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Environmental Microbiology (2012)
doi:10.1111/1462-2920.12057
Adaptation of spherical multicellular magnetotactic
prokaryotes to the geochemically variable habitat of
an intertidal zone
Ke Zhou,1,2 Wen-Yan Zhang,1,2 Hong-Miao Pan,1,2
Jin-Hua Li,2,3 Hai-Dong Yue,1,2 Tian Xiao1,2* and
Long-Fei Wu2,4**
1
Key Laboratory of Marine Ecology & Environmental
Sciences, Institute of Oceanology, Chinese Academy of
Sciences, Qingdao 266071, China.
2
France–China Bio-Mineralization and Nano-Structures
Laboratory, Beijing 100193, China.
3
Paleomagnetism and Geochronology Laboratory, Key
Laboratory of the Earth’s Deep Interior, Institute of
Geology and Geophysics, Chinese Academy of
Sciences, Beijing 100029, China.
4
Laboratoire de Chimie Bactérienne, Aix-Marseille
Université, CNRS, F-13402 Marseille Cedex 20, France.
Summary
A combination of microscopic, molecular and biogeochemical methods was used to study the structure,
phylogenetics and vertical distribution of spherical
multicellular magnetotactic prokaryotes (MMPs) of
intertidal sediments in the Yellow Sea. These MMPs
were 5.5 mm in diameter and composed of approximately 15–30 cells. They synthesized bullet-shaped
magnetites in chains or clusters. Phylogenetic analysis of 16S rRNA gene sequences suggested that
these MMPs represent a novel species affiliated to the
Deltaproteobacteria. To study their vertical distribution and the relationship to geochemical parameters,
sediment cores were collected after the redox potential was measured in situ. The sediments were composed of yellow, grey and black layers from the
surface to depth. The spherical MMPs were concentrated near the grey-black layer transition at a depth
of 8–12 cm, while coccoid-shaped magnetotactic bacteria near the yellow-grey layer transition at a depth of
3–5 cm. The intertidal MMPs showed a deeper distribution at more reduced environments than coccoidshaped magnetotactic bacteria, and MMPs in lagoon
Received 3 May, 2012; revised 5 November, 2012; accepted 22
November, 2012. For correspondence. *E-mail [email protected];
Tel. (+86) 532 82898586; Fax (+86) 532 82898586; **E-mail
[email protected]; Tel. (+33) 4 9116 4157; Fax (+33) 4 9171 8914.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd
sediments. Additionally the MMPs were concentrated
significantly in layers with high proportion of fine
sand and total organic carbon, rich in leachable iron
but poor in nitrate. These results show an adaptation
of spherical MMPs to the peculiar intertidal sediment
habitat.
Introduction
Among the diverse morphotypes of magnetotactic
bacteria (MTB) (Amann et al., 2006; Lefèvre et al., 2011;
Kolinko et al., 2012), multicellular magnetotactic prokaryotes (MMPs) are a unique group with respect to their
multicellular forms (Keim et al., 2007). Based on morphology there are two kinds of MMPs. One morphological form
is that of the ellipsoidal pineapple-like MMPs that occur in
sediments of the Yellow Sea and the Mediterranean Sea
(Lefèvre et al., 2007; Zhou et al., 2012). These MMPs,
approximately 10 mm ¥ 8 mm, are composed of 28–57
cells arranged in several interlaced circles. Magnetosomes in these MMPs are bullet-shaped magnetites.
The other morphological form is that of the spherical,
mulberry-like MMPs reported from many habitats worldwide, including the stratified water column of brackish
ponds, sediments of salt marshes, lagoons and intertidal
zones (Rodgers et al., 1990; Simmons and Edwards,
2007a; Simmons et al., 2007; Edwards and Bazylinski,
2008; Wenter et al., 2009; Lefèvre et al., 2010; Zhou
et al., 2011). These MMPs are 3–12 mm in diameter and
formed by 10–40 cells arranged in a helical symmetry
(Keim et al., 2007). Magnetosomes in these MMPs are
magnetite and/or greigite crystals of various shapes
(Mann et al., 1990; Posfai et al., 1998; Keim et al., 2004a;
2007; Lins et al., 2007; Wenter et al., 2009; Zhou et al.,
2011). In addition to their morphology, MMPs are distinctive in terms of their life cycle, motility and phylogenetics.
