1
High Diversity of the δ-proteobacteria Magnetotactic Bacteria in a 1
Freshwater Niche 2
3
Yinzhao Wang,a, b, c Wei Lin,a, b Jinhua Li,a, b and Yongxin Pana, b# 4
5
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: [email protected] 17
18
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
29
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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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REFERENCES 180
1. Bazylinski DA, Frankel RB. 2004. Magnetosome formation in 181
prokaryotes. Nat. Rev. Microbiol. 2:217-230. 182
2. Faivre D, Schüler D. 2008. Magnetotactic bacteria and 183
magnetosomes. Chem. Rev. 108:4875-4898. 184
3. Lefèvre CT, Menguy N, Abreu F, Lins U, Pósfai M, Prozorov 185
T, Pignol D, Frankel RB, Bazylinski DA. 2011. A cultured 186
greigite-producing magnetotactic bacterium in a novel group of 187
sulfate-reducing bacteria. Science. 334:1720-1723. 188
4. Wenter R, Wanner G, Schüler D, Overmann J. 2009. 189
Ultrastructure, tactic behaviour and potential for sulfate reduction 190
of a novel multicellular magnetotactic prokaryote from North Sea 191
sediments. Environ. Microbiol. 11:1493-1505. 192
5. Bazylinski DA, Heywood BR, Mann S, Frankel RB. 1993. Fe3O4 193
and Fe3S4 in a bacterium. Nature. 366:218-218. 194
6. Simmons SL, Edwards KJ. 2007. Unexpected diversity in 195
populations of the many-celled magnetotactic prokaryote. Environ. 196
Microbiol. 9:206-215. 197
7. Lins U, Keim CN, Evans FF, Farina M, Buseck PR. 2007. 198
Magnetite (Fe3O4) and greigite (Fe3S4) crystals in multicellular 199
magnetotactic prokaryotes. Geomicrobiol. J. 24:43-50. 200
8. Lefèvre CT, Frankel RB, Pósfai M, Prozorov T, Bazylinski DA. 201
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
12
2011. Isolation of obligately alkaliphilic magnetotactic bacteria 202
from extremely alkaline environments. Environ. Microbiol. 203
13:2342-2350. 204
9. Kawaguchi R, Burgess JG, Sakaguchi T, Takeyama H, 205
Thornhill RH, Matsunaga T. 1995. Phylogenetic analysis of a 206
novel sulfate-reducing magnetic bacterium, RS-1, demonstrates its 207
membership of the δ-Proteobacteria. FEMS Microbiol. Lett. 208
126:277-282. 209
10. Abreu F, Cantão ME, Nicolás MF, Barcellos FG, Morillo V, 210
Almeida LGP, do Nascimento FF, Lefèvre CT, Bazylinski DA, 211
de Vasconcelos ATR. 2011. Common ancestry of iron oxide-and 212
iron-sulfide-based biomineralization in magnetotactic bacteria. 213
ISME J. 5:1634-1640. 214
11. Liu Y, Gao S, Bai K. 2011. Water quality analysis and ecological 215
restoration scheme for Moat of Xi 'an City. J. Water. Resour. 216
Water. Eng. 22:63-65. 217
12. Jogler C, Lin W, Meyerdierks A, Kube M, Katzmann E, Flies 218
C, Pan Y, Amann R, Reinhardt R, Schüler D. 2009. Toward 219
cloning of the magnetotactic metagenome: identification of 220
magnetosome island gene clusters in uncultivated magnetotactic 221
bacteria from different aquatic sediments. Appl. Environ. 222
Microbiol. 75:3972-3979. 223
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
13
13. Lin W, Wang Y, Li B, Pan Y. 2011. A biogeographic distribution 224
of magnetotactic bacteria influenced by salinity. ISME J. 225
6:475-479. 226
14. Pan Y, Lin W, Tian L, Zhu R, Petersen N. 2009. Combined 227
approaches for characterization of an uncultivated magnetotactic 228
coccus from Lake Miyun near Beijing. Geomicrobiol. J. 229
26:313-320. 230
15. Flies CB, Jonkers HM, Beer D, Bosselmann K, Böttcher ME, 231
Schüler D. 2005. Diversity and vertical distribution of 232
magnetotactic bacteria along chemical gradients in freshwater 233
microcosms. FEMS Microbiol. Ecol. 52:185-195. 234
