Monofluorophosphate Is a Selective Inhibitor of Respiratory Sulfate

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Monofluorophosphate is a selective inhibitor of respiratory sulfate-reducing microorganisms Hans K. Carlson, Magdalena K. Stoeva, Nicholas B. Justice, Andrew Sczesnak, Mark R. Mullan, Lorraine A. Mosqueda, Jennifer V. Kuehl, Adam M. Deutshbauer, Adam P. Arkin, and John D. Coates Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es505843z • Publication Date (Web): 19 Feb 2015 Downloaded from http://pubs.acs.org on February 25, 2015

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Environmental Science & Technology

1

Monofluorophosphate is a selective inhibitor of respiratory sulfate-reducing

2

microorganisms

3 4

Running title: Selective inhibition of sulfate respiration by MFP

5 6

Hans K. Carlson1, Magdalena K. Stoeva4, Nicholas B. Justice2, Andrew Sczesnak5, Mark

7

R. Mullan1, Lorraine A. Mosqueda1, Jennifer V. Kuehl2, Adam M. Deutschbauer2, Adam

8

P. Arkin2, 5, John D. Coates1,3,4,*

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1

Energy Biosciences Institute, UC Berkeley

11

2

Physical Biosciences Division, Lawrence Berkeley National Lab

12

3

Earth Sciences Division, Lawrence Berkeley National Lab

13

4

Department of Plant and Microbial Biology, UC Berkeley

14

5

Department of Bioengineering, UC Berkeley

15 16 17

*Corresponding author: John D. Coates. Mailing address: University of California,

18

Berkeley, Department of Plant and Microbial Biology, Berkeley, CA 94720. Phone 510-

19

643-8455. Fax: 510-642-4995. E-mail: [email protected].

20 21 22

Keywords: Monofluorophosphate, sulfate-reducing microorganisms, hydrogen sulfide,

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souring

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ACS Paragon Plus Environment

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Abstract

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Despite the environmental and economic cost of microbial sulfidogenesis in

29

industrial operations, few compounds are known as selective inhibitors of respiratory

30

sulfate reducing microorganisms (SRM), and no study has systematically and

31

quantitatively evaluated the selectivity and potency of SRM inhibitors. Using general,

32

high-throughput assays to quantitatively evaluate inhibitor potency and selectivity in a

33

model sulfate-reducing microbial ecosystem as well as inhibitor specificity for the sulfate

34

reduction pathway in a model SRM, we screened a panel of inorganic oxyanions. We

35

identified several SRM selective inhibitors including selenate, selenite, tellurate, tellurite,

36

nitrate, nitrite, perchlorate, chlorate, monofluorophosphate, vanadate, molydate and

37

tungstate. Monofluorophosphate (MFP) was not known previously as a selective SRM

38

inhibitor, but has promising characteristics including low toxicity to eukaryotic

39

organisms, high stability at circumneutral pH, utility as an abiotic corrosion inhibitor, and

40

low cost. MFP remains a potent inhibitor of SRM growing by fermentation, and MFP is

41

tolerated by nitrate and perchlorate reducing microorganisms. For SRM inhibition, MFP

42

is synergistic with nitrite and chlorite, and could enhance the efficacy of nitrate or

43

perchlorate treatments. Finally, MFP inhibition is multifaceted. Both inhibition of the

44

central sulfate reduction pathway and release of cytoplasmic fluoride ion are implicated

45

in the mechanism of MFP toxicity.

46 47


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48


Introduction

49

In diverse industrial ecosystems, hydrogen sulfide (H2S) production by sulfate

50

reducing microorganisms (SRM) is environmentally and economically costly 1. H2S is

51

toxic, explosive, and corrosive. It is a primary cause of pipeline leaks, and a major

52

inhalation hazard for workers in hydrocarbon recovery and municipal wastewater

53

operations

54

treatments to prevent sulfidogenesis could save lives and prevent loss of biodiversity in

55

fragile ecosystems.

1, 2

.

An understanding of the environmental controls on SRM and new

56

While some specific inhibitors of sulfidogenesis are used in industrial ecosystems

57

(e.g. oil reservoirs) 2-4, most treatment options are non-specific biocides 5. A non-specific

58

inhibitor of microbial growth may drive the emergence of resistant populations or, upon

59

cessation of treatment, regrowth of a microbial community dominated by the most

60

abundant microorganisms, which are likely SRM in sulfidogenic systems. The use of

61

inorganic oxyanions that act as inhibitors of sulfate respiration is a popular approach to

62

achieving specific inhibition of SRM 2, 6. In oil recovery systems, nitrate injection is the

63

most popular treatment 6. Nitrate inhibits SRM through a variety of mechanisms

64

and we have recently shown that nitrate is a direct, specific inhibitor of sulfidogenesis

65

and SRM growth in microbial communities

66

(per)chlorate, represent attractive alternatives to nitrate as selective inhibitors of sulfide

67

production

68

SRM

69

microorganisms (NRM) and perchlorate-reducing microorganisms (PRM), sulfide re-

70

oxidation by NRM7 and PRM13, and direct inhibition of SRM by these compounds11.

3, 11-13

through

11

2, 7-10

,

. Perchlorate and chlorate, collectively

. In microbial communities, both nitrate and (per)chlorate can inhibit biocompetitive

exclusion

and

outgrowth

of

nitrate-reducing

71

Molybdate is a widely used inhibitor of sulfate reduction in microbial ecology

72

studies, and occasionally is used to treat sulfidogenesis in oil reservoirs. In contrast to

73

the competitive inhibitors nitrate and (per)chlorate, molybdate is a substrate for ATP

74

sulfurylase/sulfate adenosyltransferase (Sat) enzymes

75

catalyzed reaction between sulfate and ATP is adenosine 5’-phosphosulfate (APS), but

76

the product of the enzymatic reaction between molybdate and ATP is adenosine 5’-

77

phosphomolybdate (APMo), which is unstable and rapidly decomposes. This drives a

78

futile cycle that consumes cytoplasmic ATP

14-18

14-18

.

