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High Diversity of the δ-proteobacteria Magnetotactic Bacteria in a 1

Freshwater Niche 2

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Yinzhao Wang,a, b, c Wei Lin,a, b Jinhua Li,a, b and Yongxin Pana, b# 4

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Biogeomagnetism Group, Paleomagnetism and Geochronology 6

Laboratory, Key Laboratory of the Earth’s Deep Interior, Institute of 7

Geology and Geophysics, Chinese Academy of Sciences, Beijing 100029, 8

China, a France-China Bio-Mineralization and Nano-Structures 9

Laboratory, Institute of Geology and Geophysics, Chinese Academy of 10

Sciences, Beijing 100029, China, b and Graduate University of Chinese 11

Academy of Sciences, Beijing 100039, China c 12

13

Address correspondence to Yongxin Pan, Institute of Geology and 14

Geophysics, Chinese Academy of Sciences, Bei Tu Cheng Xi Lu 19, 15

Chaoyang District, Beijing 100029, China 16

Email: yxpan@mail.iggcas.ac.cn 17

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Running Title: Diversity of freshwater δ-proteobacteria MTB 19

Section: Microbial Ecology 20

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Copyright © 2013, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.03635-12 AEM Accepts, published online ahead of print on 1 February 2013

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Abstract 22

Knowledge of magnetotactic bacterial diversity in natural environments is 23

crucial for understanding their contribution to various 24

biological/geological processes. Here we report a high diversity of 25

magnetotactic bacteria in a freshwater site. Ten out of eighteen OTUs 26

were affiliated with the δ-proteobacteria. Some rod-shaped bacteria 27

simultaneously synthesized greigite and magnetite magnetosomes. 28

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Magnetotactic bacteria (MTB) within the δ-proteobacteria class have 30

been shown to produce magnetite (Fe3O4) or greigite (Fe3S4) 31

magnetosomes, or both within the same cell (1, 2, 3). They have been 32

widely found in marine sediments (4), river estuaries (5), coastal salt 33

ponds (6), lagoons (7), and alkaline environments (8), but only 34

occasionally in freshwater lakes (3, 9). Because of their unique ability to 35

biomineralize both magnetite and greigite, the δ-proteobacteria MTB 36

have attracted great interest in deciphering the mechanism of 37

magnetosome biomineralization and the evolution of bacteria 38

magnetotaxis (3, 10). 39

The δ-proteobacteria MTB may play an important role in 40

biogeochemical cycling of iron and sulfur elements. Recently, a 41

cultivable strain BW-1, which was isolated from a brackish spring, was 42

found to mineralize either magnetite or greigite magnetosomes depending 43

on the concentration of environmental hydrogen sulfide (3). In nature, 44

most reported δ-proteobacteria MTB were found in saline environments. 45

Within freshwater environments, however, the overall diversity and 46

distribution of these MTB is still poorly understood. 47

Surface sediment samples (~10 cm depth) and two in situ vertical cores 48

were collected from a site (34°15′10.00″N, 108°55 ′13.41″E) in the city 49

moat in Xi'an city, China. The water pH ranged from 7.1 to 7.5 and 50

salinity was less than 0.34 ppt. The two vertical cores (about 1 m away 51

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from each other) were sampled using a gravity sampler. Geochemical 52

analyses of S2-, SO42-, PO4

3- and NH4+ indicated that the Xi’an moat was 53

slightly or moderately polluted (11). The variations of MTB abundance 54

and the concentration of S2-, SO42-, O2, PO4

3- and NH4+ with depth are 55

shown in Fig. 1 and Table S1. Live MTB were magnetically enriched 56

using the ‘MTB trap’ method as described previously (12, 13). We 57

observed that the MTB lived in a narrow layer in the most upper sediment 58

(0-2 cm) and in the water column no more than 2 cm above the sediment 59

in core water that is correlated to the critical oxic-anoxic transition zone 60

(OATZ) of the site, where chemical parameters dramatically changed 61

(Fig. 1). The composition of the MTB community was examined using 62

the Bacteriodrome (14), light microscopy, transmission electron 63

microscopy (TEM), and 16S rRNA genes. Magnetococci were the most 64

dominant MTB, occurring 2 cm both above and beneath the 65

water-sediment interface. The rod-shaped MTB were abundant in 1 cm 66

beneath the interface. Spirilla and vibrios were also occasionally found in 67

the same layer. The occurrence of MTB around the OATZ and 68

correlations to geochemical variation were in lines with previous studies 69

(15, 16, 17). Detailed information on the sampling site and methods are 70

presented in supplemental materials. 71

Morphologies of the enriched MTB cells are shown in Fig. 2. These 72

MTB include cocci, spirilla, vibrios and large rod-shaped bacteria. Within 73

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these MTB cells, different morphologies of magnetosome were found, for 74

