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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423

424

425

426

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

in sulfide-gradient cultures. Arch Microbiol 136, 262-269.

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