The product of the Sat-

. This futile cycle also occurs with


3


17

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tungstate and chromate

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sulfate-reducing microorganisms by these compounds. In support of this, molybdate

81

does not compete with sulfate uptake by a representative Desulfovibrio culture

82

Desulfovibrio cell suspensions treated with molybdate for 60 minutes had intracellular

83

ATP concentrations ~10% that of untreated controls

84

cell suspensions of nitrate reducing bacteria, which lack Sat, had ~80% the intracellular

85

ATP levels of controls

86

molybdate specifically inhibit sulfate reduction, but not methanogenesis, in marine

87

sediments 21.

20

, and is thought to be the central mechanism of inhibition of

20

19

, and

. In contrast, molybdate treated

. Finally, it has been demonstrated that appropriate doses of

88

From studies with the eukaryotic ATP sulfurylases, inorganic oxyanions have

89

been generally classified as competitive inhibitors of sulfate binding and activation (e.g.

90

perchlorate, chlorate, nitrate, thiosulfate, and fluorosulfate) or ATP-consuming futile

91

substrates

92

monofluorophosphate) 14-16. Depending on the stability of the APS analogs, the rate and

93

extent of the ATP consuming futile cycle will vary. The molybdate, tungstate, arsenate

94

and chromate analogs are all very unstable, while adenosine 5’-phosphoselenate has a

95

half-life on the order of 15 minutes. In contrast, the product of the ATP sulfurylase

96

reaction between ATP and MFP, adenosine 5’-(2-fluorodiphosphate) (ADPβF), is more

97

stable than APS, and is apparently a better APS analog than ADP analog suggesting that

98

it may also interfere with downstream steps in the sulfate assimilation or reduction

99

pathways 22.

(e.g.

molybdate,

arsenate,

chromate,

tungstate,

selenate

and

100

Though informative about the mechanism of inhibition, studies with purified

101

proteins or pure cultures do not evaluate the selectivity of inhibitors. No study has

102

systematically evaluated the potency and selectivity of inorganic oxyanions as SRM

103

inhibitors.

104

developed a generic high-throughput screening strategy to systematically assess both the

105

specificity and potency of compounds for inhibition of sulfidogenesis in the context of a

106

marine microbial community. We evaluate a panel of sulfate analogs and demonstrate

107

for the first time that monofluorophosphate (FPO32-, MFP) is a potent and selective

108

inhibitor of respiratory sulfate reduction in environmental communities.

109

synergistic with nitrite and chlorite, and less inhibitory of nitrate and perchlorate reducing

Thus, potent and selective inhibitors may have been overlooked.


We

MFP is

4

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110

organisms than SRM suggesting that MFP could be used in combination with these

111

compounds. Finally, we obtain preliminary insights into the mechanism of inhibition of a

112

model SRM by MFP, and based on our results and observations in the literature we

113

discuss possible considerations for the use of MFP as an industrial inhibitor of

114

sulfidogenesis.

115 116

Experimental

117

Media and cultivation conditions

118

Desulfovibrio alaskensis G20 was cultivated in anoxic basal Tris-buffered

119

lactate/sulfate media, pH 7.4 at 30 °C. The media contained 8 mM MgCl2, 20 mM

120

NH4Cl, 0.6 mM CaCl2, 2 mM KH2PO4, 0.06 mM FeCl2, and 30 mM Tris-HCl. 60 mM

121

sodium lactate and 30 mM sodium sulfate were added. Trace elements and vitamins

122

were added from stocks according to a previously published recipe23, 24. Desulfovibrio

123

alaskensis G20 was recovered from 1 mL freezer stocks in 10 mL anoxic basal media in

124

Hungate tubes (Hungate tubes, Bellco, Vineland, NJ, USA) with 1 g/L yeast extract and 1

125

mM sodium sulfide and washed in basal media to remove residual yeast extract prior to

126

inoculation of microplates or tubes for experiments.

127

Marine enrichment cultures were passaged anoxic planktonic communities from

128

continuous flow reactor columns inoculated from marine sediments collected from San

129

Francisco Bay

130

Scientific, Waltham, MA, USA) marine mix (35g/L) to make seawater media and

131

enrichments were grown anoxically at 30 °C in Hungate tubes. Enrichments were stored

132

as -80 °C glycerol stocks, recovered in seawater media, and washed before inoculation of

133

cultures for experiments.

12

.

2g/L yeast extract was added to Instant Ocean (Thermo Fisher

134

For IC50 determinations, microplates were inoculated in an anaerobic chamber (Coy)

135

with cultures at an initial OD 600 of 0.02. Desulfovibrio and marine enrichment cultures

136

were cultivated in both 96 well microplates (Costar, Thermo Fisher Scientific, Waltham,

137

MA, USA) and 384 well microplates (Nunc, Thermo Fisher Scientific, Waltham, MA,

138

USA) with plate seals (Thermo Fisher Scientific, Waltham, MA, USA). The sealed

139

plates were kept in anoxic BD GasPak anaerobic boxes except when timepoints were

140

being recorded (BD, Franklin Lakes, NJ, USA) (Supplemental methods for more details).


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IC50s against growth were determined at 48 hours for sulfate reducing G20 or 36 hours

142

for sulfite reducing or pyruvate fermenting G20. IC50s against growth and sulfidogenesis

143

were determined at 48 hours.

