A biogeographic distribution of magnetotactic bacteria
influenced by salinity
Wei Lin*, Yinzhao Wang, Bi Li and Yongxin Pan*
E-mail: weilin0408@gmail.com; yxpan@mail.iggcas.ac.cn
Supplemental information
Supplementary Table 1. Site and clone library information for nine sediment
samples from which MTB 16S rRNA genes were retrieved. (.xls)
Supplementary Table 2. Information on the previously reported three sites and
publicly available 16S rRNA gene sequences which are included in this analysis.
(.xls)
Supplementary Table 3. Phylogenetic distribution of potential non-MTB sequences
associated with magnetic enrichments retrieved in this study. (.xls)
Materials and methods
Site description and sample collection
Sediment samples were collected from nine sites for comparison of the diversity and
distribution of MTB communities in a different range of ecosystem types, including
freshwater lakes, mangrove swamp, estuary, and intertidal zone across northern and
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southern China (Supplementary Table 1).
Four sites are in and around Beijing City. The climate in Beijing is
characteristically temperate, with an average annual temperature of 10-12°C and
annual rainfall values of 600 mm. Lake Kunming (YHY) is located in the Summer
Palace Park and has a water surface area ca. 2.2 km2, with an average water depth
about 1.5 m. At the sampling time, the bottom water oxygen content was 0.16 mg L-1,
pH of water 8.15. Lake Beihai (BH) is situated in Beihai Park in the city center. The
lake has a water surface area of about 0.39 km2 with a water depth of 1-3 m.
Temperature and pH during the sampling time was 29°C and 7.66, respectively;
whereas the dissolved concentration of oxygen in surface sediment is 0.12 mg L-1.
The Lake Yuyuantan (YY) is in the Yuyuantan Park, which has water surface area
about 0.61 km2. The temperature, pH, and oxygen concentration are determined to be
28°C, 8.08, and 0.10 mg L-1, respectively.
Lake Miyun (MY) is located in front of the Yanshan Mountain, about 80 km
northeast of Beijing. Its physical characteristics were described previously (Lin and
Pan, 2010; Pan et al., 2009). Briefly, MY has a water body exceeding 1 billion m3 and
a maximum water depth of 40 m. The pH of water is determined to be 7.50 in this
study. The salinities of above-mentioned 4 lakes are all below 0.5 ppt, indicating that
they are freshwater lakes.
Two sediment samples (HSL2 and HSL4) were collected from mangrove swamp in
Wenchang County in Hainan Province, China. Hainan Island is characterized by a
tropical climate with an average annual rainfall of 1600 mm and an average
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temperature of 23-25°C. The pH and salinity of samples ranged from 7.92 to 8.07, and
13.8 to 20.0 ppt, respectively (Table 1). Another two estuarine sediment samples
(BMW1 and BMW2) were collected from Bamen Bay, near Wenchang. Two
watersheds drain into the bay: Wenchang River and Wenjiao River
(http://www.marsh.csdb.cn/survey/hainan.htm). The area of water surface and tidal
flat is about 11.85 km2. The water depth of each sampling station was approximately
3-4 m. Salinities of BMW1 and BMW2 were determined to be 15.4 and 18.8 ppt,
respectively.
The last sampling site (WH) was located in the intertidal zone of the Yellow Sea
near Weihai City, Shandong Province. The average temperaure of Weihai is 26°C in
August. Annual rainfall is about 665 mm. The pH and salinity of sea water were 7.71
and 28.6 ppt, respectively.
At each sampling site, sediments from the top 5-20 cm were collected and then
were aliquoted into 4-14 plastic bottles (600 ml) covered with about 100 ml of in situ
water. The oxygen concentrations of surface sediment were determined using an
HQ40d Oxygen Meter (HACH, Loveland, Colorado, USA). The existence of MTB in
all sediment samples was checked through the “hanging-drop” method (Greenberg et
al., 2005). MTB in the sediment were magnetically enriched using a double-ended
open magnetic separation apparatus (MTB trap) as previously described (Jogler et al.,
2009; Lin and Pan, 2010). Briefly, 200 mL of surface sediments from each site were
scratched and directly transferred to the “MTB trap”. A homogeneous magnetic field,
about seven times that of the Earth’s magnetic field, was applied for cell enrichment
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for 6 h. The enriched north- and south-seeking MTB for each location were then
pooled for further TEM observations and phylogenetic analyses. After enrichment, the
pore water was separated from the sediments by centrifugation at 1000 g for 20 min
and was further filtered through 0.45-μm 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),
respectively.
TEM observation
Twenty microlitres of MTB enrichments were deposited on Formvar-carbon-coated
copper grids. After waiting for 1 h, the remaining solution was wicked away using a
piece of filter paper. The samples were then rinsed twice with sterile distilled water.
