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TRANSCRIPT
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Running Title: Innate NDFO capacity of nitrate-reducing bacteria 1
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Fe(II) oxidation is an innate capability of nitrate-reducing bacteria involving 3
abiotic and biotic reactions 4
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Hans K. Carlson1, Iain C. Clark2, Steven J. Blazewicz3, Anthony T. Iavarone4, 6
and John D. Coates1,# 7
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Department of Plant and Microbial Biology1, Department of Civil and 9
Environmental Engineering2, Department of Environmental Science, Policy 10
and Management3, QB3/Chemistry Mass Spectrometry Facility4, University 11
of California, Berkeley, CA 94720. 12
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#Corresponding author: John D. Coates. Mailing address: University of 14
California, Berkeley, Department of Plant and Microbial Biology, Berkeley, CA 15
94720. Phone 510-643-8455. Fax: 510-642-4995. E-mail: 16
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Author contributions: H.K.C., I.C.C., and J.D.C. designed research. H.K.C., 19
A.T.I., I.C.C., and S.B. performed research and analyzed data. H.K.C., I.C.C. 20
and J.D.C. wrote the paper. 21
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The authors declare no conflict of interest. 23
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Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.00058-13 JB Accepts, published online ahead of print on 17 May 2013
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Abstract 25
Phylogenetically diverse species of bacteria can catalyze the oxidation of ferrous 26
iron (Fe(II)) coupled to nitrate (NO3-) reduction, often referred to as nitrate-27
dependent iron oxidation (NDFO). Very little is known about the biochemistry of 28
NDFO, and though growth benefits have been observed, mineral encrustations 29
and nitrite accumulation likely limit growth. Acidovorax ebreus, like other species 30
in the Acidovorax genus, is proficient at catalyzing NDFO. Our results suggest 31
that the induction of specific Fe(II) oxidoreductase proteins is not required for 32
NDFO. No upregulated periplasmic or outer membrane redox active proteins, 33
like those involved in Fe(II) oxidation by acidophilic iron oxidizers or anaerobic 34
photoferrotrophs, were observed in proteomics experiments. We demonstrate 35
that while “abiotic” extracellular reactions between Fe(II) and biogenic NO2-/NO 36
can be involved in NDFO, intracellular reactions between Fe(II) and periplasmic 37
components are essential to initiate extensive NDFO. We present evidence that 38
an organic co-substrate inhibits NDFO, likely by keeping periplasmic enzymes in 39
their reduced state or by stimulating metal efflux pumping, or both, and that 40
growth during NDFO relies on the capacity of a nitrate-reducing bacterium to 41
overcome the toxicity of Fe(II) and reactive nitrogen species. Based on our data 42
and evidence in the literature, we postulate that all respiratory nitrate-reducing 43
bacteria are innately capable of catalyzing NDFO. Our findings have implications 44
for a mechanistic understanding of NDFO, the biogeochemical controls on 45
anaerobic Fe(II) oxidation, and the production of NO2-, NO and N2O in the 46
environment. 47
Introduction 48
Although microbial nitrate-dependent Fe(II) oxidation (NDFO) has been 49
known for over two decades and has been demonstrated to play a critical role in 50
both the extant (1) and ancient (2) global iron cycle, almost nothing is known of 51
the underlying biochemical or genetic mechanisms involved (3). NDFO microbes 52
have been demonstrated to oxidize both soluble and insoluble Fe(II) (1, 4-6), and 53
to produce a variety of insoluble mixed valence iron mineral products (1, 5, 7, 8). 54
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Some evidence already exists that the capacity for NDFO is widespread 55
and likely innate to all nitrate-reducing bacteria. Bacterial species that couple the 56
oxidation of Fe(II) to nitrate reduction have been isolated from a wide range of 57
habitats and are phylogenetically diverse indicating their environmental 58
prevalence and importance (5, 9, 10). Recently, species from the genus 59
Acidovorax have dominated enrichment cultures and been isolated as proficient 60
nitrate-dependent Fe(II) oxidizers (11-14), however, even E. coli has been 61
demonstrated to be capable of Fe(II) oxidation coupled to nitrate reduction (15). 62
Although NDFO is based on thermodynamically favorable redox reactions (16, 63
17), only a few studies suggest a growth enhancement from this metabolism (12, 64
18). This suggests that NDFO may primarily be inadvertent or a detoxification 65
strategy. 66
While the toxicity of Fe(II) to biological systems under aerobic conditions is 67
widely appreciated, less is understood about Fe(II) toxicity under anaerobic 68
conditions. Some studies have demonstrated that Fe(II) is toxic at low 69
concentrations to anaerobic bacteria, such as anoxygenic phototrophs (19) or 70
streptococci (20). Several possibilities for anaerobic Fe(II) toxicity include 71
inhibition of the F-ATPase (20), binding to membranes (21), disrupting protein 72
stability or replacing active-site metal cofactors (22), and oxidation and 73
precipitation of insoluble Fe(III) on cellular components to impair nutrient uptake 74
(13). Under NDFO conditions, redox transformations of nitrogen oxides can 75
produce intermediates, such as nitric oxide, which is capable of binding to and 76
reacting with heme cofactors (23) or Fe-S clusters (24) or, in the presence of 77
transition metals, nitrosating protein thiols to inhibit or alter protein activity (25). 78
Here we present the results of proteomic and physiological experiments 79
used to understand the mechanism of NDFO in the model organism Acidovorax 80
ebreus. Like several other species in the Acidovorax genus (12-14), A. ebreus 81
was isolated based on its capacity for NDFO (11). Previous studies have 82
reported a growth advantage from NDFO by Acidovorax species in both batch 83
and continuous flow cultures (12, 14), in addition to the accumulation of nitrite 84
(NO2-) and periplasmic mineral encrustations (26). Based on our findings with A. 85
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ebreus, we postulate that all nitrate respiring organisms are innately capable of 86
catalyzing NDFO, but the survival and growth benefit obtained is dependent on 87
the ability to overcome Fe(II), NO2- and NO toxicity. Our findings have 88
implications for understanding the evolution of Fe(II) oxidation as a microbial 89
metabolism, the impact of Fe(II) on nitrate reducing communities and the 90
influence of Fe(II) on the product distribution of microbial nitrate reduction. 91
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Materials and Methods: 93
Media and culture conditions 94
Anaerobic cultures of Acidovorax ebreus strain TPSY, formerly 95
Diaphorobacter sp. TPSY (11) and Azospira suillum strain PS were grown 96
organotrophically in anoxic bicarbonate buffered basal medium (BBM) (27), pH 97
6.8 at 37 °C with 10 mM sodium nitrate and varying concentrations of sodium 98
acetate and harvested in late log-phase. BBM contained the following 99
components (per Liter): 0.25 g NH4Cl, 0.6 g NaH2PO4, 0.1 g KCl, and 2.52 g 100
NaHCO3 with the addition of vitamins and minerals according to Bruce et 101