144

All inorganic oxyanions were sodium salts (Sigma-Aldrich, St. Louis, MO, USA).

145

Data analysis for inhibition experiments was carried out in GraphPad Prism 6 (GraphPad

146

Software, Inc., La Jolla, CA, USA) and curves were fit to a standard inhibition log dose-

147

response curve to generate IC50 values. 95% confidence intervals are reported. All IC50s

148

are the mean of at least three biological replicates. Synergy was assessed using the

149

equation for Fractional Inhibitory Concentration Index (FICI) (Supplemental Methods)

150

25

.

151 152

16S rRNA gene amplicon sequencing of marine enrichment cultures

153

For 16S rRNA gene amplicon sequencing, marine enrichment cultures were

154

grown in 96-well plates in the presence of 2-fold serial dilutions of nitrate or perchlorate

155

(gradient plates). The gradient plate cultures were inoculated at an initial OD 600 of 0.02

156

in a volume of 200 µL. After 48 hours (OD 600 ~ 0.3-0.4), cultures were harvested by

157

centrifugation, 180 µL supernatant was removed, and genomic DNA was extracted from

158

the remaining pellet and the V3V4 region of the 16S rRNA gene was amplified using

159

unique dual indexed primers with attached Illumina adaptors, similar to previously

160

published primers

161

Diego, CA, USA). Reads were analyzed by a combination of custom scripts, PEAR

162

and

163

https://github.com/polyatail/arkin).

the

26, 27

, and sequenced using the 600 bp MiSEQ V3 kit (Illumina, San

QIIME

pipeline

29

(Supplemental

Methods

28

and

164 165

qPCR assay for quantifying dsrA

166

DNA was pooled from 4 replicate 96-well gradient plates (~800 µL of culture)

167

and Taqman (Life Technologies, Grand Island, NY, USA) qPCR was used to quantify

168

dsrA gene abundance using previous methods with some modifications

169

(Supplemental Methods).

30,

31

170 171


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Results

174

Evaluating the potency and selectivity of monomeric inorganic oxyanions for

175

inhibition of sulfidogenesis in complex microbial communities

176

We prepared serial dilutions of compounds in microplates.

The plates were

177

inoculated with a sulfidogenic marine enrichment culture11 amended with the complex

178

electron donor, yeast extract (2g/L) to ensure maintenance of a phenotypically and

179

phylogenetically diverse community membership with sulfate as the sole electron

180

acceptor. By comparing the IC50s against growth as measured by OD 600 with the IC50s

181

against sulfide production as measured by the colorimetric Cline assay we identified

182

compounds that were selective inhibitors of sulfidogenesis versus growth (Table 1,

183

Figure 1). We evaluated a panel of inorganic oxyanion analogs using this assay (Table 1)

184

and calculated selectivity indices (SI = growth IC50:sulfide IC50).

185

compounds with SI > 2 as selective inhibitors of sulfidogenesis in the marine enrichment,

186

and confirmed that the growth and sulfide IC50s were different with ANOVA. This

187

approach could be adapted to a variety of microbial systems and allows quantification of

188

both the inhibitory potency and selectivity of compounds against sulfidogenic

189

populations.

190

Transition metal oxyanions

We classified

191

Of the transition metal oxyanions, chromate was non-specific, but vanadate,

192

molybdate and tungstate were specific inhibitors of sulfide production (Table 1). The

193

selectivity indices for molybdate (SI = 100) and tungstate (SI = 589) were among the

194

highest for the panel of compounds we screened, but concentrations above 1 mM

195

inhibited all growth in our marine enrichment cultures. In some studies, concentrations

196

in the range of 1-100 mM have been used to inhibit sulfidogenesis in environmental

197

systems 21, 32. Based on our results (Table 1) the application of lower concentrations may

198

have yielded different results in these previous studies (i.e. higher methane titers or

199

higher growth yields of other non-SRM). To our knowledge this is the first observation

200

of selective inhibition of SRM by vanadate.

201

Chalcogen (Group 16) oxyanions

202

Both sulfate and sulfite are growth substrates for sulfate reduction and only

203

inhibited growth and sulfidogenesis at high millimolar concentrations. The substituted


8

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sulfate analogs ammonium sulfamate, methanesulfonate, and dimethyl sulfone were all

205

very weak and non-specific inhibitors (Table 1). In contrast, selenate, selenite, tellurate

206

and tellurite were very potent and selective inhibitors of sulfate reduction (Table 1). Both

207

selenate and tellurate are isolectronic with sulfate and are known to be toxic and reduced

208

by SRM and other bacteria

209

and tellurium oxyanions, we observed visual color changes and precipitates consistent

210

with reduction of these compounds.

211

competitive inhibitors of the yeast ATP sulfurylase

212

for logistical reasons. Fluorosulfate decomposes in water into the toxic compounds

213

sulfuric acid and hydrogen fluoride while thiosulfate is a growth substrate for many

214

SRM.

215

Halogen (Group 17) oxyanions

33, 34

. In our cultures, at high concentrations of the selenium

Although fluorosulfate and thiosulfate are 16

we did not test these compounds

216

Of the halogen oxyanions, only perchlorate and chlorate, collectively

217

(per)chlorate, were selective inhibitors of sulfidogenesis (Table 1). While (per)chlorate

218

were less potent inhibitors of sulfidogenesis compared to the other selective inhibitors we

219

identified, they were the only monoanionic compounds that were selective inhibitors.

