Two genera of magnetococci with bean-like morphology from intertidal sediments of the Yellow Sea, China.
ABSTRACT Magnetotactic bacteria have the unique capacity of being able to swim along geomagnetic field lines. They are Gram-negative bacteria with diverse morphologies and variable phylogenetic relatedness. Here, we describe a group of uncultivated marine magnetococci collected from intertidal sediments of Huiquan Bay in the Yellow Sea. They were coccoid-ovoid in morphology, with an average size of 2.8 ± 0.3 μm by 2.0 ± 0.2 μm. Differential interference contrast microscopy, fluorescence microscopy, and transmission electron microscopy revealed that each cell was apparently composed of two hemispheres. The cells synthesized iron oxide-type magnetosomes that clustered on one side of the cell at the interface between the two hemispheres. In some cells two chains of magnetosomes were observed across the interface. Each cell had two bundles of flagella enveloped in a sheath and displayed north-seeking helical motion. Two 16S rRNA gene sequences having 91.8% identity were obtained, and their authenticity was confirmed by fluorescence in situ hybridization. Phylogenetic analysis revealed that the magnetococci are affiliated with the Alphaproteobacteria and are most closely related to two uncultured magnetococci with sequence identities of 92.7% and 92.4%, respectively. Because they display a >7% sequence divergence to all bacteria reported, the bean-like magnetococci may represent two novel genera.
- SourceAvailable from: Richard B. Frankel[show abstract] [hide abstract]
ABSTRACT: Magnetotactic bacteria were discovered almost 30 years ago, and for many years and many different reasons, the number of researchers working in this field was few and progress was slow. Recently, however, thanks to the isolation of new strains and the development of new techniques for manipulating these strains, researchers from several laboratories have made significant progress in elucidating the molecular, biochemical, chemical and genetic bases of magnetosome formation and understanding how these unique intracellular organelles function. We focus here on this progress.Nature Reviews Microbiology 04/2004; 2(3):217-30. · 22.49 Impact Factor
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ABSTRACT: Various experimental techniques for study of magnetosensitive bacteria are described and the experimental results are presented and discussed.Chinese Journal of Oceanology and Limnology 02/2009; 27(1):6-12. · 0.58 Impact Factor
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ABSTRACT: Magnetic sensitivity has been found in bacteria from freshwater sites around Pavia, Italy.Chinese Journal of Oceanology and Limnology 01/2009; 27(1):3-5. · 0.58 Impact Factor
Two Genera of Magnetococci with Bean-like Morphology from
Intertidal Sediments of the Yellow Sea, China
Wen-Yan Zhang,a,cKe Zhou,aHong-Miao Pan,aHai-Dong Yue,aMing Jiang,cTian Xiao,a,dand Long-Fei Wub,d
Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Chinaa; Laboratoire de Chimie
Bactérienne, Aix-Marseille Université, CNRS, Marseille, Franceb; College of Marine Life Science, Ocean University of China, Qingdao, Chinac; and Laboratoire International
Associé de la Bio-Minéralisation et Nano-Structures, CNRS, Marseille, Franced
thenticity was confirmed by fluorescence in situ hybridization. Phylogenetic analysis revealed that the magnetococci are affili-
They are a morphologically, metabolically, and phylogenetically
igate along geomagnetic field lines (2, 28). MTB contain intracel-
lular membrane-bound, nano-sized, single-domain crystals
termed magnetosomes, which usually consist of iron oxide (mag-
netite, Fe3O4) or iron sulfide (greigite, Fe3S4) (2). Magnetosome
species-specific morphologies and specific arrangements within
the cell (2). Magnetosomes usually organize in chains and form a
cell to align to the Earth’s magnetic field, which enables the bac-
terium to find and maintain an optimum position in the oxygen
and chemical gradient (10, 11). Magnetotactic bacteria are ubiq-
uitous in the water column and sediments of freshwater and ma-
rine habitats and are believed to play an important role in iron
cycling (2, 7).
