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Magnetovibrio blakemorei, gen. nov. sp. nov., a new magnetotactic bacterium 1
(Alphaproteobacteria: Rhodospirillaceae) isolated from a salt marsh. 2
3
Dennis A. Bazylinski,1 Timothy J. Williams,2 Christopher T. Lefèvre,3 Denis Trubitsyn,1 Jiasong 4
Fang4, Terrance J. Beveridge,5 Bruce M. Moskowitz,6 Bruce Ward,7 Sabrina Schübbe,1 Bradley 5
L. Dubbels,8 Brian Simpson9 6
7
Running title: Magnetovibrio blakemorei, new magnetotactic bacterium 8
9
1 School of Life Sciences, University of Nevada at Las Vegas, 4505 Maryland Parkway, Las 10
Vegas, NV 89154, USA 11
2 School of Biotechnology and Biomolecular Sciences, The University of New South Wales, 12
Sydney, NSW 2052, Australia 13
3 CEA Cadarache/CNRS/Aix-Marseille Université, UMR7265 Service de Biologie Végétale et 14
de Microbiologie Environnementale, Laboratoire de Bioénergétique Cellulaire, 13108, Saint Paul 15
lez Durance, France 16
4 Department of Natural Sciences, Hawaii Pacific University, 45-045 Kamehameha Highway, 17
Kaneohe, HI 96744, USA 18
5 Department of Molecular and Cellular Biology, College of Biological Science, University of 19
Guelph, Guelph, Ontario N1G 2W1, Canada 20
6 Institute for Rock Magnetism, Department of Earth Sciences, University of Minnesota-Twin 21
Cities, 310 Pillsbury Dr, SE, Minneapolis, MN 55455, USA 22
IJSEM Papers in Press. Published September 14, 2012 as doi:10.1099/ijs.0.044453-0
7 Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Kings 23
Buildings, Edinburgh, EH9 3JR, United Kingdom 24
8 Life Technologies Corporation, 29851 Willow Creek Road, Eugene, OR 97402, USA 25
9 United States Navy, Helseacombatron Two Three, San Diego, CA 92135, USA 26
27
Correspondence: Dennis A Bazylinski, School of Life Sciences, University of Nevada at Las 28
Vegas, 4505 Maryland Parkway, Las Vegas, NV 89154-4004, USA. E-mail: 29
dennis.bazylinski@unlv.edu 30
31
The GenBank accession number for the 16S rRNA gene sequence of Magnetovibrio blakemorei 32
strain MV-1T is L06455. 33
34
The GenBank accession number for both the cbbM gene sequence and putative post-translational 35
RubisCO activator cbbQ gene sequence of Magnetovibrio blakemorei strain MV-1T is 36
AF442518. 37
38
Abbreviations 39
CBB, Calvin-Benson-Bassham; RubisCO, ribulose bisphosphate carboxylase/oxygenase 40
41
42
43
44
45
A magnetotactic bacterium, designated strain MV-1T, was isolated from sulfide-rich sediments in 46
a salt marsh near Boston, Massachusetts. Cells of strain MV-1T are Gram-negative, and vibrioid 47
to helicoid in morphology. Cells are motile by means of single polar flagellum. Cells appear to 48
display a transitional state between axial and polar magnetotaxis: cells swim in both directions 49
but generally have longer excursions in one direction than the other. Cells possess a single chain 50
of magnetosomes containing truncated hexa-octahedral crystals of magnetite, positioned along 51
the long axis of the cell. Strain MV-1T is a microaerophile that is also capable of anaerobic 52
growth on some nitrogen oxides. Salinities greater than 10% of seawater are required for growth. 53
Strain MV-1T exhibits chemolithoautotrophic growth on thiosulfate and sulfide with oxygen as 54
the terminal electron acceptor (microaerobic growth), and on thiosulfate using nitrous oxide 55
(N2O) as the terminal electron acceptor (anaerobic growth). Chemoorganoautotrophic and 56
methylotrophic growth is supported by formate under microaerobic conditions. Autotrophic 57
growth occurs via the Calvin-Benson-Bassham cycle. Chemoorganoheterotrophic growth is 58
supported by various organic acids and amino acids, under microaerobic and anaerobic 59
conditions. Optimal growth occurs at pH 7.0 and between 26-28°C. The genome of strain MV-1T 60
consists of a single, circular chromosome about 3.7 Mb in size with a G + C content of 52.9-53.5 61
mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicates that strain MV-1T 62
belongs to the family Rhodospirillaceae within the Alphaproteobacteria, but is not closely 63
related to the genus Magnetospirillum. The name Magnetovibrio blakemorei gen. nov., sp. nov. 64
is proposed for strain MV-1T, which is designated as the type strain (= ATCC BAA-1436 T = 65
DSM 18854T). 66
67
Magnetotactic bacteria were first described in 1963 by Salvatore Bellini (Bellini, 2009), and then 68
independently by Richard Blakemore in 1975 (Blakemore, 1975) when microorganisms were 69
observed to rapidly migrate within freshwater mud samples on microscope slides in response to 70
magnetism. The term “magnetotaxis” was coined for this behavior (Blakemore, 1975) and was 71
found to be the result of a permanent magnetic dipole moment conferred by internal magnetite 72
(Fe3O4) or greigite (Fe3S4) crystals, which causes the cell to be oriented along geomagnetic field 73
lines as it swims (Bazylinski & Frankel, 2004). Magnetotaxis is hypothesized to expedite the 74
search for optimal concentrations of certain nutrients within water columns and sediments, by 75
simplifying a three-dimensional search to a linear search (Frankel et al., 1997; Bazylinski & 76
Frankel, 2004). The first magnetotactic bacterium to be isolated in pure culture and cultivated 77
was the magnetite-producing strain Magnetospirillum magnetotacticum (basonym Aquaspirillum 78
magnetotacticum) strain MS-1 (Blakemore et al., 1979; Maratea & Blakemore, 1981; Schleifer 79
et al., 1991). Cells of MS-1 were found to require molecular oxygen for growth and magnetite 80
biosynthesis (Blakemore et al., 1985). Biogenic, single-magnetic-domain magnetite crystals in 81
magnetosomes were therefore assumed to be formed only under microaerobic conditions and 82
were indicative of oxygenated sediments at the time of deposition. This was refuted by the 83
discovery and axenic cultivation of the magnetotactic bacterium strain MV-1T (“magnetic vibrio 84
