Published Ahead of Print 1 March 2013. 10.1128/AEM.03869-12.

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

AlphaproteobacteriaBelonging to the Class Novel Rod-Shaped Magnetotactic Bacteria

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Novel Rod-Shaped Magnetotactic Bacteria Belonging to the ClassAlphaproteobacteria

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 Wue,f

Key Laboratory of Marine Ecology & Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, Chinaa; College of Resource andEnvironment, Qingdao Agricultural University, Qingdao, Chinab; Laboratory of Fiber Materials and Modern Textile, the Growing Base for State Key Laboratory, QingdaoUniversity, Qingdao, Chinac; University of Chinese Academy of Sciences, Beijing, Chinad; Laboratoire de Chimie Bactérienne, Aix-Marseille Université, CNRS, Marseille,Francee; Laboratoire International Associé de la Bio-Minéralisation et Nano-Structures (LIA-BioMNSL), CNRS, Marseille, Francef

Novel large, rod-shaped magnetotactic bacteria (MTB) were discovered in intertidal sediments of the Yellow Sea, China. Theybiomineralized more than 300 rectangular magnetite magnetosomes per cell. Phylogenetic analysis based on the 16S rRNA genesequence revealed that they are affiliated with the Alphaproteobacteria and may represent a new genus of MTB.

Magnetotactic bacteria (MTB) are ubiquitous in the watercolumn 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 (3–5).MTB benefit from these magnetosomes, which confer an abilityto orient and navigate along geomagnetic field lines, a uniqueform of motility referred as magnetotaxis (1, 3, 6, 7). MTB havebeen identified in Proteobacteria, Nitrospirae, and candidatedivision OP3 (5, 8–12), and a variety of morphological typeshave been found (6), of which coccoid is the dominant mor-phology (13–19). Here, we report a novel group of large, ma-rine, rod-shaped MTB collected from Huiquan Bay (36°03=N,120°21=E), China.

Sediments and water were collected and stored in 500-ml plas-tic bottles, and MTB collected using the capillary racetrackmethod were observed by optical microscopy (BX51; Olympus)using the hanging-drop method in an applied magnetic field (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 MTB increased in numbers and became the dominantmorphotype. Previous reports have described temporal variationsin MTB communities in microcosms under laboratory conditions(2, 22).

The laboratory-enriched rod-shaped MTB cells from sedi-ments were homogeneous in morphology, with an average lengthof 10.07 � 1.87 �m and an average width of 3.51 � 0.49 �m.Differential interference contrast (DIC) microscopy revealed thatthese cells usually possessed an interstice dividing the cell into twoparts (Fig. 1B). An analysis of the ratio distribution of the lengthsof the two parts showed that the lengths were usually unequal (n �357; Fig. 1C). When exposed to an applied magnetic field, therod-shaped MTB display north-seeking polarity (Fig. 1A).

Fluorescence microscopy of cells revealed membrane-likesepta between the two parts (Fig. 1D), consistent with the mor-phology observed using DIC microscopy. When observed bytransmission electron microscope (TEM; Hitachi H8100), twoelectron-dense structures were found around the center(Fig. 2A1). However, no obvious interstice was observed on theouter membrane of cells (Fig. 2A1), and energy-dispersive X-ray

spectroscopy (EDXS) analysis showed no obvious differences inelemental composition. It may be that the membrane layer is notreadily detected, possibly accounting for the observation of aninterstice 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 fourto six parallel chains across the interface between the two partsof the cell (Fig. 2A2). A similar bundle of magnetosomes hasbeen observed in large, freshwater, rod-shaped Nitrospiraestrains (23) and large, watermelon-shaped MTB (12). Eachmagnetosome had a rectangular projected shape, with a lengthof 113 � 16 nm and a width of 66 � 11 nm (n � 370). Thisproduced a shape factor of approximately 0.58 � 0.07 (Fig.2B). EDXS analysis indicated that the magnetosome crystalswere composed of iron and oxygen (Fig. 2C). Consistently, theanalysis by high-resolution TEM (HRTEM) identified 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 purified samples, per-formed amplification by PCR using a pair of universal primers, 27fand 1492r (18, 24), and constructed a 16S rRNA gene library of 92clones as previously reported (18). One dominant operationaltaxonomic unit (OTU) (86 clones, 93%) was identified by restric-tion fragment length polymorphism (RFLP) analysis and se-quenced (13, 18).