MTB comprise a variety of morphological types (including
coccoid, spiral, vibroid, rod-like, or aggregated) (8, 27, 29) and
have a great phylogenetic diversity. MTB have been identified in
Proteobacteria and Nitrospirae, and a recent study indicates the
existence of large ovoid-shaped MTB belonging to candidate di-
31, 32), and the interactions of bacterial species in a given habitat
with the prevailing environmental conditions are likely to shape
the dominant phylogenetic pattern. In a previous study we re-
ported the almost homogeneous occurrence of magnetotactic
magnetotactic cocci appear to be extremely diverse. We report
here a novel group of bean-like magnetococci collected from the
observed a hemispheric morphology, and phylogenetic analysis
agnetotactic bacteria (MTB) were first discovered indepen-
dently by Bellini in 1964 and Blakemore in 1975 (3–5, 9).
suggests that they belong to two novel genera of Alphaproteobac-
MATERIALS AND METHODS
Sampling and collection of MTB. Surface sediments and water (approx-
imate ratio, 1:1) were collected during low tide from the Huiquan Bay
(36°03= N, 120°21= E) of the Yellow Sea and were stored in 500-ml plastic
35‰ and a pH of 7.6. As previously reported, magnetotactic bacteria in
the samples were magnetically enriched by attaching the south pole of
permanent magnets outside the bottles (25) and were purified using the
racetrack method (34).
analyzed by optical microscopy (Olympus BX51, equipped with a DP71
camera system) using the hanging-drop method in an applied magnetic
field, and the motility track was recorded using dark-field microscopy.
For scanning electron microscopy (SEM), the samples were fixed in
filters with high-density pores 0.2 ?m in diameter (Whatman), and then
dehydrated with ethanol and isoamyl acetate. The dried and gold-coated
samples were examined using a KVKV-2800B SEM operating at 25 kV.
For transmission electron microscopy (TEM), the samples were
(operating at 75 kV) and a JEM 2100 high-resolution transmission elec-
tron microscope ([HRTEM] operating at 200 kV) equipped for energy-
dispersive X-ray spectroscopy (EDXS). The length and width of magne-
Received 11 January 2012 Accepted 24 May 2012
Published ahead of print 1 June 2012
Address correspondence to Tian Xiao, firstname.lastname@example.org, or Long-Fei Wu,
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
aem.asm.orgApplied and Environmental Microbiologyp. 5606–5611August 2012 Volume 78 Number 16
tosomes were measured using Adobe Photoshop software. The size and
shape factor of the magnetosomes were calculated as (length ? width)/2
and width/length, respectively (22).
Sequence analysis of the 16S rRNA gene. The 16S rRNA genes of
collected bacterial cells were amplified with the universal primers 27f (5=-
AGA GTY TGA TCC TGG CTC AG-3=) and 1492r (5=-GGT TAC CTT
GTT ACG ACT T-3=) using PCR (16). The PCR amplification and con-
struction of a 16S rRNA gene clone library were performed as previously
reported (25). After eliminating four false-positive clones from library of
200 clones, we further screened the other clones using restriction frag-
ment length polymorphism (RFLP) analysis. Clones with identical pat-
terns were defined as an operational taxonomic unit (OTU), and then
(21). After we eliminated the sequences for chimeras by using the Pintail
program (http://www.bioinformatics-toolkit.org/Web-Pintail/), the se-
.ncbi.nlm.nih.gov/BLAST/]). The related sequences were aligned using
CLUSTAL W multiple alignment. Identity was calculated using the
BioEdit program, and phylogenetic trees were constructed using MEGA,
version 4.0, applying a neighbor-joining method. Bootstrap values were
calculated with 1,000 replicates.
tide probes p-3 (5=-TCT TTG AGG AGG GAG CCG TTG-3=; nucleotide
G-3=; nucleotide positions 112 to 133) were designed using the probe
design tool in Primer Premier, version 5.0 software. The probes were
labeled with Cy3 as the fluorescent dye, and the general probe EUB338
(5=-GCT GCC TCC CRT AGG AGT-3=; nucleotide positions 338 to 355)
was labeled with 6-carboxyfluorescein (FAM) and used as the positive
control in the hybridization. Escherichia coli Top 10 cells and magnetot-
the hybridization with specific probes.
and Pernthaler et al. (26). The racetrack-purified samples were fixed with
(PBS), and then stored in ethanol-PBS (1:1) at ?20°C. The samples were
dried on prepared glass slides, dehydrated in an ethanol series, immersed
for 10 min at 48°C. The hybridizations were analyzed by fluorescence
microscopy (Olympus BX51 fluorescence microscope).
Nucleotide sequence accession numbers. The sequences of the 16S
rRNA genes in clones 1-3 and 1-9 were deposited in GenBank under
accession numbers JF421219 and JF421220, respectively.
RESULTS AND DISCUSSION
Morphology and motility of the bean-like magnetococci. The
mogeneous in morphology and numbered up to 103to 104cells/
microscopy. They survived in an aquarium for more than 1 year
under laboratory conditions.