Specimens were imaged using a JEM-2010 microscope operating at 200 kV (JEOL
Corporation, Japan).
PCR amplification of 16S rRNA genes and construction of clone libraries
16S rRNA genes were directly amplified from the magnetically enriched MTB using
the bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and
1492R (5'-GGTTACCTTGTTACGACTT-3') (Lane, 1991) as previously described
(Lin et al., 2008; Lin et al., 2009). Each 20-μl PCR mixture contained 1 μl of template,
10 μl of DreamTaq PCR Master Mix (MBI Fermentas), and 8 pmol of each primer.
The PCR conditions were 95°C for 5 min, 30 cycles of 92°C for 1.5 min, 50°C for 1
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min, and 72°C for 2 min, and a final 10-min extension at 72°C. To avoid potential
sample biases, triplicate PCR products for each sample were pooled and purified by
0.8% (w/v) agarose gel electrophoresis. PCR controls with no template were negative.
Purified PCR products were ligated with the pMD19-T vector (TaKaRa, Japan) and
cloned into the chemically DH5 competent cells (Tiangen, Beijing, China) according
to the manufactures’ instructions. Randomly selected clones were sequenced using a
27F primer (Beijing Genomics Institute, China).
Sequence analysis
After removing sequences of insufficient length or low quality, in total, nearly 400
sequences were retrieved in this study. The length of sequences were about 450-500
bp, covering V1 to V3 hypervariable regions. The sequences were aligned using the
NAST aligner at the Greengenes web site and were then taxonomically classified
according to the best match with the Greengenes reference database (DeSantis et al.,
2006). The presence of chimeras was checked using the Greengenes chimera-check
tool (Bellerophon server) (Huber et al., 2004). NCBI BLAST was used to find the
most closely related 16S rRNA gene sequences in the public database. The sequences
unrelated to known MTB (<80% sequence identity) in the database were attributed to
non-magnetotactic contaminations and were removed in this study (Supplementary
Table 3). In this way, a total of 334 high quality 16S rRNA gene sequences, with
21-47 sequences per sample, were retrieved.
The number of OTUs was estimated using the FastGroupII algorithm with
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similarity threshold of 98% (Percentage Sequence Identity with Gaps algorithm) (Yu
et al., 2006). Rarefaction curves were calculated using the FastGroupII algorithm (Yu
et al., 2006) or the freeware program aRarefactWin (available at
http://www.uga.edu/~strata/software/Software.html). To estimate the coverage (Good,
1953) of clone libraries, sequences were collected into OTUs based on 98% sequence
identity. We chose the 98% threshold because this is the similarity threshold between
two well-studied model MTB organisms Magnetospirillum magnetotacticum MS-1
and M. magnetotacticum AMB-1 (Lin et al., 2009). The coverage estimators were
calculated by the equation C = [1 - (n/N)]×100, where n is the number of unique
clones and N is the total number of clones examined (Good, 1953).
A representative sequence from each OTU (98% sequence identity) was aligned
using CLUSTAL W (Thompson et al., 1994) software and corrected by manual
inspection. The phylogenetic tree was constructed using MEGA version 4.0 (Tamura
et al., 2007) through the neighbor-joining method and was linearized assuming equal
evolutionary rates in all lineages. The bootstrap resamplings were repeated 1000
times.
Statistical analyses
The unweighted UniFrac approach was used to determine the overall phylogenetic
distance between each pair of MTB communities from distinct locations (Hamady et
al., 2010; Lozupone and Knight, 2005). The UniFrac algorithm estimates the
phylogenetic distance between communities and can reflect the occurrence of distinct
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microbial lineages based on phylogenetic information. In addition to sequences
retrieved in the present study, publicly available sequence sets of MTB communities
from globally distinct locations were also included in the UniFrac analysis in order to
compare their phylogenetic relationships to the MTB communities retrieved here.
These 16S rRNA gene sequences were extracted from the most updated Database of
Magnetotactic Bacteria (http://database.biomnsl.com/, last check on October 5, 2010)
(Lin et al., 2011). The selection was restricted to studies that matched each one of the
following criteria: (i) investigation of the overall diversity of MTB communities in
single locations; (ii) use of bacterial universal primers; (iii) high-quality sequences at
least covering the V1 to V3 hypervariable regions. Studies performed at 3 sites were
met these criteria, including sequences from Itaipu lagoon (saline) in Rio de Janeiro,
Brazil (Spring et al., 1998), Jiaozhou Bay (saline) in Shandong Province, China (Pan
et al., 2008; Xing et al., 2008), and Lake Chiemsee (freshwater) near Munich,
Germany (Spring et al., 1992; Spring et al., 1993; Spring et al., 1994). A detailed
information list of these sequences and environmental sources is given in the
Supplementary Table 2. Spearman rank correlation was performed to examine the
correlations between the phylogenetic distance of nine MTB communities in China
(UniFrac distance matrix) and the measured environmental factors (Euclidean
distances). For all statistical analyses, a value of P<0.05 was considered significant.