They remain multicellular during division and no viable
unicellular cells similar to that of MMPs are observed
(Keim et al., 2004b; Abreu et al., 2006). MMPs exhibit a
specific escape motility which involves a rapid backward
movement with continuous deceleration and a slow
forward movement with uniform acceleration (Greenberg
et al., 2005; Keim et al., 2007). According to analysis of
16S rRNA gene sequences and fluorescence in situ
2
K. Zhou et al.
hybridization (FISH), all cells from a MMP have the same
phylotype, and all reported MMPs are affiliated to the
Deltaproteobacteria and are closely related to the sulfatereducing bacteria (Delong et al., 1993; Abreu et al., 2007;
Simmons and Edwards, 2007a; Wenter et al., 2009; Zhou
et al., 2012). MMPs may represent as sinks of iron, sulfur
and organic content, and play an important role in the
geochemical cycles of iron and sulfur (Simmons et al.,
2004; Martins et al., 2007; Simmons and Edwards,
2007b).
Since the first report of their occurrence in 1983 (Farina
et al., 1983), spherical MMPs have been found to be
broadly distributed worldwide (Rodgers et al., 1990; Keim
et al., 2004a,b; Silva et al., 2007; Simmons and Edwards,
2007a; Simmons et al., 2007; Edwards and Bazylinski,
2008; Martins et al., 2009; Wenter et al., 2009; Lefèvre
et al., 2010; Shapiro et al., 2010; Zhou et al., 2011; 2012).
However, knowledge of their vertical distribution has been
limited to few studies in a stratified salt pond in the USA
and in a hypersaline lagoon in Brazil (Simmons et al.,
2004; Abreu et al., 2007; Sobrinho et al., 2011). The intertidal region is an important model system containing a
high diversity of species, and the zonation created by the
tides causes species ranges to be compressed into very
narrow bands (Manivanan, 2008). Our previous study
reported the occurrence of spherical MMPs in sediments
of the Yellow Sea (Zhou et al., 2011), but little is known
about the phylogeny and vertical distribution of these
MMPs. Here we report a more detailed investigation of the
structure, phylogenetic affiliation of spherical MMPs of the
Yellow Sea, and their vertical distribution with respect to
the profiles of geochemical parameters in intertidal sandy
sediments, which implying an adaptation of these MMPs
in vertical distribution to the ever-changing intertidal environment.
Results
Occurrence and structure of spherical MMPs
There were two kinds of sediments in the site under study
(Supplemental Fig. S1). One was the gravel sediments
where the ellipsoidal MMPs were obtained (Zhou et al.,
2012), the other was the sandy sediment where only
spherical, mulberry-like MMPs were commonly observed
with a maximum abundance approximately of 500 ind cm-3
in summer. When inspected in the hanging drop, more than
90% of the MMPs migrated parallel to the magnetic field
lines (north-seeking) (Fig. 1A). The average speed during
the forward movement was 55 mm s-1 (n = 74) within the
drop of filtered seawater. Typical escape motility was also
observed in these MMPs (Supplemental Movie S1). In
addition, spherical MMPs exhibited negative phototaxis,
quickly swim away from the illumination sources in the
opposite direction to the applied magnetic field, when they
were exposed to the light beam with wavelength ranges of
460–490 nm, 400–410 nm or 330–385 nm.
The spherical MMPs were composed of approximately
15–30 cells arranged in a helix (Fig. 1B and C). Elongated
MMPs in the process of division stage, as evidenced by
the central constriction at the septum, were occasionally
observed (Fig. 1D). On reduction of the osmolarity of the
hanging drop by addition of distilled water, the MMPs
Fig. 1. Morphology of spherical MMPs in the Yellow Sea.
A. Spherical MMPs were observed in intertidal sediments of the Huiquan Bay, in the Yellow Sea. The maximum concentration observed was
500 ind cm-3. More than 90% of the MMPs had north-seeking polarity (the direction of the magnetic filed is indicated at the bottom left).
B. The spherical morphology of the MMPs.
C. The helical arrangement of MMP cells.
D. A dividing MMP showing its elongated shape.
E. A partially disaggregated MMP showing the ovoid cells.
F and G. Size histograms of MMPs and individual cells respectively.
(A), (B), (D) and (E) were obtained using differential interference contrast microscopy, while (C) was obtained by scanning electron
microscopy. Bars = 10 mm in (A), 2 mm in (B), (D) and (E), and 500 nm in (C).