220

Previously, we have demonstrated that nitrate, perchlorate and chlorate were not reduced

221

in our marine enrichments

222

not due to outgrowth of respiratory (per)chlorate reducing microorganisms. Bromate,

223

iodate and periodate were also non-specific, but potent inhibitors of all growth in our

224

marine enrichment cultures. Bromine and iodine oxyanions are strong oxidizing agents

225

that rapidly react with sulfide, Fe(II) minerals, and cellular components.

226

Pnictogen (Group 15) oxyanions

11

, implying that the effect of these compounds is direct and

227

Of the pnictogen oxyanions, nitrate, nitrite and monofluorophosphate were

228

selective inhibitors. Nitrate was the least potent of the selective inhibitors in our panel,

229

and nitrite was only weakly selective (SI ~ 2). In contrast to monofluorophosphate, the

230

other phosphate derivatives, thiophosphate and phosphite, were weak inhibitors and non-

231

selective. Arsenate was not selective, likely because it interferes with phosphate

232

metabolism in all organisms in the enrichment.

233


9


234

16S

235

monofluorophosphate against growth of SRM

amplicon

sequencing

and

dsrA

qpcr

to

Page 10 of 29

confirm

selectivity

of

236

In the marine enrichment cultures, MFP inhibited sulfidogenesis with an IC50 =

237

1.9 (0.29-4.4) mM, but the growth IC50 was greater than 100 mM (Figure 1A, Table 1).

238

The selective inhibition by MFP (SI = 53) could potentially shift a facultative SRM

239

population to fermentative or syntrophic growth coupled to methanogenesis. However,

240

in our marine enrichment culture, Desulfovibrionales was the only proteobacterial genus

241

observed and 16S amplicon sequencing of cultures grown in varying concentrations of

242

MFP revealed dramatic depletion of proteobacteria (dominated by Desulfovibrionales) at

243

concentrations above the sulfide IC50 with little change in the relative abundances of

244

other phyla (Figure 1B). Furthermore, the IC50 values for MFP against sulfidogenesis,

245

Desulfovibrionales abundance, and abundance of the dsrA gene copy number were

246

identical (Figure 1C) indicating that the SRM do not persist and grow during MFP

247

treatment by using alternative metabolisms.

248 249

Resistance

250

monofluorophosphate

251

of

a

nitrate

and

perchlorate

respiring

microorganism

to

At present, the dominant strategy to combat sulfidogenesis in industrial

252

ecosystems is nitrate treatment 2. Perchlorate treatment is a promising alternative

12, 13

253

and we have previously demonstrated its greater potency and selectivity against SRM11,

254

12

255

we sought to evaluate whether or not MFP was inhibitory of these organisms and could

256

be used as an additive or synergistic treatment during nitrate or perchlorate injection. We

257

grew Azospira suillum PS, a model organism capable of both nitrate and (per)chlorate

258

respiration

259

inhibited the growth of Desulfovibrio alaskensis G20 with an IC50 of 1.2 (0.88-1.6) mM

260

while A. suillum PS grown under either nitrate or perchlorate reducing conditions

261

tolerated much higher concentrations (IC50 > 50 mM) (Figure 2A, B).

. These two treatments may rely, in part, on the activity of NRM and PRM, and thus,

35

, in the presence of varying concentrations of MFP (Figure 2).

MFP

262 263

Synergistic

264

monofluorophosphate

inhibition

of

sulfidogenesis

by

nitrite


or

chlorite

and

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265

Synergistic SRM inhibitors can decrease the inhibitory concentrations needed to

266

treat sulfidogenesis 4. In drug combinations, synergy can increase not only the potency,

267

but also the selectivity of compounds

268

used in treating sulfidogenesis in industrial systems at lower cost and with greater

269

efficacy. We evaluated the potential for synergy between MFP and nitrate, perchlorate,

270

nitrite, and chlorite in inhibition of sulfidogenesis in the marine enrichment culture.

271

Synergy was assessed using the equation for Fractional Inhibitory Concentration Index

272

(FICI) based on the IC50 for each inhibitor A and B in the absence or presence of the

273

other inhibitor (Supplemental methods). Combinations of MFP with nitrate (FICI=1) or

274

perchlorate (FICI = 0.95) were additive indicating no synergistic impact (Figures 2C &

275

D). In contrast, combinations of MFP with nitrite (FICI = 0.3) or chlorite (FICI = 0.06)

276

were highly synergistic (Figure 2E & 2F). Together with the observation that MFP is

277

only weakly inhibitory of nitrate and perchlorate reducing bacteria (Figure 2A,B), this

278

result suggests that in a stratified system in which nitrate or perchlorate reduction (and

279

potentially nitrite and chlorite accumulation) spatially precede sulfate reduction,

280

synergistic inhibition of SRM could occur through combined nitrate or perchlorate and

281

MFP amendments. It is proposed that biogenic nitrite is the most important inhibitor of

282

sulfate reduction produced in nitrate treated oil reservoirs, but this has not been

283

conclusively demonstrated 2. Thus, evaluating the potential for MFP synergy with nitrate

284

or perchlorate amendments could be a powerful diagnostic tool to understand whether

285

inhibition in a complex community is due to the parent ions or the reactive respiratory

286

intermediates.