1”), which is capable of growth and magnetite production under strictly anoxic conditions 85
(Bazylinski et al., 1988). This was an important discovery, because strain MV-1T demonstrated 86
that magnetite biosynthesis was not dependent upon the availability of molecular oxygen, and 87
therefore anaerobic magnetotactic bacteria could be important contributors to the magnetic 88
remanence in sediments (Bazylinski et al., 1988). Since then, other magnetotactic bacteria have 89
been demonstrated to grow anaerobically, including the obligate anaerobe Desulfovibrio 90
magneticus RS-1, Candidatus Desulfamplus magnetomortis strain BW-1 and the obligately 91
alkaliphilic strains ML-1, ZZ-1 and AV-1, using sulfate (Sakaguchi et al., 1993, 2002; Lefèvre et 92
al., 2011a,b), and the facultative anaerobes Magnetospirillum sp. strain AMB-1 and 93
Magnetospirillum gryphiswaldense MSR-1, using nitrate (Matsunaga et al., 2005; D. Schüler, 94
personal communication). 95
96
Strain MV-1T was originally isolated from sulfide-rich sediments from a salt marsh pool at the 97
Neponset River Estuary near Boston, Massachusetts (Bazylinski et al., 1988). Strain MV-1T has 98
been the subject of several previous studies, centered mostly upon the production and 99
morphology of magnetite by this strain in culture (Bazylinski et al., 1988, 1995; Thomas-Keprta 100
et al., 2001; Dubbels et al., 2004), including the generation of spontaneous non-magnetotactic 101
mutants (Dubbels et al., 2004), and autotrophic growth of strain MV-1T via the Calvin-Benson-102
Bassham (CBB) pathway (Bazylinski et al., 2004). Nevertheless, this important magnetotactic 103
strain has not yet been characterized in the literature. The aim of the current study is to 104
characterize and formally name the magnetic vibrio strain MV-1T. 105
106
Cells of strain MV-1T were isolated from a natural enrichment of magnetotactic bacteria at the 107
oxic-anoxic interface (OAI) within a bottle containing water and sediment from a salt marsh pool 108
near the Neponset River Estuary (Fig. S1). For a complete description of the collection, 109
enrichment and isolation procedure please refer to Supplementary Material. After isolation, cells 110
were routinely grown in oxygen-free liquid cultures of diluted artificial seawater (ASW) medium 111
containing 16.4 g L-1 NaCl, 3.5 g L-1 MgCl2•6H2O, 2.7 g L-1 Na2SO4, 0.47 g L-1 KCl, 0.39 g L-1 112
CaCl2•2H2O, 5 mL of modified Wolfe’s mineral elixir (Frankel et al., 1997), 0.5 mL vitamin 113
solution (Frankel et al., 1997), 0.5 g sodium succinate, 0.5 g Casamino acids, 0.2 g sodium 114
acetate•3H2O, 0.2 g NH4Cl, 2 mL of freshly prepared, filter-sterilized, neutralized 0.43 M 115
cysteine•HCl•H2O as the reducing agent, 1.8 mL of 0.5 M KHPO4 buffer pH 6.9, and 2.4 mL 0.8 116
M NaHCO3 per L of diluted ASW. Nitrous oxide (N2O) at 1 atm was used as the terminal 117
electron acceptor. 118
119
Analytical electron microscopy was performed on cells using a VG Microscopes (Fisons 120
Instrument Surface Systems, East Grinstead, UK) model HB-5 scanning transmission electron 121
microscope (STEM) operating at 100 kV linked to a field-emission electron gun, a Link LZ-5 X-122
ray detector (Link Analytical, Gif Sur Yvette, France), and an AN10000 X-ray analysis system. 123
Motility of strain MV-1T was determined using a Zeiss AxioImager M1 light microscope (Carl 124
Zeiss MicroImaging Inc., Thornwood, NY) equipped with phase-contrast and differential 125
interference contrast capabilities. Cells of strain MV-1T are vibrioid to helicoid, 1-3 µm long by 126
0.2-0.4 µm wide (Fig. 1a; Fig. S2) and motile by means of a single polar flagellum 127
(monotrichous; Fig. 1a). Cells have a more vibrioid (comma-shaped) morphology than helicoid 128
when grown microaerobically (Fig. S2). They possess a typical Gram-negative cell wall and 129
some cells have a polar membrane or organelle (Fig. 1c) that has been associated with the 130
flagellar apparatus and ATPases, and cytochrome oxidase activities in other bacteria (Tauschel, 131
1985). Cells contain sulfur-rich inclusions when grown on sulfide (Bazylinski et al., 2004). 132
Some cells also contained intracellular inclusions that were rich in phosphorus, and likely 133
represent deposits of polyphosphate (Bazylinski et al., 2004). The parallelepipedal-shaped 134
magnetite crystals are arranged as a single chain of around ten crystals positioned along the long 135
axis of the cell, which sometimes have large gaps between them (Fig. 1b). 136
137
Cells of strain MV-1T appear to display aspects of both polar and axial magnetotaxis (Frankel et 138
al., 1997); that is, they swim in both directions spontaneously changing direction without turning 139
around, similar to magnetotactic species of Magnetospirillum (Bazylinski et al., 1988). However, 140
in hanging drop assays, many cells appeared to accumulate at the edge of the drop suggesting 141
that some cells have a polar preference in their swimming direction and that they swim longer 142
distances in one direction than another (Bazylinski & Williams, 2007). Thus, cells of strain MV-143
1T appear to display a transitional state between axial and polar magnetotaxis. 144
145
Anaerobic growth (using sodium succinate, sodium acetate, and Casamino acids as the electron 146
donors) was tested in liquid medium using the following as terminal electron acceptors (sodium 147
salts, where appropriate): NO3- (2, 5 and 10 mM); NO2
- (1, 2 and 5 mM); N2O (1 atm); fumarate 148
(20 mM); SO42- (5 and 10 mM); trimethylamine oxide (15 mM); and dimethylsulfoxide (15 149
mM). Cells grew anaerobically with NO3- at 2 and 5 but not 10 mM, NO2
- and N2O but none of 150
the other tested terminal electron acceptors supported anaerobic growth. 151
152
During growth on N2O, oxidation of completely reduced resazurin to resorufin in the growth 153
medium caused the color of the medium to change from initially colorless to pink. This did not 154