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Adaptation of spherical MMPs to intertidal habitat
3
Fig. 2. Structure of spherical MMPs.
A. A whole mount of MMP, showing chains and clusters of magnetosomes near the periphery of each cell.
B. Magnification of a selected area in (A), showing the bullet-shaped magnetosomes.
C and D. Histograms of the length and width of magnetosomes respectively.
E. Electron diffraction pattern showing the magnetite composition of crystals.
F. Peritrichous flagella.
G. Nile red staining showing lipid granules and the outmost layer (arrow) of the MMP.
Bars = 500 nm in (A), 100 nm in (B), 1 mm in (F) and 2 mm in (G).
rapidly disaggregated and adopted the ovoid shape of
individual cells (Fig. 1E). The average diameter of the
MMPs was 5.5 ⫾ 0.8 mm (n = 384) and the average size
of individual cells was 1.4 ⫾ 0.2 mm (n = 153) in the
largest dimension (Fig. 1F and G).
In the spherical MMPs from the Yellow Sea the magnetosomes were arranged in chains or in clusters and distributed near the periphery of the cell (Fig. 2A). The
numbers of magnetosomes per MMP ranged from 282 to
1155; average number of magnetosomes in one cell
was 41 ⫾ 20 (n = 188). The magnetosomes were bulletshaped and 92 ⫾ 20 nm long by 35 ⫾ 4 nm wide
(n = 406) (Fig. 2B–D). X-ray elemental mapping with
scanning TEM and selected area electron diffraction
(SAED) analyses determined the mineral phase of the
magnetosomes as magnetites (Supplemental Fig. S2 and
Fig. 2E). TEM analyses showed the presence of peritrichous flagella of the MMPs (Fig. 2F). Nile red staining
revealed the presence of lipid granules and an outmost
layer in the spherical MMPs (Fig. 2G).
Phylogenetic analysis
We cloned and sequenced the 16S rRNA genes from
magnetically purified samples dominated by spherical
MMPs. Only one phylotype closely related to reported
MMPs was obtained in present study. Specific probe for
ellipsoidal MMPs did not hybridize spherical MMPs. FISH
analysis with specific probe M97 confirmed the authenticity of the obtained sequence (HQ857737) being that of the
spherical MMPs from the Yellow Sea (Supplemental
Fig. S3) and the same phylotype of cells in a MMP. Phylogenetic analysis of the 16S rRNA gene sequences indicated that the spherical MMPs were affiliated to the
Deltaproteobacteria and were most closely related to
Candidatus Magnetomorum litorale isolated from the
North Sea (96% sequence identity). In addition, they
shared 95% sequence identity with Candidatus Magnetoglobus multicellularis from the Araruama lagoon of
Brazil (Fig. 3). We propose the name Candidatus Magnetomorum tsingtaoroseum for this novel species of
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
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K. Zhou et al.
Fig. 3. Phylogenetic tree of 16S rRNA gene sequences constructed based on neighbour-joining analysis. The sequence determined in this
study is shown in bold. GenBank accession numbers of the sequences used are indicated in parentheses. The sample origins of the
downloaded, uncultured sequences are: GU784824 – Salton Sea, CA, USA; EF014726 – Araruama lagoon, Brazil; EU717681 – intertidal
sandy flats of the North Sea, Germany; GU732821 – Great Boiling Springs, NV, USA; others – the Little Sipperwissett salt marsh, MA, USA.
Scale bar indicates 0.02 substitutions per nucleotide position.
spherical, mulberry-like MMPs detected in the intertidal
sediments at Qingdao (Tsingtao) city.
Vertical distribution of spherical MMPs and the
relationship to geochemical parameters
Biogeochemical characteristics of sediment cores and
pore water. At each time of the analyses the temperature
was in the range of 15.8–17.2°C. The pH of the pore water
varied from 7.56 to 8.08, and the salinity ranged from 14‰
to 29‰ (Supplemental Fig. S4).
The cores were composed of three layers distinguished on the basis of colour (Fig. 4). The upper layer
(extending to 3 cm depth from the surface in cores A, B
and D, and 5 cm from the surface in core C) was yellow
sand, while the deeper two layers were grey and black
sand respectively. The black layer, which may be due to
the precipitation of FeS, occurred at a depth of approximately 8–10 cm. Redox potential profiles showed that
the redoxcline was located at the yellow-grey layers
transition (Fig. 4), implying the oxic–anoxic transition.