al.,1999 (27) and the appropriate concentration of electron donors and acceptors. 102
Addition of millimolar Fe(II) to the growth medium immediately formed a 103
white precipitate leaving 1-2 mM soluble Fe(II). This precipitate was shown to be 104
predominantly vivianite by Kappler et al., 2005 (13). Unless otherwise indicated, 105
our growth and cell suspension experiments utilized this mixed soluble and 106
insoluble (vivianite) Fe(II) form. 107
Fe(II) oxidation cell suspensions. Anaerobic cultures of Acidovorax ebreus 108
were grown organotrophically in bicarbonate-buffered basal media (BBM) with 10 109
mM sodium nitrate and 6.25 mM sodium acetate and harvested in late log-phase. 110
Cells were washed three times in BBM without donors or acceptors under an 111
N2:CO2 (80%:20% v:v) headspace. In cell suspensions, cells were inoculated into 112
fresh media at ~5x108 cells/mL with electron donor and acceptor concentrations 113
as indicated in the text. When indicated, chloramphenicol was added to cell 114
suspensions at a concentration of 100 μg/mL. Chloramphenicol was found to 115
completely inhibit growth of A. ebreus at concentrations above 50 μg/mL. When 116
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indicated, Fe(II) in the form of Fe(II)-NTA, Fe(II)-NTA-agarose or synthetic 117
vivianite was added from anoxic stock solutions. Fe(II)-NTA-agarose was 118
prepared by adding FeCl2 from sterile anoxic stock solutions to NTA-agarose 119
beads (Qiagen). The beads were washed 5x with sterile water under anoxic 120
conditions. Synthetic vivianite was made by chemical precipitation. FeCl2 was 121
added to 10 mM phosphate buffer. The precipitate was washed with 10 mM 122
phosphate buffer and kept as a suspension under anoxic conditions. To assess 123
the capacity of NO and NO2- to oxidize and solubilize Fe(II) from synthetic 124
vivianite, anoxic aqueous stock solutions of sodium nitrite or diethylamine 125
NONOate (Cayman chemical) in 0.1 M NaOH were added to BBM containing 126
synthetic vivianite. NONOates, or diazeniumdiolates, are stable at alkaline pH, 127
but decompose at neutral pH to release nitric oxide (28). DEA-NONOate decays 128
at pH 7.4 and 25 °C with a 16-minute half-life to release 2 moles of NO per mole 129
of parent compound. Appropriate controls for the effect of diethylamine (the 130
product of NONOate decay) and sodium hydroxide were used to show that 131
neither of these compounds, in the concentrations added to generate NO, altered 132
the oxidation state or solubility of Fe(II) in synthetic vivianite. 133
Analytical techniques 134
Nitrate, nitrite, and acetate were measured with an ion chromatograph 135
(ICS-2100, Dionex, Sunnyvale, California) using a method developed to quantify 136
all three analytes simultaneously. Samples for ion chromatography were diluted 137
in 10 mM NaOH, filtered and stored at -20 °C until analysis. The guard and 138
analytical columns were IonPac AG16 (4 x 50mm) and IonPac AS16 (4 x 139
250mm), respectively, with an ASRS-300 4-mm suppressor system and a DS6 140
heated conductivity cell. A KOH gradient was generated using the EGC III KOH 141
generator at an isocratic flow rate of 1.5 ml/min. The KOH concentration was 1.5 142
mM from 0-7 min, ramped to 10 mM from 7-13 min, was held at 10 mM from 13-143
16 min, ramped to 35 mM from 16-17 min, was held at 35 mM from 17-27 min 144
and ramped back down to 1.5 mM from 27-30 min. 145
Soluble and insoluble Fe(II) were measured separately to characterize the 146
oxidation of each Fe(II) form in cultures and to avoid analytical artifacts due to 147
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rapid reactions which occur between NO2- and Fe(II) in acidic solution and the 148
regeneration of NO2- through reactions between NO and O2 under oxic 149
conditions. Klueglein and Kappler (29) provide an excellent discussion of this 150
problem and other alternatives to avoid it. At each time point, 0.5 mL of culture 151
media was removed in the anaerobic chamber (Coy) and centrifuged to separate 152
insoluble Fe(II) from soluble Fe(II) and NO2- in solution. Soluble Fe(II) was 153
directly measured with the ferrozine assay and insoluble Fe(II) was resuspended 154
in 0.5 M HCl for 24 hrs and Fe(II) was quantified using the ferrozine assay (30). 155
Control experiments in which the insoluble Fe(II) was washed with anoxic buffer 156
to remove residual nitrite prior to resuspension in 0.5 M HCl gave identical 157
ferrozine results. 158
Two techniques were used for measurement of nitric oxide (NO) gas. For 159
headspace NO analysis, culture headspace was anaerobically sampled and 160
injected into the purge cell of a Nitric Oxide Analyzer 280i (Sievers). For solution 161
NO, samples of culture solution were sampled and injected into a purge solution 162
in anoxic vials (31). The headspace from these vials was injected into the NOA. 163
The purge solution releases NO from iron complexes in solution and contains 5.5 164
mL glacial acetic acid, 1.5 mL 0.8 M potassium ferricyanide and 1 mL 0.1 M 165
sulfanilamide in 2 M glacial acetic acid. The ferricyanide oxidizes iron and 166
releases NO and sulfanilamide reacts rapidly with NO2- to prevent further NO 167
release in the acid solution. NO gas standards were used to generate a standard 168
curve for quantification. 169
For measurement of N2O, culture headspace (2 mL) was collected with a 170
gas tight syringe and injected directly into a Hewlett Packard HP6890 (Agilent 171
Technologies Inc., Santa Clara, CA) gas chromatograph fitted with a Hayesep 172
DB 100/120 (1/16” x 1.5 m) column which fed into a Hayesep DB 120/140 (1/16” 173
x 2.0 m) column leading to a pulse discharge detector (PDD). The PDD was 174
calibrated for N2O using a 5 point-standard curve, and a single standard was 175
analyzed hourly thereafter to correct for instrumental drift. 176
Growth was monitored in cultures by direct counts of acridine orange-177
stained cells. At timepoints, 500 μL of cultures was fixed in 3.7% formaldehyde. 178
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Cells were then diluted in water or 100 mM oxalate buffer. 1 mL of diluted cells 179
were stained with acridine orange (final concentration 1 μg/mL), and applied to a 180
polycarbonate black filter in a glass vacuum-filter apparatus (Fisher). The filter 181
was washed with 5 mL of oxalate buffer and cells counted by microscope 182
inspection with a 100x magnification oil-immersion lens using an Axioimager M1 183
microscope (Zeiss). 184
Trypsin shaving proteomics sample preparation 185
For proteomics, cells from NDFO and organoheterotrophic cultures were 186
harvested anaerobically at timepoints between 8-48 hours. At each timepoint, 50 187
mL of culture was centrifuged anaerobically, treated with oxalate buffer to 188
remove Fe(II) from cells and washed 2x with 100 mM ammonium bicarbonate 189
buffer. Cells were resuspended in two aliquots of 500 μL. One sample was 190
lysed (for a whole cell lysate proteome) and the other was kept intact (for a 191
trypsin-shaved proteome) under anaerobic conditions. 200 nanograms of 192
Trypsin Gold porcine protease (Pierce) was added to lysed and intact cells and 193
samples were incubated for 45 minutes at 37 °C . After 45 minutes, both the 194
lysed and intact cells were centrifuged at 10,000 rcf and the supernatant was 195
treated in two sequential 30-minute steps with 1 mM dithiothreitol and 1 mM 196
