Published Ahead of Print 1 March 2013.

10.1128/AEM.03869-12.
2013, 79(9):3137. DOI: Appl. Environ. Microbiol.
Xiao and Long-Fei Wu
Yi-Ran Chen, Rui Zhang, Wanneng Ye, Chaojing Lu, Tian
Wen-Yan Zhang, Ke Zhou, Hong-Miao Pan, Hai-Jian Du,

Alphaproteobacteria Belonging to the Class

Novel Rod-Shaped Magnetotactic Bacteria
http://aem.asm.org/content/79/9/3137
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Novel Rod-Shaped Magnetotactic Bacteria Belonging to the Class
Alphaproteobacteria
Wen-Yan Zhang,
a,f
Ke Zhou,
b,f
Hong-Miao Pan,
a,f
Hai-Jian Du,
a,d,f
Yi-Ran Chen,
a,d,f
Rui Zhang,
a,d,f
Wanneng Ye,
c
Chaojing Lu,
c
Tian Xiao,
a,f
Long-Fei Wu
e,f
Key Laboratory of Marine Ecology & Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
a
; College of Resource and
Environment, Qingdao Agricultural University, Qingdao, China
b
; Laboratory of Fiber Materials and Modern Textile, the Growing Base for State Key Laboratory, Qingdao
University, Qingdao, China
c
; University of Chinese Academy of Sciences, Beijing, China
d
; Laboratoire de Chimie Bactrienne, Aix-Marseille Universit, CNRS, Marseille,
France
e
; Laboratoire International Associ de la Bio-Minralisation et Nano-Structures (LIA-BioMNSL), CNRS, Marseille, France
f
Novel large, rod-shaped magnetotactic bacteria (MTB) were discovered in intertidal sediments of the Yellow Sea, China. They
biomineralized more than 300 rectangular magnetite magnetosomes per cell. Phylogenetic analysis based on the 16S rRNA gene
sequence revealed that they are afliated with the Alphaproteobacteria and may represent a new genus of MTB.
M
agnetotactic bacteria (MTB) are ubiquitous in the water
column and in sediments of freshwater and marine hab-
itats (1, 2). MTB can form intracellular crystals termed magne-
tosomes, usually consisting of magnetite or greigite (35).
MTB benet fromthese magnetosomes, which confer an ability
to orient and navigate along geomagnetic eld lines, a unique
form of motility referred as magnetotaxis (1, 3, 6, 7). MTB have
been identied in Proteobacteria, Nitrospirae, and candidate
division OP3 (5, 812), and a variety of morphological types
have been found (6), of which coccoid is the dominant mor-
phology (1319). Here, we report a novel group of large, ma-
rine, rod-shaped MTB collected from Huiquan Bay (3603=N,
12021=E), China.
Sediments and water were collected and stored in 500-ml plas-
tic bottles, and MTB collected using the capillary racetrack
method were observed by optical microscopy (BX51; Olympus)
using the hanging-drop method in an applied magnetic eld (20,
21). Freshly collected MTB exhibited various cell shapes, includ-
ing rods, vibrios, spirilla, and the dominant coccoid morphotype.
After incubation in the dark at room temperature for 6 months
(15), the MTB community varied greatly, and a group of large,
rod-shaped MTBincreased in numbers and became the dominant
morphotype. Previous reports have described temporal variations
in MTBcommunities in microcosms under laboratory conditions
(2, 22).
The laboratory-enriched rod-shaped MTB cells from sedi-
ments were homogeneous in morphology, with an average length
of 10.07 1.87 m and an average width of 3.51 0.49 m.
Differential interference contrast (DIC) microscopy revealed that
these cells usually possessed an interstice dividing the cell into two
parts (Fig. 1B). An analysis of the ratio distribution of the lengths
of the two parts showedthat the lengths were usually unequal (n
357; Fig. 1C). When exposed to an applied magnetic eld, the
rod-shaped MTB display north-seeking polarity (Fig. 1A).
Fluorescence microscopy of cells revealed membrane-like
septa between the two parts (Fig. 1D), consistent with the mor-
phology observed using DIC microscopy. When observed by
transmission electron microscope (TEM; Hitachi H8100), two
electron-dense structures were found around the center
(Fig. 2A1). However, no obvious interstice was observed on the
outer membrane of cells (Fig. 2A1), and energy-dispersive X-ray
spectroscopy (EDXS) analysis showed no obvious differences in
elemental composition. It may be that the membrane layer is not
readily detected, possibly accounting for the observation of an
interstice between the two parts when using DIC microscopy.
The large, rod-shaped MTB contained 320 to 567 magneto-
somes per cell, arranged in a rope-like bundle formed by four
to six parallel chains across the interface between the two parts
of the cell (Fig. 2A2). A similar bundle of magnetosomes has
been observed in large, freshwater, rod-shaped Nitrospirae
strains (23) and large, watermelon-shaped MTB (12). Each
magnetosome had a rectangular projected shape, with a length
of 113 16 nm and a width of 66 11 nm (n 370). This
produced a shape factor of approximately 0.58 0.07 (Fig.
2B). EDXS analysis indicated that the magnetosome crystals
were composed of iron and oxygen (Fig. 2C). Consistently, the
analysis by high-resolution TEM (HRTEM) identied magne-
tosome crystals as magnetite (Fig. 2D).
To determine the 16S rRNA gene sequence of the large, rod-
shaped MTB, we extracted the DNA from puried samples, per-
formedamplicationby PCRusing a pair of universal primers, 27f
and 1492r (18, 24), and constructed a 16S rRNAgene library of 92
clones as previously reported (18). One dominant operational
taxonomic unit (OTU) (86 clones, 93%) was identied by restric-
tion fragment length polymorphism (RFLP) analysis and se-
quenced (13, 18).
The sequence obtained was analyzed by BLAST and
CLUSTAL W multiple alignment (25), and a phylogenetic tree
was constructed using MEGA 4.0 (26, 27), applying the neigh-
bor-joining method. Analysis revealed that the 16S rRNA gene
sequence showed maximum sequence identity (91.7%) with
uncultured magnetococci collected from intertidal sediments
of the China Sea (EF371486) and afliated with the class Alp-
Received 13 December 2012 Accepted 26 February 2013
Published ahead of print 1 March 2013
Address correspondence to Tian Xiao, txiao@qdio.ac.cn, or Long-Fei Wu,
wu@imm.cnrs.fr.
Copyright 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.03869-12
May 2013 Volume 79 Number 9 Applied and Environmental Microbiology p. 31373140 aem.asm.org 3137