The composition of all four cores was predominantly
sand (82.4% to 99.9%). Fine-grained sand (0.10–
0.25 mm) dominated most layers. The percentage of fine
sand was not uniform in the vertical profile; but
increased with increasing depth in the profile, maximum
in the black layer (Fig. 5). No obvious variation in the
proportion of fine sand was observed in the bottom parts
of black layer.
The total organic carbon (TOC) varied from 0.04% to
0.88% in the sediment cores. The proportion of TOC in
each core increased with depth, and peaked at a depth of
approximately 6–8 cm in cores A and B, and 8–10 cm in
cores C and D, in all cases in or near the upper boundary
of the black sediment layer. At greater depth the concentration of TOC decreased sharply (Fig. 5).
The concentration of nitrate was the greatest in the
surface layer and declined rapidly with the depth, which
was the opposite of the pattern observed for the concentration of ammonium (Fig. 5). The sulfate profiles varied
greatly among the four sediment cores (Supplemental
Fig. S4). In core A and core D, the sulfate concentration
decreased with depth. In core B there were two peaks of
sulfate concentration, one in the surface layers and
another in the upper part of the black sediment layer. In
core C the sulfate concentration ranged from 2168 mg l-1
to 2322 mg l-1 throughout the core.
The concentration of leachable iron in the sediment
ranged from 132 mg kg-1 to 1756 mg kg-1. The leachable
Fig. 4. Characteristics of sediment core.
A. Two transitions (indicated by the white dash lines) according to
the sediment colour.
B. Redox potential profile showing that the redoxcline was located
at the yellow-grey layers transition.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Adaptation of spherical MMPs to intertidal habitat
iron concentration generally increased from the surface
yellow sand and peaked near the upper boundary of the
black sediment layer. For all four cores the concentration
of leachable iron declined abruptly below the depth of
peak concentration (Fig. 5). The proportion of ferrous iron
in the leachable iron was least in the surface layer and
greatest in the deeper layers (Fig. 5). In the surface sediments (0–2 cm), ferrous iron comprised < 50% of the
leachable iron, while at the sites with peak leachable iron
concentration, ferrous iron comprised > 82%.
Vertical distribution of MMPs. Vertically, MMPs were heterogeneously distributed in all four core profiles, and their
distribution differed from that of the unicellular MTB
(Fig. 5). In the surface layers of the yellow sediments
there were fewer MMPs (maximum density 2 ind cm-3).
MMPs were more abundant in the grey sediments, and
the maximum concentrations were detected at a depth of
8–10 cm in cores A, B and D, and 10–12 cm in core C, in
all cases near the upper boundary of the black sediment.
The peak concentration in these layers was 14 ind cm-3,
which was 10 times greater than that in the surface layers.
In contrast, the coccoid-shaped magnetotactic bacteria
mainly occurred at the oxic–anoxic transition layer at a
depth of 3–5 cm (cores A, B and D), or in the layer above
that having the maximum concentration of MMPs (core
C). Occasionally, spirillum-shaped magnetotactic bacteria
occurred in the grey and black sediments of the profiles
(cores C and D) and were the dominant type
of unicellular MTB in the layers where MMPs were
abundant.
Although no obvious correlations were evident between
the distribution of MMPs and the profiles of temperature,
pH, salinity and sulfate (Supplemental Fig. S4), the variation in MMPs density was closely related to the grain size
and the availability of organic matter and iron.
The concentration profiles of the MMPs were typically
coincident with the fine sand and TOC profiles. Both the
peaks of fine sand proportion and TOC concentration
were in or near the peaks of MMPs concentration. In the
layers where the MMPs were concentrated, fine sand
dominated the cores (> 80% in cores B, C and D, and
65.0% in core A) and the TOC proportion ranged from
0.15% to 0.88%. Pearson’s correlation analysis showed
that the concentration of MMPs was positively related to
the proportion of fine sand in core C (r = 0.786, P < 0.05)
and to the availability of TOC in core D (r = 0.989,
P < 0.01).
In the deeper layers where MMPs were most abundant,
there was abundant leachable iron (587–942 mg kg-1)
that was dominated by ferrous iron (Fig. 5). A positive
correlation (r = 0.943, P < 0.01) was found between the
distribution of MMPs and the proportion of leachable iron
in core D.