36

. Therefore, synergistic combinations could be

287 288

Potency of inorganic oxyanions for inhibition of Desulfovibrio alaskensis G20 wild-

289

type and tn5::rex mutant

290

To gain insights into the mechanism of inhibition of a model SRM by the panel of

291

inorganic oxyanions, we conducted several assays with D. alaskensis G20. First, we

292

compared the inhibitory potencies against wild-type D. alaskensis G20 and against a

293

tn5::rex mutant strain that overproduces the central pathway of sulfate reduction

294

particular, the tn5::rex strain overproduces the core Rex regulon consisting of qmoABCD

295

(Dde_1111:Dde_1114), sat (Dde_2265), adenylate kinase (Dde_2028), pyrophosphatase


37

. In

11


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(Dde_1178), a sulfate transporter (Dde_2406), an ATP synthase, atpFFHAGD

297

(Dde_0990:Dde_0984) and atpIIBE (Dde_2698:Dde_2701)

298

the enzymes in the core Rex regulon may be more or less inhibitory to tn5::rex than to

299

wild-type G20. Previously, we and others have demonstrated that Rex mutants are

300

resistant to competitive inhibitors of sulfate reduction including nitrate

301

(per)chlorate11, which strongly suggests that these compounds directly target the sulfate

302

reduction pathway in D. alaskensis G20.

37

. Compounds that target

11, 38

and

303

Strikingly, of our panel of compounds, Rex mutants were only resistant to nitrate

304

and (per)chlorate, which are the only confirmed competitive inhibitors of eukaryotic ATP

305

sulfurylase

306

arsenate (Table 2). While overproduction of sulfate reduction enzymes may overcome

307

competitive inhibition, non-competitive inhibitors or alternative/futile substrates of the

308

ATP

309

monofluorophosphate) are likely to be equally or slightly more potent inhibitors of Rex

310

mutants. For example, Rex mutants express higher levels of sulfate transporters, and are

311

therefore equally if not more permeable to these toxic compounds. Higher levels of Sat

312

may also catalyze more non-productive catalysis by futile/alternative substrates and more

313

rapidly consume ATP pools in Rex mutants.

16

that we tested (Table 2). Rex mutants were only sensitive to MFP and

sulfurylase

(e.g.

molybdate,

tungstate,

chromate,

arsenate,

selenate,

314

Arsenate was not selective against sulfidogenesis in the marine enrichment

315

cultures, likely because it functions as a toxic phosphate mimic in all organisms.

316

However, arsenate is a more potent inhibitor of Rex mutants than wild-type G20 (Table

317

2). Arsenate is believed to enter D. alaskensis G20 through sulfate transporters

318

higher levels of the transporters in Rex mutants might lead to higher intracellular arsenate

319

concentrations that could overwhelm the G20 arsenate detoxification mechanisms

320

Arsenate is also a futile substrate for ATP sulfurylase in eukaryotes

321

similarly in G20. The sensitivity of Rex mutants to MFP may be through a similar

322

mechanism. More MFP may be transported into the cytoplasm by Rex mutants and

323

intracellular MFP may be the active inhibitor. Also, MFP could potentially be converted

324

into other inhibitory compounds, (i.e. ATPβF, F-) by components of the sulfate reduction

325

pathway. Molybdate and tungstate were equally potent inhibitors of Rex mutants and

326

wild-type cells.

16

39

, and 39

.

and may function

This is consistent with molybdate and tungstate functioning as


12

Page 13 of 29


327

alternative substrates for ATP sulfurylases

328

nitrite

329

upregulated in Rex mutants 37.

40

14, 16, 17

. Competitive inhibition of Dsr by

would likely similarly impact wild-type G20 and Rex mutants as Dsr is not

330 331

Potency of selected inorganic oxyanions for inhibition of G20 growth under

332

fermentative and sulfite-reducing conditions

333

In the marine enrichment cultures, 16S amplicon sequencing and dsrA qpcr

334

analyses indicate that the sulfidogenic Desulfovibrionales did not switch to fermentative

335

or syntrophic growth in the presence of MFP (Figure 1), nitrate or perchlorate11.

336

However, because the capacity for fermentative growth could theoretically confer

337

resistance of SRM to sulfate reduction specific inhibitors, we evaluated the inhibitory

338

potency of selected inorganic oxyanions against D. alaskensis G20 growing by pyruvate

339

fermentation (Table 3).

340

central pathway of sulfate reduction under pyruvate fermenting conditions relative to

341

sulfate reducing conditions 41. Thus, as with Rex mutants, compounds that target sulfate

342

reduction could have altered potencies against pyruvate fermenting cells. Of the

343

compounds we tested, only the competitive inhibitors, nitrate and (per)chlorate, were less

344

potent inhibitors of pyruvate grown G20 than lactate/sulfate grown G20. In contrast, the

345

other selective inhibitors of sulfate reduction from our marine enrichment cultures (Table

346

1) were equally potent inhibitors of pyruvate fermenting G20. This observation has

347

implications for choosing an inhibitor to apply to an environmental or industrial setting.

348

In a system with varying fluxes of sulfate, SRM may shift to a fermentative lifestyle. A

349

competitive inhibitor of sulfate reduction would be less effective at preventing SRM

350

growth by fermentation, but compounds such as MFP may retain efficacy.

Furthermore, D. alaskensis G20 apparently upregulates the

351

Comparison of the inhibitory potency of compounds against sulfate reduction

352

versus sulfite reduction can distinguish compounds that target sulfate transport, sulfate

353

activation and APS reduction from compounds that target sulfite reduction

354

observed that several compounds proposed to target sulfate reduction including nitrate,

355

(per)chlorate, monofluorophosphate and molybdate were less inhibitory to sulfite grown

356

cells than sulfate grown cells (Table 3). In contrast, nitrite was more inhibitory of sulfite

357

reduction, and is known to be a competitive/alternative substrate of the dissimilatory


42

.

We

13


40

Page 14 of 29

358

sulfite reductase, Dsr

359

Hildenborough, this organism possesses an NrfA, nitrite reductase, and detoxification

360

systems for reactive nitrogen species (RNS)

361

associated with nitrite resistance were for mutants in RNS resistance proteins, and

362

evaluating the true cellular target(s) of nitrite inhibition has been difficult.