iodoacetamide then further digested overnight with 200 nanograms of trypsin at 197
37 °C. Peptides from overnight digests were concentrated with 100 μL C18 OMIX 198
tips (Agilent) and eluted with 85% acetonitrile:0.1% trifluoroacetic acid in water. 199
Acetonitrite was removed by vacuum centrifugation (speed-vac) and peptides 200
analyzed by LC-MS/MS (see supplemental methods). 201
Proteomics Data Analysis 202
Normalized peptide counts were used as semi-quantitative measure of 203
relative protein abundance. Validation of this approach has been published (32, 204
33). Peptide counts were normalized by dividing the observed number of 205
peptides for a given protein by the total number of peptides observed in a 206
sample. For statistical analysis, normalized peptide counts in intact and lysed 207
samples were pooled for a given growth condition by combining all timepoints 208
from across the growth curve. Student’s t-test was used to compare the 209
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normalized peptide counts from NDFO and organotrophic growth conditions. P-210
values≤0.05 were considered to be significant. 211
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Results and Discussion 213
Evidence against an inducible Fe(II) oxidoreductase in A. ebreus 214
Previous studies on anaerobic Fe(II) oxidation by phototrophic organisms 215
demonstrated induction of putative Fe(II) oxidoreductase proteins and enhanced 216
Fe(II) oxidation when cells were pre-cultured in the presence of Fe(II)-NTA (34). 217
In contrast, pre-culturing of A. ebreus on Fe(II)-NTA was not required for NDFO 218
of mixed insoluble (vivianite/siderite) and soluble Fe(II) in chloramphenicol-219
treated washed cell suspensions without acetate, indicating a basal capacity for 220
NDFO (Fig. 1a). Similar results were obtained in the absence of chloramphenicol 221
(Supp. Fig. S1). Furthermore, pre-culturing in the presence of Fe(II)-NTA did not 222
enhance the rate or extent of Fe(II) oxidation of mixed soluble and insoluble 223
Fe(II) (Fig. 1a). 224
In the model acidophilic Fe(II) oxidizer Acidothiobacillus ferroxidans, outer 225
membrane multiheme c-type cytochromes and rusticyanin, a periplasmic copper 226
protein, are proposed to be involved in Fe(II) oxidation (35). In phototrophic iron 227
oxidizers Rhodobacter sp. SW2 and Rhodopseudomonas palustris TIE-1, outer 228
membrane porins (PioB), periplasmic multiheme c-type cytochromes 229
(PioA/FoxE), and periplasmic high-potential iron proteins (PioC) or quinoproteins 230
(FoxY) are involved (35). Similar c-type cytochrome-based mechanisms are 231
proposed for microaerophilic Fe(II) oxidation (36). While a number of c-type 232
cytochrome genes are present in the A. ebreus genome, there are no multiheme 233
c-type cytochromes or close homologs of the pio/fox genes (35). The genes 234
Dtpsy_1207, Dtpsy_1208, and Dtpsy_2198 have homology with multicopper 235
oxidase (MCO) family proteins, some of which are known to be involved in Mn(II) 236
oxidation, but these proteins typically utilize oxygen as a co-substrate to oxidize 237
metals (37), and were never observed in proteomics experiments; as such, they 238
are unlikely to be involved in NDFO by A. ebreus. 239
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To search for an inducible Fe(II) oxidoreductase, we compared the whole 240
cell lysate and trypsin-shaved proteomes of organotrophic (5 mM acetate/10 mM 241
nitrate) and NDFO (10 mM Fe(II)/5 mM acetate/10 mM nitrate) growth cultures of 242
A. ebreus at 8, 18, 24, 32 and 48 hrs. Representative growth curves and data on 243
the transformation of Fe(II), acetate, and nitrogen oxides are presented in 244
Supplementary Figure S2. Trypsin shaving has previously been shown to be 245
useful for identifying surface exposed proteins, including cytochromes, in other 246
bacteria (38, 39), and we used it to increase our chance of identifying outer 247
membrane proteins which may be contacting Fe(II) and catalyzing cell surface 248
Fe(II) oxidation. When the proteomes of cells harvested at timepoints between 8 249
and 48 hours from NDFO and organotrophic A. ebreus growth cultures are 250
compared, significantly increased abundance (p≤0.05) is observed for heavy 251
metal RND (resistance-nodulation-division) family efflux pumps, Dtpsy_1460 252
(p=0.018), Dtpsy_1461 (p=0.0030) and Dtpsy_1462 (p= 0.00070), (Fig. 2, 253
Dataset S1). In the set of extracytoplasmic proteins that were observed 254
exclusively in NDFO cultures (Dataset S1E), only a few peptides were observed, 255
but efflux pumps are highly represented. The expression of heavy metal efflux 256
pumps in response to anaerobic Fe(II) stress is a novel finding, and suggests 257
that these proteins may be involved in exporting metals other than those 258
commonly considered to be substrates (40, 41). Furthermore, because the RND 259
family efflux pumps identified in A. ebreus are proton/solute antiporters, the 260
energetic cost is putatively less than an ATP-consuming transporter, and RND 261
family efflux pumps are able to load substrate from either the periplasm or 262
cytoplasm (42). 263
The proteomics data also suggest a cytoplasmic response to 264
redox/nitrosative stress. Although the p-values are higher (Dataset S1), 265
dihydrolipoamide dehydrogenase subunits of the pyruvate dehydrogenase 266
complex, Dtpsy_1140 (p=0.074) and Dtpsy_1658 (p=0.070), which are known to 267
be sensitive to nitric oxide (NO) (43), were more abundant. Also more abundant 268
were proteins involved in fatty acid synthesis, enoyl-CoA hydratase, Dtpsy_2933 269
(p=0.11), and a member of the 2-nitropropane dioxygenase family, Dtpsy_2296 270
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(p=0.055) which is closely related to a Neisseria gonorrhoeae homolog 271
(NGO1024) putatively involved in anaerobic, NO-dependent fatty acid synthesis 272
(44). Aconitase, Dtpsy_1066 (p= 0.25), which is sensitive to nitrosative stress 273
(45), and the large subunit of NO reductase Dtpsy_0109 (p=0.13) (46), were also 274
observed more often in Fe(II) cultures, but the p-values are higher (Dataset S1). 275
Two proteins, Dtpsy_0452 (p=0.030) and Dtpsy_1098 (p=0.045), were 276
significantly less abundant in NDFO cultures of A. ebreus (Dataset S1). Given 277
that peptides from core periplasmic and inner-membrane respiratory proteins 278
were observed in the proteomics experiments, it is unlikely that a respiratory 279
ferroxidase was missed. Several periplasmic c-type cytochromes were observed 280
(Dataset S1), including Dtpsy_0754, a component of the cytochrome bc1 281
complex, but there was no significant difference in their abundance between 282
organotrophic and NDFO proteomes (Dataset S1). Overall, the proteomic 283
response to Fe(II) is consistent with a cell envelope and cytoplasmic stress 284
response to Fe(II) and nitrogen oxides rather than induction of a respiratory 285
metabolism. 286
287
Fe(II) is toxic to nitrate reducing cultures of A. ebreus 288
In support of Fe(II) toxicity to nitrate-reducing cells, when 0.1 mM Fe(II) 289
was added to organotrophic cultures of A. ebreus, a growth lag of between 12-18 290
hours was observed (Fig. 1b). Similar concentrations of Fe(II) are inhibitory to 291
the growth of Rhodobacter capsulatus grown under humic acid oxidizing 292
phototrophic conditions (19). A lag was also observed in NDFO cultures of A. 293
ebreus containing higher Fe(II) concentrations (8 to 10 mM) (Supp. Fig. S2b, c), 294