5
Discussion
The finding of spherical, mulberry-like MMPs in intertidal
sediments of the Yellow Sea is consistent with their worldwide distribution (Bazylinski et al., 1990; Simmons and
Edwards, 2007a; Wenter et al., 2009; Lefèvre et al., 2010;
Sobrinho et al., 2011; Zhou et al., 2011). The bulletshaped magnetite crystals in these spherical MMPs were
also reported for the spherical MMPs from the lagoons in
Brazil and China, and the ellipsoidal MMPs from gravel
sediments of the Yellow Sea (Lins et al., 2007; Zhou et al.,
2011; 2012). Phylogenetically, the spherical MMPs in the
Yellow Sea intertidal sediments represent a novel species
within a known genus of other spherical MMPs.
The finding of different distributions of spherical MMPs
and the unicellular MTB in the profiles of coastal sediment
is consistent with the reported distribution of MTB in the
stratified water column of a salt pond, where the coccoidshaped magnetotactic bacteria were concentrated in the
upper, oxic region while the MMPs and the rod-shaped
magnetotactic bacteria were associated with the anoxic
area (Simmons et al., 2004). Flies et al. have carried out
thorough studies about the vertical distribution of magnetotactic bacteria along various physicochemical gradients
in freshwater microcosms (Flies et al., 2005). They found
that different species of magnetotactic bacteria were
detected at different position within vertical gradients
although the largest proportion of magnetotactic bacteria
was detected within the suboxic zone. Importantly, within
very narrow distance the population may have very important change. In our study we did not find significant difference of the distribution profiles when the slices were cut at
every 2 cm compared with those at every 1 cm (data not
shown). It might be because the sediments in the microcosms are more stable than the intertidal sites. Nevertheless, the current studies show that MTB are typically
concentrated within a few centimetres of the sediment
surface in aquatic environments (Petermann and Bleil,
1993; Flies et al., 2005; Lin and Pan, 2009; Sobrinho
et al., 2011). In the sediments of Araruama lagoon
(Brazil), the MMPs were concentrated in the anoxic zone
at a depth of approximately 4–5 cm (Abreu et al., 2007;
Sobrinho et al., 2011). In present study, the spherical
MMPs in intertidal sediments were concentrated at a
greater depth of 8–12 cm, deeper than that of coccoidshaped magnetotactic bacteria in the profile and that of
MMPs in lagoons. The deeper distribution of spherical
MMPs at more reduced environments in present study
may result from an adaptation to the intertidal sediments,
of which the upper layers are ever-changing and the
stable transition zones are located deeper than those in
lagoons (Fig. 5).
The distribution of MMPs was closely related to the
grain size and the concentration of TOC (Fig. 5). The
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
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K. Zhou et al.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Adaptation of spherical MMPs to intertidal habitat
7
Fig. 5. Vertical distribution of spherical MMPs and the relationship with geochemical parameters.
Panel 1. MMPs were concentrated in the deeper layer at more reduced environments (Fig. 4) while coccoid-shaped magnetotactic bacteria
( ) were concentrated in the upper layers, at the oxic–anoxic layers transition indicated by the redox potential profile (Fig. 4). Occasionally,
spirillum-shaped magnetotactic bacteria ( ) occurred in the grey and black sediments of the profiles (cores C and D) and was the dominant
type of unicellular MTB in the layers where MMPs were abundant. The symbols ( , ) near the bars of unicellular MTB indicate the dominant
morphotypes of unicellular MTB.
Panel 2. Vertical distribution of fine sand (FS) proportion, nitrate and ammonium concentration. Generally, MMPs peaked in the layers
dominated by fine sand and containing high levels of ammonium, low concentration of nitrate.
Panel 3. Vertical variation in the concentration of leachable iron, the proportion of Fe(II) and the TOC in the sediment cores.
In the layers in which MMPs were most abundant, the leachable iron was abundant and dominated by ferrous iron. The peaks of MMPs were
partially overlapped with that of TOC.
spherical MMPs in the intertidal zone of the Yellow Sea
were concentrated in sediments primarily composed of
fine sand. Fine sand is generally less permeable than
medium sand and coarse sand, and has a lower pore
water advection, and consequently there is less advection
and shallower penetration of oxygen into such sediments
(Janssen et al., 2005). This would be advantageous for
MMPs that are sensitive to oxygen exposure. In addition,
the smaller grain size of fine sediments provides greater
specific surface area, which results in greater capacity
with respect to organic matter adsorption (Keil et al.,
1994). This is consistent with the findings of the present
study, where the TOC concentration peaked in or near the
layer with the maximum proportion of fine sand. As illustrated in the partial overlap of the peaks of MMPs with that
of TOC, one of the advantages of MMPs relative to the
unicellular MTB, may be their better ability to access
high-nutrient environments.