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haproteobacteria (Fig. 3). This sequence exhibited a divergence
of 8% from those of all previously reported bacteria. There-
fore, the rod-shaped MTB described here may represent a new
genus of MTB.
We further corroborated the authenticity of the novel se-
quence as being that of rod-shaped MTB by uorescence in situ
hybridization (FISH). The specic oligonucleotide probe p-774
(5=-CCAACAACCAGCACTCATCG3=; positions 774 to793)
FIG 1 Morphology of large, rod-shaped MTB based on optical microscopy. DIC images of large, rod-shaped MTB are shown in panels A and B. The ratio
distribution of the lengths of the two parts is shown in panel C. Panel Dshows the uorescence of large, rod-shaped MTB exposed to blue light (wavelength, 450
to 480 nm). Scale bars, 20 m in panel A and 10 m in panels B and D.
FIG2 Characteristics of large, rod-shaped MTBcells and intracellular features determined by TEM. (A1 and A2) Morphology of large, rod-shaped MTBcells (A1) and
magnetosome chains (A2). (B1 andB2) Characteristics of magnetosomes: size histograms (B1) andshape factor distribution(B2). (C) EDXS analysis of magnetosomes.
Top trace, magnetosomes (note the peaks of iron and oxygen); bottom trace, cell. a.u., arbitrary units. (D) High-resolution TEM images of a magnetsome (D1) and
magnication of a selected area (D2). Scale bars, 2 min panel A1, 200 nmin panel A2, 5 nmin panel D1, and 1 nmin panel D2.
Zhang et al.
3138 aem.asm.org Applied and Environmental Microbiology

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was designed using Primer Premier 5.0 software and labeled with
Cy3. The general probe EUB 338 (5=-GCT GCC TCC CRT AGG
AGT-3=) labeled with 6-carboxyuorescein (FAM) was used as a
positive control. Escherichia coli cells were used as a negative con-
trol in hybridizations with specic probes. FISHwas carried out in
hybridization buffer containing 30% formamide according to a
previously published protocol (28, 29). As expected, the general
probe EUB338 hybridized with all bacteria in the samples (Fig.
4A). However, the specic probe p774 designed in this study rec-
ognized the rod-shaped MTB (Fig. 4B, white arrows) but not
other bacteria (Fig. 4B, red arrow).
Although the 16S rRNA gene sequence of this large, rod-
shaped MTB is most closely related to that of uncultured magne-
tococci, they are morphologically distinct. In fact, the relatively
poor sequence identity (91.7%) between the marine rods and
magnetococci clearly suggests that these organisms belong to dif-
ferent genera. Genome amplication fromcells of low-abundance
MTB, an approach pioneered by Schler and colleagues (9, 30),
has revealed the existence of novel and extended phylogenetic di-
versity that may escape detection by conventional cloning strate-
gies. This approach, which uses a micromanipulator to separate
single cells in combination with whole-genome amplication to
determine the phylogenetics of MTB in environmental samples,
represents a future trend for evaluating the phylogenetic diversity
of environmental bacteria.
Nucleotide sequence accession numbers. The sequence ob-
tained in this study was deposited in GenBank under accession
number JX515337.
ACKNOWLEDGMENTS
We thank Jianhong Xu for assistance insampling and Ming Jiang for TEM
observations.
This work was supported by the National Natural Science Foundation
of China (NSFC 41206150, 41276170, 41106135, and 40776094) and the
Special Construction Engineering Foundation for Taishan scholar.
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Characterization of Novel Rod-Shaped MTB
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