example, elongated prismatic magnetosome in magnetotactic cocci (Figs. 75

2A-C), cuboidal magnetosome in magnetotactic spirilla (Fig. 2D), 76

bullet-shaped and irregular magnetosome in vibrioid, rod-shaped to 77

oval-shaped MTB (Figs. 2G-K). The magnetosomes were arranged in 78

single chains (Figs. 2D-E), multiple chains (Figs. 2A, 2F, 2J), clusters of 79

randomly oriented grains concentrated on one side of the cell (Fig. 2C), 80

or multiple short chains parallel to the short axis of the cells (Fig. 2G). 81

Phylogenetic analysis based on 16S rRNA gene sequences was 82

performed to determine the community structure of MTB. All sequences 83

(50 sequences) were composed of 18 operational taxonomic units (OTUs), 84

which were defined at the 98% similarity level (Fig. 3A and Table S2). 85

Ten OTUs (OTU 9 to 18) were identified as belonging to sulfate-reducing 86

bacteria (SRB) within the δ-proteobacteria, which accounted for more 87

than 50% of all retrieved sequences. Although three OTUs (OTU 16-18) 88

were closely related to previously described MTB (3), sequences 89

belonging to OTUs 9-15 were much divergent from known MTB species 90

(Fig. 3A) and might therefore represent so far novel branches. 91

Furthermore, phylogenetic analysis has demonstrated that OTU 8 was 92

92% identical to cultured γ-proteobacteria MTB BW-2 belonging to the 93

family Thiotrichales (18); whereas OTU 7, which had a relatively low 94

similarity with the other cultured strain SS-5 (90% similarity), was 95

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affiliated with the family Chromatiales. Both families Thiotrichales and 96

Chromatiales possess the ability to oxidize sulfur and known as 97

sulfur-oxidizing bacteria (SOB) (18, 19, 20). Additionally, six OTUs 98

(OTU 1 to 6) belong to the α-proteobacteria, three of which were most 99

similar to three different known magnetotactic cocci, whereas the others 100

had high similarities with cultured Magnetospirillum gryphiswaldense 101

MSR-1 (96%-99%). 102

Fluorescence in situ hybridization (FISH) analysis was performed to 103

confirm whether the δ-proteobacteria 16S rRNA gene sequences truly 104

originated from the MTB enrichment. Probe SRB385Db (21) specific for 105

SRB in the δ-proteobacteria was found to match all the δ-proteobacteria 106

sequences retrieved in this study, and therefore was selected for FISH 107

analysis. The enriched MTB sample was stained with DAPI, hybridized 108

with the universal bacterial probe EUB338 and the specific probe 109

SRB385Db. As shown in Figs. 3B-D, large rod-shaped bacteria (2.5-5.7 110

µm in length) and small cocci (1-2 µm in diameter) can be robustly 111

hybridized with the specific probe. TEM analysis on the rod-shaped 112

bacteria revealed that they contain both bullet-shaped and irregular 113

rectangular magnetosomes in the same cell (Fig. 3E-F). High resolution 114

transmission electron microscopy (HRTEM) imaging (Figs. 3G-H), fast 115

Fourier transform (FFT) patterns (Fig S1), and energy dispersive X-ray 116

spectroscopy (EDX) analyses (Figs. 3I-J) indicate that the mineral phase 117

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of bullet-shaped magnetosomes were magnetite, while the irregular 118

rectangular magnetosomes were greigite. MTB which exclusively 119

produce either greigite or magnetite sharing the same cell shape and size 120

were also observed in the same microcosm. 121

MTB cells simultaneously producing magnetite and greigite 122

magnetosomes are of great interest for studies of microbiology, 123

environmental magnetism and biomineralization (3, 5, 22). These bacteria 124

were first identified in the Pettaquamscutt Estuary USA, a saline 125

environment (5). The authors discovered that these rod-shaped bacteria 126

form bullet-shaped magnetite magnetosome when found within the upper 127

sediment layers, but when found in the deeper, hydrogen sulfide-rich 128

layers, most of rod-shaped bacteria synthesize greigite magnetosome (23). 129

The present study has shown that diverse, large rod-shaped 130

δ-proteobacteria MTB that can produce either magnetite or greigite 131

magnetosomes, or both, can be found in the surface sediments of 132

freshwater Xi’an city moat (Fig. 3). Microbial community analysis based 133

on 16S rRNA genes and FISH in this study identified that these MTB 134

belonging to the sulfate-reducing δ-proteobacteria, which have a 135

similarity with the pure cultured strain BW-1 (90%-92%). Recently, 136

Lefèvre and coworkers found that the mineralization of greigite and 137

magnetite magnetosomes by BW-1 depends on the concentration of 138

hydrogen sulfide (3). Two candidate gene clusters may control the 139

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biomineralization of greigite or magnetite magnetosome (3). Further 140