occur during anaerobic growth with nitrate or nitrite. Growth and N2O reduction appeared to be 155
necessary for the oxidation of resazurin because the addition of 1% acetylene, a known inhibitor 156
of N2O reduction (Yoshinari et al., 1977), to these cultures completely inhibited growth, N2O 157
reduction, and resazurin oxidation. This suggests that the completely reduced form of resazurin 158
under these conditions acts as an electron donor during N2O reduction although it is not 159
necessary for growth on this terminal electron acceptor. To our knowledge, reduced resazurin 160
has never been shown to act as an electron donor in nitrogen oxide reduction, although a method 161
was developed that utilized this reaction to detect NO- or N2O-producing bacteria in solid or 162
liquid medium (Jenneman et al., 1986). Alternatively, the oxidation of Fe(II) might support N2O 163
reduction during growth and the resulting Fe(III), in turn, could cause the oxidation of resazurin 164
and the change of colorless to pink. Strain MV-1T is known to possess an Fe(II) oxidase 165
(Dubbels et al., 2004). Our results show that strain MV-1T is a facultatively anaerobic 166
microaerophile. 167
168
Carbon sources at a concentration of 0.1% (w/v) were tested for microaerobic growth in semi-169
solid (1.5 g Agar Noble (Difco) L-1) [O2] gradient media as well as for anaerobic growth with 170
N2O as the terminal electron acceptor. Screw cap test tubes were used to test for microaerobic 171
growth. Each tube contained 10 mL of growth medium and was inoculated with approximately 172
106 cells mL-1 distributed throughout the growth medium while the medium was still liquid (kept 173
at about 44ºC). A carbon source was deemed positive for the support of aerobic growth of strain 174
MV-1T if three conditions were met: (a) a band of cells formed in the tube; (b) the cell count 175
after growth was significantly greater than the control containing no carbon source; and (c) cells 176
continued to grow in the same medium in three successive transfers. Anaerobic growth on 177
specific carbon sources was determined using N2O as the terminal electron acceptor the same 178
way, except that the test tubes had septum stoppers. After inoculation as described above, the air 179
headspace of these tubes was quickly replaced with 1.2 atm (121.6 kPa) of N2O. Growth was 180
determined as described above except that bands of cells did not form and N2 formation as 181
bubbles from the reduction of N2O was monitored. The semi-solid agar tended to trap bubbles of 182
N2 thereby making them visible. 183
184
A range of compounds was tested as carbon sources; sodium salts were used for all acids, 185
neutralized when appropriate. The list of organic acids, amino acids, alcohols, aldehydes, 186
carbohydrates, and complex substrates that were tested is shown in Table S1. Those compounds 187
that resulted in growth indicate chemoorganoheterotrophic growth by strain MV-1T, except for 188
formate, which is interpreted as supporting chemoorganoautotrophic growth (see below). 189
190
Cells grow autotrophically under microaerobic conditions in semi-solid [O2] gradient cultures 191
using sulfide, thiosulfate and formate as electron donors but not tetrathionate or ferrous iron (as a 192
sulfide or carbonate) (Bazylinski et al., 2004). However, in further studies of these electron 193
donors, cells only grew on thiosulfate anaerobically with N2O as the terminal electron acceptor. 194
Strain MV-1T utilizes the CBB cycle for CO2 fixation and autotrophy and possesses a form II 195
ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) gene (cbbM) (Bazylinski et al., 196
2004). Thus, strain MV-1T is capable of chemolithoautotrophic and chemoorganoautotrophic 197
(with formate as electron donor) growth. 198
199
Because strain MV-1T was found in a brackish-to-marine environment, we examined whether 200
concentrations of salts close to that of marine waters were required for growth. Cells were grown 201
anaerobically with N2O as the terminal electron acceptor as described above except that different 202
concentrations of Lyman and Fleming’s ASW (Lyman & Fleming, 1940), a formulation that has 203
a salinity very close to natural seawater (~35 ppt) was used as the diluent. Cells grew well in 204
concentrations of this ASW ranging from 25 to 125% with the shortest doubling time during 205
exponential growth, ~11 h, at a 50% concentration. Cells grew very poorly (1-2 doublings) at 206
10% and 150% ASW and not at all with distilled water as the diluent or when the concentration 207
of ASW was >150%. Thus, like many marine bacteria (Kaye & Baross, 2004), strain MV-1T is 208
euryhaline but has a growth requirement for salts, as it will not grow at very low concentrations 209
of ASW or in freshwater media. 210
211
The magnetosomes of strain MV-1T contain crystals of magnetite surrounded by a membrane 212
(Figs. 1b, c, d) (Bazylinski et al., 1988). Greigite was never observed in magnetosomes. The 213
specific crystal habit of magnetite is truncated hexa-octahedral, with eight {111} octahedral 214
faces, six {110} hexagonal faces, and six {100} cubic faces (Thomas-Keprta et al., 2001). The 215
dimensions of each crystal are approximately 35 x 35 x 53 nm, with the axis of elongation 216
corresponding to the alignment of crystals within the chain (Figs. 1b, d) (Sparks et al., 1990). 217
218
Cells produced magnetosomes and were magnetotactic under both microaerobic and anaerobic 219
conditions. When grown anaerobically with N2O as the terminal electron acceptor, cells 220
produced an average of 10.4 ± 3.9 magnetosomes per cell (n = 125), while 2.4 % of the cells did 221
not contain magnetosomes. Interestingly, cells still produced magnetosomes even when the 222
major source of iron (ferric quinate) was omitted from the growth medium, but with an average 223
of 4.9 ± 3.7 magnetosomes per cell (n = 111). In this latter case, 10.8% of the cells did not 224
contain magnetosomes. However, the final cell yield of these cultures was only 23-28% of the 225
high-iron containing culture based on direct cell counts, which suggests that cells in these low-226
iron cultures continue producing magnetosomes thereby starving themselves of iron and limiting 227
their growth yield. This further indicates that magnetosome iron in this strain is not biologically 228
available for growth and thus magnetosomes are not used for iron storage (Dubbels et al., 2004). 229
Further support for this notion comes from the fact that the addition of ferric quinate to these 230
cultures resulted in continued growth and a final yield similar to that when ferric quinate was 231
added prior to inoculation (data not shown). Cells of strain MV-1T grown anaerobically with 232
nitrate produced less magnetosomes than those grown with N2O, on average 6.7 ± 4.8 233
magnetosomes per cell (n =150). When grown on nitrate, 10.5% of cells did not contain 234
magnetosomes. Cells grown microaerobically produced slightly less magnetosomes than cells 235
grown on N2O but more than on nitrate, an average of about 8 per cell when the major source of 236
iron was included in the growth medium (Bazylinski, 1991) 237
238
Nitrogenase activity of whole cells was determined as acetylene (C2H2) reduction to ethylene 239
(C2H4) in [O2]-gradient cultures as described in Bazylinski et al. (2000) using Magnetospirillum 240
magnetotacticum as a positive control (Bazylinski & Blakemore, 1983a; Bazylinski et al., 2000). 241
Cells of strain MV-1T showed relatively high nitrogenase activities (Table S2) as measured by 242
acetylene (C2H2) reduction to ethylene (C2H4) in semi-solid [O2] gradient cultures with succinate 243
and acetate as electron donors. C2H2 reduction was inhibited by the addition of the fixed nitrogen 244
sources 4 mM NH4Cl and 4 mM NaNO3. The fact that NO3- inhibited C2H2 reduction suggests 245
that strain MV-1T is capable of assimilatory nitrate reduction. 246
247
Cells of strain MV-1T for chemical analyses were grown to late exponential phase, harvested by 248
centrifugation and freeze-dried. These cells were analyzed for % carbon, hydrogen and nitrogen 249
as described by Bazylinski & Blakemore (1983b) and % protein (Lowry et al., 1951) using 250
bovine serum albumin as the standard; and for % iron and magnetite (see below). Measurements 251
were performed in triplicate. Whole freeze-dried cells of strain MV-1T from late exponential 252
anaerobic growth cultures consisted of (% on a dry weight basis): 42.9 ± 0.5 C; 10.5 ± 0.4 N; 5.7 253
± 0.2 H; and 51.4 ± 1.0 protein. These values are generally comparable to those of another 254
magnetotactic bacterium, Magnetospirillum magnetotacticum strain MS-1 (Bazylinski & 255
Blakemore, 1983b). 256
257
In the case of iron and magnetite, two separate batches of cells grown with and without the major 258
source of iron (ferric quinate) were analyzed. All glassware for the determination of total iron in 259
cells was soaked in 10% HCl overnight and then rinsed 5 times with Nanopure water (Millipore, 260
Billerica, MA). All acids used for iron determinations were trace metal grade (Mallinckrodt 261
Baker Inc., Phillipsburg, NJ). Approximately 15 mg of freeze-dried cells were acid digested as 262
described in Dubbels et al. (2004). Samples of growth medium were acidified and diluted 263
directly with concentrated HCl to a final concentration of 1%. Total iron was determined using 264
the ferrozine reagent (Stookey, 1970) and/or atomic absorption spectroscopy (Dubbels et al., 265
2004). The percentage of magnetite was determined from the saturation magnetization (Ms) 266
obtained from room temperature hysteresis loops measured with a vibrating sample 267
magnetometer using an electromagnet to produce fields up to 1.5 T (Jackson & Solheid, 2010). 268
Saturation magnetization was determined after the loops were corrected for high-field slopes to 269
remove the effects of diamagnetic or paramagnetic contributions to the magnetization. The 270
weight percent magnetite in samples consisting of a known amount of whole freeze-dried cells 271
(~10-30 mg) was determined from the measured Ms values and the known value for pure 272
magnetite (Ms=92 Am2/kg) as follows: wt% magnetite = 100% Ms/(92 Am2/kg). 273
274
Percentages of total iron and magnetite of whole freeze-dried cells is shown in Table S3. The % 275
of total iron in cells grown with the major iron source appears variable from culture to culture 276
and we obtained values of about 1.9 and 1.3 in two separate cultures grown the same way. Cells 277
from cultures grown without the major source of iron appear to be more consistent containing 278
about 0.5% total iron. Values for the % of magnetite were similar. The presence of magnetite in 279
cells grown with low iron is consistent with the previously discussed magnetosome results in 280
which these cells produced a significant number of magnetosomes under low iron conditions. It 281
is noteworthy that the amount of total iron calculated from the % of magnetite, in all cases, was 282
less than the measured total iron values showing that there is a significant amount of iron 283
associated with cells that is not in the form of magnetite. 284
285
Cells for lipid analyses were grown anaerobically with N2O and were harvested at mid to late 286
exponential phase of growth by centrifugation at 4°C. Membrane lipids from cells of strain MV-287
1T were extracted at room temperature in test tubes containing a monophasic mixture of 288
methanol:dichloromethane:phosphate buffer (50 mM, pH 7.4) (2:1:0.8) (Fang & Findlay, 1996). 289
The extraction mixture was allowed to stand overnight in the dark at 4oC. Crude lipids were 290
collected after phase partitioning by adding dichloromethane and deionized water to a final ratio 291
of methanol:dichloromethane:water 1:1:0.9. Total lipids were fractionated into different lipid 292
classes using miniature columns (Supelco, Inc., Bellefonte, PA) containing 100 mg silicic acid. 293
Neutral lipids, glycolipids, and phospholipids were obtained by sequential elution with 5 mL 294
aliquots of chloroform, acetone, and methanol, respectively. 295
296
Ester-linked phospholipid fatty acids were subjected to a mild alkaline trans-methylation 297
procedure to produce fatty acid methyl esters (Fang & Findlay, 1996). The fatty acid methyl 298
esters were analyzed on an Agilent 6890 GC interfaced with an Agilent 5973N mass selective 299
detector. Analytical separation of the compounds was accomplished using a 30 m x 0.25 mm i.d. 300
DB-5 MS fused-silica capillary column (J&W Scientific, Folsom, CA). The column temperature 301
was programmed from 50ºC to 140oC at 20ºC/min, then to 300ºC at 5ºC/min and then held 302
isothermally for 15 min. Individual compounds were identified from their mass spectra. 303
Response factors were obtained for each compound using duplicate injections of quantitative 304
standards at five different concentration levels. Concentrations of individual compounds were 305
obtained based on the GC/MS response relative to that of an internal standard (C18:0 fatty acid 306
ethyl ester). Method blanks were extracted with samples and were assumed to be free of 307
contamination if chromatograms of the blanks contained no peaks. A standard containing known 308
concentrations of 26 fatty acids was analyzed daily on the GC/MS to check analytical accuracy 309
(>90%). Replicate analyses of samples were done to ensure reproducibility (variation ≤10%). 310
The following fatty acid profile was obtained for strain MV-1T (mol%): C18 : 1ω7 (49.6%), C16 : 311
1ω7 (30.6%), C16 : 0 (13.7%), C16 : 1 ω5 (4.1%), C14 : 0 (0.8%), C18 : 0 (0.3%), C18 : 1 ω7 (0.4%) and 312
C16:1ω7 (0.4%). Analysis of cellular fatty acids and polar lipids was carried out by the 313
Identification Service and Dr. B. J. Tindall, DSMZ, Braunschweig. Germany. The total fatty acid 314
profile from the DSMZ was similar to the profile described above. Major polar lipids were 315
phosphatidylethanolamine and phosphatidylglycerol, as well as an unknown phospholipid and 316
two aminophospholipids (Fig. S3). 317
318
The genome of strain MV-1T consists of a single, circular chromosome about 3.7 Mb in size, and 319
extrachromosomal elements (e.g., plasmids) were not detected (Dean & Bazylinski, 1999). The 320
mol % guanine plus cytosine (G + C) of the genomic DNA of strain MV-1T was determined by 321
high-performance liquid chromatography (HPLC) by the DSMZ GmbH, according to the method 322
of Mesbah et al. (1989) and by the thermal melt technique (Herdman et al., 1979) after 323
purification of genomic DNA using the method described by Kimble et al. (1995). The mol % G 324
+ C of the genome as determined by HPLC is 52.9 ± 0.1 (three measurements); that determined 325
by thermal melt was 53.5 ± 0.1 mol % (three measurements). 326
327
Phylogenetic analysis based on a partial 16S rRNA gene sequence (1,128 nucleotides out of 328
1,361 nucleotides) obtained as described by DeLong et al. (1993), shows strain MV-1T as a 329
member of the Rhodospirillaceae within the Alphaproteobacteria (Fig. 2). Strain MV-1T does 330
not share a close relationship with Magnetospirillum, or with Magnetococcus (Bazylinski et al., 331
2012). Strain MV-1T is most closely related to Magnetospira thiophila (89.2% 16S rRNA gene 332
sequence identity with MV-1T; Williams et al., 2012) and strain QH-2 (89.2% sequence identity; 333
Zhu et al., 2010) among magnetotactic bacteria, and to the non-magnetotactic species 334
Terasakiella pusilla (87.6% sequence identity; Satomi et al., 2002), Thalassospira lucentensis 335
(85.9% sequence identity; López-López et al., 2002) and an undescribed marine bacterium 336
Candidatus Kopriimonas byunsanensis (88.1% sequence identity). This cluster may constitute a 337
new clade within the Rhodospirillaceae, although the node currently has weak bootstrap support 338
(Fig. 2). Within this cluster, strain MV-1T shares magnetotactic abilities with Ms. thiophila and 339
strain QH-2. Strain MV-1T is capable of anaerobic growth using several nitrogen species as 340
terminal electron acceptors (N2O, nitrate, nitrite). The type strains of Te. pusilla, Th. lucentensis, 341
and Ms. thiophila all require oxygen as the terminal electron acceptor (Satomi et al., 2002; 342
López-López et al., 2002; Williams et al., 2012), although other Thalassospira species are 343
facultative anaerobes that are capable of growth on nitrate (Liu et al., 2007; Kodama et al., 2008; 344
Zhao et al., 2010). Characterized species of Terasakiella and Thalassospira are obligate 345
chemoorganoheterotrophs, unlike strain MV-1T and Ms. thiophila, both of which are capable of 346
chemolithoautotrophic growth using the CBB cycle (Williams et al., 2012). 347
348
Among characterized magnetotactic members of the Alphaproteobacteria, strain MV-1T shows 349
the greatest metabolic versatility in the compounds that can be used as potential electron donors 350
and carbon sources for growth, during microaerobic and anaerobic growth (Table 1). This is in 351
contrast to its closest characterized relative, Ms. thiophila strain MMS-1T, which is an obligate 352
microaerophile that can use only a relatively small number of organic acids as carbon and energy 353
sources (Table 1). Chemoorganoheterotrophic growth of strain MV-1T is supported by a large 354
number of organic acids (including formate for methylotrophic growth) and certain amino acids 355
(Table S1). Unique among characterized magnetotactic bacteria, strain MV-1T is also capable of 356
using N2O for respiration. N2O is available in the marine environment as an alternative terminal 357
electron acceptor to oxygen: under suboxic or anaerobic conditions, heterotrophic denitrifying 358
bacteria produce N2O as an intermediate, and ammonia-oxidizing archaea and bacteria generate 359
N2O by nitrite reduction (Santoro et al., 2011). Thus, we posit an important role for MV-1T in 360
nitrogen cycling in its environment. In light of the characteristics of this magnetotactic strain, a 361
new genus is warranted for reception of strain MV-1T, within the family Rhodospirillaceae. 362
363
Description of Magnetovibrio gen. nov. 364
Magnetovibrio (Ma.gne.to.vi'bri.o. Gr. n. magnês -êtos, a magnet; N.L. pref. magneto-, 365
pertaining to a magnet; N.L. masc. n. vibrio, a vibrio; N.L. masc. n. Magnetovibrio, the magnetic 366
vibrio, which references the vibrioid morphology and magnetotactic behavior of this bacterium.) 367
368
Cells are Gram-negative and vibroid to helicoid in morphology, and motile by means of a single 369
polar flagellum. Cells assimilate inorganic carbon (as CO2) chemolithoautotrophically with 370
thiosulfate and sulfide as the electron donors, using a form II ribulose-1,5-bisphosphate 371
oxygenase/carboxylase (CbbM) and the CBB cycle. Cells of strain MV-1T exhibit characteristics 372
of both axial and polar magnetotaxis, and biomineralize a single chain of magnetosomes that 373
contain magnetite crystals of truncated hexa-octahedral habit, positioned along the long axis of 374
the cell. Major polar lipids identified include phosphatidylethanolamine and 375
phosphatidylglycerol. Strain MV-1T belongs to the Rhodospirillaceae, within the 376
Alphaproteobacteria. The type species for genus Magnetovibrio is M. blakemorei. 377
378
Description of Magnetovibrio blakemorei sp. nov. 379
Magnetovibrio blakemorei (blake.mo're.i. N.L. gen. masc. n. blakemorei, of Blakemore, named 380
in honor of United States microbiologist Richard P. Blakemore, who was the first to 381
scientifically describe and publish on magnetotactic behavior in bacteria.) 382
383
Exhibits the following characteristics in addition to those for the genus. Cells 1-3 µm long by 384
0.2-0.4 µm wide. Facultatively microaerophilic and anaerobic. Catalase-negative. Oxidase-385
positive. Mesophilic, with a growth temperature range of 4°C to 31°C, and optimal growth 386
temperature of 26-28°C. Cells produce internal sulfur-rich globules when grown on sulfide. 387
Terminal electron acceptors include oxygen, N2O, nitrate, and nitrite. Capable of 388
chemolithoautotrophic growth on thiosulfate and sulfide with oxygen as the terminal electron 389
acceptor, and on thiosulfate using N2O, as the terminal electron acceptor. Capable of 390
chemoorganoautotrophic and methylotrophic growth on formate under microaerobic (but not 391
anaerobic) conditions. Capable of chemoorganoheterotrophic growth under microaerobic 392
conditions using certain organic acids (acetate, fumarate, lactate, malate, maleate, 2-oxoglutarate, 393
proprionate, pyruvate, succinate), amino acids (aspartate, glutamate, alanine, glutamine, serine), 394
Casamino acids, peptone, yeast extract, and tryptone. Capable of chemoorganoheterotrophic 395
growth under anaerobic conditions (N2O as the terminal electron acceptor) using certain organic 396
acids (malate, malonate, oxaloacetate, 2-oxoglutarate, succinate) and casamino acids. Cellular 397
fatty acids dominated by C18 : 1ω7, C16 : 1ω7, C16 : 0, and C16 : 1ω5. The genome consists of a 398
single, circular chromosome with a size of 3.7 Mb with a G + C content of 52.9-53.5 mol%. The 399
type strain is MV-1T (= ATCC BAA-1436 T = DSM 18854T), originally isolated from a salt 400
marsh pool at the Neponset River Estuary near Boston, Massachusetts. 401
402
Acknowledgements 403
This work was supported by US National Science Foundation (NSF) Grant EAR-0920718 to 404
D.A.B. C.T.L. was the recipient of an award from the Fondation pour la Recherche Médicale 405
(FRM: SPF20101220993) and funded by the French national research agency ANR-P2N entitled 406
MEFISTO. We thank A. López-López for providing us with a culture of Thalassospira 407
lucentensis strain QMT2. Magnetic measurements were performed at the Institute for Rock 408
Magnetism, which is supported by grants from the Instruments and Facilities Program, Division 409
of Earth Science, NSF. This is publication 1202 of the Institute for Rock Magnetism. 410
411
412
413
414
415
416
417
418
419
420
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424
425
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427
428
429
430
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Figure Legends 563
564
Fig. 1. Transmission electron microscope (TEM) images of strain MV-1T. (a) TEM image of a 565
negatively stained dividing cell showing chain of magnetosomes in one cell and the single 566
polar flagellum (at arrow). (b) TEM image of a thin-section of a chain of magnetosomes 567
showing membrane-like structure (at arrows) along the length of the magnetosome chain. (c) 568
TEM image of a thin-section of the end of a cell showing polar membrane (PM) and the 569
magnetosome membrane (MM). (d) TEM image of negatively stained purified preparation of 570
magnetosomes from strain MV-1T. The “halo” around the magnetosome crystals represents 571
the magnetosome membrane. 572
573
Fig. 2. Phylogenetic relationships of strain MV-1T (type strain of Magnetovibrio blakemorei) 574
within the Alphaproteobacteria based on 16S rRNA sequences. 575
576
577
578
579
580
581
582
583
584
585
Table 1. Characteristics that differentiate Magnetovibrio blakemorei MV-1T from other magnetotactic species within the Alphaproteobacteria. +, Positive ; -,
negative; Magnetospira thiophila strain MMS-1T (Williams et al., 2012); Magnetospirillum gryphiswaldense strain MSR-1T (Schleifer et al., 1991; Bazylinski
and Williams, 2007; D. Schüler, pers. comm); Magnetospirillum magnetotacticum strain MS-1 (Maratea and Blakemore, 1981; Bazylinski and Blakemore,
1983b; Bazylinski and Williams, 2007); Magnetococcus marinus strain MC-1T (Meldrum et al., 1993; Bazylinski and Williams, 2007; Bazylinski et al., 2012).
Characteristic MV-1T Magnetospira thiophila
MMS-1T
Magnetospirillum
gryphiswaldense MSR-1T
Magnetospirillum
magnetotacticum MS-1
Magnetococcus marinus
MC-1T
Cell
morphology vibrioid-helical vibrioid-helical spiral-helical spiral-helical coccoid
Flagella monotrichous amphitrichous amphitrichous amphitrichous bilophotrichous
Magnetotaxis axial & polar? polar axial & polar axial & polar polar
Magnetite
shape truncated hexa-octahedral elongated octahedral cuboctahedral cuboctahedral
elongated
pseudo-hexahedral
Electron donors S2O3
2-, S2-,
organic C (= C sources) S2O3
2- organic C
(= C sources)
organic C
(= C sources) S2O3
2-, S2-
Terminal
electron
acceptors
O2 (microaerobic), N2O,
NO3- and NO2
- O2 (microaerobic)
O2 and NO3- (both
microaerobic) O2 (microaerobic) O2 (microaerobic)
C sources
(autotrophic)
Microaerobic: CO2,
formate
Anaerobic: CO2
CO2 none known none known CO2
C sources
(heterotrophic)*
Microaerobic: acetate,
formate, fumarate, lactate,
malate, maleate, 2-
oxoglutarate, proprionate,
pyruvate, succinate,
aspartate, glutamate,
alanine, glutamine, serine.
Anaerobic (N2O): malate,
malonate, oxaloacetate,
2-oxoglutarate, succinate.
acetate, fumarate, malate,
succinate
acetate,
lactate, malate, pyruvate
succinate
fumarate, lactate, malate,
pyruvate, oxaloacetate,
malonate, tartrate,
succinate
acetate
Metabolism
chemolithoautotrophic,
chemoorganoheterotrophic,
chemoorganoautotrophic
chemolithoautotrophic,
chemoorganoheterotrophic chemoorganoheterotrophic chemoorganoheterotrophic
chemolithoautotrophic,
chemoorganoheterotrophic
Nitrogenase + + + + +
Oxidase + + + - +
Catalase - - + - -
G + C (mol%) 53.5 47.2 62.7 64.5 55.8
* Defined carbon sources only.
Magnetovibrio blakemorei, gen. nov. sp. nov., a new magnetotactic bacterium
(Alphaproteobacteria: Rhodospirillaceae) isolated from a salt marsh.
Supplementary Material
Collection, enrichment and isolation of strain MV-1T. Bottles of water and sediment were
collected from shallow, brackish, salt marsh pools near the Neponset River Estuary in Milton,
Massachusetts, USA, and placed under dim light at room temperature. After several days, a
horizontal “plate” of microorganisms formed in the water column of one of the bottles (Fig.
S1). Microscopic examination of microorganisms within the plate showed that the dominant
organism was a small, magnetotactic, vibrioid-to-helicoid-shaped bacterium. In order to
understand the environmental conditions favoring the enrichment of this bacterium, sulfide
concentrations were determined using the method of Cline (1969). The concentration of
sulfide was 2.2 mM just over the sediment in the bottle which decreased to 0.4 mM 1-2 mm
below the plate. Sulfide was at undetectable levels at and above the plate. From this data, we
inferred the presence of an opposing gradient of oxygen diffusing from the surface and thus
the plate likely formed at the oxic-anoxic interface (OAI) within the bottle. The pH of the
sample was 7.5 and the salinity 23 parts per thousand (ppt) as determined with a Palm Abbe
PA203 hand-held refractometer (MISCO Refractometer, Cleveland, OH).
After chemical analysis of the microcosm in the bottle was completed, cells were removed
from the plate and used to inoculate sulfide-O2 concentration ([O2]) gradient media, prepared
following the recipe of Nelson and Jannasch (1983) but modified by replacing a diluted
artificial seawater (ASW) solution for natural seawater and by the addition of 25 mM ferric
quinate (Blakemore et al., 1979) and 200 µl of 0.2% aqueous resazurin per liter. The ASW
was adjusted to approximately 23 ppt and consisted of (in g L-1): NaCl, 16.4; MgCl2•6H2O,
3.5; Na2SO4, 2.7; KCl, 0.47; and CaCl2•2H2O, 0.39. Growth, as microaerophilic bands of
cells, occurred in all tubes although the magnetotactic bacterium under study was only
present in small amounts in a low percentage of the cultures. For isolation of the strain, cells
from these enrichment gradient culture were inoculated in a dilution series of solid agar (13 g
L-1 Agar Noble (Difco Laboratories, Detroit, MI)) shake tubes of an [O2]-gradient medium
containing: 5 mL of modified Wolfe’s mineral elixir (Frankel et al., 1997); 0.5 mL vitamin
solution (Frankel et al., 1997); 0.5 g sodium succinate, 0.5 g Casamino acids and 0.2 g of
sodium acetate•3H2O as electron donors; 0.2 g NH4Cl as the nitrogen source; 2 mL of freshly
prepared, filter-sterilized, neutralized 0.43 M cysteine•HCl•H2O as the reducing agent; 1.8
mL of 0.5 M KHPO4 buffer pH 6.9; and 2.4 mL 0.8 M NaHCO3 per liter of diluted ASW.
The cysteine and NaHCO3 were added after autoclaving and the pH adjusted to 7.1-7.2. The
medium was dispensed as 10 mL aliquots into sterile 15 x 125 mm test tubes which were kept
at 44°C, inoculated, inverted several times and then quickly cooled on ice. In some tubes,
cells of this strain grew as dark, lens-shaped colonies at the OAI of this medium after about 2
weeks. However, the tubes were highly contaminated with non-magnetotactic bacteria and it
was difficult to remove colonies aseptically without these contaminants. In an attempt to
eliminate contamination, cells from several colonies were aseptically extracted and serially
diluted in anoxic shake tubes series containing one of a variety of electron acceptors
including nitrate (2 mM), nitrite (2 mM) and nitrous oxide (N2O) at a pressure of 2 atm
(202.7 kPa) in the headspace. Similar contamination problems occurred in the nitrate and
nitrite tubes. After 2-3 weeks, the N2O shake tubes all contained black lens-shaped colonies
consisting of the magnetotactic vibrio. Contamination was only observed in the lowest
dilution tubes indicating that the dominant bacterium in the inoculum was the magnetotactic
vibrio. Individual colonies were removed from tubes that did not contain contaminants and
used as inocula for a second series of shake tubes and the process repeated one more time to
ensure purity of the culture. After isolation, cells were routinely grown in oxygen-free liquid
cultures of the medium described above with N2O as the terminal electron acceptor.
References
Cline, J. D. (1969). Spectrophotometric determination of hydrogen sulfide in natural waters,
Limnol Oceanogr 14, 454-458.
Frankel, R. B., Bazylinski, D. A., Johnson, M. S. & Taylor, B. L. (1997). Magneto-
aerotaxis in marine, coccoid bacteria. Biophys J 73, 994-1000.
Nelson, D. C. & Jannasch, H. W. (1983). Chemoautotrophic growth of a marine Beggiatoa
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Fig. S1. Natural enrichment of strain MV-1T from sediment collected from the Neponset
River Estuary (Milton, MA) from, showing some of the chemical conditions, and the zone
(“plate”) of enriched microorganisms that formed at the oxic-anoxic interface, and the sulfide
concentrations within the bottle. Ppt, parts per thousand.
Fig. S2. Phase contrast light microscope images of cells of strain MV-1T grown microaerobically (a) and anaerocially with N2O as the terminal
electron acceptor (b). Cells tended to be more comma-shaped when grown microaerobically compared to the much more helical cells present in
anaerobic cultures.
Fig. S3. Polar lipid analysis of strain MV-1T. PE = phosphatidylethanolamine, PG = phosphatidylglycerol, PL1 = phospholipid, PN1-PN2 =
aminophospholipids.
Table S1. Substrate utilization by strain MV-1T during chemoorganoheterotrophic growth.
+, strong growth; -, no growth; w, weak growth, as determined by band formation in tubes. g, growth as indicated by N2 gas production in tubes.
Substrate Utilization
(microaerobic)
Utilization
(anaerobic)*
Organic acids Acetate + -
Butyrate - -
Citrate - -
Formate † + -
Fumarate + g
Gluconate - -
Glycolate - -
Glyoxylate - -
Hippurate - -
Lactate + -
Malate + g
Maleate + -
Malonate - g
Oxalate - -
Oxaloacetate - g
2-Oxoglutarate + g
Propionate + -
Pyruvate + -
Succinate + g
Quinate - -
Tartrate - -
Valerate - -
Amino acids
L-Alanine, w -
L-Arginine - -
L-Aspartate + -
L-Cysteine - -
L-Glutamate + -
L-Glutamine w -
Glycine - -
L-Isoleucine - -
L-Leucine - -
L-Methionine - -
L-Ornithine - -
L-Phenylalanine - -
L-Proline - -
L-Serine w -
L-Tryptophan - -
L-Valine - -
Alcohols and aldehydes
Methanol - -
Ethanol - -
Butanol - -
Ribitol (adonitol) - -
Formaldehyde - -
Acetaldehyde - -
Carbohydrates (D- or mixed enantiomers used)
Glycerin (glycerol) - -
Amygdalin - -
Arabinose - -
Cellobiose - -
Dulcitol - -
Fructose - -
Galactose - -
Gelatin - -
Glucose - -
Glycogen - -
Inulin - -
Lyxose - -
Maltose - -
Mannitol - -
Mannose - -
Melezitose - -
Melibiose - -
Raffinose - -
Rhamnose - -
Ribose - -
Salicin - -
Sedoheptulose - -
Sorbitol - -
Sorbose - -
Sucrose - -
Trehalose - -
Xylose - -
Undefined
Casamino acids + g
Peptone + -
Tryptone + -
Yeast extract + -
* N2O used as terminal electron acceptor.
† Chemoorganoautotrophic growth.
Table S2. Acetylene (C2H2) reduction to ethylene (C2H2) by cells of strain MV-1T grown heterotrophically in semi-solid, oxygen concentration
gradient medium containing 3.7 mM succinate and 1.4 mM acetate as the electron source. Magnetospirillum magnetotacticum strain MS-1 was
used as positive control and grown with 3.7 mM succinate. Results are the mean ± standard deviation from triplicate or quadruplicate cultures.
______________________________________________________________________________________________________________________________________________ Bacterial Electron Fixed N Total cell protein Rate of C2H2 Duration r Value (from strain source source per culture reduction (nmol of assay linear regression (4 mM when (μg) C2H4 produced min-1 calculations)
present mg cell protein-1)
______________________________________________________________________________________________________________________________________________
MV-1T Succinate-acetate None 27.3 ± 3.1 25.2 ± 2.3 5 h ≥0.9957 (n = 5-6) NH4Cl 32.4 ± 5.9 ND 5 h Not applicable NaNO3 2.5 ± 0.4 ND 5 h Not applicable Magnetospirillum Succinate None 16.3 ± 5.2 22.3 ± 4.4 7 h ≥0.9995 (n = 4-6) magnetotacticum
NH4Cl 240.4 ± 25.1 0 7 h Not applicable NaNO3 256.3 ± 7.2 0 7 h Not applicable ______________________________________________________________________________________________________________________________________________
Table S3. Percentages of iron and magnetite (Fe3O4) of freeze-dried whole cells of strain
MV-1T grown with and without the major source of iron, 25 μM ferric quinate, in the growth
medium. For total iron, the result is the mean ± standard deviation of triplicate measurements.
AA, atomic absorption spectroscopy.
Total Fe in
growth
medium
Batch % Total Fe
(AA)
% Total Fe
(ferrozine
assay)
% Fe3O4 % Fe as Fe3O4
(calculated)
High: 26.3 μM 1 1.90 ± 0.01 1.85 ± 0.01 1.83 1.32
2 1.34 ± 0.00 1.33 ± 0.01 1.38 1.00
Low: 1.5 μM 1 0.49 ± 0.01 0.46 ± 0.01 0.50 0.36
2 0.52 ± 0.00 0.51 ± 0.01 0.61 0.44