16. Jogler C, Niebler M, Lin W, Kube M, Wanner G, Kolinko S, 235
Stief P, Beck A, De Beer D, Petersen N. 2010. 236
Cultivation-independent characterization of ‘Candidatus 237
Magnetobacterium bavaricum’via ultrastructural, geochemical, 238
ecological and metagenomic methods. Environ. Microbiol. 239
12:2466-2478. 240
17. Simmons SL, Sievert SM, Frankel RB, Bazylinski DA, 241
Edwards KJ. 2004. Spatiotemporal distribution of marine 242
magnetotactic bacteria in a seasonally stratified coastal salt pond. 243
Appl. Environ. Microbiol. 70:6230-6239. 244
18. Lefèvre CT, Viloria N, Schmidt ML, Pósfai M, Frankel RB, 245
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
14
Bazylinski DA. 2011. Novel magnetite-producing magnetotactic 246
bacteria belonging to the Gammaproteobacteria. ISME J. 247
6:440-450. 248
19. Schauer R, Røy H, Augustin N, Gennerich HH, Peters M, 249
Wenzhoefer F, Amann R, Meyerdierks A. 2011. Bacterial sulfur 250
cycling shapes microbial communities in surface sediments of an 251
ultramafic hydrothermal vent field. Environ. Microbiol. 252
13:2633-2648. 253
20. Sylvan JB, Toner BM, Edwards KJ. 2012. Life and death of 254
deep-sea vents: bacterial diversity and ecosystem succession on 255
Inactive hydrothermal sulfides. mBio. 3:e00279-00211. 256
21. Rabus R, Fukui M, Wilkes H, Widdle F. 1996. Degradative 257
capacities and 16S rRNA-targeted whole-cell hybridization of 258
sulfate-reducing bacteria in an anaerobic enrichment culture 259
utilizing alkylbenzenes from crude oil. Appl. Environ. Microbiol. 260
62:3605-3613. 261
22. Posfai M, Buseck PR, Bazylinski DA, Frankel RB. 1998. Iron 262
sulfides from magnetotactic bacteria; structure, composition, and 263
phase transitions. Am. Mineral. 83:1469-1481. 264
23. Bazylinski DA, Frankel RB, Heywood BR, Mann S, King JW, 265
Donaghay PL, Hanson AK. 1995. Controlled biomineralization of 266
magnetite (Fe3O4) and greigite (Fe3S4) in a magnetotactic 267
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
15
bacterium. Appl. Environ. Microbiol. 61:3232-3239. 268
24. Lin W, Li J, Schüler D, Jogler C, Pan Y. 2009. Diversity 269
analysis of magnetotactic bacteria in Lake Miyun, northern China, 270
by restriction fragment length polymorphism. Syst. Appl. 271
Microbiol. 32:342-350. 272
25. Lin W, Pan Y. 2010. Temporal variation of magnetotactic 273
bacterial communities in two freshwater sediment microcosms. 274
FEMS Microbiol. Lett. 302:85-92. 275
26. Jørgensen BB. 1982. Mineralization of organic matter in the sea 276
bed-the role of sulphate reduction. Nature. 296:643-645. 277
27. Zhou J, Xia B, Treves DS, Wu LY, Marsh TL, O’Neill RV, 278
Palumbo AV, Tiedje JM. 2002. Spatial and resource factors 279
influencing high microbial diversity in soil. Appl. Environ. 280
Microbiol. 68:326-334. 281
28. Mußmann M, Ishii K, Rabus R, Amann R. 2005. Diversity and 282
vertical distribution of cultured and uncultured Deltaproteobacteria 283
in an intertidal mud flat of the Wadden Sea. Environ. Microbiol. 284
7:405-418. 285
29. Bienhold C, Boetius A, Ramette A. 2011. The energy-diversity 286
relationship of complex bacterial communities in Arctic deep-sea 287
sediments. ISME J. 6:724-732. 288
30. Zhou K, Zhang W, Yu-Zhang K, Pan H, Zhang S, Zhang W, 289
on April 12, 2019 by guest
http://aem.asm
.org/D
ownloaded from
16
Yue H, Li Y, Xiao T, Wu L. 2011. A novel genus of multicellular 290
magnetotactic prokaryotes from the Yellow Sea. Environ. 291
Microbiol. 14:405-413. 292
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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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