363

observation that nitrite is a more potent inhibitor of sulfite reduction relative to sulfate

364

reduction by D. alaskensis G20 (Table 3) is the first clear evidence that Dsr inhibition is

365

implicated in nitrite toxicity in an SRM.

.

Although nitrite toxicity has been studied in D. vulgaris 8, 43

.

Therefore, the only phenotypes

Our

366 367

Fluoride ion toxicity is associated with MFP toxicity

368

Little is known about the mechanism of inhibition of SRM by MFP aside from

369

the observation that it is competitive inhibitor of sulfate reduction at low concentrations

370

but non-competitive at higher concentrations

371

alternative substrate for ATP sulfurylases from eukaryotes 16. We can gain some insight

372

into the mechanism of SRM inhibition by comparing the inhibitory potency and

373

selectivity of MFP against growth and sulfidogenesis in marine enrichments with the

374

closely related compounds phosphite and thiophosphate. Though these compounds have

375

similar ionic radii and charge state at neutral pH and are stable to hydrolysis, these other

376

phosphate analogs were very weak, non-selective inhibitors of sulfidogenesis.

377

implies a unique structural feature of the intact MFP ion or toxicity associated with F-

378

release.

3

and the observation that MFP is an

This

379

We sought to evaluate the role of F- in the mechanism of MFP toxicity. While

380

MFP has an IC50 = 1.2 (0.88-1.6) mM against D. alaskensis G20, fluoride ion has an IC50

381

of 34 (21-54) mM (Figure 3A). Fluoride ion is highly toxic to microbial cells, but

382

generally higher concentrations are inhibitory because fluoride only traverses cell

383

membranes as HF 44. Recently, a class of fluoride specific efflux pumps, crcB, has been

384

identified in diverse bacteria, further emphasizing the importance of developing

385

resistance mechanisms to F-

386

homolog is Dde_2102. We grew the tn5::dde_2102 strain in the presence of 30mM F- or

387

1 mM MFP, and observed that relative to wild-type G20, this mutant strain grew well in

388

the absence of stress, but was sensitive to F- and MFP (Figure 3B-D). Because the

45

. In D. alaskensis G20, the fluoride efflux pump (CrcB)


14

Page 15 of 29


389

fluoride efflux pump is involved in the efflux of cytoplasmic fluoride ion, this result

390

confirms that cytoplasmic fluoride is present in D. alaskensis G20 cells treated with MFP,

391

and is strong evidence that intracellular MFP hydrolysis occurs in D. alaskensis G20 and

392

contributes to the mechanism of inhibition.

393 394 395

Discussion We quantitatively compared the potency and selectivity of a panel of inorganic

396

oxyanions as inhibitors of sulfate reduction in a marine enrichment culture.

397

subsequent screens with D. alaskensis G20 wild-type and the G20 tn5::rex mutant, we

398

observed distinct inhibition patterns for the presumed competitive inhibitors of the sulfate

399

reduction pathway and futile substrates of Sat. The susceptibility of pyruvate fermenting

400

and sulfite reducing G20 to inorganic oxyanions provided further insights into the

401

mechanism of action of selected compounds.

In

402

Of the compounds we identified as selective inhibitors of sulfide production in

403

our screen, other considerations limit their worth as possible industrial treatments.

404

Selenate, selenite, tellurate, and tellurite are toxic to diverse microorganisms and their

405

abiotic reactivity may prevent their penetration into sulfidogenic environments in the

406

desired redox state. Molybdate and tungstate are essential nutrients for many SRM 46, are

407

reactive with metals and sulfides in the environment, and are toxic to aquatic

408

organisms47. This is the first observation of vanadate as an SRM selective compound.

409

Vanadium porphyrins and other transition metals have been observed in oil reservoirs 48.

410

It is unknown the extent to which transition metal oxyanions may be present in oil

411

reservoirs and control sulfidogenesis.

412

Mechanistic insights into MFP inhibition

413

Taken together, our results support a model for MFP as a competitive substrate

414

for the sulfate reduction pathway and as a vehicle for F- delivery to the cytoplasm of

415

actively sulfate respiring cells. Monofluorophosphate is isoelectronic with SO42- and is a

416

competitive inhibitor of sulfate respiration but also non-competitive at higher

417

concentrations 3. MFP is a futile substrate of eukaryotic ATP sulfurylases that forms a

418

relatively stable product

419

marine enrichment cultures, and the susceptibility of tn5::rex mutants and resistance of

16

. In our experiments, MFP is a selective inhibitor of SRM in


15


Page 16 of 29

420

sulfite grown G20 to MFP suggest that it targets the initial steps of sulfate reduction. In

421

support of a non-competitive inhibition mechanism, MFP inhibition of Desulfovibrio

422

alaskensis G20 was partially alleviated by fluoride efflux pumping suggesting that

423

intracellular fluoride accumulation is associated with MFP toxicity. Fluoride is a potent

424

inhibitor of microorganisms, largely due to its competition with catalytic hydroxide ions

425

in enzyme active sites (i.e. enolase)

426

50 times higher than MFP suggesting that its potency is offset by active efflux from the

427

cell.

49, 50

, but the IC50 of F- against D. alaskensis G20 is

428 429

Considering MFP as an inhibitor of sulfate reduction in natural microcosms and

430

engineered ecosystems

431

MFP is a more potent inhibitor of sulfidogenesis in our marine enrichments (IC50

432

= 1.9 (0.29-4.4) mM) than nitrate or (per)chlorate, and while nitrite and chlorite are

433

similarly inhibitory, MFP is more selective than any of the nitrogen or chlorine oxyanions

434

(Table 1).