investigations, such as metagenomics or single cell analysis, of these 141

newly isolated δ-proteobacteria MTB are needed in future to detect and 142

reveal the detailed function of the magnetosome genes and their 143

regulation networks. 144

It was interesting to find a high phylogenetic diversity of MTB, 145

especially in the δ-proteobacteria, in Xi’an city moat (Fig. 3). These 146

MTB mainly occupied the top layer (less than 2 cm) of the sediments, 147

where chemical gradients were steep, as indicated by the concentration of 148

S2-, SO42-, NH4

+, PO43-, and O2 (Fig. 1 and Table S1). The sampling site 149

contained high amounts of nutrients and can be classified as an eutrophic 150

environment (11). Therefore, a possible explanation for the highly 151

diversified δ-proteobacteria MTB in this sampling site could be that the 152

high nutrient loading, steep vertical chemical gradient and fast changes 153

associated with sewage pollution provide diverse micro-ecological niches 154

for different bacteria lineages and helps to stimulate their growth when 155

compared with other studies on freshwater MTB (15, 24, 25). The 156

availability of nutrients, and hence energy supply, as well as sharp 157

vertical redox environments have been shown to be important drivers of 158

microbial diversity (6, 26, 27, 28, 29). The distribution of the 159

δ-proteobacteria MTB have been documented in saline environments (3, 160

4, 5, 7, 30), and fresh water niches (3, 9). Altogether, our results may 161

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suggest that the δ-proteobacteria MTB, which includes greigite 162

producing varieties, may widely exist in both saline and freshwater 163

environments. 164

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Nucleotide sequence accession numbers 166

The sequence data has been submitted to the DDBJ/EMBL/GenBank 167

databases under accession numbers JX134734-JX134751. 168

169

ACKNOWLEDGEMENTS 170

We thank Haitao Chen, Qinyang Wang and Liming Wang for help with 171

field sampling. The authors also thank Greig A. Paterson for improving 172

the English, Mo Huang and Wenfang Wu for useful discussion and 173

Xin’an Yang and Jingnan Liang for TEM analyses. We also thank for 174

three anonymous referees for valuable comments on an earlier version. 175

This work was supported by the CAS/SAFEA International Partnership 176

Program for Creative Research Teams (KZCX2-YW-T10), the CAS 177

project, and NSFC grants 40821091 and 41104041. 178

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Legends to figures: 294

Figure 1 The vertical variation of MTB cells in relation to the 295

concentrations of S2-, SO42-, O2, PO4

3- and NH4+ in the vertical core (Core 296

1) from Xi’an city moat. The depth was measured relatively to the 297

water-sediment boundary. 298

299

Figure 2 Representative TEM micrographs of the MTB cells collected 300

from the freshwater Xi'an city moat. These MTB include cocci (A-C), 301

spirillium (D) and vibrios, rod-shaped to oval-shaped magnetotactic 302

bacteria (E-K). In all figures the scale bars = 500 nm. 303

304

Figure 3 (A) Phylogenetic tree of 16S rRNA gene sequences, which was 305

constructed based on the neighbor-joining method. Bootstrap values at 306

each node are based on 1000 replicates. The same microscopic field after 307

staining with DAPI (B), after hybridization with 5’-6-carboxyfluorescein 308

(FAM)-labeled bacterial universal probe EUB338 (C), and after 309

hybridization with 5’-Cy3-labeled probe SRB385Db (D). In (B) to (D), 310

arrows indicate MTB vibrio and MTB cocci cells as negative control. 311

TEM images (E), which indicate magnetite and greigite magnetosome 312

mineralizing rod-shaped bacteria from the Xi’an city moat. (F) The 313

rod-shaped bacteria synthesize both irregular rectangular and 314

bullet-shaped magnetosomes. HRTEM analyses of an irregular 315

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rectangular magnetosome and a bullet-shaped magnetosome are shown in 316

(G) and (H), respectively. (I and J) EDX analyses of the irregular 317

rectangular magnetosome (I) and the bullet-shaped magnetosome (J) 318

shown in (G) and (H), respectively. EDX analyses and HRTEM imaging 319

on individual particles indicate that the mineral phase of irregular 320

rectangular and bullet-shaped magnetosome is greigite and magnetite, 321

respectively. 322

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