In the presence of an applied magnetic field, the freshly col-
lected magnetococci in a hanging drop displayed north-seeking
taxis (Fig. 1A). Using long-time-exposure photography of the
swimming magnetococci, we observed a clockwise helical trajec-
tory during forward swimming. The cells completed approxi-
FIG 1 Morphology and motility of bean-like magnetococci (BMC) based on optical microscopy. Differential interference contrast (DIC) images of BMC are
were recorded using dark-field microscopy. BMC cells display a clockwise helical trajectory during forward swimming (B1) and a U-like track in response to
reversal of the magnetic field (B2). The exposure time was 3 s for panel B1 and 5 s for panel B2. Panel D shows the fluorescence of BMC exposed to blue light
(wavelength 450 to 480 nm). Scale bars, 5 ?m (A), 100 ?m (B), and 2 ?m (C and D).
Characterization of Two New Magnetococcus Genera
August 2012 Volume 78 Number 16 aem.asm.org 5607
398) and a Gaussian speed distribution (data not shown). This
50 ?m/s) isolated from Huiquan Bay (36) but slower than MO-1
(approximately 250 ?m/s) from the Mediterranean Sea (17).
Differential interference contrast (DIC) microscopy revealed
FIG 2 Characteristics of BMC cells determined by SEM and TEM. (A) Morphology of BMC cells by SEM as shown by a top view (A1) and a lateral view (A2).
The two types of cells were observed in the examples shown in panel B: BMC with two chains of magnetosomes (C1) and flagella (D1) and BMC with clustered
magnetosomes (C2) and flagella (D2). Scale bars, 2 ?m (A) and 500 nm (B to D).
FIG 3 Intracellular features of the bean-like magnetococci. (A) Characteristics of cluster-forming magnetosomes: morphology (A1), size histograms (A2), and
Energy dispersive X-ray (EDX) analysis of magnetosomes (C1) and granules (C2) is also shown. Line 1, cell; line 2, magnetosomes (note the peaks of iron and
oxygen); line 3, granules (note the peaks of phosphorus and oxygen). Scale bars, 50 nm.
Zhang et al.
aem.asm.org Applied and Environmental Microbiology
morphology (similar to that of a bean) with a space between the
two hemispheres (Fig. 1C, white arrows). Fluorescence micros-
between the two hemispheres (Fig. 1D, white arrows), consistent
with the observed space under DIC microscopy. In view of their
remarkable morphology, we termed them bean-like magneto-
cocci (BMC). We used SEM and TEM to analyze their morphol-
ogy. SEM revealed an overall spherical morphology without an
obvious double hemisphere (Fig. 2A1 and 2A2). In contrast, the
hemispherical morphology was evident using TEM, which re-
vealed two large electron-dense granules (Fig. 2B, labeled P), a
groove around the center confirming that the cell comprised two
parts (Fig. 2B, black arrow), and an intracellular hemispherical
architecture. We hypothesize that the intracellular hemispherical
architecture of the BMC cells may be a consequence of the distri-
Itaipu Lagoon, Brazil (32), and from a salt pond in Falmouth,
two magnetosome arrangements (Fig. 2B). Most cells in the pop-
gular projected shape in a disorganized cluster on one side of the
cell at the interface between the two hemispheres (Fig. 2C1). De-
spite the apparent disorganization of the magnetosomes, the cells
displayed the capacity of north-seeking magnetotaxis, implying
that the magnetosomes still form an overall magnetic dipole mo-
ment. The size of the magnetosome crystals varied from 47 to 145
nm, and the shape factor was 0.60 (average length and width,
percent of the BMC cells contained an average of 22 ? 4 (n ? 64)
between the hemispheres (Fig. 2C2). Each magnetosome had a
rectangular projected shape of length 125 ? 32 nm and width
99 ? 25 nm (n ? 118). This produced a shape factor of approxi-
mately 0.80 ? 0.11 (Fig. 3B), which is similar to that of the mag-
netotactic coccus QHL, collected from the low-tide zone in
Huiquan Bay, China (25). Relative to the distribution of magne-
tosomes in BMC cells that had magnetosomes arranged in a dis-
organized cluster, distribution of magnetosomes in cells having
much smaller and had lower contrast relative to the background
than the other crystals in the chains (Fig. 2C2, black arrows). The
smaller crystals may be early-stage immature magnetosomes. A
similar distribution of magnetosomes has been observed for the
cultivated magnetotactic spirillum strains AMB-1 and MSR-1
TEM analysis revealed that each bean-like magnetococcus had
two flagellar bundles approximately 80 nm diameter (Fig. 2D),
which are substantially larger than the diameter of 10 to 30 nm
FIG 4 Fluorescence in situ hybridization analyses of magnetically enriched BMC cells. The same microscopic field is shown following staining with 4=,6=-
labeled probe p-3 (C) and p-9 (D). Scale bars, 5 ?m.