and NO2- and NO both accumulated to at least ten-fold higher levels in the NDFO 295
cultures relative to organotrophic (no Fe) cultures (Supp. Fig. S2d, e). The 296
growth lag in NDFO cultures was partially relieved by addition of NTA with an 297
accompanying increase in the rate of Fe(II) oxidation (Supp. Fig. S3a, b). We 298
also observed less NO accumulation in the headspace of NDFO cultures grown 299
with Fe(II)-NTA compared with unchelated Fe(II) (Supp. Fig. S3c). NO toxicity is 300
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likely less of a problem in the presence of Fe(II)-NTA which forms a stable NO 301
complex with a sub-micromolar Kd (47). 302
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Possible mechanisms for NDFO by A. ebreus and other nitrate-reducing 304
bacteria 305
In the absence of an inducible Fe(II) oxidoreductase, one or a combination 306
of several mechanisms for NDFO are possible, including (i) direct electron 307
donation from Fe(II) to respiratory complexes if the appropriate electron acceptor 308
is available (i.e. NO3- for Nar, NO2
- for Nir, etc), (ii) extracellular or intracellular 309
mineral phase Fe(II) oxidation coupled to the reduction of NO3- or biogenic NO2
- 310
and NO (48) (green rusts have been shown to form during NDFO by Acidovorax 311
sp. BoFeN1 (49), (iii) proton dependent abiotic Fe(II) reduction of NO2- at lower 312
pH conditions that exist in the bacterial periplasm, and (iv) Fe(II) oxidation 313
coupled to the reduction of NO2- and NO catalyzed by periplasmic components 314
such as protein thiols (3, 41, 50) or membrane bound iron (51, 52). 315
316
Fe(II) must enter the periplasm for extensive oxidation by A. ebreus 317
NDFO catalysis in the periplasm requires that Fe(II) cross the outer 318
membrane, which, though porous, is negatively charged and will bind Fe(II) (52). 319
A long lag (~6hrs) precedes Fe(II) oxidation (Fig. 1a) and nitrate reduction (Supp. 320
Fig. S5a) in cell suspensions with mixed soluble and insoluble Fe(II), consistent 321
with the outer membrane acting as a barrier to Fe(II) entry into the periplasm 322
(Fig. 1a, Supp. Fig. S1, Supp. Fig. S5a). When A. ebreus cells are lysed, 323
releasing periplasmic components, NDFO commences earlier (Fig. 3a). Fe(II)-324
NTA, which will not adsorb to cell surfaces because the Fe(II) is bound by a 325
chelator, is oxidized rapidly in the first 6 hours of the incubation (Fig. 3b). A more 326
extensive treatment of the effect of metal ligands on accelerating the rate of 327
microbial Fe(II) oxidation was recently published (53), and the authors 328
demonstrate that NO2- will react more rapidly with Fe(II)-NTA than un-chelated 329
Fe(II), suggesting another possible explanation for the rapid rate of Fe(II)-NTA 330
oxidation by A. ebreus (Fig. 3b) (53). However, Fe(II)-NTA-agarose beads, 331
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which cannot penetrate the outer-membrane, showed no oxidation by A. ebreus 332
cell suspensions (Fig. 3b). This result suggests that Fe(II)-NTA must enter the 333
periplasm to be oxidized, and argues against the involvement of inducible outer-334
membrane redox active proteins or a secreted oxidant in NDFO of Fe(II)-NTA by 335
A. ebreus. Previous observations from a scanning transmission x-ray 336
microscopy (STXM) study found that the rate of Fe(II) oxidation by Acidovorax 337
BoFeN1 is faster at the cells, and specifically in the periplasm, compared to at 338
the extracellular Fe(II) (26). This is consistent with periplasmic access of Fe(II) 339
being required for robust NDFO by members of the Acidovorax genus. 340
We sought to understand how NDFO occurs in the periplasm. 341
Cytochrome complexes of intact cells are reduced by Fe(II)-NTA (Supp. Fig. S4), 342
as has previously been observed with other organisms (5, 18) and acetate is not 343
required for NDFO in cell suspensions (Fig 1a). Thus, we hypothesized that 344
respiratory complexes could directly mediate NDFO. To test this, we deprived 345
cells of molybdenum, the metal cofactor of the nitrate reductase, Nar. A. ebreus 346
cells grown anaerobically in the absence of molybdenum under nitrite-reducing 347
conditions, but with NO3- available to induce Nar expression, were unable to 348
either grow by organotrophic NO3- reduction (data not shown) or oxidize Fe(II) 349
coupled to NO3- reduction in cell suspensions (Fig. 3c). These results suggest 350
that either Nar directly catalyzes NO3- reduction coupled to Fe(II) oxidation (54) 351
and/or NO2- is produced to further react with Fe(II) through the abiotic and biotic 352
mechanisms previously discussed. 353
354
Both nitrite and nitric oxide can oxidize extracellular solid phase Fe(II) 355
Previously, it was observed that synthetic solid-phase iron phosphate 356
(vivianite) in the absence of soluble Fe(II) is not oxidized by growth cultures of 357
Acidovorax species (13, 26). In contrast, we found that small amounts of 358
synthetic vivianite (~0.5 mM Fe(II) over the course of 24 hours) can be oxidized 359
by cell suspensions of A. ebreus, but only in the presence of an organic co-360
substrate (Fig. 4a). As the cell suspensions were treated with chloramphenicol, it 361
is unlikely that the cells produced an organic chelator as a result of acetate 362
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amendment. An alternative option is that intermediates of nitrate respiration 363
escape the periplasm and solubilize and/or oxidize solid-phase iron. Candidates 364
for a secreted oxidant include NO2- or, when soluble Fe(II) is also present in the 365
system, Fe(III) (13, 26). Some NDFO organisms, such as Pseudogulbenkiania 366
ferroxidans (11) or Pseudogulbenkiania sp. MAI-1 (53), release higher 367
concentrations of extracellular NO2- during organotrophic nitrate reduction, which 368
can react with extracellular Fe(II) especially in the presence of catalytic mineral 369
phases, such as green rusts (21, 48, 49), or chelating ligands (53). However, NO 370
should also be considered. NO is produced during organotrophic nitrate 371
reduction by A. ebreus (Supp. Fig. S2e), and other nitrate reducers (55, 56). NO 372
oxidizes Fe(II) and is a good Fe(II) ligand that will displace a number of anionic 373
ligands (57). NO oxidizes synthetic vivianite Fe(II) faster than NO2- (Fig. 4c). 374
After 15 minutes, ~0.5 mM synthetic vivianite Fe(II) is oxidized by 0.5 mM DEA 375
NONOate, but no noticeable synthetic vivianite Fe(II) oxidation occurred in the 376
presence of 1 mM NO2- (Fig. 4c). Even low steady-state concentrations of NO 377
may be enough to account for the small amount of synthetic vivianite oxidized 378
when acetate is added to dense cell suspensions (Fig. 4a). NO can also 379
solubilize Fe(II) from synthetic vivianite (Fig. 4b) and increase the rate of Fe(II) 380
oxidation via the periplasmic reactions previously discussed. We see 381
accumulation of solution NO (Supp. Fig. S6a) in later phases of cultures along 382
with a rebound in the concentration of soluble Fe(II) (Supp. Fig. S6b). The 383
observation that NO, and to a lesser extent NO2- can oxidize solid phase 384
extracellular Fe(II) (Fig. 4c) is ostensibly at odds with the observation that Fe(II)-385
NTA agarose was not oxidized by A. ebreus (Fig. 3b). However, NO binds to 386
Fe(II)-NTA with nanomolar affinity to form stable Fe(II)-NTA-NO complexes (58), 387
but does not oxidize the chelated Fe(II) (47). Although abiotic oxidation of Fe(II)-388
NTA by NO2- is faster than the un-chelated form at neutral pH (53), negligible 389