Generally, the occurrence of fine-grained, less permeable sand is associated with the occurrence of ammonium
rather than nitrate in intertidal sandy sediment (Janssen
et al., 2005). Indeed, there was more ammonium and less
nitrate in the fine sand layer where the MMPs were concentrated. In previous studies in freshwater and marine
habitats, nitrogen (mainly nitrate) has been found to affect
the distribution and genetic variability of the MTB communities (Petermann and Bleil, 1993; Lin and Pan, 2010;
Sobrinho et al., 2011). This relationship with nitrogen
availability may be a result of the physiological characteristics. Several cultivated MTB strains have been found to
use nitrate or nitrous oxide as terminal electron acceptors
for respiration (Bazylinski and Williams, 2007). However,
Desulfovibrio magneticus RS-1 and strain BW-1, two
magnetotactic strains also in the Deltaproteobacteria, are
unable to respire nitrate (Sakaguchi et al., 2002; Lefèvre
et al., 2011). This may explain why the presence of MMPs
in the Yellow Sea did not coincide with the availability of
nitrate.
Taken together, the combination of geochemical analysis and the viable cell counting confirmed the hallmark
feature of the intertidal sediments that are composed of
an ever-changing upper layers and a deeper location of
MMPs in this habitat. This adaptive distribution may result
from multiple strategies. In addition of magneto-aerotaxis,
negative phototactic behaviour was also observed in the
spherical MMPs, unicellular magnetotactic bacteria, nonmagnetotactic multicellular prokaryotes (nMMPs), spherical MMPs and ellipsoidal MMPs (Frankel et al., 1997;
Lefèvre et al., 2010; Shapiro et al., 2010; Zhu et al., 2010;
Zhou et al., 2012). We confirmed that MMPs may exhibit
a fine balance between magnetotaxis and phototaxis, and
benefit from both in their survival in the ever-changing
habitats (Shapiro et al., 2010).
Experimental procedures
Sampling and in situ measurements
The sampling site is located at the intertidal zone of Huiquan
Bay (36°03′N, 120°21′E) in the Yellow Sea, China. The
topography varies rapidly with the tide. The site is also
affected by freshwater and organic matter from sewerage
effluents. From 2008 to 2010, during the low tide when there
was little water above the sediment (Supplemental Fig. S1),
subsurface sediment was collected in the sandy area, along
with the water penetrated after the sediment removing (ratio
1:1), and stored in 500 ml plastic bottles.
To study the vertical distribution of MMPs and the relationship to environmental parameters, sediment cores were collected in a Plexiglass tube (10 cm in diameter) at the
beginning of the outgoing tide, when there was little water
above the sediment, over four consecutive days in November
2011, from within a 1 m2 site. Following insertion of the tube
into the sediment the top opening of the tube was sealed with
a foam plug to avoid compression of the sediment. The redox
potential was measured in situ from the depth of 1 cm to
approximately 15 cm with an interval of 2 cm, using a pair of
custom electrodes (Nanjing Zhongkeyuan Kuake Sci.Tech,
China), which include a platinum electrode and a reference
calomel electrode. The value was presented in millivolts
versus the reference electrode H+/H2. Simultaneously, the
temperature of the sediment was determined at the same
depth, using a pH/mV/temperature benchtop meter (JENCO
6173, Shanghai, China) equipped with a stainless steel temperature probe (6230 AST, Shanghai, China). Prior to removal
of the core, the pore water was collected from immediately
outside the tube at the same depth using a modified multiple
pore water sampler (Hüttel, 1990, Supplemental Fig. S5).
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
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K. Zhou et al.
Collection and enumeration of magnetotactic bacteria
MMPs in the sample bottles were magnetically concentrated,
and purified using the race-track method, as previous
reported (Wolfe et al., 1987).