The sequence obtained was analyzed by BLAST andCLUSTAL W multiple alignment (25), and a phylogenetic treewas constructed using MEGA 4.0 (26, 27), applying the neigh-bor-joining method. Analysis revealed that the 16S rRNA genesequence showed maximum sequence identity (91.7%) withuncultured magnetococci collected from intertidal sedimentsof the China Sea (EF371486) and affiliated 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

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haproteobacteria (Fig. 3). This sequence exhibited a divergenceof �8% from those of all previously reported bacteria. There-fore, the rod-shaped MTB described here may represent a newgenus of MTB.

We further corroborated the authenticity of the novel se-quence as being that of rod-shaped MTB by fluorescence in situhybridization (FISH). The specific oligonucleotide probe p-774(5=-CCA ACA ACC AGC ACT CAT CG �3=; positions 774 to 793)

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 ratiodistribution of the lengths of the two parts is shown in panel C. Panel D shows the fluorescence of large, rod-shaped MTB exposed to blue light (wavelength, 450to 480 nm). Scale bars, 20 �m in panel A and 10 �m in panels B and D.

FIG 2 Characteristics of large, rod-shaped MTB cells and intracellular features determined by TEM. (A1 and A2) Morphology of large, rod-shaped MTB cells (A1) andmagnetosome chains (A2). (B1 and B2) Characteristics of magnetosomes: size histograms (B1) and shape 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) andmagnification of a selected area (D2). Scale bars, 2 �m in panel A1, 200 nm in panel A2, 5 nm in panel D1, and 1 nm in panel D2.

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was designed using Primer Premier 5.0 software and labeled withCy3. The general probe EUB 338 (5=-GCT GCC TCC CRT AGGAGT-3=) labeled with 6-carboxyfluorescein (FAM) was used as apositive control. Escherichia coli cells were used as a negative con-trol in hybridizations with specific probes. FISH was carried out inhybridization buffer containing 30% formamide according to apreviously published protocol (28, 29). As expected, the generalprobe EUB338 hybridized with all bacteria in the samples (Fig.4A). However, the specific probe p774 designed in this study rec-ognized the rod-shaped MTB (Fig. 4B, white arrows) but notother 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 relativelypoor sequence identity (91.7%) between the marine rods andmagnetococci clearly suggests that these organisms belong to dif-

ferent genera. Genome amplification from cells of low-abundanceMTB, an approach pioneered by Schüler 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 separatesingle cells in combination with whole-genome amplification todetermine the phylogenetics of MTB in environmental samples,represents a future trend for evaluating the phylogenetic diversityof environmental bacteria.

Nucleotide sequence accession numbers. The sequence ob-tained in this study was deposited in GenBank under accessionnumber JX515337.

ACKNOWLEDGMENTS

We thank Jianhong Xu for assistance in sampling and Ming Jiang for TEMobservations.

This work was supported by the National Natural Science Foundationof China (NSFC 41206150, 41276170, 41106135, and 40776094) and theSpecial Construction Engineering Foundation for “Taishan scholar.”

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FIG 3 Phylogenetic tree showing the relationship between the novel, large, rod-shaped MTB and related magnetotactic bacteria. The tree was constructed basedon neighbor-joining analysis using the sequence region from position 27 to position 1492 (E. coli numbering), and bootstrap values were calculated using 1,000replicates. The description and accession number of the MTB whose sequence was determined in this study are shown in bold. GenBank accession numbers ofthe sequences used are indicated in parentheses. Scale bar, 0.01 substitution per nucleotide position.

FIG 4 FISH analyses of magnetically enriched, rod-shaped MTB cells. Thesame microscopic field is shown following hybridization with the 5=-FAM-labeled universal bacterial probe EUB338 (A) and with the 5=-Cy3-labeledprobe (B). Scale bars, 5 �m.

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