amounts of NO2- are produced by A. ebreus during organotrophic denitrification 390
(Supp. Fig. S2d). 391
It has been proposed that a local low pH around iron oxidizing cells could 392
keep Fe(III) soluble and allow it to diffuse out of cells to react with extracellular 393
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vivianite (26). However, we have direct evidence for NDFO-associated efflux 394
pumps from proteomics (Fig. 2, Dataset S1), suggesting a possible active 395
mechanism for secreting Fe(III) from cells to react with extracellular Fe(II) 396
minerals through an electron exchange mechanism. RND family efflux pumps 397
have relaxed specificity and can transport a wide range of metal ions (40). This 398
electron exchange mechanism may involve reactions between extracellular 399
colloidal iron and extracellular minerals (13, 26). Previous reports that found 400
NDFO of solid phase Fe(II) in batch cultures could be explained by a combination 401
of the solubilization of Fe(II) by NO, oxidation by NO, NO2- and Fe(III), and lysis 402
and cryptic growth in long incubations (4, 5, 59). 403
404
Acetate is utilized before Fe(II) as an electron donor for nitrate-reduction. 405
Excess acetate inhibits NDFO and NO2-, NO and N2O accumulation 406
Previously, it was noted that acetate is consumed before Fe(II) in NDFO 407
growth cultures (5, 60) and this is also true for A. ebreus (Supp. Fig. S2b). For 408
other Acidovorax species in growth culture under donor-limited conditions, 409
increasing acetate concentrations increased the extent of iron oxidation (12), 410
likely due, at least in part, to increasing biomass. However, we observed that 411
under acceptor-limited conditions in growth cultures, excess acetate decreased 412
the rate and extent of Fe(II) oxidation and NO2-, NO and N2O accumulation by A. 413
ebreus (Supp. Fig. S2, S7a). To understand the effect of acetate on iron 414
oxidation without growth or cell number as confounding variables, we performed 415
cell suspensions with chloramphenicol. Remarkably, the presence of acetate 416
also controls Fe(II) oxidation and NO2- and NO accumulation in cell suspensions 417
(Fig. 5, Supp. Fig. S5). In donor/acceptor balanced conditions (10 mM Fe(II), 5 418
mM acetate, 10 mM nitrate), the most rapid phase of Fe(II) oxidation occurred 419
once acetate was depleted to below 0.5 mM (between 24-36 hrs) (Fig. 5a). A 420
similar rapid phase of Fe(II) oxidation at acetate concentrations below 0.5 mM is 421
observed in NDFO growth cultures (Supp. Fig. S2b). However, in acceptor-422
limited conditions (10 mM Fe(II), 10 mM acetate, 10 mM nitrate), this effect was 423
greatly diminished because acetate was consumed prior to over Fe(II) (Fig. 5b, 424
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Supp. Fig. S2c, Supp. Fig. S5b). Consistent with the observations made with A. 425
ebreus, acetate is consumed before Fe(II) in NDFO growth cultures of Azospira 426
suillum strain PS (5) (Supp. Fig. S7b) and excess acetate inhibits Fe(II) oxidation 427
under acceptor-limited conditions (Supp. Fig. S7c). 428
A recent paper from Klueglein and Kappler (29) makes the claim that 429
enzymatic NDFO is unlikely to occur in Acidovorax sp. BoFeN1 in part based on 430
the fact that the organism does not oxidize an initial concentration of 7 mM Fe(II) 431
in a growth culture with 5 mM acetate (40 mM electron equivalents), 100 μM 432
NO3- (0.5 mM electron equivalents) and ~20mM N2O (40 mM electron 433
equivalents) (5% added to headspace). If acetate is preferentially consumed by 434
Acidovorax sp. BoFeN1, very few electron equivalents of N2O will remain for 435
Fe(II) oxidation to proceed coupled to N2O reduction. The authors mention, but 436
do not show, the data from a similar growth experiment with 0.5 mM acetate 437
instead of 5 mM acetate. Of course, in this instance, very little growth is 438
expected and the concentration of cells available to catalyze NDFO would be 439
very low. While our results suggest that NDFO, particularly of chelated Fe(II) 440
(Figure 2b), can be catalyzed in the periplasm of nitrate-reducing bacterial cells, 441
more work is needed to define precisely the catalytic sites. 442
443
A model for NDFO as an innate capability of nitrate-reducing bacteria 444
In NDFO growth cultures, when acetate is present, periplasmic respiratory 445
complexes remain reduced and NO/NO2- concentrations remain low. Also, metal 446
efflux pumps may function to keep Fe(II) out of the periplasm (Fig. 2) (61). When 447
acetate is depleted and respiratory complexes are oxidized, Fe(II) efflux is 448
slowed and periplasmic NDFO reactions can proceed. Fe(II) oxidation coupled 449
to reduction of intermediates of nitrate reduction may be directly catalyzed by 450
Nar, Nir and Nor (54), or other periplasmic components or through abiotic 451
reactions that can be accelerated by ligands (53). If the rate of Fe(II) oxidation 452
and Fe(III) mineral precipitation is faster than the rate of efflux, precipitation of 453
Fe(II) minerals on periplasmic proteins will lead to their inhibition and NO2-, NO, 454
and N2O accumulation (Supp. Fig. S2d-f) (13, 14, 62). In our data, NO2-, NO and 455
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N2O did not accumulate to their maximum steady-state level and acetate was not 456
completely consumed in NDFO cultures or cell suspensions until after the more 457
rapid phase of Fe(II) oxidation (Supp. Fig. S2b, S2d-f, Fig. 5, Supp. Fig. S5). 458
This result suggests that a catalyst of Fe(II) oxidation is depleted alongside a 459
catalyst of NO2-, NO and N2O reduction. As no inducible Fe(II) oxidoreductase 460
appears to be present in A. ebreus (Dataset S1), the simplest explanation of our 461
results is that an important site of Fe(II) oxidation is the same as the site of NO2-, 462
NO and N2O reduction (i.e. the redox-active proteins Nar, Nir, Nor and Nos and 463
other redox-active components of the periplasm). A model for NDFO consistent 464
with our results and results in the literature is presented in Figure 6. 465
We postulate that the periplasmic components of all nitrate-reducing 466
bacteria are innately capable of catalyzing a baseline level of NDFO. The rate 467
and extent of NDFO will depend on differences in the respiratory chain 468
components, the Fe(II) form, the accumulation of respiratory intermediates (NO2-, 469
NO), the concentration of the organic co-substrate and the capacity of the 470
bacteria to overcome Fe(II) and nitrogen oxide toxicity through efflux pumps and 471
detoxification mechanisms. While an inducible Fe(II) oxidoreductase may exist, 472
our results suggest that it is not necessary for a nitrate-reducing organism to be 473
proficient at catalyzing NDFO. 474
Some previous work suggests a growth benefit from NDFO. In batch 475
cultures the effect is subtle (12, 14), but continuous flow may enhance the growth 476
benefit (14). We did not observe a significant growth benefit due to NDFO in our 477
batch cultures of A. ebreus (Supp. Fig. S8), but it may be that any energetic gain 478
from Fe(II) oxidation is largely masked by the energetic cost of coping with Fe(II) 479
toxicity. We have recently proposed several possible mechanisms whereby an 480
organism could gain energetic benefit from NDFO (3). Future work should focus 481
on demonstrating which, if any, of these dominates in different bacteria. 482
Preliminary proteomics data suggest that efflux pumps are induced during 483