435

inhibitor of sulfidogenesis in combination with nitrite and chlorite, which may increase

436

the selectivity of the inhibitors (Figure 2). Inappropriate dosing can lead to elimination

437

of alternative microbial populations, drive the evolution of microbial resistance and could

438

lead to a greater corrosion risk if the inhibitor is capable of driving microbially influenced

439

corrosion (e.g. nitrate) 7.

Furthermore, MFP is tolerated by NRM and PRM and is a synergistic

440

MFP has been demonstrated to be an effective inhibitor of abiotic corrosion of

441

steel in concrete, though this could be largely due to passivization by phosphate ions after

442

fluoride hydrolysis 51, 52. Evaluating the stability of MFP in an industrial ecosystem is an

443

important first step for assessing its utility as an SRM inhibitor. At extremes of alkaline

444

and acidic pH, MFP is unstable, but is stable for months at neutral pH 53. Bacterial and

445

eukaryotic alkaline phosphatases can hydrolyze fluorophosphate bonds

446

fluorophosphate is stable in the presence of other enzymes, such as acid phosphatase

447

Also, pyruvate kinase has been demonstrated to possess fluorokinase activity,

448

synthesizing FPO32- from F- and PO42-

449

inhibited by MFP

450

source, addition of more labile forms of sulfur (e.g. cysteine) could be considered as

16

55

.

54

, but 53

.

The assimilatory ATP sulfurylases are

. Thus, in microbial ecosystems where sulfate is the primary sulfur


16

Page 17 of 29


451

additional amendments during SRM specific inhibitor treatments to help ensure

452

selectivity for dissimilatory SRM versus organisms relying on sulfate assimilation to

453

obtain sulfur. Dicationic MFP salts (i.e. calcium MFP, ferrous MFP) are more soluble

454

than the corresponding phosphate salts, and are more similar to phosphite (PO32-) salts.

455

The solubility constant for calcium MFP is ~ 30mM, thus SRM inhibitory concentrations

456

of soluble MFP should be deliverable to environments with high concentrations of

457

divalent cations

458

potency of MFP in industrial microbial ecosystems, but our results and those available in

459

the literature suggest that MFP is a promising alternative to conventional strategies for

460

inhibition of sulfate reduction.

56

. Further studies are necessary to evaluate the stability and inhibitory

461 462 463

Acknowledgements

464

We thank members of the Coates and Arkin groups for critical comments on this

465

manuscript. Work in the laboratory of JDC on biosouring is supported by the Energy

466

Biosciences Institute.

467

free of charge via the Internet at http://pubs.acs.org/.

Supporting Information Available: This information is available

468


17


469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513

Page 18 of 29

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30. Bourne, D. G.; Muirhead, A.; Sato, Y., Changes in sulfate-reducing bacterial populations during the onset of black band disease. ISME J. 2011, 5 (3), 559-64. 31. Leloup, J.; Loy, A.; Knab, N. J.; Borowski, C.; Wagner, M.; Jorgensen, B. B., Diversity and abundance of sulfate-reducing microorganisms in the sulfate and methane zones of a marine sediment, Black Sea. Environ. Microbiol. 2007, 9 (1), 131-42. 32. Tor, J. M.; Amend, J. P.; Lovley, D. R., Metabolism of organic compounds in anaerobic, hydrothermal sulphate-reducing marine sediments. Environ. Microbiol. 2003, 5 (7), 583-591. 33. Tucker, M. D.; Barton, L. L.; Thomson, B. M., Reduction of Cr, Mo, Se and U by Desulfovibrio desulfuricans immobilized in polyacrylamide gels. J. Ind. Microbiol. Biot. 1998, 20 (1), 13-19. 34. Zehr, J. P.; Oremland, R. S., Reduction of selenate to selenide by sulfate-respiring bacteria: experiments with cell suspensions and estuarine sediments. Appl. Environ. Microbiol. 1987, 53 (6), 1365-9. 35. Achenbach, L. A.; Michaelidou, U.; Bruce, R. A.; Fryman, J.; Coates, J. D., Dechloromonas agitata gen. nov., sp. nov. and Dechlorosoma suillum gen. nov., sp. nov., two novel environmentally dominant (per)chlorate-reducing bacteria and their phylogenetic position. Int. J. Syst. Evol. Microbiol. 2001, 51 (Pt 2), 527-33. 36. Lehar, J.; Krueger, A. S.; Avery, W.; Heilbut, A. M.; Johansen, L. M.; Price, E. R.; Rickles, R. J.; Short, G. F., 3rd; Staunton, J. E.; Jin, X.; Lee, M. S.; Zimmermann, G. R.; Borisy, A. A., Synergistic drug combinations tend to improve therapeutically relevant selectivity. Nat. Biotechnol. 2009, 27 (7), 659-66. 37. Kuehl, J. V.; Price, M. N.; Ray, J.; Wetmore, K. M.; Esquivel, Z.; Kazakov, A. E.; Nguyen, M.; Kuehn, R.; Davis, R. W.; Hazen, T. C.; Arkin, A. P.; Deutschbauer, A., Functional genomics with a comprehensive library of transposon mutants for the sulfatereducing bacterium Desulfovibrio alaskensis G20. mBio 2014, 5 (3), e01041-14. 38. Korte, H. L.; Fels, S. R.; Christensen, G. A.; Price, M. N.; Kuehl, J. V.; Zane, G. M.; Deutschbauer, A. M.; Arkin, A. P.; Wall, J. D., Genetic basis for nitrate resistance in Desulfovibrio strains. Front. Microbiol. 2014, 5, 153. 39. Li, X.; Krumholz, L. R., Regulation of arsenate resistance in Desulfovibrio desulfuricans G20 by an arsRBCC operon and an arsC gene. J. Bacteriol. 2007, 189 (10), 3705-11. 40. Wolfe, B. M.; Lui, S. M.; Cowan, J. A., Desulfoviridin, a multimericdissimilatory sulfite reductase from Desulfovibrio vulgaris (Hildenborough). Purification, characterization, kinetics and EPR studies. Eur. J. Biochem. 1994, 223 (1), 79-89. 41. Meyer, B.; Kuehl, J. V.; Price, M. N.; Ray, J.; Deutschbauer, A. M.; Arkin, A. P.; Stahl, D. A., The energy-conserving electron transfer system used by Desulfovibrio alaskensis strain G20 during pyruvate fermentation involves reduction of endogenously formed fumarate and cytoplasmic and membrane-bound complexes, Hdr-Flox and Rnf. Environ. Microbiol. 2014, 16 (11), 3463-86. 42. Lie, T. J.; Godchaux, W.; Leadbetter, E. R., Sulfonates as terminal electron acceptors for growth of sulfite-reducing bacteria (Desulfitobacterium spp.) and sulfatereducing bacteria: effects of inhibitors of sulfidogenesis. Appl. Environ. Microbiol. 1999, 65 (10), 4611-7.