The cores collected in the field were returned to the laboratory within 30 min and sliced into 2 cm sections. For enumeration of MMPs and unicellular MTB, sediments of each
section were mixed with filtered in situ seawater. After magnetic concentration, samples near the magnet were transferred to 1.5 ml tubes and the volumes were determined. A 50
ml sample of each concentrate was used to prepare a hanging drop (Schüler, 2002) for microscopic examination. The
numbers of MMPs and unicellular MTB, and the dominant
morphotype of unicellular MTB were recorded. MMPs were
easily distinguished because of their unique morphology. Unicellular bacteria accumulated at the edge of the drop and with
the magnetotactic response were considered to be magnetotactic bacteria. As the numbers of MMPs and unicellular
MTB were markedly affected by the time of magnetic concentration and of exposure to the magnetic field (data not
shown), we removed the concentrated samples at one time,
placed all the hanging drops (seven or eight) from the same
sediment core almost simultaneously into the magnetic field,
and left them for approximately 30 min before counting. The
slide used for the hanging drop contained four O-rings (Supplemental Fig. S5), which can support four samples at one
time. Two microscopes (OLYMPUS BX51 and BH-2, Tokyo,
Japan) were used to perform the counts at each time, after
which the density (ind cm-3, i.e. individual cm-3) of MMPs and
unicellular MTB in sediments was calculated (see Supplemental text for the calculation formula).
Structural and motility observations
Race-track purified samples were used to observe the structure and motility of MMPs by optical microscopy (OLYMPUS
BX51), scanning electron microscopy (KVKV-2800B, Beijing,
China) and transmission electron microscopy (HITACHI
H8100, Tokyo, Japan, and JEOL2100F, Tokyo, Japan), as
described previously (Zhou et al., 2012). The optical microscope was equipped with LED white light and mercury arc
UV/visible light sources (Olympus, Tokyo). In order to
observe their phototaxis, MMPs were exposed to light at
wavelengths of 460–490 nm, 400–410 nm and 330–385 nm,
respectively, by selecting different fluorescence mirror unit
(Olympus, Tokyo).
DNA extraction and sequence analysis of 16S
rRNA genes
The DNA of MMPs was extracted by heating the magnetically
collected and purified samples at 100°C for 15 min, and was
amplified by performing the polymerase chain reaction (PCR)
in an Eppendorf Mastercycler (Heidelberg, Germany), using
the universal primers 27f and 1492r (Sangon Biotech, Shanghai, China). The purified PCR products were cloned into the
pMD19-T vector and chemically Escherichia coli Top 10 competent cells. After screening the positive clones by using
restriction fragment length polymorphism (RFLP) analysis
with MspI restriction endonucleases (Sangon Biotech,
Shanghai, China), clones with identical patterns were defined
as an operational taxonomic unit (OTU), and then representative clones of each OTU were randomly selected for
sequencing (Nanjing Genscript Biotechnology, Nanjing,
China). The obtained sequence HQ857737 and sequences of
other MMPs available through GenBank were used to construct a phylogenetic tree, using MEGA software version 4.
The neighbour-joining method was applied and the bootstrap
values were calculated based on 1000 replicates.
Fluorescence in situ hybridization
The specific oligonucleotide probe M97 (5′-GCAGAAGATA
GTAAAGTGGCG-3′, corresponding to position 77–97 of
HQ857737) was designed and labelled with the fluorescent
dye Cy3 (Sangon Biotech, Shanghai, China). The specificity of
the probe was checked by the probe match tool of the RDP-II
database. No hybridization to any other sequence of the
databases was predicted even when allowing for two mismatches. The sample for hybridization was magnetically collected and treated as previously (Zhang et al., 2012). The
bacterial probe EUB 338 (5′-GCTGCCTCCCRTAGGAGT-3′)
was labelled with the fluorophore FAM (Sangon Biotech,
Shanghai, China) and used as a positive control in hybridization. The unicellular magnetotactic bacteria in the enriched
sample were used as a negative control for the probe M97.
Another negative control was the hybridization with the specific probe for ellipsoidal MMPs. Hybridization with 30% formamide was carried out according to a previously reported
protocol (Pernthaler et al., 2001; Simmons and Edwards,
2007a; Zhang et al., 2012). Hybridizations were analysed
using an Olympus BX51 microscope equipped with epifluorescence capability and a DP71 camera system (Tokyo, Japan).