References
DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K et al. (2006).
Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible
7
with ARB. Appl Environ Microbiol 72: 5069-5072.
Good IJ. (1953). The population frequencies of species and the estimation of population
parameters. Biometrika 40: 237-264.
Greenberg M, Canter K, Mahler I, Tornheim A. (2005). Observation of magnetoreceptive
behavior in a multicellular magnetotactic prokaryote in higher than geomagnetic
fields. Biophys J 88: 1496-1499.
Hamady M, Lozupone CA, Knight R. (2010). Fast UniFrac: facilitating high-throughput
phylogenetic analyses of microbial communities including analysis of
pyrosequencing and PhyloChip data. Isme J 4: 17-27.
Huber T, Faulkner G, Hugenholtz P. (2004). Bellerophon: a program to detect chimeric
sequences in multiple sequence alignments. Bioinformatics 20: 2317-2319.
Jogler C, Lin W, Meyerdierks A, Kube M, Katzmann E, Flies C et al. (2009). Towards cloning
the magnetotactic metagenome: identification of magnetosome island gene clusters in
uncultivated magnetotactic bacteria from different aquatic sediments. Appl Environ
Microbiol 75: 3972-3979.
Lane DJ. (1991). 16S/23S rRNA sequencing. In: Stackenbrandt E, Goodfellow M (eds).
Nucleic Acid Techniques in Bacterial Systematics. Wiley: New York. pp 115-175.
Lin W, Tian L, Li J, Pan Y. (2008). Does capillary racetrack-based enrichment reflect the
diversity of uncultivated magnetotactic cocci in environmental samples? FEMS
Microbiol Lett 279: 202-206.
Lin W, Li J, Schüler D, Jogler C, Pan Y. (2009). Diversity analysis of magnetotactic bacteria
in Lake Miyun, northern China, by restriction fragment length polymorphism. Syst
Appl Microbiol 32: 342-350.
Lin W, Pan Y. (2010). Temporal variation of magnetotactic bacterial communities in two
freshwater sediment microcosms. FEMS Microbiol Lett 302: 85-92.
Lin W, Li B, Pan Y. (2011). DMTB: a comprehensive online resource of 16S rRNA genes,
ecological metadata, oligonucleotides, and magnetic properties of magnetotactic
bacteria. Chin Sci Bull In press.
Lozupone CA, Knight R. (2005). UniFrac: a new phylogenetic method for comparing
microbial communities. Appl Environ Microbiol 71: 8228-8235.
8
Pan H, Zhu K, Song T, Yu-Zhang K, Lefevre C, Xing S et al. (2008). Characterization of a
homogeneous taxonomic group of marine magnetotactic cocci within a low tide zone
in the China Sea. Environ Microbiol 10: 1158-1164.
Pan Y, Lin W, Tian L, Zhu R, Petersen N. (2009). Combined approaches for characterization
of an uncultivated magnetotactic coccus from Lake Miyun near Beijing.
Geomicrobiol J 26: 313-320.
Spring S, Amann R, Ludwig W, Schleifer KH, Petersen N. (1992). Phylogenetic diversity and
identification of nonculturable magnetotactic bacteria. Syst Appl Microbiol 15:
116-122.
Spring S, Amann R, Ludwig W, Schleifer KH, van Gemerden H, Petersen N. (1993).
Dominating role of an unusual magnetotactic bacterium in the microaerobic zone of a
freshwater sediment. Appl Environ Microbiol 59: 2397-2403.
Spring S, Amann R, Ludwig W, Schleifer KH, Schüler D, Poralla K et al. (1994).
Phylogenetic analysis of uncultured magnetotactic bacteria from the alpha-subclass of
Proteobacteria. Syst Appl Microbiol 17: 501-508.
Spring S, Lins U, Amann R, Schleifer K, Ferreira L, Esquivel D et al. (1998). Phylogenetic
affiliation and ultrastructure of uncultured magnetic bacteria with unusually large
magnetosomes. Arch Microbiol 169: 136-147.
Tamura K, Dudley J, Nei M, Kumar S. (2007). MEGA4: molecular evolutionary genetics
analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:
1596-1599.
Thompson JD, Higgins DG, Gibson TJ. (1994). CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting,
positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:
4673–4680.
Xing S, Pan H, Zhu K, Wu L, Xiao T. (2008). Diversity of marine magnetotactic bacteria in
the Huiquan bay near Qingdao city. Chinese High Technology Letters 18: 312-317.
Yu YN, Breitbart M, McNairnie P, Rohwer F. (2006). FastGroupII: a web-based
bioinformatics platform for analyses of large 16S rDNA libraries. BMC
Bioinformatics 7: 57.
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