NDFO in both A. suillum and P. ferroxidans, but genetic knockouts and other 484
experiments are needed to precisely define their role. Also, more studies are 485
needed on the reactivity of NO and NO2- with various geochemically important 486
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Fe(II) forms. An awareness of the accumulation of NO is important to a thorough 487
understanding of the influence of Fe(II) on biological systems. For example, 488
recent work has emphasized the importance of NO in regulating bacterial motility 489
and biofilm formation by environmentally important species (25, 63), including the 490
environmentally important Fe(III) reducer, Shewanella oneidensis (64), but 491
almost nothing is known about the biogeochemical controls on NO production in 492
the environment. Finally, both NO and N2O are well-known atmospheric 493
pollutants, and the emissions of NO and N2O from diverse environments have 494
been compared (65, 66). However, to our knowledge, no study has assessed 495
the importance of organic electron donors in controlling NO and N2O emissions in 496
Fe(II) rich systems. Our results suggest that NDFO may be a major contributor 497
to the flux of nitrogen oxide intermediates in Fe(II)-rich systems limited for 498
organic electron donor. 499
Acknowledgements 500
The authors would like to thank members of the Coates lab for stimulating 501
discussions and critical comments on the experiments and manuscript, Mary 502
Firestone for advice on experiments, Michael Marletta, Lars Plate and other 503
members of the Marletta lab for assistance with measuring nitric oxide and 504
stimulating discussions, the Energy Biosciences Institute for funding, and the 505
National Institutes of Health for LC-MS instrumentation (grant 1S10RR022393-506
01). We would also like to thank anonymous reviewers for helpful insights and 507
comments. 508
509
510
511
Figure Legends: 512 Fig. 1. NDFO of mixed soluble and insoluble Fe(II) is not inducible, but 513
Fe(II) is toxic to nitrate-reducing A. ebreus. a. Concentration of Fe(II) 514
(soluble and vivianite) in chloramphenicol treated washed cell suspensions of 515
Acidovorax ebreus. Cells were pre-grown organotrophically (10 mM acetate, 10 516
mM nitrate) in the absence of Fe(II) or under NDFO conditions in the presence of 517
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Fe(II)-NTA (10 mM Fe(II)-NTA, 5 mM acetate, 10 mM nitrate) b. Growth 518
expressed as OD 600 in organotrophic cultures (5 mM acetate, 10 mM nitrate) of 519
Acidovorax ebreus containing 0 mM, 0.01 mM or 0.1 mM Fe(II). Points represent 520
averages and error bars represent the standard deviations of triplicates. 521
Fig. 2. Heavy metal efflux pumps are more abundant in NDFO cultures. 522
Normalized peptide counts for Dtpsy_1460, Dtpsy_1461 and Dtpsy_1462 in 523
NDFO and organotrophic cultures at 8, 18, 24, 36, and 48 hours. Top row: Box 524
plots showing the lower extreme, lower quartile, median, upper quartile and 525
upper extreme of normalized peptide counts. Data from the time courses were 526
pooled from trypsin-shaving proteomics experiments. Data is overlayed on the 527
box plots. Bottom row: Normalized peptide count data at 8, 18, 24, 36, and 48 528
hours in NDFO and organotrophic cultures. 529
Fig. 3. Periplasmic reactions are essential to initiate extensive NDFO. a. 530
Concentration of Fe(II) in NDFO cell suspensions of lysed or intact Acidovorax 531
ebreus in the presence of chloramphenicol in media containing 10 mM Fe(II), 10 532
mM nitrate. b. Concentration of Fe(II) as Fe(II)-NTA or Fe(II)-NTA agarose 533
beads in NDFO cell suspensions (10 mM Fe(II), 5 mM acetate, 10 mM nitrate) in 534
the absence of chloramphenicol. c. Concentration of Fe(II) in NDFO cell 535
suspensions of molybenum-depleted Acidovorax ebreus in the presence of 536
chloramphenicol in either molybdenum-free or molybdenum-replete media (10 537
mM Fe(II), 10 mM nitrate). Cells of A. ebreus were pre-grown in molybdenum-538
free media (5 mM acetate, 10 mM nitrite, 10 mM nitrate) and were incapable of 539
growth by organotrophic nitrate reduction (not shown). Points represent 540
averages and error bars represent the standard deviations of triplicates. 541
Fig. 4. Extracellular reactions between NO2- and NO and mineral-phase 542
Fe(II) can contribute to NDFO. a. Concentration of Fe(II) with synthetic 543
vivianite as the sole Fe(II) source in NDFO cell suspensions of Acidovorax 544
ebreus (4 mM Fe(II), 10 mM nitrate and 0 mM acetate or 5 mM acetate) in the 545
presence of chloramphenicol. b. Concentration of soluble Fe(II) after 15 minutes 546
in cell-free basal media containing 4 mM Fe(II) in the form of synthetic vivianite 547
and either 1 mM sodium nitrite (NO2-) or 0.5 mM diethylamine NONOate (NO) or 548
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both compounds. Controls contained diethylamine and sodium hydroxide and 549
showed no Fe(II) oxidation over the time of the experiment. c. Concentration of 550
total Fe(II) after 15 minutes and 24 hours in cell-free basal media containing 4 551
mM Fe(II) in the form of synthetic vivianite and either 1 mM sodium nitrite or 0.5 552
mM diethylamine NONOate (NO releasing compound) or both compounds. 553
Controls contained diethylamine and sodium hydroxide and showed no Fe(II) 554
oxidation over the time of the experiment. Points represent averages and error 555
bars represent the standard deviations of triplicates. 556
Fig. 5. Acetate is consumed prior to Fe(II) as an electron donor in nitrate-557
reducing cultures. a. Concentrations of Fe(II), acetate, nitrate, and nitrite in 558
NDFO cell suspensions of Acidovorax ebreus (10 mM Fe(II), 5 mM acetate, 10 559
mM nitrate) in the presence of chloramphenicol. Points represent averages and 560
error bars represent the standard deviations of triplicates. b. Fe(II) 561
concentrations in NDFO cell suspensions of Acidovorax ebreus with 10 mM 562
Fe(II), 10 mM nitrate and 0 mM acetate, 5 mM acetate or 10 mM acetate. Points 563
represent averages and error bars represent the standard deviations of 564
triplicates. 565
Fig. 6. A mechanistic model for nitrate-dependent Fe(II) oxidation by A. 566
ebreus. Across a representative NDFO growth curve dominant electron flow 567
pathways are proposed for before (a), during (b) and after (c) the most rapid 568
phase of Fe(II) oxidation. Dominant reactions in each panel are indicated by bold 569
arrows, minor/inhibited reactions are indicated by dashed arrows. a. In the 570
presence of acetate, respiratory complexes remain reduced by electrons from the 571
quinone pool and enzymatic NO3-, NO2
-, NO and N2O reduction proceeds. 572
Periplasmic Fe(II) concentrations are low due to metal efflux pumping , and few 573
reactions between Fe(II) and reduced respiratory complexes occur. Some 574
extracellular reactions between Fe(II) and NO2-/NO may proceed depending on 575
how much NO2- and NO accumulate outside of the cell. b. Once acetate has 576
dropped to a low level, Fe(II) will accumulate in the periplasm and be oxidized 577
coupled to nitrogen oxide reduction at respiratory complexes and other 578
periplasmic components in the most rapid phase of Fe(II) oxidation. c. As Fe(II) 579
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is oxidized at respiratory complexes, mineral precipitates will begin to form in 580
association with these proteins, leading to their inhibition and the accumulation of 581
NO2-, NO and N2O. Extracellular NO2
- and NO can oxidize solid phase Fe(II), but 582
NO can also solubilize Fe(II) which could diffuse back into cells to be oxidized. 583
Ultimately, periplasmic Fe(III) precipitation damages respiratory complexes and 584
other periplasmic sites, leading to a slower rate of NDFO, primarily driven by 585
abiotic reactions. 586
587 588 589 590
References Cited 591 592
1. Weber, K. A., L. A. Achenbach, and J. D. Coates. 2006. 593 Microorganisms pumping iron: anaerobic microbial iron oxidation and 594 reduction. Nat. Rev. Microbiol. 4:752-64. 595
2. Canfield, D. E., M. T. Rosing, and C. Bjerrum. 2006. Early anaerobic 596 metabolisms. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361:1819-34; 597 discussion 1835-6. 598
3. Carlson, H. K., I. C. Clark, R. A. Melnyk, and J. D. Coates. 2012. 599 Toward a mechanistic understanding of anaerobic nitrate-dependent iron 600 oxidation: balancing electron uptake and detoxification. Front. Microbiol. 601 3:57. 602
4. Weber, K. A., F. W. Picardal, and E. E. Roden. 2001. Microbially 603 catalyzed nitrate-dependent oxidation of biogenic solid-phase Fe(II) 604 compounds. Environ. Sci. Technol. 35:1644-1650. 605
5. Chaudhuri, S. K., J. G. Lack, and J. D. Coates. 2001. Biogenic 606 magnetite formation through anaerobic biooxidation of Fe(II). Appl. 607 Environ. Microbiol. 67:2844-2848. 608
6. Widdel, F., S. Schnell, S. Heising, A. Ehrenreich, B. Assmus, and B. 609 Schink. 1993. Ferrous iron oxidation by anoxygenic phototrophic bacteria. 610 Nature 362:834-836. 611
7. Machulla, G., J. Thieme, and J. Niemeyer. 1998. Interaction of 612 microorganisms with soil colloids observed by X-ray microscopy, p. II-21 – 613 II-28. In J. Thieme, G. Schmahl, D. Rudolph, and E. Umbach (ed.), X-Ray 614 Microscopy and Spectroscopy. Springer-Verlag, Heidelberg. 615
8. Straub, K. L., and B. E. E. Buchholz-Cleven. 1998. Enumeration and 616 detection of anaerobic ferrous iron-oxidizing, nitrate-reducing bacteria 617 from diverse European sediments. Appl. Environ. Microbiol. 64:4846-618 4856. 619
9. Straub, K. L., M. Benz, B. Schink, and F. Widdel. 1996. Anaerobic, 620 nitrate-dependent microbial oxidation of ferrous iron. Appl. Environ. 621 Microbiol. 62:1458-1460. 622
on May 2, 2020 by guest
http://jb.asm.org/
Dow
nloaded from
21
21
10. Hafenbradl, D., M. Keller, R. Dirmeier, R. Rachel, P. Robnagel, S. 623 Burggraf, H. Huber, and K. O. Stetter. 1996. Ferroglobus placidus gen. 624 nov., sp. nov. a novel hyperthermophilic archaeum that oxidizes Fe2+ at 625 neutral pH under anoxic conditions. Arch. Microbiol. 166:308-314. 626
11. Byrne-Bailey, K. G., K. A. Weber, A. H. Chair, S. Bose, T. Knox, T. L. 627 Spanbauer, O. Chertkov, and J. D. Coates. 2010. Completed genome 628 sequence of the anaerobic iron-oxidizing bacterium Acidovorax ebreus 629 strain TPSY. J. Bacteriol. 192:1475-6. 630
12. Muehe, E. M., S. Gerhardt, B. Schink, and A. Kappler. 2009. 631 Ecophysiology and the energetic benefit of mixotrophic Fe(II) oxidation by 632 various strains of nitrate-reducing bacteria. FEMS Microbiol. Ecol. 70:335-633 343. 634
13. Kappler, A., B. Schink, and D. K. Newman. 2005. Fe(III)-mineral 635 formation and cell encrustation by the nitrate-dependent Fe(II)-oxidizer 636 strain BoFeN1. Geobiology 3:235-245. 637
14. Chakraborty, A., E. E. Roden, J. Schieber, and F. Picardal. 2011. 638 Enhanced growth of Acidovorax sp. strain 2AN during nitrate-dependent 639 Fe(II) oxidation in batch and continuous-flow systems. Appl. Environ. 640 Microbiol. 77:8548-8556. 641
15. Brons, H. J., W. R. Hagen, and A. J. Zehnder. 1991. Ferrous iron 642 dependent nitric oxide production in nitrate reducing cultures of 643 Escherichia coli. Arch. Microbiol. 155:341-7. 644
16. Koppenol, W. H. 1996. Thermodynamics of reactions involving nitrogen-645 oxygen compounds. Methods Enzymol. 268:7-12. 646
17. Dutton, A. S., J. M. Fukuto, and K. N. Houk. 2005. Theoretical reduction 647 potentials for nitrogen oxides from CBS-QB3 energetics and (C)PCM 648 solvation calculations. Inorg. Chem. 44:4024-8. 649
18. Weber, K. A., D. B. Hedrick, A. D. Peacock, J. C. Thrash, D. C. White, 650 L. A. Achenbach, and J. D. Coates. 2009. Physiological and taxonomic 651 description of the novel autotrophic, metal oxidizing bacterium, 652 Pseudogulbenkiania sp. strain 2002. Appl. Microbiol. Biotechnol. 83:555-653 65. 654
19. Poulain, A. J., and D. K. Newman. 2009. Rhodobacter capsulatus 655 Catalyzes Light-Dependent Fe(II) Oxidation under Anaerobic Conditions 656 as a Potential Detoxification Mechanism. Appl. Environ. Microbiol, 657 75:6639-6646. 658
20. Dunning, J. C., Y. Ma, and R. E. Marquis. 1998. Anaerobic killing of oral 659 streptococci by reduced, transition metal cations. Appl. Environ. Microbiol. 660 64:27-33. 661
21. Picardal, F. 2012. Abiotic and Microbial Interactions during Anaerobic 662 Transformations of Fe(II) and NOx. Front. Microbiol. 3:112. 663
22. Gurd, F. R., and P. E. Wilcox. 1956. Complex formation between metallic 664 cations and proteins, peptides and amino acids. Adv. Protein Chem. 665 11:311-427. 666
23. McCleverty, J. A. 2004. Chemistry of nitric oxide relevant to biology. 667 Chem. Rev. 104:403-18. 668
on May 2, 2020 by guest
http://jb.asm.org/
Dow
nloaded from
22
22
24. Harrop, T. C., Z. J. Tonzetich, E. Reisner, and S. J. Lippard. 2008. 669 Reactions of synthetic [2Fe-2S] and [4Fe-4S] clusters with nitric oxide and 670 nitrosothiols. J. Am. Chem. Soc. 130:15602-10. 671
25. Spiro, S. 2007. Regulators of bacterial responses to nitric oxide. FEMS 672 Microbiol. Rev. 31:193-211. 673
26. Miot, J., K. Benzerara, G. Morin, A. Kappler, S. Bernard, M. Obst, C. 674 Ferard, F. Skouri-Panet, J. M. Guigner, N. Posth, M. Galvez, G. E. 675 Brown, and F. Guyot. 2009. Iron biomineralization by anaerobic 676 neutrophilic iron-oxidizing bacteria. Geochim. Cosmochim. Acta 73:696-677 711. 678
27. Bruce, R. A., L. A. Achenbach, and J. D. Coates. 1999. Reduction of 679 (per)chlorate by a novel organism isolated from paper mill waste. Environ. 680 Microbiol. 1:319-29. 681
28. Keefer, L. K., R. W. Nims, K. M. Davies, and D. A. Wink. 1996. 682 "NONOates" (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide 683 donors: convenient nitric oxide dosage forms. Methods Enzymol. 268:281-684 93. 685
29. Klueglein, N., and A. Kappler. 2012. Abiotic oxidation of Fe(II) by 686 reactive nitrogen species in cultures of the nitrate-reducing Fe(II) oxidizer 687 Acidovorax sp. BoFeN1 - questioning the existence of enzymatic Fe(II) 688 oxidation. Geobiology 11(2):180-190. 689
30. Stookey, L. L. 1970. Ferrozine - a New Spectrophotometric Reagent for 690 Iron. Anal. Chem. 42:779-&. 691
31. Nagababu, E., S. Ramasamy, D. R. Abernethy, and J. M. Rifkind. 692 2003. Active nitric oxide produced in the red cell under hypoxic conditions 693 by deoxyhemoglobin-mediated nitrite reduction. J. Biol. Chem. 278:46349-694 56. 695
32. Zhu, W., J. W. Smith, and C. M. Huang. 2010. Mass spectrometry-based 696 label-free quantitative proteomics. J. Biomed. Biotechnol. 2010:840518. 697
33. Zhang, B., N. C. VerBerkmoes, M. A. Langston, E. Uberbacher, R. L. 698 Hettich, and N. F. Samatova. 2006. Detecting differential and correlated 699 protein expression in label-free shotgun proteomics. J. Proteome Res. 700 5:2909-18. 701
34. Jiao, Y., and D. K. Newman. 2007. The pio operon is essential for 702 phototrophic Fe(II) oxidation in Rhodopseudomonas palustris TIE-1. J. 703 Bacteriol. 189:1765-1773. 704