Geochemical analysis of the sediments and pore water
For the sediment analysis, the water content and the grain
size were measured using the gravity method and sieving
method respectively. The total organic carbon (TOC) was
determined by the combustion/non-dispersive infrared
(NDIR) method using a vario TOC cube (Elementar, Hanau,
Germany). The leachable iron was extracted from approximately 0.5 g wet sediment by adding 0.5 M HCl and standing
for 30 min at room temperature (Kostka & Luther, 1994); iron
was then determined using the revised ferrozine method
(Viollier et al., 2000). Briefly, after filtering and different dilution, 200 ml of ferrozine was added into 2000 ml of extracted
solution, and then the absorbance (A1) at the 562 nm was
determined. Then 300 ml of reducing buffer hydroxylamine
hydrochloride (1.4 mol l-1 prepared in a solution of hydrochloric acid 2 mol l-1) was added. After 10 min reaction,
100 ml of 10 M ammonium acetate buffer at pH 9.5 was
added, and then the absorbance (A2) was determined. A1
represented to Fe(II) and A2 represented to Fe(III). The
standard curve was constructed from 1000 mg ml-1 FeCl3
diluted in 3% NaCl solution.
The salinity and pH of pore water were determined using a
hand-held refractometer (YW100, Chengdu, China) and a
pH/mV/temperature benchtop meter (JENCO 6173) respectively. Sulfate concentrations were determined using ion
chromatography (ICS-90, DIONEX, California, USA). The
concentrations of ammonium and nitrate were measured
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Adaptation of spherical MMPs to intertidal habitat
using an autoanalyser (QuAAtro, Bran & Luebbe Corporation, Heidelberg, Germany), by salicylic acid spectrophotometry and cadmium-reduction method respectively.
Acknowledgements
We thank Jian-hong Xu, Qun Liu and Hai-jian Du for the kind
assistance in sampling, Zeng-xia Zhao in Jiao Zhou Bay
Marine Ecosystem Research Station for the determination of
nitrate and ammonium concentration, Ming Jiang and Wei Liu
for the assistance in the operation of TEM and SEM, and Min
Liu and Yi Dong for the helpful advice for the molecular
experiments, Yi-ran Chen and Rui Zhang for discussion. This
research was supported by the National Science Foundation
of China (NSFC 41106135 & 40906069), the Special Construction Engineering Foundation for ‘Taishan scholar’, the
China Postdoctoral Science Foundation (20110491625) and
Special Fund for Postdoctoral Innovation Program of Shandong Province (201102034).
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Supporting information
Additional Supporting Information may be found in the online
version of this article:
Movie S1. Escape motility of spherical MMPs.
Fig. S1. Two kinds of sediment in the intertidal zones of the
Huiquan Bay. During the sampling at the low tide, there was
little water above the sediments. Typically, the ellipsoidal
MMPs were only detected in the gravel sediments (red
arrow). In the sandy sediments (blue arrow) in our present
study, only spherical MMPs were observed.
Fig. S2. Elemental mapping of magnetosomes in spherical
MMPs. High angle annular dark field (HAADF) image
showing the location of magnetsomes. Elemental maps of
iron (B), oxygen (C) and sulfur (D) revealed the iron oxide
composition of crystals.
Fig. S3. Fluorescence in situ hybridization analysis. Fluorescence in situ hybridization analysis showing that bacterial
probe EUB 338 (A) was hybridized with both spherical MMPs
(red arrows) and unicellular bacteria (white arrows), while
specific probe M97 (B) was only hybridized with spherical
MMPs (red arrows). All cells in a MMP exhibited the hybridization signals (C). Bars = 5 mm in (A) and (B), and 2 mm
in (C).
Fig. S4. Vertical variation of temperature, salinity, pH and
sulfate concentration. The temperature of the sediment and
the pH of the pore water varied little in the vertical direction,
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
Adaptation of spherical MMPs to intertidal habitat
with a maximum variation of 1.2°C (core B) and 0.52 (core C)
respectively. The salinity, ranging from 14‰ to 29‰, varied
greatly from the surface to the deep layers. The sulfate profiles varied greatly among the four sediment cores. In the
layers with maximum MMPs, the temperature, pH, salinity
and the sulfate concentration were in the range of
16.0–16.4°C, 7.83–7.88, 22.0–29.0‰, 1497–2257 mg l-1
respectively.
11
Fig. S5. Instruments used for pore water sampling and cell
counting.
A. The pore water sampler was revised from Hüttel (1990).
B. The slide with four O-rings for hanging drop observation.
Supplemental text. Formula used to calculate the density of
MMPs and unicellular MTB in sediments.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology