2.7 Fluorescence In Situ Hybridization
An oligonucleotide probe (YDC69, 50 -CGCCAGCACCTTTCGGCCTGCTGC-30 )

that specifically targets all 16S rRNA gene sequences from Pond Xiaoyue in
Yuandadu Park was designed. The specificity was evaluated by using the PRO-
BE_MATCH program in the RDP-II (Cole et al. 2003). Bacterial universal probe
EUB338 (50 -GCTGCCTCCCGTAGGAGT-30 ) was used as control (Amann et al.
1990). Probes YDC69 and EUB338 were synthesized and fluorescently labeled
with hydrophilic sulfoindocyanine dye Cy3 and fluorescein phosphoramidite FAM
at the 50 end, respectively. Escherichia coli cells were used as a negative control.
FISH analysis was carried out according to Pernthaler et al. (2001) with some
modifications. Briefly, 20 ll of MTB enrichment from Yuandadu Park was placed
directly on an agarose-coated (0.02 %) welled slide and air-dried. The sample was
fixed with 2 % paraformaldehyde in phosphate-buffered saline (PBS) at room
temperature for 30 min and was then dehydrated for 3 min each in 50, 80, and
100 % ethanol, respectively. In situ hybridization was performed at 46 °C for 3 h
in hybridization buffer with 20 % formamide. Then, 1 ll probe (50 ng/ll) was
mixed with 9 ll hybridization buffer and applied for in situ hybridization. A
fluorescent microscope (Olympus Optical, Tokyo, Japan) was used to record
optical sections.
3.2.8 Statistical Analyses
For spatiotemporal variation analysis, the unweighted UniFrac algorithm (Lozu-

pone et al. 2006, 2007) was used to compare MTB communities from microcosms
MY8 and MY11 from Lake Miyun. Unweighted UniFrac considers the phyloge-
netic distance between taxa and reflects the occurrence of distinct microbial lin-
eages among different communities based on phylogenetic information. For the
UniFrac analysis, phylogenetic tree of 16S rRNA gene sequences of MTB was
generated by PHYLIP program (http://evolution.genetics.washington.edu/
phylip.html) using the neighbor-joining method and exported as NEWICH for-
mat, which was submitted to the UniFrac web interface (http://bmf2.colorado.edu/
unifrac/index.psp) with the environment file. Principal Coordinates Analyses
(PCoA) and Jackknife Environment Clusters were performed to separate MTB
communities (Lozupone et al. 2007). Jackknife Environment Clusters tree was
projected using the TreeView software (http://taxonomy.zoology.gla.ac.uk/rod/
treeview.html). Pearson’s Correlations were performed to correlate the physical–
chemical factors with the main component of genetic variability of MTB (PC1
factor of PCoA).
3.3 Results
3.3.1 MTB Cell Morphologies and Magnetosomes
MTB cells were detected in all sampling locations under light microscope
(Fig. 3.2), with cell densities as high as approximately 103–107 cells/ml. These
bacteria were collected and purified through the MTB trap by using the two-step
magnetic enrichment strategy. A great morphological variability, such as cocci,
vibrios, rods and spirilla, was observed (as shown in Fig. 2.4). The shapes of
magnetosomes found in these MTB were diverse, including cuboidal, elongated
prismatic and rod-shaped magnetosomes (Fig. 3.3). Energy-dispersive X-ray
analysis has revealed that these magnetosomes were composed of iron oxide
(Fig. 3.3). Large rod-shaped magnetotactic bacteria (5–10 lm) were found in
some microcosms from Lake Miyun, which was morphologically similar to the
previously described ‘‘Candidatus Magnetobacterium bavaricum’’ found in the
Lake Chiemsee in Germany (Spring et al. 1993). These large rods contain up to
600 bullet-shaped magnetosomes in a single cell (Fig. 2.4g). Besides these large
rods, bullet-shaped magnetosomes were also found in some small rods and
peculiar cocci (Fig. 2.4h and i).
3.3 Results 37
Fig. 3.2 Micrographs of enriched magnetotactic bacteria from (a) Lake Miyun and (b) Yuandadu
Park as observed under a light microscope. Bars = 10 lm. b is reprinted with the permission
from Ref. Lin and Pan (2009). Copyright 2009 by American Society for Microbiology
Fig. 3.3 a–d Representative transmission electron micrographs of different morphologies of

magnetosomes. Bars = 100 nm. e EDX spectrum obtained from magnetosome crystals
3.3.2 Phylogenetic Diversity of MTB
In order to assess their phylogenetic diversity, 16S rRNA gene sequences were
determined for MTB communities. Low quality sequences, potential chimeras and
sequences potentially from non-MTB contaminations were removed from further
analyses. Phylogenetic analysis has revealed that all identified MTB are affiliated
within the Alphaproteobacteria and the phylum Nitrospirae (Fig. 3.4).
For MTB in Lake Miyun, the majority of retrieved sequences belonged to
Alphaproteobacteria magnetotactic cocci, while 3 OTUs (samples 11, 20 and 28)
were found to affiliate with the phylum Nitrospirae, which were clustered into two
phylogenetic groups (Fig. 3.4). OTU sample 11 had a maximum identity of
Fig. 3.4 Neighbor-joining phylogenetic tree of representative MTB 16S rRNA genes retrieved
from Lake Miyun and Pond Xiaoyue with related sequences from the GenBank database
97.8 % with ‘‘Ca. Magnetobacterium bavaricum’’ (Spring et al. 1993); while,

OTUs sample 20 and 28 were 97.8 and 95.3 % similar to another Nitrospirae
magnetotactic bacterium MHB-1 (Flies et al. 2005a), respectively.
MTB populations in Pond Xiaoyue are affiliated with magnetotactic cocci in the
Alphaproteobacteria. All MTB sequences from Pond Xiaoyue in Yuandadu Park
were very similar ([99 % similarity) and likely from a single MTB population.
The closest cultivated relative of this bacterium was magnetotactic coccoid strain
MC-1 (88 % sequence identity) (Meldrum et al. 1993). Although from a single
population, the number of magnetosome chains in MTB cells from Pond Xiaoyue
changed from 2 to 4 (Fig. 3.5a and b). In addition, about 26.2 % characterized
cells contained clustered magnetosomes adjacent to chains, and these disorganized
magnetosomes were distinctly smaller than their counterparts aligned in chains
(Fig. 3.5c and d).
3.3.3 FISH Analysis of MTB Population from Yuandadu

Park
Since MTB community from Yuandadu Park is dominant of one population, FISH
analysis was performed to confirm whether this sequence is truly from identified
magnetotactic coccus. A specific oligonucleotide probe, YDC69, was designed to
be complementary to the retrieved sequences. Comparative sequence analysis
using the PROBE_MATCH function in the RDP-II showed that this probe had at
3.3 Results 39
Fig. 3.5 a–c Representative transmission electron micrographs of MTB cells from Pond
Xiaoyue, Yuandadu Park. Bars = 400 nm. d Size distributions of magnetosomes in chains and
disorganized magnetosomes in enriched magnetotactic cocci. Modified after the permis-
sion from Ref. Lin and Pan (2009). Copyright 2009 by American Society for Microbiology
least two mismatches with all other 16S rRNA genes. Fluorescent microscopy
revealed a strong signal for all collected magnetotactic cocci after hybridization
with the bacterial universal probe EUB338 and the specific probe YDC69
(Fig. 3.6). This result suggested that the enriched magnetotactic cocci from Yu-
andadu Park belonged to a homogeneous population.
3.3.4 Spatiotemporal Variation of MTB Communities

from Lake Miyun
MTB communities from two separate sites (MY8 and MY11) in Lake Miyun were
compared over a 3 month period (in total six samples) (Lin and Pan 2010). The
geochemical properties of pore water of six samples from two microcosms were
summarized in Table 3.1. The pH of each microcosm ranged from 7.35 to 7.64,
Fig. 3.6 a–c In situ hybridization of magnetically enriched coccoid cells, and d–f negative
control of clones of E. coli with the inserted 16S rRNA genes of the uncultured Magnetococcus
sp. clone 37. Reprinted with the permission from Ref. Lin and Pan (2009). Copyright 2009
by American Society for Microbiology
suggesting that all samples were circumneutral or slight alkaline. The temperature
ranged from 15 to 17 °C. The concentrations of oxygen in surface sediments in
which MTB were enriched were 0.29 and 0.10 mg/L, respectively, for microcosms
MY8 and MY11 in April, indicating the microaerobic conditions. Overall, the
concentrations of most anions and cations of MY8 decreased over time, yet the
corresponding changes of MY11 were rather irregular. MY8a had higher con-
centrations of Cl- (18.8 lg/mL), Na+ (24.5 lg/mL), K+ (4.25 lg/mL), Mg2+
(20.5 lg/mL) and iron (626 lg/L) than the other samples; whereas, MY11c highly
enriched in SO42- (128 lg/mL) and Ca2+ (42.4 lg/mL). The concentrations of
NO3- of MY8 (0.39 to 0.74 lg/mL) were higher than that of MY11 (B0.24 lg/
mL). The concentrations of F- were relatively constant for all samples.
MTB communities were diverse and a total of 13 different OTUs were iden-
tified in all samples (Fig. 3.7). Five OTUs were found in MY8 (libraries MY8a,
MY8b and MY8c). The average distance between them was 15 %, and all
sequences were B94 % identical. All OTUs except OTU 1 were in the Alpha-
proteobacteria and most related to magnetotactic cocci. OTU 2 was the closest
relative to magnetococcus clone CF22 recovered from freshwater habitat in
3.3 Results
Table 3.1 Chemistry of pore water collected from microcosms MY-8 and MY-11 at different time
pH Temp. Oxygen F- Cl- NO3- SO42- Na+ K+ Mg2+ Ca2+ Total iron
(°C) (mg/L) (lg/mL) (lg/mL) (lg/mL) (lg/mL) (lg/mL) (lg/mL) (lg/mL) (lg/mL) (lg/L)
MY8a (February) 7.35 15 NMa 0.33 18.8 0.74 91.4 24.5 4.52 20.5 39 626
MY8b (March) 7.64 17 NM 0.29 6.67 0.56 41.1 12.3 2.14 11.7 26.2 42.1
MY8c (April) 7.59 17 0.29 0.26 4.95 0.39 72.9 5.8 1.53 7 17.5 BDb
MY11a (February) 7.44 15 NM 0.35 6.27 0 97.3 8.3 3.18 15.9 34.4 9.39
MY11b (March) 7.61 17 NM 0.35 7.25 0 63.4 4.7 2.48 10.9 25.9 BD
MY11c (April) 7.43 17 0.1 0.33 8.45 0.24 128 8.3 3.32 18.1 42.4 BD
a
NM not measured
b
BD below detection
Reproduced from Ref. Lin and Pan (2010) by permission of John Wiley & Sons Ltd
41
Fig. 3.7 Neighbor-joining phylogenetic tree of MTB 16S rRNA genes retrieved from samples MY8
and MY11. Modified after Ref. Lin and Pan (2010) by permission of John Wiley & Sons Ltd
northern Germany (Flies et al. 2005a) with 97.25 % similarity. OTU 8 and 50 were
96.64 and 97.38 %, respectively, similar to magnetococcus clone CF2 which was
detected in lake ‘‘Waller See’’ in Bremen (Flies et al. 2005a). OTU 29 was found
to share high similarity (98.36 %) with magnetococcus clone MYG-22 which was
recovered from the same place (Lin et al. 2008). Phylogenetic analysis of OTU 1
had shown that it clustered within the phylum Nitrospirae. Eight OTUs were
identified from microcosm MY11 (libraries MY11a, MY11b and MY11c), which
were all affiliated with the Alphaproteobacteria and were B98 % similarity
(Fig. 3.7). OTUs 13 and 14 had 97.47 and 96.92 % sequence identities, respec-
tively, with magnetotactic coccus CS308 (Spring et al. 1992). OTUs 52 and 53
were closely related to magnetococcus clone CF23 (98.76 and 97.74 %, respec-
tively) (Flies et al. 2005a). OTUs 15, 17 and 21 were 96.85, 89.04 and 97.06 %
identical with magnetococcus clones MYG-22, XSE-42 and CF2, respectively.
Furthermore, OTU 51 was found to share high identity (99.66 %) to magneto-
coccus clone sample 17 (see Fig. 3.4). Although the majority of clones in
microcosm MY8 (64.29 %) and all clones in microcosm MY11 belonged to the
Alphaproteobacteria magnetotactic cocci, no identical OTU was found between
them. The most related OTUs from MY8 to MY11 were OTU 29 and OTU 51 with
98.89 % similarity. Other OTUs from MY8 were B97 % similarity with that from
MY11 (Fig. 3.7).
MTB communities from each site showed a clear temporal variation. For MY8,
OTU 1 in the phylum Nitrospirae was dominant in February (MY8a, 84.21 %), but
its relative abundance dramatically decreased in March (MY8b) and April (MY8c)
3.3 Results 43
Fig. 3.8 The distributions of OTUs in each clone library from samples MY8 and MY11 collected at
different times. Modified after Ref. Lin and Pan (2010) by permission of John Wiley & Sons Ltd
(Fig. 3.8). Although, OTU 8 only comprised 5.26 % in MY8a, it significantly

increased to 79.17 and 77.78 % in MY8b and MY8c, respectively. For MY11,
OTU 14 was the dominant group in February (MY11a, 52.94 %), but it was not
observed in MY11b and MY11c (Fig. 3.8). In contrast, OTU 51, which was not
found in MY11a, became the most dominant OTU in March (MY11b, 82.60 %)
and April (MY11c, 80.95 %). OTU 17 was relatively evenly distributed over time
(4.35–14.29 %). OTU 15 was detected only in MY11a (5.88 %) and MY11b
(4.35 %), while OTU 53 was only found in MY11b (4.35 %) and MY11c
(4.76 %).
All six MTB communities were further compared using the unweighted Uni-
Frac analysis. The PCoA plot clearly showed that MTB communities clustered by
microcosms rather than collection time (Fig. 3.9a). Samples from microcosm
MY11 clustered together to the left along PC1 that accounted for 66.7 % of the
variation, while samples from microcosm MY8 grouped to the right. This result
Fig. 3.9 a Principal coordinate analysis and b Jackknife Environment Clusters of MTB
sequences among six clone libraries as calculated using unweighted UniFrac analysis. Circles
represent Jackknife support at the nodes in (b), solid circles, [99.9 %; open circle, [70.0 %.
Modified after Ref. Lin and Pan (2010) by permission of John Wiley & Sons Ltd
was further supported by Jackknife Environment Clusters with high Jackknife

values (Fig. 3.9b). Pearson’s correlation analysis between the unweighted PC1
factors and the measured physical–chemical variables demonstrated that the for-
mer significantly correlated with the concentrations of NO3- (Table 3.2,
P \ 0.05).
3.4 Discussion
In this chapter, cocci in the Alphaproteobacteria are the dominant groups of MTB
in Lake Miyun and Pond Xiaoyue (Yuandadu Park), which is similar to previous
reports (Spring et al. 1994, 1998; Simmons et al. 2004; Flies et al. 2005a). It is
interesting to find a homogeneous taxonomic MTB population in Yuandadu Park.
Competitive interactions between bacteria is thought to shape a dominant diversity
pattern in particular locations (Zhou et al. 2002), which may suggest that the MTB
population in Pond Xiaoyue outperform other MTB group through competition. It
was reported by Pan et al. (2008) that a single population of ovioid-coccoid MTB
was dominant in the low tide zone of the East China Sea near Qingdao due to the
specific habitat selection. This may be true for Pond Xiaoyue sample as well. The
sediments in this pond are from time to time exposed to air because of pumping
and irrigation.
In spite of a homogenous taxonomic population, it is noted that the number of
magnetosome chains in cells from Yuandadu Park changed from 2 to 4. This
perhaps indicates that no strict correlation exists between magnetosome arrange-
ments and phylogenetic positions of magnetotactic cocci (Spring et al. 1994; Cox
3.4 Discussion
Table 3.2 Pearson’s correlations showing relationships between physical–chemical parameters and PC1 factors that resulted from principal coordinates
analyses in Fig. 3.9
Pearson’s correlation
(significant P)
pH Temp. F- Cl- NO3- SO42- Na+ K+ Mg2+ Ca2+
PC1 factors 0.060 (0.909) -0.115 (0.829) -0.0761 (0.079) 0.293 (0.573) 0.824 (0.044)* -0.406 (0.425) 0.510 (0.301) -0.120 (0.820) -0.207 (0.694) -0.400 (0.432)
*
Correlation is significant at the 0.05 level
Reproduced from Ref. Lin and Pan (2010) by permission of John Wiley & Sons Ltd.
45
et al. 2002). In addition, the disorganized magnetosomes in these cells are

apparently smaller than that arranged in chain structure. One explanation is that
those disorganized smaller magnetosome are early-stage non-mature ones, as
growing they may gradually form chains. If this is true, it implies that the
mechanism involving in the spatial arrangement of magnetosomes in these bacteria
may be different from that proposed for cultivated Magnetospirillum strains AMB-
1 and MSR-1, where empty magnetosome vesicles first assembled into a chain
structure and then the magnetite crystals start to biomineralize in the chain-
arranged vesicles (Komeili 2007; Schüler 2008). However, other possibilities
cannot be excluded, such as the empty magnetosome vesicles are aligned into
multiple chains and the formation of magnetosomes occurs randomly, or cells with
different morphologies are from different microenvironments.
Based on the recommendations of Murray and Schleifer (1994), and Murray
and Stackebrandt (1995), the sequence-based potential new microorganisms could
be recorded by a ‘‘Candidatus’’ designation. The magnetotactic cocci enriched
from Yuandadu Park are therefore designated ‘‘Candidatus Magnetococcus yu-
andaducum’’. The bacteria are magnetically collected from freshwater pond sed-
iment. Cells are coccoid with a diameter between 1.5 and 2.5 lm. They contain
31–62 truncated hexahedral prisms of magnetite magnetosomes (shape factor
0.64), which are arranged into 2–4 chains per cell that sometimes appear partially
disorganized. The cells are north-seeking MTB with a motility speed
of *112 lm/s under 4 Oe magnetic fields. The bacteria belong to a new genus
within the Alphaproteobacteria. The assignment is based on the 16S rRNA gene
sequence; on GenBank accession numbers FJ667777 and FJ667778, and on the
16S rRNA-targeted oligonucleotide probe YDC69 (50 - CGCCAG-
CACCTTTCGGCCTGCTGC-30 ). The organism has yet to be cultivated.
Three OTUs affiliated within the phylum Nitrospirae are identified in Lake
Miyun. MTB in the phylum Nitrospirae were originally described as comprising
only ‘‘Ca. Magnetobacterium bavaricum’’ (Spring et al. 1993). However, due to
efforts made by several labs, it has been recently recognized that Nitrospirae MTB
contain members showing a broad range of morphological properties with phy-
logenetic diversity. For example, a small magnetotactic rod (MHB-1) from Bre-
men in Germany is identified as Nitrospirae bacterium that is phylogenetically
similar to ‘‘Ca. Magnetobacterium bavaricum’’ based on 16S rRNA gene sequence
and FISH analysis (Flies et al. 2005a). In addition, a population of moderately
thermophilic MTB within the Nitrospirae phylum was discovered in a brackish hot
spring in Nevada, USA, which contained a single chain of magnetosomes per cell
(Lefèvre et al. 2010). Recently, a coccoid bacterium was identified to contain
multiple chains of bullet-shaped magnetosomes and represents a novel lineage of
Nitrospirae MTB (Lefèvre et al. 2011a). In this study, those three OTUs could be
cluster into two phylogenetic groups, which has expanded the distribution and
diversity of MTB in this phylum. These results, together with previously reports,
suggest that Nitrospirae MTB may ubiquitously distribute in various environments
and their phylogenetic diversity is more complicated than previously expected.
3.4 Discussion 47
Because few efforts have been made to explore the distribution and ecology of
MTB in nature, our knowledge on spatiotemporal variations of MTB communities
is still scarce. In this chapter, the temporal changes of MTB communities in
microcosms from two different locations in Lake Miyun were compared. The
diversity of MTB in each microcosm is not stable within a 3-month incubation,
and shows rapid short-term variations (Fig. 3.8). The changes are prominent from
February to March for both microcosms. Considering their incubation in the
laboratory without disturbance, these results suggest that MTB populations may be
very sensitive to the imperceptible changes in microenvironments. Our results are
consistent with the previous report which showed a dynamic change of MTB
communities in a microcosm during a long-term incubation (Flies et al. 2005b).
Therefore, MTB communities are microenvironment-sensitive and may act as
potential proxies for changes of ecology and climate.
UniFrac analysis clearly shows that these MTB communities cluster by
microcosms rather than by collection time, indicating that phylogenetic discrep-
ancy of MTB communities collected from distinct microcosms exceeds the tem-
poral variation in each microcosm. This observation suggests a potential adaption
of different MTB populations to their respective microenvironments. A significant
correlation between phylogenetic distance of MTB communities and nitrate con-
centrations is noted here (Table 3.2), suggesting that nitrate may play a role in
MTB community variation here. Petermann and Bleil (1993) reported that nitrate
or other nitrous oxides could be reduced by most MTB in deep marine environ-
ments and might contribute to their vertical distribution. This speculation was
recently supported by observations that the majority of cultivated MTB could
utilize nitrous compounds as terminal electron acceptors for respiration (Flies et al.
2005b). Similar situation is expected for Nitrospirae MTB as well, because the
phylogenetic nonmagnetic relatives of these MTB in Nitrospirae phylum are
nitrite-oxidizing bacteria that can oxidize nitrite to nitrate in environments (Daims
et al. 2001). Together, these results suggest that nitrate may play an important role
in the occurrence and distribution of MTB lineages in distinct microenvironments.
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Chapter 4
Metagenomic Analysis of Uncultivated
Magnetotactic Bacteria Within
the Phylum Nitrospirae
4.1 Introduction
MTB within the phylum Nitrospirae were first identified through a 16S rRNA
gene-based survey in Lake Chiemsee near Munich, Germany. A giant rod-shaped
bacterium named ‘‘Candidatus Magnetobacterium bavaricum’’ is found to syn-
thesize hundreds of bullet-shaped magnetite magnetosomes arranged in several
braid-like bundles of chains within cells (Vali et al. 1987). Several years later, the
phylogenetic position of this MTB was identified within the phylum Nitrospirae
by a molecular approach and fluorescence in situ hybridization (FISH) technique
(Spring et al. 1993). This rod-shaped bacterium is one of the largest MTB,
approximately 8–10 lm in length and 1.5–2 lm in width (Spring et al. 1993). The
number of ‘‘Ca. Magnetobacterium bavaricum’’ is up to 7 9 105 cells per cm3 in
the first few millimeters of Lake Chiemsee sediment, which represents approxi-
mately 30 % of the microbial biomass and may play a dominant ecological role in
this sediment layer (Spring et al. 1993). Recent magnetic measurements of pure
cells and bulk sediments from Lake Chiemsee suggest that magnetosomes of ‘‘Ca.
Magnetobacterium bavaricum’’ and other MTB populations are reliable remanence
carriers in surface sediments (Pan et al. 2005a, b).
MTB that are phylogenetically similar to ‘‘Ca. Magnetobacterium bavaricum’’
(here called ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria) are not confined to
Lake Chiemsee. In a recent study, a slowly moving magnetotactic rod, designated
MHB-1, is detected in the sediments of Waller See near Bremen (Flies et al. 2005).
The 16S rRNA gene of this bacterium is 91 % similar to ‘‘Ca. Magnetobacterium
bavaricum’’. Unlike its giant counterpart, MHB-1 is much smaller (1.5–3 lm in
length) and only contains 30–60 magnetite magnetosomes per cell (Flies et al.
2005). Besides ‘‘Ca. Magnetobacterium bavaricum’’ and MHB-1, several different
types of MTB affiliated within the phylum Nitrospirae have also been discovered
from sediments in southern Nevada (Lefèvre et al. 2011) and Great Boiling
Springs, USA (Lefèvre et al. 2010). In addition, bacteria that are morphologically
similar to ‘‘Ca. Magnetobacterium bavaricum’’ and other Nitrospirae MTB have
also been found from a world-wide sediments, including Brazil (Lins et al. 2000),

52 4 Metagenomic Analysis of Uncultivated Magnetotactic
France (Isambert et al. 2007), Japan (Thornhill et al. 1994), and USA (Mann et al.
1987), but their phylogenetic affiliations have not been identified yet. These results
suggest that MTB of the phylum Nitrospirae are likely globally distributed.
However, since attempts to isolate these MTB in pure culture have not been
successful, our present knowledge of the diversity and phylogenetic breadth of the
Nitrospirae MTB is still limited.
MTB in the phylum Nitrospirae have attracted great interests because of their
peculiar phylogenetic position, unique products of mineralization and potential
contributions to local or global iron and sulfur cycling (Spring et al. 1993; Flies
et al. 2005; Pan et al. 2005a). However, despite intense efforts, all ‘‘Ca. Magne-
tobacterium bavaricum’’-like bacteria have resisted isolation in pure culture, and
thus little information about their metabolic potential and functional traits has been
acquired to date (Jogler et al. 2010, 2011). Recently developed metagenomic
approach provides an effective way to investigate the genetic information about
uncultivated bacteria directly from various environments (Wommack et al. 2008).
Construction of a metagenomic library and subsequent screening for rRNA gene
and linked genomic sequences was first reported by Stein et al. (1996), and was
successfully performed on various environmental samples, which indeed provided
increasing information and new insights into the microbial ecology (Quaiser et al.
2002; Liles et al. 2003; Moreira et al. 2004; Elshahed et al. 2005; Fieseler et al.
2006; Xu et al. 2007; Massana et al. 2008).
As mentioned in Chap. 3, we have analyzed the diversity of MTB in Lake
Miyun near Beijing, China, and determined at least three operational taxonomic
units (OTUs) of MTB that were in the phylum Nitrospirae and showed
91.8–98.4 % similarity to ‘‘Ca. Magnetobacterium bavaricum’’. To elucidate more
phylogenetic and genetic information of these uncultivated bacteria, here we have
attempted to perform a combination of cultivation-independent molecular
approaches, including metagenomic analysis and FISH. We present five fosmid
sequences (representing a total of 172 kb of genomic information) isolated from
uncultivated members of MTB in the phylum Nitrospirae and identify two novel
magnetotactic bacterial populations affiliated within this phylum.
4.2.1 Sampling and Collection of MTB
Sediments at the topmost 5–10 cm from Lake Miyun in the northeast of Beijing
were sampled (Lin et al. 2011). The collected surface sediments were subsequently
divided into 600 ml plastic bottles (microcosms) in the laboratory, overlaid
with *100-ml lake water and incubated at room temperature under dim condition.
Sediment in each microcosm was checked periodically using hanging-drop method
(for details see Chap. 2) under Bacteriodrome (Pan et al. 2009) for the existence of
‘‘Ca. Magnetobacterium bavaricum’’-like bacteria (Pan et al. 2005a). Nineteen of

more than 100 microcosms were found rich in ‘‘Ca. Magnetobacterium bavari-
cum’’-like bacteria and therefore were selected for further analyses. MTB in the
sediment were magnetically enriched using the MTB trap and the two-step mag-
netic enrichment strategy as described in Chap. 2. The enrichments were centri-
fuged and stored at -20 °C.
4.2.2 Preparation of High Molecular DNA for Library

Construction
Metagenomic DNA of enriched MTB was extracted according to Zhou and col-
leagues (Zhou et al. 1996) with some modifications. Briefly, MTB enrichment was
mixed with 675 ll of DNA extraction buffer (100 mM Tris–HCl [pH 8.0],
100 mM sodium EDTA [pH8.0], 100 mM sodium phosphate [pH 8.0], 1.5 M
NaCl, 1 % CTAB) and 2.5 ll of Proteinase K (20 mg/ml) by shaking at 225 rpm
at 37 °C for 30 min. Then, 75 ll of 20 % SDS was added and the sample was
incubated at 65 °C for 2 h with gentle inversions every 20 min. After incubation,
the sample was mixed with 750 ll of Chloroform/Isoamyl alcohol (24:1, vol/vol)
and centrifuged at 6,000 9 g for 20 min at room temperature. The supernant
aqueous phase was transferred to a new 1.5-ml Eppendorf tube and combined with
350 ll of Isopropanol, incubated at room temperature for 1 h. The aqueous phase
was subsequently removed by centrifugation at the maximum speed for 20 min at
room temperature. Crude nucleic acids was washed with cold 70 % ethanol and
resuspended in sterile deionized water in a final volume of 50 ll.
4.2.3 Fosmid Library Construction, Screening

and Sequencing
Extracted DNA was end-repaired by End-Repair Enzyme Mix (Epicenter, Madi-

son, USA). DNA fragments bigger than 25 kb were selected by running 1 % low
melting point agarose gel electrophoresis at 30–35 V overnight. The gel slice
containing 25–40 kb DNA fragments was recovered from gel by using GELase
Enzyme preparation (Epicenter, Madison, USA). Then the purified DNA was
blunt-ligated to pCC1FOS Vector and the ligation mixture was applied for in vitro
packaging into lambda phages and for infection of EPI300-T1 Escherichia coli
according to the manufacturer’s protocol (CopyControl Fosmid Library Production
Kit, Epicenter, Madison, USA). The colonies were transferred to 96-well micro-
titre plates containing 200 ll of LB medium with 12.5 lg/ml chloramphenicol and
incubated at 37 °C for 20–24 h. The average insert size was calculated from 10
randomly selected clones after BamHI restriction analyses.
In order to identify fosmids containing 16S rRNA genes of MTB in the phylum
Nitrospirae, high throughput screens of fosmid library clone pools were performed
by PCR using the bacterial universal primer 27F (50 -AGAGTTTGATCCTG
GCTCAG-30 ) (Lin et al. 2009) as forward primer and a ‘‘Ca. Magnetobacterium
bavaricum’’ specific probe (50 -GCCATCCCCTCGCTTACT-30 , named BaP in this
study) (Spring et al. 1993) as reverse primer. The PCR reaction was set in 50-ll
volume containing 0.2 mM of each of the four dNTPs, 1.25 U Taq DNA poly-
merase (TaKaRa Ex Taq, Daliang, China) with 1 9 Taq buffer and 8 pmol of each
primer. Each template clone was first cultivated in liquid Luria–Bertani medium
with ampicillin at 37 °C overnight. Then the culture was boiled in 10 min and 1 ll
of the boiled culture was applied in PCR reaction. The PCR processes were
optimized using a T-Gradient thermocycler (Whatman-Biometra, Göttingen,
Germany) using the following conditions: 5 min of initial denaturation at 95 °C,
followed by 30 cycles of 1.5 min at 92 °C, 1 min at 45–60 °C, and 2 min at 72 °C.
Final extension was carried out at 72 °C for 10 min. Amplified product was
analyzed by gel electrophoresis in agarose gel (0.8 %, w/v) stained with ethidium
bromide. The plates, which had positive PCR product, were further amplified by
columns and rows. PCR products of positive clones were transformed and
sequenced for phylogenetic identification. Five fosmid clones containing ‘‘Ca.
Magnetobacterium bavaricum’’-like bacterial 16S rRNA genes were identified and
chosen for complete shotgun sequencing.
4.2.4 Fosmids Annotation and Phylogenetic Analyses
Open reading frame (ORF) prediction of fosmid sequences was performed with
ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). All predicted ORFs
were characterized using BLASTp program and the Clusters of Orthologous
Groups of proteins (COGs) on the NCBI database (Altschul et al. 1997; Tatusov
et al. 2000). The tRNA gene was identified with the tRNAscan-SE Search Server
(http://lowelab.cse.ucsc.edu/tRNAscan-SE/). All fosmids were compared to each
other via the BLASTn Suite by choosing the ‘‘Align two or more sequences’’
option (Tatusova and Madden 1999; Benson et al. 2005).
Complete 16S rRNA genes from all fosmids and complete 23S rRNA genes
from 4 fosmids (except fosmid MY4-5C) were BLAST against the NCBI GenBank
database to identify similar sequences. Then the CLUSTALW (Thompson et al.
1994) was used for alignments and the MEGA software version 4.0 was applied
for phylogenetic analyses based on the neighbor-joining method (Tamura et al.
2007).
4.2.5 Oligonucleotide Probes and Fluorescence In Situ

Hybridization
The following rRNA-targeted oligonucleotides were used in this study: (1)

EUB338 (Amann et al. 1990); (2) BaP (Spring et al. 1993); (3) Mba1000 (50 -TTA
TCCTTTCGGACTCCTACCACT-30 ); and (4) Mba1019 (50 -CTCCCTTACGG-
GAAAGTCGCTCA-30 ). Probes Mba1000 and Mba1019 were manually designed
for ‘‘Ca. Magnetobacterium bavaricum’’-like bacterial sequences retrieved in this
study. The specificities of these probes were tested by submission to the PRO-
BE_MATCH algorithm of the Ribosomal Database Project II (RDP-II) (Cole et al.
2009). All probes were synthesized and fluorescently labeled with a hydrophilic
sulfoindocyanine dye CY3 or fluorescein phosphoramidite (FAM) at the 50 end.
In situ hybridizations were performed according to Pernthaler et al. (2001).
Briefly, the enriched bacteria were fixed in 2 % paraformaldehyde solution at 4 °C
for 3–4 h. 10 ll of the fixed cell suspension was spot on a gelatin-coated slide, air-
dried, and subsequently dehydrated in 50, 80 and 100 % ethanol for 3 min at each
concentration. The hybridization was performed at 46 °C for 3 h and the final
probe concentration was 5 ng/ll. Hybridization was followed by a stringent
washing at 48 °C for 25 min. The slide was rinsed with distilled water and air-
dried. Then the sample was counterstained with 10 ll of 4,6-diamidino-2-phen-
ylindole (DAPI, 1 lg/ml) for 3 min. The slide was examined with an Olympus
epifluorescence microscope (Olympus Optical, Tokyo, Japan).
4.2.6 Transmission Electron Microscope (TEM)
A drop (10 ll) of magnetic enrichment was placed on a formvar-coated grid and
air-dried. The grid was then rinsed with distilled water twice and air-dried.
Electron micrographs were done on a FEI Tecnai 20 with accelerating voltage
120 kV.
4.2.7 Nucleotide Sequence Accession Numbers
The fosmid sequences retrieved in this study were deposited in the GenBank
database under accession numbers HM454279 to HM454283.
4.3 Results
4.3.1 Library Construction and Screening
MTB trap approach allowed collecting pure MTB from many microcosms at one
time. By using this approach, large amounts of MTB cells were acquired which were
sufficient for genomic DNA extraction and fosmid library construction. ‘‘Ca.
Magnetobacterium bavaricum’’ specific probe (50 -GCCATCCCCTCGCTTACT-30 ,
named BaP in this study) was designed by Spring et al. (1993) previously. PRO-
BE_MATCH analyses performed on BaP against the 16S rRNA gene sequences
available in the RDP-II databases (Cole et al. 2009) showed that this probe could also
specifically detect all published ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria
(till October, 2008). Thus we attempted to combine the BaP with one universal
bacterial primer 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) to specifically
amplified the ‘‘Ca. Magnetobacterium bavaricum’’-like bacterial sequences from
the metagenomic library constructed here. Five fosmids (MY4-5C, MY3-11A,
MY2-1F, MY3-5B, and MY2-3C) showed positive signals, the PCR products of
which were subsequently sequenced for verification and were finally chosen for
complete sequencing. We additionally screened the library by using specific primers
for Alphaproteobacteria magnetotactic cocci (Thornhill et al. 1995), which were
normally found to coexist with ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria in
Lake Miyun sample (Lin et al. 2009). However, no positive clone was detected,
which might suggest that most of fosmid clones in this metagenomic library con-
tained the genomic fragments from ‘‘Ca. Magnetobacterium bavaricum’’-like
bacteria.
4.3.2 Analyses of Five Fosmid Clones
All fosmids except MY4-5C contained typical bacterial-like rRNA operon orga-
nizations that consisted of 16S rRNA gene, tRNA Ile, tRNA Ala, 23S rRNA gene
and 5S rRNA gene (Fig. 4.1). The sizes of the fosmid inserts were between
30,297 bp and 41,797 bp, with the average G ? C content varying from 45.1 to
50.2 % (Table 4.1). Between 23 and 35 predicted ORFs were identified from each
fosmids (Fig. 4.1). The majority of ORFs on all fosmids had most similar ortho-
logues from the Deltaproteobacteria and the phylum Nitrospirae (Table 4.1). The
number of ORFs with no significant similarities to protein encoding genes in the
NCBI database (designated hypothetical proteins in Table 4.1) ranged from 2 to 9
per fosmids. Between 0 and 9 genes per clone were identified to potentially encode
transposases. BLAST and COG analyses of other ORFs revealed that the predicted
gene products are mostly involved in cellular processes and metabolism (Fig. 4.1
and Supplementary Table S1-5 in Appendix). For example, one predicted protein
encoded on fosmid MY3-5B was found to be a polysulphide reductase NrfD
4.3 Results 57
Fig. 4.1 Organizations of predicted genes located on ‘‘Ca. Magnetobacterium bavaricum’’-like

fosmids MY4–5C, MY3–11A, MY2–1F, MY3–5B and MY2–3C, and the fosmid fragment isolated
from ‘‘Ca. Magnetobacterium bavaricum’’ (Jogler et al. 2010). Modified after the permission
from Ref. Lin et al. (2011). Copyright 2011 by American Society for Microbiology
Table 4.1 General genomic features of the fosmids MY2-1F, MY3-11A, MY4-5C, MY2-3C and
MY3-5B
Feature MY4–5C MY3–11A MY2–1F MY3–5B MY2–3C
Size (bp) 35,154 34,185 30,297 41,797 30,357
G ? C content (%) 45.1 47.5 47.3 50.0 50.2
Number of predicted ORFs 35 23 23 32 28
Number of tRNA genes 1 2 2 3 3
Number of hypothetical proteins 9 2 2 4 5
Number of transposases 9 1 1 2 0
Number of proteins with most similar 5 3 4 7 5
ortholog in Nitrospirae
Number of proteins with most similar 8 8 7 8 5
ortholog in Deltaproteobacteria
Reprinted with the permission from Ref. Lin et al. (2011). Copyright 2011 by American Society
for Microbiology
(Fig. 4.1), an integral transmembrane protein that was thought to interact with
menaquinone during electron transfer, suggesting a potential anaerobic respiratory
system of this bacterium (Jormakka et al. 2008; Simon and Kern 2008). Another
gene encoding a highly conserved selenophosphate synthetase (SelD)was found
adjacent to the 16S rRNA gene on fosmid fragments MY4-5C, MY3-11A, MY2-
1F and MY3-5B (Fig. 4.1), which generates the selenium donor for selenocysteine
biosynthesis in bacteria and suggests the trait of selenium utilization (Romero et al.
2005). Furthermore, we found four genes encoding putative fimbrial proteins and
type IV pilins on fosmid MY3-11A (Fig. 4.1), which might involve in pili bio-
synthesis (Hobbs and Mattick 1993).
Phylogenetic analysis with the 16S rRNA genes has revealed that the affiliations
of these fosmids are in the Nitrospirae phylum (Fig. 4.2). 16S rRNA genes
identities of these five fosmids ranged between 92.2 and 99.9 %. Fosmid clones
MY2-1F, MY3-11A and MY4-5C were [99 % identical to ‘‘Ca. Magnetobacte-
rium bavaricum’’-like bacteria sample 20 found in Lake Miyun (Chap. 3). While,
the 16S rRNA genes of fosmids MY2-3C and MY3-5B showed 97.6 and 98.5 %
identity to ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria sample 11 (Chap. 3)
and MHB-1 (Flies et al. 2005), respectively. Results from 23S rRNA genes also
indicated that these fosmids were clustered within the Nitrospirae phylum and
were most similar to ‘‘Ca. Magnetobacterium bavaricum’’ and Thermodesulf-
ovibrio yellowstonii DSM 11347 (Fig. 4.2). Phylogenetic trees of 16S rRNA genes
were calculated by using maximum-parsimony and maximum-likelihood methods
(data not shown), which are identical in topology to the neighbor-joining tree
(Fig. 4.2). MTB of the Nitrospirae phylum within this study formed two distin-
guishable groups (groups 1 and 2) defined by nearly 5 % sequence divergence and
high bootstrap values (Fig. 4.2). Group 1 contained fosmid MY2-3C, while group
2 included fosmids MY2-1F, MY3-5B, MY3-11A and MY4-5C. This result is
corroborated by the phylogenetic analysis of 23S rRNA genes, which revealed a
congruent branching pattern (Fig. 4.2).
Clones MY2-1F and MY3-11A contained an overlapping region of 14 507 bp
with only two nucleotides mismatches and identical 16S/23S/5S rRNA operons
(Fig. 4.1), indicating that these two fosmids might be from the same species,
which could be assembled into a contig of about 50 kb, representing so far the
largest continuous genomic stretch determined from the Nitrospirae MTB. The
genomic organizations, however, appeared very divergent on other clones,
including MY4-5C whose 16S rRNA gene was 99.98 % identical to MY2-1F and
MY3-11A.
Fig. 4.2 Neighbor-joining phylogenetic trees of 16S and 23S rRNA genes encoded on the
fosmids compared to other related bacteria. Sequences determined in this study are written in
blue. GenBank accession numbers are given in parentheses. Modified after the permission from
Ref. Lin et al. (2011). Copyright 2011 by American Society for Microbiology
4.3 Results 59
4.3.3 In Situ Characterization of ‘‘Ca. Magnetobacterium

bavaricum’’-Like Bacteria
Two probes (Mba1000 and Mba1019) specific for each group were developed for
in situ characterization of MTB in this study. Most collected MTB cells detectable
with probe EUB338 could simultaneously be visualized with ‘‘Ca. Magnetobac-
terium bavaricum’’-like bacteria specific probe BaP, including giant rod-shaped
bacteria and a population of small coccoid-to-ovoid bacteria (Fig. 4.3). However,
only rod-shaped bacteria were detected if we hybridized the sample with probe
Mba1000; whereas, probe Mba1019 was able to exclusively reveal the coccoid-to-
ovoid bacteria (Fig. 4.3). It should be noted that a few of coccoid bacteria could
not be hybridized by either probes BaP, Mba1000 or Mba1019. We attributed
these non-targeted bacteria to the Alphaproteobacteria MTB which often coex-
isted with ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria in Lake Miyun as
revealed by Chap. 3 and by transmission electron micrograph analysis in this study
(data not shown). Transmission electron micrograph of the giant bacteria showed
that they were morphologically similar to ‘‘Ca. Magnetobacterium bavaricum’’
detected in Lake Chiemsee (Fig. 4.3). While the coccoid-to-ovoid bacteria are
smaller than the rod-shaped bacteria (1–2 lm in length), which contained 50–80
bullet-shaped magnetosomes arranged in 4–5 braid-like bundles of chains
(Fig. 4.3).
4.4 Discussion
Since most MTB observed in nature cannot be cultivated by using standard

approaches, molecular phylogenetic studies have opened new opportunities to
exploit their diversity. MTB that are phenotype or genotype similar to ‘‘Ca.
Magnetobacterium bavaricum’’ are likely much more diverse and widespread than
previously suspected (Lins et al. 2000; Flies et al. 2005; Isambert et al. 2007; Nash
2008). However, due to the cultivation of these bacteria is intractable, besides 16S
rRNA genes, little genomic information about them is known (Jogler et al. 2010,
2011). In this study, we have combined the metagenomic analysis and fluorescence
in situ hybridization to investigate the natural MTB belonging to the phylum
Nitrospirae in Lake Miyun.
We have constructed a fosmid library that contains fractions of genomes of
‘‘Ca. Magnetobacterium bavaricum’’-like bacteria from Lake Miyun sediments.
Five fosmids containing 16S rRNA genes of ‘‘Ca. Magnetobacterium bavaricum’’-
like bacteria are detected and are subsequently completely sequenced. All fosmids
are most close to MTB in the phylum Nitrospirae based on 16S rRNA genes as
well as 23S rRNA genes (Fig. 4.2). All fosmids except MY4-5C have a typical
rRNA operon structure for bacteria, with complete 16S rRNA gene, tRNA Ile,
tRNA Ala, 23S rRNA gene and 5S rRNA gene adjacent to each other. In addition,
Fig. 4.3 Fluorescence in situ hybridization (a–i) and transmission electron micrographs (j and
k) of ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria from the Lake Miyun sediments. (a–
c) the same microscopic field is shown in (a) after staining with DAPI, (b) after hybridization
with 50 -FAM-labelled bacterial universal probe EUB338, and (c) after hybridization with 50 -Cy3-
labelled probe BaP specific for all ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria; d–f the
same microscopic field is shown in (d) after staining with DAPI, e after hybridization with 50 -
FAM-labelled bacterial universal probe EUB338, and (f) after hybridization with 50 -Cy3-labelled
probe Mba1000 specific for group 1 in ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria; only
the giant rod-shaped bacteria (j) hybridized with the specific probe Mba1000, whereas the
accompanying cells were not stained; (g–i) the same microscopic field is shown in (g) after
staining with DAPI, h after hybridization with 50 -FAM-labelled bacterial universal probe
EUB338, and (i) after hybridization with 50 -Cy3-labelled probe Mba1019 specific for group 2 in
‘‘Ca. Magnetobacterium bavaricum’’-like bacteria; only the coccoid-to-ovoid bacteria containing
bullet-shaped magnetosomes (k) were stained with the specific probe Mba1019, the arrows
indicated the giant rod-shaped cells as negative control. The bars in (a) to (i) indicated a size of
50 lm. Reprinted with the permission from ref. (Lin et al. 2011). Copyright 2011 by American
Society for Microbiology
the G ? C contents are quite similar among these five fosmids (45.1–50.2 %). In
spite of phylogenetic similarity, the genomic organization of the regions sur-
rounding the rRNA operons appears very divergent (Fig. 4.1). The genomic het-
erogeneity of close related microorganisms was previously detected in planktonic
4.4 Discussion 61
crenarchaeotes (Beja et al. 2002) and Acidobacteria in soils (Quaiser et al. 2003).
The poor conservation may suggest that multiple rearrangements occurred in these
regions (Lopez-Garcia et al. 2004). While we cannot exclude the possibility that
the genome of ‘‘Ca. Magnetobacterium bavaricum’’-like bacterium may contain
more than one 16S/23S/5S rRNA genes.
The deduced ORFs acquired here are identified to involve in diverse functional
categories based on their distribution in different COG clusters (Supplementary
Table S1-5 in Appendix). Although the current-known closely related sequenced
relative of Nitrospirae MTB, based on 16S and 23S rRNA genes, is Thermodes-
ulfovibrio yellowstonii DSM 11347, a thermophilic sulfate-reducing bacterium
affiliated within phylum Nitrospirae (accession number: NC_011296) (Henry et al.
1994), much more ORFs on the five fosmids had the most similar orthologues from
the Deltaproteobacteria (36 ORFs in total) than in the phylum Nitrospirae (24
ORFs in total) (Table 4.1). This result suggests that MTB in the phylum Nitros-
pirae may have a specific relationship with the Deltaproteobacteria. Alternatively,
this may also due to more genomes from the Deltaproteobacteria are available in
public database than that from the phylum Nitrospirae. Our results presented here
have additionally shown that the genomic information of ‘‘Ca. Magnetobacterium
bavaricum’’-like bacteria are distinct with that of T. yellowstonii DSM 11347. For
example, the five fosmids show higher G ? C content than the 34 % of T. yel-
lowstonii DSM 11347. Additionally, in the fosmids, 16S, 23S and 5S rRNA genes
are organized in one operon, which, however, is different from T. yellowstonii
DSM 11347 whose 16S and 23S rRNA genes arrange together, but the 5S rRNA
gene locates elsewhere in the genome. In addition, no tRNA gene was identified
between 16S and 23S rRNA genes in T. yellowstonii DSM 11347 (based on the
information from http://www.ncbi.nlm.nih.gov/nuccore/206889172).
Phylogenetic trees of the publicly available 16S rRNA sequences related to the
‘‘Ca. Magnetobacterium bavaricum’’-like bacteria are calculated by using maxi-
mum-parsimony and maximum-likelihood methods (data not shown), which are
identical in topology to the neighbor-joining tree (Fig. 4.2). It is clearly shown that
‘‘Ca. Magnetobacterium bavaricum’’-like bacteria retrieved in this study consist of
two distinguishable groups defined by nearly 5 % sequence divergence and high
bootstrap values (Fig. 4.2). Two oligonucleotide probes targeting 16S rRNA genes
of defined groups were developed. Probe Mba1000 designed to detect group 1 is
fully complementary to 16S rRNA sequences in this group. This probe exclusively
stains a giant rod-shaped bacterium that has typical morphology same to ‘‘Ca.
Magnetobacterium bavaricum’’ (Fig. 4.3), indicating that this bacterium is a
member of group 1. Probe Mba1019 is specific for group 2 except a single mis-
match with MY3-5Bsequence. By using this probe, a novel, as-yet-uncultured
coccoid-to-ovoid bacterium that are 1–2 lm long and 0.5–1 lm in diameter and
containing 4–5 braid-like bundles of chains of bullet-shaped magnetosomes is
identified (Fig. 4.3). Both of these two bacteria were detected in Chap. 3, the
corresponding phylogenetic positions have now been identified. So far, all iden-
tified ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria contain bullet-shaped
magnetite magnetosomes, which supports the hypothesis that formation of these
magnetosomes may be a common trait of MTB in the phylum Nitrospirae (Flies

et al. 2005). However, it should be noted that not all MTB contained such type of
magnetosomes belong to ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria. For
example, some multicellular magnetotactic prokaryotes can mineralize magnetite
in bullet-shaped magnetosomes, yet they are affiliated to the Deltaproteobacteria
(Lins et al. 2007).
4.5 Conclusions
The survey of phylogenetically linked genomic sequences from a metagenomic

library as well as fluorescence in situ hybridization provide a strong molecular tool
to study the diversity and genomic information of uncultivated MTB in the phylum
Nitrospirae. We have shown analyses of five fragments of genomes from ‘‘Ca.
Magnetobacterium bavaricum’’-like bacteria in Lake Miyun. Complete rRNA
operon in each fosmid allows a high resolution of phylogenetic analysis. A
comparison of ORFs organization in the fosmids shows a variable genomic
structure, which may due to the rearrangements of these regions or the existence of
multiple rRNA operons within the chromosome. In situ characterizations have
revealed two groups of ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria in Lake
Miyun, including a rod-shaped bacterium morphologically similar to ‘‘Ca.
Magnetobacterium bavaricum’’ and a new member of coccoid-to-ovoid bacterium
containing several rows of bullet-shaped magnetosomes, which expands our
knowledge of the diversity of these bacteria. All together, a combination of me-
tagenomic analysis and in situ hybridization gives us novel insights into the
genome of ‘‘Ca. Magnetobacterium bavaricum’’-like bacteria and opens a new
route for exploration of these, and perhaps other uncultivated MTB.
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Chapter 5
Magnetic Properties of Uncultivated
5.1 Introduction
Magnetite is a stable carrier of natural remanent magnetization in sedimentary

rocks and sediments. Studies of magnetite preserved in sediments provide novel
knowledge in paleomagnetic directions and paleointensity of the geomagnetic
field. However, our understanding of magnetic properties of ultrafine-grained
magnetite particles is still very poor. Recent discovery of bacterial magnetite, such
as magnetosomes in magnetotactic bacteria (MTB), provides a unique opportunity
for studying the formation, transportation and preservation of ultrafine-grained
magnetite and its magnetic properties in natural biogeochemical cycles (Egli 2004;
Pan et al. 2004; Muxworthy and Williams 2009).
Magnetite magnetosomes are potentially useful in bio- and nano-technological
applications because of their narrow sizes and uniform morphologies (Lang et al.
2007; Staniland et al. 2008; Xie et al. 2009; Yoshino et al. 2010; Alphandéry et al.
2011). Fully understanding their magnetic characteristics will have an impact on
future applications of magnetosomes. In addition, putative MTB original magnetite
has been discovered in the Martian meteorite ALH84001, which provides the
probable evidence of ancient life on Mars (McKay et al. 1996). The origin of these
magnetic minerals is still under a heated debate, and a variety of techniques have
been applied to determine its origin (e.g. Thomas-Keprta et al. 2001; Barber and
Scott 2002; Golden et al. 2004; Weiss et al. 2004a; Thomas-Keprta et al. 2009;
Jimenez-Lopez et al. 2010; Martel et al. 2012). Magnetic measurement is a fast and
nondestructive method that only needs small amount of samples. Therefore,
understanding the magnetic properties of magnetosomes in MTB will provide clues
to extraterrestrial probable biogenic magnetic materials (Weiss et al. 2004a, b).
In spite of their wide distribution, MTB are intractable and so far only a few of
them are grown in pure culture, which makes our knowledge of the general
magnetic properties of MTB far from complete. Although several studies on
magnetic properties of cultivated MTB are presented (Moskowitz 1988; Mosko-
witz et al. 1989; Moskowitz et al. 1993; Weiss et al. 2004b; Kopp et al. 2006;
Pósfai et al. 2006; Prozorov et al. 2007; Fischer et al. 2008), further information on

68 5 Magnetic Properties of Uncultivated Magnetotactic Bacteria
uncultivated MTB, especially those with multiple chains of magnetosomes that are
commonly encountered in natural sediments, are needed, because nearly all cur-
rently cultivable MTB only harbor single magnetosomes chain. In this chapter, we
investigate the uncultivated magnetotactic bacteria with multiple magnetosome
chains from two freshwater sites, Pond Xiaoyue in Yuan Dynasty Capital City
Wall Relics (Yuandadu) Park and Lake Kunming in Beijing, China, aiming to
characterize the room- and low-temperature magnetic properties of these pure
natural MTB cells. The results acquired here are additionally compared with
magnetic properties of pure MTB samples reported in the literature.
5.2.1 Sample Sites and MTB Collection
Surface sediments (5–10 cm) were collected from Pond Xiaoyue in Yuandadu
Park (Lin and Pan 2009) and Lake Kunming as described in Chaps. 2 and 3,
respectively, and divided into 600-ml plastic bottles covered with about 100-ml
lake water. The hanging-drop method (see Chap. 2 for detail) was used to check
the existence of MTB in each bottle. Pure MTB cells were magnetically enriched
using the MTB trap and the two-step magnetic enrichment strategy as described in
Chap. 2. The purified MTB cells were washed by sterilized distilled water twice
and resuspend in sterilized distilled water.
5.2.2 Transmission Electron Microscopy Observation
A drop of MTB enrichment (20 ll) from each enriched MTB sample was
deposited on formvar/carbon-coated copper grid and allowed to air dry. Exami-
nation was performed with a FEI Tecnai 20 transmission electron microscope with
an accelerating voltage 120 kV.
5.2.3 Magnetic Measurements of MTB
MTB enrichments from Yuandadu Park (named YDC) and Lake Kunming (named
KM) were placed into gelatin capsules, respectively, and dried overnight, which
were then immediately performed on several types of magnetic measurements.
Isothermal remanent magnetization (IRM) was measured using an Alternating
Gradient Force Magnetometer Model MicroMag 2900 (Princeton Measurements
Corporation, Princeton, USA). MTB samples were first demagnetized and then
given a stepwise IRM up to 100 mT in a direct field. IRM curves were normalized
to the maximum saturation IRM for each sample. The back-field demagnetization
curve was transformed as [1 ? IRM(-H)/SIRM]/2.
Hysteresis loop and the first-order reversal curve (FORC) analyses of MTB
samples were measured on an Alternating Gradient Force Magnetometer Model
MicroMag 2900 (Princeton Measurements Corporation, Princeton, USA). Hys-
teresis loop was measured on 0–1 T, which gave saturation magnetization (Ms),
saturation remanence (Mrs) and coercive force (Bc). Coercivity of remanence (Bcr)
was acquired by measuring the back-field demagnetization curve by applying
static field up to -300 mT. For FORC analysis, MTB sample was first saturated in
1 T, and then the field was reversed to a number of negative fields and subse-
quently returned to the positive saturation. These processes generated a number of
curves that were then transformed into contour plots named FORC diagrams. This
diagram is thought to represent coercivity as well as grain size range along the
horizontal axis, and magnetic interaction along vertical axis (Roberts et al. 2000;
Chen et al. 2007).
Low-temperature magnetic characteristics were measured on a Magnetic
Property Measurement System (Quantum Design, San Diego, USA). The samples
were first cooled to 5 K in a zero field environment and then saturated by applying
a 2.5 T of magnetic field. After applied for 60 s, the field was removed and the
thermal demagnetization curve (ZFC) was measured from 5 to 300 K during zero
field. The sample was then cooled from 300 to 5 K in the presence of a 2.5 T field.
After keeping sample at 5 K in a 2.5 T for 60 s, the field was turned off and the
thermal demagnetization curve (FC) was measured. The first derivatives (dM/dT)
of FC were calculated in order to better identify changes during warming pro-
cesses. Here, we defined the Verwey transition temperature (Tv) was at which the
dM/dT is minimum. The d ratio, which reflected the amount of remanence lost
during warming through the Verwey transition, was calculated from FC and ZFC
data based on Moskowitz et al. (1993).
5.3 Results
5.3.1 Room-Temperature Properties
IRM curves of samples YDC and KM were saturated at a field of 100 mT

(Fig. 5.1), indicating that MTB cells in both samples contained magnetosomes
with low coercivities. The Bcr of samples YDC and KM were 50.2 mT and 46.3
mT, respectively. The acquisition and demagnetization curves for each sample
were nearly symmetrical, and the Wohlfarth-Cisowski test (Cisowski 1981)
revealed that values of crossover point Rdf (df refers to the DC field) for both
samples were less than 0.5 (0.41 for sample YDC and 0.44 for sample KM,
Fig. 5.1). The Wohlfarth-Cisowski test is normally used to characterize magnetic
interactions among magnetic particles, and the R-value should be 0.5 for non-
interacting uniaxial single-domain (SD) particles.
At room temperature, hysteresis loops of both samples showed a pot-bellied
shape and were saturated at a field of 100–200 mT (Fig. 5.2), suggesting that a
single magnetic component of ferromagnetic minerals contributed to the rema-
nence. The values of Bc of samples YDC and KM are 41 and 42.2 mT, respec-
tively. The remanence ratio Mrs/Ms was 0.49 and the coercivity ratio Bcr/Bc was
1.22 for sample YDC, and these ratios were 0.55 and 1.1, respectively, for sample
KM (Fig. 5.2). These parameters suggested that magnetosomes in these two
samples were probably SD ferromagnetic minerals.
The FORC diagrams of both samples spread along Hc = 0 axis with a distri-
bution along the Hu axis (Fig. 5.3). The FORC distribution of sample KM had a
smaller vertical spread compared to the FORC of sample YDC. Both contours
closed and peaked on the Hu = 0 axis.
Fig. 5.1 Room-temperature

IRM acquisition (blue lines)
and demagnetization (red
lines) curves of samples YDC
and KM
5.3 Results 71
Fig. 5.2 Room-temperature

hysteresis loops of samples
YDC and KM. Ms, Mrs, Bc,
and Bcr represent the
saturation magnetization,
saturation remanence,
coercive force, and coercivity
of remanence, respectively.
Modified after the permission
from Ref. (Lin and Pan
2009). Copyright 2009 by
American Society for
Microbiology
5.3.2 Low-Temperature Properties
The FC and ZFC curves of two MTB samples were presented in Fig. 5.4. SIRM
curves of both FC and ZFC decreased as a function of increasing temperature. The
Tv of samples YDC and KM were 108 and 104 K, respectively, which were within
the range of Tv measured from other strains of MTB with magnetite magnetosomes
(86–117 K) but were lower than that of stoichiometric magnetite (120–125 K)
(Moskowitz et al. 1993; Pan et al. 2005; Pósfai et al. 2006; Prozorov et al. 2007).
The FC and ZFC curves also experienced a less distinct drop around 10 K for both
samples, which perhaps attributed to the nonstoichiomentry of magnetite mag-
netosomes (Pan et al. 2005). Above Tv, the magnetization curves presented rela-
tively flat, indicating SD-sized magnetite were formed by bacteria in these
samples. These low-temperature results were consistent with results of hysteresis
and FORC data. dFC and dZFC defined by Moskowitz et al. (1993) could quantify
the divergences of FC and ZFC below and above the Tv. The ratio between them
(dFC/dZFC, the Moskowitz test) was found sensitive to the presence of
Fig. 5.3 First-order reversal

curve (FORC) diagrams of
samples YDC and KM. The
smoothing factors of FORC
diagrams is 4 for YDC and 1
for KM. Modified after the
permission from Ref. (Lin
and Pan 2009). Copyright
2009 by American Society
for Microbiology
magnetosomes chain structure, as dFC/dZFC of MTB cells were normally bigger

than 2 (Moskowitz et al. 1993; Moskowitz et al. 2008). In this study, the dFC/dZFC
of YDC and KM samples were 2.03 and 3.06, respectively.
5.4 Discussion
Day plot analysis is a useful approach to characterize the domain states of mag-
netite-dominant magnetic particles. In Day plot, magnetic particles with SD
behavior are normally defined by Mrs/Ms C 0.5 and Bcr/Bc B 2.0 (Dunlop 2002a,
b). As shown in Fig. 5.5, both MTB samples YDC and KM fall near the SD box,
suggesting that magnetosomes in these samples represent SD behaviors. These
results are consistent with TEM observations that sizes of magnetosomes in these
MTB cells are between 30 nm and 120 nm (Fig. 5.6). The results presented here
are comparable with previous results from cultivated and uncultivated MTB
samples (Fig. 5.5). However, due to high interactions between magnetosomes
extracted from cells, extracted magnetosomes normally show reduced Mrs/Ms and
increased Bcr/Bc ratios, which result in them falling in the PSD box in the Day plot
(Fig. 5.5) (Moskowitz et al. 1993; Pan et al. 2005).
5.4 Discussion 73
Fig. 5.4 Low-temperature

demagnetization behaviors of
MTB samples YDC and KM.
DFC, the first derivatives of
the FC curve (dashed line).
TV, the Verwey transition
temperature. Modified after
the permission from Ref. (Lin
and Pan 2009). Copyright
2009 by American Society
for Microbiology
Fig. 5.5 Day plot for

samples YDC and KM, data
from literature (Moskowitz
et al. 1993; Pan et al. 2005;
Lippert and Zachos 2007)
Magnetic properties of MTB samples could be affected by magnetostatic

interactions between magnetosomes. Cells with multiple magnetosomes chains
may have stronger interactions than those with single chain structure. Wohlfarth-
Cisowski test that compares the IRM acquisition and demagnetization behavior is
able to characterize magnetostatic interactions between magnetic minerals. For
non-interacting uniaxial SD particles, the R-value of Wohlfarth-Cisowski test
Fig. 5.6 a Transmission electron micrograph of MTB cells from sample YDC, and b transmis-
sion electron micrographs of MTB cells from sample KM. Bars = 500 nm
should be 0.5. The R-values for samples YDC and KM are 0.41–0.44 which are
lower than 0.5, indicating moderate magnetostatic interactions in these MTB
samples. It used to be suggested that the interaction among magnetic particles
might reduce Mrs/Ms and increase Bcr/Bc (Cisowski 1981; Dankers 1981; Sprowl
1990). However, this is not the case for the YDC and KM samples compared with
the MTB strains harbored single chain (for example, MTB strains MS-1, AMB-1,
MSR-1 and MV-1 in Table 5.1, and Fig. 5.5). This result may suggest that the
hysteresis parameters are not sensitive enough to characterize the magnetostatic
interactions among magnetosomes chains in MTB cells. Comparisons of magnetic
results from MTB samples with multiple chains (e.g., YDC, KM, MYR-1, P2, and
P3 in Table 5.1) and MTB with single chain structure imply that interactions
among multiple chains were not significantly affected the ratios of Mrs/Ms and Bcr/
Bc (Pan et al. 2005). It is interesting to note that Bc and Bcr for uncultivated MTB
samples with multiple chain structures are normally higher than those reported
from MTB samples with single magnetosome chain (Table 5.1). Although, the
multiple and tortuous chain structure may increase the magnetostatic interaction
and therefore be negative to the values of Bc and Bcr, the high elongation of
individual magnetosomes in uncultivated MTB sample may increase the coercivity
of bulk sample (Fig. 5.6).
FORC diagrams are a powerful tool to provide detailed information about
magnetostatic interactions, crystal size distribution and particle switching fields
(Pike et al. 1999; Roberts et al. 2000). The FORC diagrams of samples YDC and
KM are peaked on Hu = 0 axis with closed contours, indicating that SD mag-
netosomes exist in these samples. No SP signal is found, which is consistent with
the TEM results that all characterized magnetosomes are larger than 30 nm
(Fig. 5.6). In addition, previous study on MTB harbored multiple chains indicated
that the FORC diagram was able to reflect chain–chain interactions within cells
rather than the intra-chain (particle–particle interaction within single chain) or
5.4 Discussion
Table 5.1 Comparison of magnetic parameters of the cultivated and uncultivated MTB
MTB Mrs/Ms Bc (mT) Bcr (mT) Bcr/Bc Tv (K) Delta ratio Reference
Sample
Uncultivated MTB YDC 0.49 41 50.2 1.22 108 2.03 This study
KM 0.55 42.2 46.3 1.1 104 3.06 This study
MYR-1 0.59 54.5 61.0 1.12 *100 3.1 (Li et al. 2010)
P2 0.47 26.7 40 1.50 *110 2.30 (Pan et al. 2005)
P3 0.51 33.4 45.5 1.36 *110 3.70 (Pan et al. 2005)
Cultivated MTB MS-1 0.53 26.7 37.8 1.42 100–110 [2 (Moskowitz et al. 1989, 1993)
AMB-1 0.45 18.1 23.3 1.29 106 2 (Li et al. 2009)
MSR-1 0.44 16.27 20.34 1.25 *100 1.16 (Fischer et al. 2008)
MC-1 / / / / 102 / (Prozorov et al. 2007)
MV-1 0.49 / / 1.1 117 [2 (Moskowitz et al. 1993; Prozorov et al. 2007)
RS-1-AF 0.44 / 46.7 / 86 5.54 (Pósfai et al. 2006)
75
cell–cell interactions (Pan et al. 2005). The vertical spread of FORC contours of
YDC sample is wider than that of KM sample, which perhaps indicates that MTB
cells in YDC have stronger interactions within cells than the MTB from KM.
The values of Tv of YDC and KM samples are 108 and 104 K, respectively,
similar as other characterized MTB (Table 5.1), but lowers than that of stoichi-
ometric magnetite (120–125 K). The lower Tv of MTB is perhaps due to the
nonstoichiometry of bacterial magnetite as suggested by Pan et al. (2005) and
Moskowitz et al. (1993), but it is still unclear whether the nonstoichiometry is an
intrinsic property of magnetosomes or arises from the preparation of samples (e.g.,
post oxidation). However, the former interpretation seems more convincible
considering similar results were acquired from different handling of MTB samples
(Moskowitz et al. 1993; Pan et al. 2005). The dFC/dZFC of both samples are bigger
than 2, which provide additional support from the uncultivated MTB for the power
of the Moskowitz test as an indicator of MTB, as dFC/dZFC greater than 2 is a
characteristic of MTB with magnetite magnetosomes arranged in chain(s) (Mo-
skowitz et al. 1993). Our result additionally suggests that multiple as well as
partially tortuous chains configuration seems to have negligible influence on the
Moskowitz test (Pan et al. 2005; Moskowitz et al. 2008). So far, Moskowitz tests
of most reported MTB samples are bigger than 2 except for one report (Table 5.1).
All together, the Moskowitz test is a potentially powerful tool for rapid detection
of MTB with magnetite magnetosomes in nature, though the underlying reasons
for this unique feature are still not fully clear (Carter-Stiglitz et al. 2001, 2002).
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Chapter 6
Concluding Remarks
This thesis focuses on improving our knowledge on the diversity, distribution,

genomic information, and magnetic properties of magnetotactic bacteria (MTB),
which are a diverse group of microorganisms synthesized intracellular magnetic
minerals of magnetite (Fe3O4) and/or greigite (Fe3S4) magnetosomes. MTB not
only play significant roles in global iron cycling and bulk magnetic signals of
sediments, but also are a perfect model system to study fundamental and general
biomineralization mechanisms. The conclusions of this thesis are summarized as:
1. A double-ended open magnetic separation apparatus MTB trap and a two-step
magnetic enrichment strategy are developed, which allow mass collection of
pure environmental MTB for further molecular, genomic and magnetic
analyses.
2. A set of specific primers for freshwater Alphaproteobacteria magnetotactic
cocci is designed, which could amplify longer products (about 864 bp) and
target more MTB populations.
3. The resolving powers of different restriction endonucleases are assessed by in
silico experiments. Of different sets of enzymes, MspI plus RsaI was identified
to be most effective for correctly differentiating the selected MTB populations.
Therefore, the MspI plus RsaI restriction fragment length polymorphism
(RFLP) analysis could be applied to characterize the structures and composi-
tional changes of MTB communities, which may facilitate future studies on the
ecology and biogeography of MTB in nature.
4. The morphological and phylogenetic diversity of MTB communities in Lake
Miyun and Pond Xiaoyue near Beijing, China are investigated. MTB from Lake
Miyun are affiliated within the Alphaproteobacteria and the phylum Nitrospi-
rae. To our knowledge, this is the first report of the occurrence of Nitrospirae
MTB in China, which suggests that these bacteria are more ubiquitous in
environmental samples than previously imagined. MTB from pond Xiaoyue are
composed of a single population that belongs to the Alphaproteobacteria
magnetotactic coccus. Based on the morphological structure and phylogenetic
position, this magnetotactic coccus is provisionally named as ‘‘Candidatus
Magnetococcus yuandaducum’’.

80 6 Concluding Remarks
5. The spatiotemporal variation of MTB communities from two sampling sites in

Lake Miyun are characterized and compared over a 3-month period. Although
the temporal variation of MTB communities was evident, the phylogenetic
discrepancy of MTB communities between two microcosms is more prominent
than that of the same microcosm at different times, implying adaptation of
MTB lineages to specific microenvironments. Among the physical–chemical
parameters measured, a significant correlation was shown between nitrate and
the main genetic variability of MTB communities, indicating that nitrate may
influence the occurrence of MTB phylogenetic lineages in this lake.
6. A targeted metagenomic approach was applied to investigate the phylum Ni-
trospirae MTB in Lake Miyun. Five fosmids containing rRNA operons were
identified. Comparative sequence analysis of a total of 172 kb provided new
insights into their genome organization and revealed unexpected subgenomic
diversity of uncultivated MTB in this phylum. Affiliation of two novel MTB
populations with the phylum Nitrospirae was verified by fluorescence in situ
hybridization. One of them was morphologically similar to ‘‘Candidatus
Magnetobacterium bavaricum’’, but the other differed substantially in cell
shape and magnetosome organization from all previously described ‘‘Candid-
atus Magnetobacterium bavaricum’’-like bacteria.
7. Both room- and low-temperature magnetic properties of ‘‘Candidatus Magne-
tococcus yuandaducum’’ from Pond Xiaoyue (sample YDC) and MTB cells
from Lake Kunming (sample KM) in Beijing are characterized and compared.
The magnetosomes in cells are dominated by single-domain magnetite. The
Verwey transition temperatures (Tv) of samples YDC and KM were 108 and
104 K, respectively, which were within the range of Tv measured from other
strains of MTB with magnetite magnetosomes (86–117 K) but were lower than
that of stoichiometric magnetite (120–125 K). The dFC/dZFC of both samples
are bigger than 2, which support the power of Moskowitz test as dFC/dZFC
greater than 2 is a characteristic of MTB with magnetite magnetosomes
arranged in chain(s). These results provide new insights into the magnetic
properties of bacterial magnetite and may advance the use of magnetic proxies
for decoding the magnetic signals of sediments containing magnetosome
magnetite.
Appendix
Supplementary Table S1 Identified RNA genes and predicted ORFs in the fosmid MY4-5C
ORF Locus Predicted protein or RNA (COG accession number if Length (aa) e-Value Best BLASTp hit (accession no.) Phylogenetic distribution of best hit
available)
1 132-290 hypothetical protein 52 \ \ \
2 757-1152 NHL repeat containing protein 131 5.00E-16 Methanosphaerula palustris E1-9c (YP_002466473) Archaea; Euryarchaeota; Methanomicrobia
4 2160-3893 NAD+ synthetase (COG0171 H) 577 0 Sulfurihydrogenibium sp. YO3AOP1 (YP_001931710) Bacteria; Aquificae; Aquificales

6 4221-4700 Rubrerythrin (COG1633 S) 159 7.00E-15 Desulfohalobium retbaense DSM 5692 Bacteria; Proteobacteria;
(YP_003197071) Deltaproteobacteria
7 4701-5549 hypothetical protein THEYE_A0454 (COG0313 R) 282 9.00E-94 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002248299)
8 5542-5892 phosphoribosylglycinamide formyltransferase PurN 116 8.00E-34 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(COG0299 F) (YP_002248646)
W. Lin, Diversity, Biomineralization and Rock Magnetism

9 5849-6148 phosphoribosylglycinamide formyltransferase PurN 99 9.00E-38 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(COG0299 F) (YP_002248646)
11 6481-7926 transposase (COG3666 L) 481 1.00E-71 Lentisphaera araneosa HTCC2155 (ZP_01874497) Bacteria; Lentisphaerae; Lentisphaerales
12 8334-9263 transposase (COG3547 L) 309 2.00E-89 Geobacter metallireducens GS-15 (YP_383020) Bacteria; Proteobacteria;
Deltaproteobacteria
of Magnetotactic Bacteria, Springer Theses, DOI: 10.1007/978-3-642-38262-8,

15 10649-15268 Tfp pilus assembly protein tip-associated adhesin 1539 5.00E-159 Geobacter bemidjiensis Bem (YP_002138350) Bacteria; Proteobacteria;
PilY1-like protein (COG3419 N) Deltaproteobacteria
(continued)
81
82
Supplementary Table S1 (continued)

available)
17 15532-15948 general secretion pathway protein G (COG2165 N) 138 1.00E-43 Burkholderia sp. H160 (ZP_03267299) Bacteria; Proteobacteria;
Betaproteobacteria
18 16060-16839 CO dehydrogenase\acetyl-CoA synthase complex, 259 5.00E-72 Carboxydothermus hydrogenoformans Z-2901 Bacteria; Firmicutes; Clostridia
accessory protein CooC (COG3640 D) (YP_360654)
19 16836-17432 5-formyltetrahydrofolate cyclo-ligase (COG0212 H) 198 2.00E-31 Rubrobacter xylanophilus DSM 9941 (YP_644097) Bacteria; Actinobacteria; Rubrobacteridae
20 17534-17986 GTP cyclohydrolase I (COG0302 H) 150 4.00E-50 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002248515)
21 18237-18854 methenyltetrahydrofolate cyclohydrolase (COG0190 205 2.00E-72 Magnetococcus sp. MC-1 (YP_867603) Bacteria; Proteobacteria;
H) Alphaproteobacteria
22 18885-19553 hypothetical protein THEYE_A0675 222 9.00E-58 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002248517)
24 21684-22079 transposase (COG1662 L) 131 2.00E-25 Verrucomicrobia bacterium N20 (ABV66281) Bacteria; Verrucomicrobia; Opitutae
25 21947-22357 transposase (COG3677 L) 136 8.00E-16 Synechocystis sp. PCC 6803 (NP_441309) Bacteria; Cyanobacteria; Chroococcales
26 22425-23198 IstB ATP binding domain-containing protein 257 4.00E-84 Pelobacter propionicus DSM 2379 (YP_899560) Bacteria; Proteobacteria;
(COG1484 L) Deltaproteobacteria
27 23167-24429 transposase (COG4584 L) 420 2.00E-113 Pelobacter propionicus DSM 2379 (YP_900694) Bacteria; Proteobacteria;
Deltaproteobacteria
28 24515-25603 transposase (COG3666 L) 362 8.00E-52 Candidatus Kuenenia stuttgartiensis (CAJ75132) Bacteria; Planctomycetes; Planctomycetacia
29 26121-27050 transposase (COG3547 L) 309 2.00E-89 Geobacter metallireducens GS-15 (YP_383020) Bacteria; Proteobacteria;
Deltaproteobacteria
31 27979-28872 extracellular solute-binding protein (COG1226 P) 297 4.00E-28 Syntrophobacter fumaroxidans MPOB (YP_846083) Bacteria; Proteobacteria;
Deltaproteobacteria
32 28860-29954 extracellular solute-binding protein (COG1226 P) 364 2.00E-38 Syntrophobacter fumaroxidans MPOB (YP_846083) Bacteria; Proteobacteria;
Deltaproteobacteria
(continued)
Appendix
Appendix
available)
35 32110-33108 selenophosphate synthase (COG0709 E) 332 1.00E-72 Hydrogenobacter thermophilus TK-6 Bacteria; Aquificae; Aquificales
(YP_003433030)
16S rRNA 33312-34841 \ \ \ \ \
tRNA Ile 34949-35022 \ \ \ \ \

83
Supplementary Table S2 Identified RNA genes and predicted ORFs in the fosmid MY3-11A
84
ORF Locus Predicted protein or RNA (COG accession Length (aa) e-Value Best BLASTp hit (accession no.) Phylogenetic distribution of best
number if available) hit
1 1-860 conserved hypothetical protein 286 1.00E-27 Campylobacterales bacterium GD 1 Bacteria; Proteobacteria;
(ZP_05070903) Epsilonproteobacteria
2 875-1888 lipopolysaccharide heptosyltransferase I 337 2.00E-82 Syntrophobacter fumaroxidans MPOB Bacteria; Proteobacteria;
(COG0859 M) (YP_844510) Deltaproteobacteria
3 2183-2743 fimbrial protein 186 1.00E-08 Thermocrinis albus DSM 14484 Bacteria; Aquificae; Aquificales
(YP_003472807)
4 2903-3457 type IV pilin 184 1.00E-07 Vibrio vulnificus (ACJ54284) Bacteria; Proteobacteria;
Gammaproteobacteria
5 3495-4040 prokaryotic N-terminal methylation motif domain 181 5.00E-07 Dictyoglomus thermophilum H-6-12 Bacteria; Dictyoglomi;
protein (COG2165 N) (YP_002250594) Dictyoglomales
6 4423-4983 fimbrial protein 186 6.00E-09 Thermocrinis albus DSM 14484 Bacteria; Aquificae; Aquificales
(YP_003472807)
7 5070-6863 TPR-repeat-containing protein (COG0457 R) 597 1.00E-40 NC10 bacterium ’Dutch sediment’ Bacteria; candidate division NC10
(CBE67393)
8 6956-7438 type IV pilin (COG2165 N) 160 2.00E-10 Gloeobacter violaceus PCC 7421 Bacteria; Cyanobacteria;
(NP_925201) Gloeobacteria
9 7450-9540 TPR-repeat-containing protein (COG0457 R) 696 8.00E-81 Desulfovibrio sp. FW1012B Bacteria; Proteobacteria;
(ZP_06370037) Deltaproteobacteria
10 9680-9874 hypothetical protein MAE_42900 (COG1598 S) 64 1.00E-09 Microcystis aeruginosa NIES-843 Bacteria; Cyanobacteria;
(YP_001659304) Chroococcales
12 10498-15498 Tfp pilus assembly protein tip-associated adhesin 1666 9.00E-161 Geobacter bemidjiensis Bem Bacteria; Proteobacteria;
PilY1-like protein (COG3419 N) (YP_002138350) Deltaproteobacteria
13 15800-16954 transposase (COG3385 L) 384 2.00E-93 Nitrosococcus halophilus Nc4 Bacteria; Proteobacteria;
(YP_003526825) Gammaproteobacteria
14 17404-18591 UDP-glucose/GDP-mannose dehydrogenase 395 3.00E-129 Brachyspira hyodysenteriae WA1 Bacteria; Spirochaetes;
(COG0677 M) (YP_002721867) Spirochaetales
15 18628-20007 FAD linked oxidase domain protein (COG0277 459 1.00E-125 Geobacter sp. FRC-32 Bacteria; Proteobacteria;
C) (YP_002536297) Deltaproteobacteria
16 20292-20483 protein of unknown function 63 5.00E-06 Leptospirillum ferrodiazotrophum Bacteria; Nitrospiraee;
(EES52562) Nitrospiraeles
(continued)
Appendix
ORF Locus Predicted protein or RNA (COG accession Length (aa) e-Value Best BLASTp hit (accession no.) Phylogenetic distribution of best
Appendix
number if available) hit

18 21381-22376 selenophosphate synthase (COG0709 E) 331 8.00E-75 Geobacter metallireducens GS-15 Bacteria; Proteobacteria;
(YP_385850) Deltaproteobacteria
16S rRNA 22585-24114 \ \ \ \ \
tRNA Ile 24222-24295 \ \ \ \ \
tRNA Ala 24431-24503 \ \ \ \ \
23S rRNA 24565-27578 \ \ \ \ \
5S rRNA 27669-27791 \ \ \ \ \
19 28095-28457 tetratricopeptide domain protein 120 1.00E-33 Desulfonatronospira thiodismutans Bacteria; Proteobacteria;
ASO3-1 (ZP_03736812) Deltaproteobacteria
20 28630-29034 hypothetical protein GM18DRAFT_3460 134 7.00E-13 Geobacter sp. M18 (ZP_05312801) Bacteria; Proteobacteria;
Deltaproteobacteria
21 29167-30033 tyrosine recombinase XerC (COG0582 L) 288 7.00E-88 Thermodesulfovibrio yellowstonii DSM Bacteria; Nitrospiraee;
11347 (YP_002249196) Nitrospiraeles
22 30047-31279 tRNA (uracil-5-)-methyltransferase Gid, involved 410 2.00E-124 Geobacter sulfurreducens PCA Bacteria; Proteobacteria;
in cell division (COG1206 D) (NP_953593) Deltaproteobacteria
23 31445-32632 tryptophan synthase, beta subunit (COG0133 E) 395 4.00E-162 Thermodesulfovibrio yellowstonii DSM Bacteria; Nitrospiraee;
11347 (YP_002248404) Nitrospiraeles
85
Supplementary Table S3 Identified RNA genes and predicted ORFs in the fosmids MY2-1F
86
available)
1 615-806 protein of unknown function 63 5.00E-06 Leptospirillum ferrodiazotrophum (EES52562) Bacteria; Nitrospiraee; Nitrospiraeles
3 1704-2699 selenophosphate synthase (COG0709 E) 331 8.00E-75 Geobacter metallireducens GS-15 (YP_385850) Bacteria; Proteobacteria; Deltaproteobacteria
16S rRNA 2908- 4437 \ \ \ \ \
tRNA Ile 4545- 4618 \ \ \ \ \
tRNA Ala 4754- 4826 \ \ \ \ \
23S rRNA 4888- 7901 \ \ \ \ \
5S rRNA 7992- 8114 \ \ \ \ \
4 8418-8780 tetratricopeptide domain protein 120 1.00E-33 Desulfonatronospira thiodismutans ASO3-1 Bacteria; Proteobacteria; Deltaproteobacteria
(ZP_03736812)
5 8953-9357 hypothetical protein GM18DRAFT_3460 134 7.00E-13 Geobacter sp. M18 (ZP_05312801) Bacteria; Proteobacteria; Deltaproteobacteria
6 9490-10356 tyrosine recombinase XerC (COG0582 L) 288 7.00E-88 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002249196)
7 10370-11602 tRNA (uracil-5-)-methyltransferase Gid, involved in cell 410 2.00E-124 Geobacter sulfurreducens PCA (NP_953593) Bacteria; Proteobacteria; Deltaproteobacteria
division (COG1206 D)
8 11768-12955 tryptophan synthase, beta subunit (COG0133 E) 395 4.00E-162 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002248404)
9 13169-15064 Phosphomannomutase (COG1109 G) 631 2.00E-146 Roseburia inulinivorans DSM 16841 (ZP_03754451) Bacteria; Firmicutes; Clostridia
10 15072-16253 HI0933 family protein (COG2081 R) 393 5.00E-114 bacterium Ellin514 (ZP_03630885) Bacteria; Verrucomicrobia; Verrucomicrobiae
11 16228-16620 hypothetical protein HS_1381 130 1.00E-07 Haemophilus somnus 129PT (YP_719593) Bacteria; Proteobacteria;
Gammaproteobacteria
13 16870-17913 N-acylneuraminate-9-phosphate synthase (COG2089 M) 347 1.00E-130 Thermoanaerobacter mathranii subsp. mathranii str. Bacteria; Firmicutes; Clostridia
A3 (ZP_05380252)
14 17900-18535 HAD family hydrolase (COG1011 R) 211 2.00E-37 Candidatus Desulforudis audaxviator MP104C Bacteria; Firmicutes; Clostridia
(YP_001717901)
Appendix
(continued)
Appendix
available)
15 18580-19554 CarB protein (COG0458 EF) 324 8.00E-89 Anaerobaculum hydrogeniformans ATCC BAA-1850 Bacteria; Synergistetes; Synergistia
(ZP_06440163)
16 19551-20510 NAD-dependent epimerase/dehydratase (COG0451 MG) 319 2.00E-70 Candidatus Desulforudis audaxviator MP104C Bacteria; Firmicutes; Clostridia
(YP_001717904)
17 20507-21505 GCN5-related N-acetyltransferase (COG0438 M) 332 7.00E-62 Roseiflexus castenholzii DSM 13941 (YP_001430911) Bacteria; Chloroflexi; Chloroflexales
18 21778-23610 methyl-accepting chemotaxis sensory transducer 610 8.00E-97 Geobacter uraniireducens Rf4 (YP_001232123) Bacteria; Proteobacteria; Deltaproteobacteria
(COG0840 N)
19 23801-24955 transposase IS4 family protein (COG3385 L) 384 2.00E-93 Nitrosococcus halophilus Nc4 (YP_003526825) Bacteria; Proteobacteria;
Gammaproteobacteria
20 25076-26413 putative sensor histidine kinase (COG0642 T) 445 2.00E-66 uncultured archaeon (CBH37099) Archaea; environmental samples
21 26416-27303 two component signal transduction response regulator 295 2.00E-36 Pelobacter carbinolicus DSM 2380 (YP_358115) Bacteria; Proteobacteria; Deltaproteobacteria
(COG0784 T)
22 27329-28930 glycosyl transferase family protein 533 2.00E-41 Syntrophobacter fumaroxidans MPOB (YP_844281) Bacteria; Proteobacteria; Deltaproteobacteria
23 28944-30110 NADH oxidase (COG1902 C) 388 1.00E-127 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002248973)
87
Supplementary Table S4 Identified RNA genes and predicted ORFs in the fosmid MY3-5B
88
available)
1 92-508 adenylate/guanylate cyclase 138 7.00E-11 Beggiatoa sp. PS (ZP_01999918) Bacteria; Proteobacteria;
Gammaproteobacteria
2 438-1430 4-hydroxythreonine-4-phosphate dehydrogenase 330 1.00E-77 Thermoanaerobacter pseudethanolicus ATCC 33223 Bacteria; Firmicutes; Clostridia
(COG1995 H) (YP_001665647)
3 1421-1861 hypothetical protein Pcar_1743 (COG2246 S) 146 6.00E-07 Pelobacter carbinolicus DSM 2380 (YP_357156) Bacteria; Proteobacteria;
Deltaproteobacteria
4 2062-3045 selenophosphate synthase (COG0709 E) 327 3.00E-74 Geobacter metallireducens GS-15 (YP_385850) Bacteria; Proteobacteria;
Deltaproteobacteria
16S rRNA 3244-4773 \ \ \ \ \
tRNA Ile 4883- 4956 \ \ \ \ \
tRNA Ala 5086- 5158 \ \ \ \
23S rRNA 5258-8255 \ \ \ \ \
5S rRNA 8336-8458 \ \ \ \ \
6 8784-9584 4Fe-4S ferredoxin iron-sulfur binding domain protein 266 3.00E-81 Chloroflexus aggregans DSM 9485 (YP_002464939) Bacteria; Chloroflexi; Chloroflexales
(COG0437 C)
7 9616-10806 polysulphide reductase NrfD 396 3.00E-31 Chloroflexus aurantiacus J-10-fl (YP_001633668) Bacteria; Chloroflexi; Chloroflexales
8 10803-14021 molybdopterin oxidoreductase Fe4S4 region (COG0243 1072 0.00E+00 Geobacter sp. M18 (ZP_05313625) Bacteria; Proteobacteria;
C)Anaerobic dehydrogenases, typically Deltaproteobacteria
selenocysteine-containing
9 14238-14735 nucleotide-binding protein (COG1666 S) 165 5.00E-47 Candidatus Solibacter usitatus Ellin6076 (YP_824456) Bacteria; Acidobacteria; Solibacteres
11 15247-16041 probable inorganic polyphosphate/ATP-NAD kinase 264 2.00E-50 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(Poly(P)/ATP NAD kinase) (COG0483 G) (YP_002248805)
(continued)
Appendix
Appendix
available)
13 16719-18725 long-chain-fatty-acid CoA ligase (COG1022 I) 668 1.00E-117 Hydrogenivirga sp. 128-5-R1-1 (ZP_02177080) Bacteria; Aquificae; Aquificales
15 19174-20988 potassium uptake protein Kup (COG3158 P) 604 0.00E+00 Methanosarcina acetivorans C2A (NP_618072) Archaea; Euryarchaeota;
Methanomicrobia
16 21064-22074 putative flagellin (COG1344 N) 336 4.00E-61 Stenotrophomonas maltophilia K279a (YP_001972099) Bacteria; Proteobacteria;
Gammaproteobacteria
(YP_002248859)
tRNA Met 23022- 23097 \ \ \ \ \
18 23307-23468 hypothetical protein SBO_4335 53 4.00E-07 Shigella boydii Sb227 (YP_410579) Bacteria; Proteobacteria;
Gammaproteobacteria
19 23479-23844 transposase (COG2963 L) 121 2.00E-63 Shigella sonnei Ss046 (AAZ87103) Bacteria; Proteobacteria;
Gammaproteobacteria
20 23916-24707 transposase (COG2801 L) 263 2.00E-154 Escherichia (P51026) Bacteria; Proteobacteria;
Gammaproteobacteria
21 24692-29941 Tfp pilus assembly protein tip-associated adhesin PilY1- 1749 6.00E-174 Geobacter bemidjiensis Bem (YP_002138350) Bacteria; Proteobacteria;
like protein (COG3419 N) Deltaproteobacteria
22 29943-30584 conserved hypothetical protein 213 4.00E-06 Geobacter sp. M18 (ZP_05311815) Bacteria; Proteobacteria;
Deltaproteobacteria
23 30523-30972 hypothetical protein Geob_3075 149 5.00E-11 Geobacter sp. FRC-32 (YP_002538519) Bacteria; Proteobacteria;
Deltaproteobacteria
24 30969-32078 conserved hypothetical protein 369 2.00E-49 Geobacter sp. M18 (ZP_05311813) Bacteria; Proteobacteria;
Deltaproteobacteria
25 32092-32679 hypothetical protein HG1285_04009 195 6.00E-14 Hydrogenivirga sp. 128-5-R1-1 (ZP_02179789) Bacteria; Aquificae; Aquificales
26 32729-35116 peptidase S8 and S53 subtilisin kexin sedolisin (COG1404 795 7.00E-79 Streptomyces sp. ACT-1 (ZP_06278998) Bacteria; Actinobacteria;
O) Actinobacteridae
27 35307-36041 3-oxoacyl-(acyl-carrier-protein) reductase (COG1028 QR) 244 2.00E-87 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002247953)
28 36107-36337 acyl carrier protein (COG0236 IQ) 76 1.00E-24 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002247954)
(continued)
89
90

available)
29 36373-37617 beta-ketoacyl-acyl carrier protein synthase II (COG0304 414 6.00E-168 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
IQ) (YP_002247955)
30 37620-38411 RNase III (COG0571 K) 263 2.00E-50 Pelobacter carbinolicus DSM 2380 (YP_356819) Bacteria; Proteobacteria;
Deltaproteobacteria
31 38404-39450 phosphoribosylformylglycinamidine cyclo-ligase PurM 348 8.00E-119 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(COG0150 F) (YP_002248725)
32 39575-41383 molybdenum cofactor biosynthesis protein (COG0303 H) 602 0.00E+00 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002249113)
Appendix
Supplementary Table S5 Identified RNA genes and predicted ORFs in the fosmid MY2-3C
available)
Appendix
1 117-323 hypothetical protein GobsU_18857 68 1.00E-07 Gemmata obscuriglobus UQM 2246 (ZP_02733868) Bacteria; Planctomycetes;
Planctomycetacia
5 1515-2708 phosphoglycerate kinase (COG0126 G) 397 4.00E-148 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002248096)
6 2711-3712 glyceraldehyde-3-phosphate dehydrogenase, type I 333 7.00E-141 Carboxydothermus hydrogenoformans Z-2901 Bacteria; Firmicutes; Clostridia
(COG0057 G) (YP_359152)
7 3778-4995 hypothetical protein THEYE_A0249 405 7.00E-73 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002248098)
tRNA Leu 5180- 5261 \ \ \ \ \
8 5373-5597 hypothetical protein ssr1765 (COG1598 S) 74 2.00E-11 Synechocystis sp. PCC 6803 (NP_440250) Bacteria; Cyanobacteria; Chroococcales
9 5928-6446 abortive infection protein (COG1266 R) 172 5.00E-07 Hydrogenobacter thermophilus TK-6 (YP_003433468) Bacteria; Aquificae; Aquificales
10 6720-8906 hypothetical protein L8106_04436 (COG2199 T) 728 1.00E-86 Lyngbya sp. PCC 8106 (ZP_01618684) Bacteria; Cyanobacteria; Oscillatoriales
11 8907-9722 conserved hypothetical methylase protein 272 4.00E-56 Candidatus Kuenenia stuttgartiensis (CAJ71796) Bacteria; Planctomycetes;
Planctomycetacia
12 9759-10406 methyltransferase type 11 (COG0500 QR) 215 9.00E-40 Alkalilimnicola ehrlichii MLHE-1 (YP_742469) Bacteria; Proteobacteria;
Gammaproteobacteria
13 10746-12059 protein of unknown function 437 4.00E-30 Leptospirillum ferrodiazotrophum (EES52810) Bacteria; Nitrospiraee; Nitrospiraeles
14 12179-12622 unknown protein 147 2.00E-38 Candidatus Kuenenia stuttgartiensis (CAJ75113) Bacteria; Planctomycetes;
Planctomycetacia
15 12634-13119 hypothetical protein Dacet_1166 161 3.00E-14 Denitrovibrio acetiphilus DSM 12809 (YP_003503894) Bacteria; Deferribacteres;
Deferribacterales
16 13212-13787 conserved hypothetical protein 191 1.00E-04 Dethiobacter alkaliphilus AHT 1 (ZP_03729088) Bacteria; Firmicutes; Clostridia
17 14772-16031 L-lysine 2,3-aminomutase (COG1509 E) 419 1.00E-117 Dehalococcoides sp. BAV1 (YP_001214064) Bacteria; Chloroflexi;
Dehalococcoidetes
91
(continued)
92

available)
19 16441-16902 molecular chaperone (COG0071 O) 153 6.00E-26 Syntrophus aciditrophicus SB (YP_460100) Bacteria; Proteobacteria;
Deltaproteobacteria
Deltaproteobacteria
Deltaproteobacteria
22 18049-19113 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase 354 3.00E-118 Geobacter metallireducens GS-15 (YP_384312) Bacteria; Proteobacteria;
(COG0821 M) Deltaproteobacteria
(YP_002248212)
24 20094-20636 hypothetical protein THEYE_A0750 (COG2365 T) 180 2.00E-50 Thermodesulfovibrio yellowstonii DSM 11347 Bacteria; Nitrospiraee; Nitrospiraeles
(YP_002248592)
25 20814-21398 ABC transporter ATP-binding protein (COG1136 R) 194 2.00E-77 Syntrophus aciditrophicus SB (YP_460093) Bacteria; Proteobacteria;
Deltaproteobacteria
26 21519-22736 similar to AcrA/AcrE family multidrug efflux protein 405 4.00E-96 Candidatus Kuenenia stuttgartiensis (CAJ73768) Bacteria; Planctomycetes;
(COG0845 Q) Planctomycetacia
16S rRNA 23108-24638 \ \ \ \ \
tRNA Ile 24813- 24886 \ \ \ \ \
tRNA Ala 24940- 25012 \ \ \ \ \
23S rRNA 25052-28039 \ \ \ \ \
5S rRNA 28088-28210 \ \ \ \ \
27 28612-29358 hypothetical protein MA2615 (COG2013 S) 248 2.00E-63 Methanosarcina acetivorans C2A (NP_617516) Archaea; Euryarchaeota;
Methanomicrobia
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Diversity, Biomineralization and Rock Magnetism of Magnetotactic Bacteria

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Diversity, Biomineralization and Rock Magnetism of Magnetotactic Bacteria

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Springer Theses

Recognizing Outstanding Ph.D. Research

Recognizing Outstanding Ph.D. Research

For further volumes:

Theses are accepted into the series by invited nomination only

• They must be written in good English.

Prof. Dr. Dirk Schüler

ISSN 2190-5053 ISSN 2190-5061 (electronic)

Library of Congress Control Number: 2013939046

Springer-Verlag Berlin Heidelberg 2013

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Research in biomineralization investigates the mechanisms and properties of

magnetosomes are important for decoding the paleomagnetic signals of sediments

Beijing Yongxin Pan

Beijing, China Wei Lin

2 Enrichment, Purification and Characterization of Environmental

3 Diversity of Magnetotactic Bacteria and Its Environmental

3.2.6 Phylogenetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 35

4 Metagenomic Analysis of Uncultivated Magnetotactic Bacteria

5 Magnetic Properties of Uncultivated Magnetotactic Bacteria . . . . . 67

W. Lin, Diversity, Biomineralization and Rock Magnetism 1

Fig. 1.1 Transmission

applications in various fields, such as magnetic separation, diagnostics, and

1.1 General Information

1.1.1 Diversity of MTB

The majority of MTB populations in nature are so far uncultivable. However,

Fig. 1.2 Representative shapes of magnetosome crystals. Bars = 100 nm

Fig. 1.3 Phylogenetic tree of representative magnetotactic bacterial strains

1.1.2 Distribution of MTB

Through comprehensive studies of MTB communities across different ecosystems,

1.1.3 Molecular Mechanism of MTB Biomineralization

Biomineralization of magnetosomes in MTB is a key issue for understanding

1.1.4 Magnetic Properties of MTB and Magnetosomes

Magnetite is one of the principal recorders of paleomagnetic information on Earth.

Table 1.1 Magnetic properties of various MTB samples

1.1.5 Fossil Magnetosomes and Paleoenvironmental

Magnetosomes formed by MTB may be preserved in sediments as fossil records.

1.2 Aims and Motivation

The general aims of this thesis are:

2. To investigate the diversity and distribution of MTB communities across var-

1.3 Outline of this Thesis

Chapter 1 contains a short introduction of MTB. Chapter 2 is devoted to develop

base of the Alphaproteobacteria. International Journal of Systematic and Evolutionary

Although magnetotactic bacteria (MTB) are ubiquitous in various aquatic or

W. Lin, Diversity, Biomineralization and Rock Magnetism 15

2.2 Novel Apparatus and Strategy for MTB Enrichment

Fig. 2.3 Schematic diagram of the hanging-drop method

2.3 Specific Primers for Freshwater Alphaproteobacteria

In the present study, we focused on the Alphaproteobacteria magnetotactic cocci

2.4 In silico Evaluation of Restriction Endonucleases

RFLP analysis enables a rapid investigation of microorganism community diver-

the same OTU

Magnetotactic bacteria (MTB) are ubiquitous in aquatic and sedimentary envi-

W. Lin, Diversity, Biomineralization and Rock Magnetism 31

(RFLP), fluorescence in situ hybridization (FISH), and phylogenetic analysis. The

3.2 Materials and Methods

3.2.1 Sampling Sites

3.2.2 Physical–Chemical Analyses

The oxygen concentrations of surface sediment were determined using an HQ40d

Miyun were conducted at the Analytical Laboratory Beijing Research Institute of

3.2.3 Magnetic Collection of MTB

3.2.4 Transmission Electron Microscope Observation