35. Bird, L. J., V. Bonnefoy, and D. K. Newman. 2011. Bioenergetic 705 challenges of microbial iron metabolisms. Trends Microbiol. 19:330-40. 706
36. Singer, E., D. Emerson, E. A. Webb, R. A. Barco, J. G. Kuenen, W. C. 707 Nelson, C. S. Chan, L. R. Comolli, S. Ferriera, J. Johnson, J. F. 708 Heidelberg, and K. J. Edwards. 2011. Mariprofundus ferrooxydans PV-1 709 the first genome of a marine Fe(II) oxidizing Zetaproteobacterium. PLoS 710 One 6:e25386. 711
37. Tebo, B. M., H. A. Johnson, J. K. McCarthy, and A. S. Templeton. 712 2005. Geomicrobiology of manganese(II) oxidation. Trends Microbiol. 713 13:421-428. 714
on May 2, 2020 by guest
http://jb.asm.org/
Dow
nloaded from
23
23
38. Carlson, H. K., A. T. Iavarone, A. Gorur, B. S. Yeo, R. Tran, R. A. 715 Melnyk, R. A. Mathies, M. Auer, and J. D. Coates. 2012. Surface 716 multiheme c-type cytochromes from Thermincola potens and implications 717 for respiratory metal reduction by Gram-positive bacteria. Proc. Natl. 718 Acad. Sci. U S A 109:1702-7. 719
39. Tjalsma, H., L. Lambooy, P. W. Hermans, and D. W. Swinkels. 2008. 720 Shedding & shaving: disclosure of proteomic expressions on a bacterial 721 face. Proteomics 8:1415-28. 722
40. Nies, D. H. 2003. Efflux-mediated heavy metal resistance in prokaryotes. 723 FEMS Microbiol. Rev. 27:313-39. 724
41. Flores-Santana, W., D. J. Salmon, S. Donzelli, C. H. Switzer, D. 725 Basudhar, L. Ridnour, R. Cheng, S. A. Glynn, N. Paolocci, J. M. 726 Fukuto, K. M. Miranda, and D. A. Wink. 2011. The specificity of nitroxyl 727 chemistry is unique among nitrogen oxides in biological systems. Antioxid. 728 Redox. Signal. 14:1659-74. 729
42. Nikaido, H., and Y. Takatsuka. 2009. Mechanisms of RND multidrug 730 efflux pumps. Biochimi. Biophys. Acta-Proteins and Proteomics 1794:769-731 781. 732
43. Richardson, A. R., E. C. Payne, N. Younger, J. E. Karlinsey, V. C. 733 Thomas, L. A. Becker, W. W. Navarre, M. E. Castor, S. J. Libby, and F. 734 C. Fang. 2012. Multiple targets of nitric oxide in the tricarboxylic acid cycle 735 of Salmonella enterica serovar typhimurium. Cell Host Microbe 10:33-43. 736
44. Isabella, V. M., and V. L. Clark. 2011. Identification of a conserved 737 protein involved in anaerobic unsaturated fatty acid synthesis in Neiserria 738 gonorrhoeae: implications for facultative and obligate anaerobes that lack 739 FabA. Mol. Microbiol. 82:489-501. 740
45. Jones-Carson, J., J. R. Laughlin, A. L. Stewart, M. I. Voskuil, and A. 741 Vazquez-Torres. 2012. Nitric oxide-dependent killing of aerobic, 742 anaerobic and persistent Burkholderia pseudomallei. Nitric Oxide 27:25-743 31. 744
46. Rock, J. D., M. J. Thomson, R. C. Read, and J. W. B. Moir. 2007. 745 Regulation of denitrification genes in Neisseria meningitidis by nitric oxide 746 and the repressor NsrR. J. Bacteriol. 189:1138-1144. 747
47. Schneppensieper, T., A. Wanat, G. Stochel, and R. van Eldik. 2002. 748 Mechanistic information on the reversible binding of NO to selected iron(II) 749 chelates from activation parameters. Inorg. Chem. 41:2565-73. 750
48. Hansen, H. C. B., S. Guldberg, M. Erbs, and C. B. Koch. 2001. Kinetics 751 of nitrate reduction by green rusts - effects of interlayer anion and Fe(II): 752 Fe(III) ratio. Appl. Clay Sci. 18:81-91. 753
49. Pantke, C., M. Obst, K. Benzerara, G. Morin, G. Ona-Nguema, U. 754 Dippon, and A. Kappler. 2012. Green rust formation during Fe(II) 755 oxidation by the nitrate-reducing Acidovorax sp. strain BoFeN1. Environ. 756 Sci. Technol. 46:1439-46. 757
50. Wong, P. S., J. Hyun, J. M. Fukuto, F. N. Shirota, E. G. DeMaster, D. 758 W. Shoeman, and H. T. Nagasawa. 1998. Reaction between S-759
on May 2, 2020 by guest
http://jb.asm.org/
Dow
nloaded from
24
24
nitrosothiols and thiols: generation of nitroxyl (HNO) and subsequent 760 chemistry. Biochemistry 37:5362-71. 761
51. Coby, A. J., and F. W. Picardal. 2005. Inhibition of NO3- and NO2- 762 reduction by microbial Fe(III) reduction: Evidence of a reaction between 763 NO2- and cell surface-bound Fe2+. Appl. Environ. Microbiol. 71:5267-764 5274. 765
52. French, S., D. Puddephatt, M. Habash, and S. Glasauer. 2012. The 766 dynamic nature of bacterial surfaces: Implications for metal-membrane 767 interaction. Crit. Rev. Microbiol 39(2):196-217. 768
53. Kopf, S. H., C. Henny, and D. K. Newman. 2013. Ligand-Enhanced 769 Abiotic Iron Oxidation and the Effects of Chemical versus Biological Iron 770 Cycling in Anoxic Environments. Environ. Sci. Technol. 47:2602-11. 771
54. Lack, J. G., S. K. Chaudhuri, R. Chakraborty, L. A. Achenbach, and J. 772 D. Coates. 2002. Anaerobic biooxidation of Fe(II) by Dechlorosoma 773 suillum. Microbiol. Ecol. 43:424-31. 774
55. Ye, R. W., B. A. Averill, and J. M. Tiedje. 1994. Denitrification: 775 production and consumption of nitric oxide. Appl. Environ. Microbiol. 776 60:1053-8. 777
56. Price, M. S., L. Y. Chao, and M. A. Marletta. 2007. Shewanella 778 oneidensis MR-1 H-NOX regulation of a histidine kinase by nitric oxide. 779 Biochemistry 46:13677-83. 780
57. Mcdonald, C. C., W. D. Phillips, and H. F. Mower. 1965. An Electron 781 Spin Resonance Study of Some Complexes of Iron Nitric Oxide and 782 Anionic Ligands. J. Am. Chem. Soc. 87:3319-&. 783
58. Kumaraswamy, R., K. Sjollema, G. Kuenen, M. van Loosdrecht, and 784 G. Muyzer. 2006. Nitrate-dependent [Fe(II)EDTA](2-) oxidation by 785 Paracoccus ferrooxidans sp nov., isolated from a denitrifying bioreactor. 786 Systematic and Applied Microbiology 29:276-286. 787
59. Shelobolina, E., H. Xu, H. Konishi, R. Kukkadapu, T. Wu, M. Blothe, 788 and E. Roden. 2012. Microbial Lithotrophic Oxidation of Structural Fe(II) 789 in Biotite. Appl. Environ. Microbiol. 78:5746-52. 790
60. Benz, M., A. Brune, and B. Schink. 1998. Anaerobic and aerobic 791 oxidation of ferrous iron at neutral pH by chemoheterotrophic nitrate-792 reducing bacteria. Arch. Microbiol. 169:159-65. 793
61. Long, F., C. C. Su, M. T. Zimmermann, S. E. Boyken, K. R. 794 Rajashankar, R. L. Jernigan, and E. W. Yu. 2010. Crystal structures of 795 the CusA efflux pump suggest methionine-mediated metal transport. 796 Nature 467:484-U140. 797
62. Miot, J., K. Maclellan, K. Benzerara, and N. Boisset. 2011. Preservation 798 of protein globules and peptidoglycan in the mineralized cell wall of nitrate-799 reducing, iron(II)-oxidizing bacteria: a cryo-electron microscopy study. 800 Geobiology 9:459-70. 801
63. Wang, Y., and E. G. Ruby. 2011. The roles of NO in microbial symbioses. 802 Cell Microbiol 13:518-26. 803
on May 2, 2020 by guest
http://jb.asm.org/
Dow
nloaded from
25
25
64. Plate, L., and M. A. Marletta. 2012. Nitric Oxide Modulates Bacterial 804 Biofilm Formation through a Multicomponent Cyclic-di-GMP Signaling 805 Network. Mol. Cell 46:449-60. 806
65. Venterea, R. T., and D. E. Rolston. 2000. Nitric and nitrous oxide 807 emissions following fertilizer application to agricultural soil: Biotic and 808 abiotic mechanisms and kinetics. J. Geophys. Res.-Atmospheres 809 105:15117-15129. 810
66. Beaulieu, J. J., J. L. Tank, S. K. Hamilton, W. M. Wollheim, R. O. Hall, 811 Jr., P. J. Mulholland, B. J. Peterson, L. R. Ashkenas, L. W. Cooper, C. 812 N. Dahm, W. K. Dodds, N. B. Grimm, S. L. Johnson, W. H. McDowell, 813 G. C. Poole, H. M. Valett, C. P. Arango, M. J. Bernot, A. J. Burgin, C. 814 L. Crenshaw, A. M. Helton, L. T. Johnson, J. M. O'Brien, J. D. Potter, 815 R. W. Sheibley, D. J. Sobota, and S. M. Thomas. 2011. Nitrous oxide 816 emission from denitrification in stream and river networks. Proc. Natl. 817 Acad. Sci. U S A 108:214-9. 818
819 820
on May 2, 2020 by guest
http://jb.asm.org/
Dow
nloaded from