Characterization of Two New Magnetococcus Genera
August 2012 Volume 78 Number 16aem.asm.org 5609
observed for single flagella (12), and dispersed individual flagella
had been observed (data not shown). These results suggested the
coid-ovoid bacteria (17, 35).
be single-domain crystals, according to theoretical predictions
(6). Energy-dispersive X-ray spectroscopy (EDXS) analysis indi-
cated that the magnetosome crystals in both groups were com-
posed of iron and oxygen (Fig. 3C1). This is consistent with the
results of high-resolution TEM (HRTEM) analysis, which identi-
fied the magnetosome crystals as magnetite (data not shown). In
dense granules were observed in each group of BMC cells. EDXS
analysis revealed that these were rich in phosphorus and oxygen
(Fig. 3C2) and may be polyphosphate, as reported for other MTB
(17, 23, 36).
Fluorescence in situ hybridization and phylogenetic analy-
sis. Nearly full-length 16S rRNA genes from the BMC cells were
amplified and cloned. After four false-positive clones were elimi-
nated from the clone library, 13 OTUs were identified by RFLP
analysis and sequenced. Two dominant OTUs (107 clones) were
putative chimeras (two OTUs, 2 clones) were eliminated. Clones
1-3 and 1-9 are representatives of the two dominant OTUs (in-
cluding 61 clones and 46 clones, respectively), and they exhibited
91.8% sequence identity.
To confirm that the two sequences were associated with the
BMC cells, two specific probes (p-3 and p-9) were designed and
used in FISH analysis. Fluorescence microscopy revealed a strong
signal for all collected cocci following hybridization with the uni-
versal bacterial probe EUB338 (Fig. 4B1 and B2). In contrast, the
specific probes p-3 and p-9 hybridized with only some of the cells
(Fig. 4C and D), confirming the presence of two types of BMC
TEM analysis revealed either the clustered or chain-forming
organization of the magnetosomes, whereas FISH experiments
lected samples. It was technically infeasible to directly relate mag-
netosome organization to the phylogenetic affiliation of the cells,
and as magnetosomes display species specificity (2), it was possi-
ble to make a connection based on the occurrence frequency.
RFLP analysis showed that the 16S rRNA gene sequence 1-3 rep-
resented 61 clones while the 1-9 represented 46 clones. In addi-
tion, most cells had cluster-type magnetosomes (Fig. 2B), and the
p-3 probe hybridized with more cells (45%) than did the p-9
probe (38%) (Fig. 4C and D). Therefore, it may be possible that
the 1-3 16S rRNA gene sequence is associated with cells that have
clustered magnetosomes and hybridize with the p-3 probe. This
bacteria are obtained or until successful separation of single cells
by various approaches, such as micromanipulation, is achieved.
Among all 16S rRNA gene sequences in GenBank, the 1-3 16S
rRNA gene sequence showed a maximum sequence identity
(92.7%) with uncultured magnetococci collected from sediments
of a freshwater lake (Miyun Lake, China; accession number
EU780674), while clone 1-9 16S rRNA gene sequence showed
92.4% sequence identity with uncultured magnetococci from in-
Phylogenetic analysis revealed that the BMC cells were affiliated
with the Alphaproteobacteria, and their 16S rRNA gene sequences
had ?7% divergence from all previously reported bacteria (Fig.
genera of magnetotactic bacteria (13). This finding highlights the
remarkable biodiversity of magnetotactic bacteria in general and
suggests that many novel magnetococcus species remain to be
We thank Jianhong Xu for assistance in sampling.
of China (NSFC 40906069, 41106135, and 40776094), the Special Con-
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FIG 5 Phylogenetic tree showing the relationship between the BMC and related magnetotactic bacteria. The tree is constructed based on neighbor-joining
analysis using the sequence region from position 27 to 1492 using E. coli numbering. The sequences determined in this study are shown in bold. GenBank
accession numbers of the sequences used are indicated in parentheses. The scale bar is 0.02 substitutions per nucleotide position.
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