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43. Yurkiw, M. A.; Voordouw, J.; Voordouw, G., Contribution of rubredoxin:oxygen oxidoreductases and hybrid cluster proteins of Desulfovibrio vulgaris Hildenborough to survival under oxygen and nitrite stress. Environ. Microbiol. 2012, 14 (10), 2711-25. 44. Breaker, R. R., New insight on the response of bacteria to fluoride. Caries Res. 2012, 46 (1), 78-81. 45. Baker, J. L.; Sudarsan, N.; Weinberg, Z.; Roth, A.; Stockbridge, R. B.; Breaker, R. R., Widespread genetic switches and toxicity resistance proteins for fluoride. Science 2012, 335 (6065), 233-5. 46. Mota, C. S.; Valette, O.; Gonzalez, P. J.; Brondino, C. D.; Moura, J. J.; Moura, I.; Dolla, A.; Rivas, M. G., Effects of molybdate and tungstate on expression levels and biochemical characteristics of formate dehydrogenases produced by Desulfovibrio alaskensis NCIMB 13491. J. Bacteriol. 2011, 193 (12), 2917-23. 47. Heijerick, D. G.; Regoli, L.; Carey, S., The toxicity of molybdate to freshwater and marine organisms. II. Effects assessment of molybdate in the aquatic environment under REACH. Sci. Total Environ. 2012, 435-436, 179-87. 48. Treibs, A., On the occurrence of chlorophyll-derivatives in an oil-slate in the upper trias. Liebigs Ann. Chem. 1934, 509, 103-114. 49. Hoorn, R. K. J.; Flikweert, J. P.; Staal, G. E. J., Purification and properties of enolase of human erythrocytes. Int. J. Biochem. 1974, 5 (11-12), 845-852. 50. Qin, R.; Chai, G. Q.; Brewer, J. M.; Lovelace, L. L.; Lebioda, L., Fluoride inhibition of enolase: Crystal structure and thermodynamics. Biochemistry-Us 2006, 45 (3), 793-800. 51. Ngala, V. T.; Page, C. L.; Page, M. M., Corrosion inhibitor systems for remedial treatment of reinforced concrete. Part 2: Sodium monofluorophosphate. Corros. Sci. 2003, 45 (7), 1523-1537. 52. Soylev, T. A.; Richardson, M. G., Corrosion inhibitors for steel in concrete: Stateof-the-art report. Constr. Build. Mater. 2008, 22 (4), 609-622. 53. Setnikar, I.; Arigoni, R., Chemical stability and mode of gastrointestinal absorption of sodium monofluorophosphate. Arzneimittelforschung 1988, 38 (1), 45-9. 54. Fernley, H. N.; Walker, P. G., Studies on alkaline phosphatase. Inhibition by phosphate derivatives and the substrate specificity. Biochem. J. 1967, 104 (3), 1011-8. 55. Tietz, A.; Ochoa, S., Fluorokinase and pyruvic kinase. Arch Biochem Biophys 1958, 78 (2), 477-93. 56. Rowley, H. H.; Stuckey, J. E., Preparation and Properties of Calcium Monofluorophosphate Dihydrate. J. Am. Chem. Soc. 1956, 78 (17), 4262-4263.

639 640 641


21


Page 22 of 29

642

Figure and Table Legends:

643

Table 1: IC50s and selectivity indices of sulfate analogs against growth and sulfide

644

production by marine enrichment cultures.

645

ANOVA (p100

Not inhibitory

CH3SO3-

Methanesulfonate

16 (chalcogen)

>100

>100

Not inhibitory

NH2SO3-

Ammonium sulfamate

16 (chalcogen)

74 (52-104)

SeO42-

Selenate

16 (chalcogen)

>10

SeO32-

Selenite

16 (chalcogen)

>10

TeO42-

Tellurate

16 (chalcogen)

>1

81 (66-99) 0.38 (0.21-0.72) 0.57 (0.56-0.59) 0.012 (0.010-0.013)

TeO32-

Tellurite

16 (chalcogen)

>1

0.053 (0.040-0.071)

>18.9

ClO4-

Perchlorate

17 (halogen)

21 (14-31)

2.3 (2.0-2.6)

9.1

ClO3-

Chlorate

17 (halogen)

44 (29-60)

1.6 (1.4-1.8)

27.5

ClO2-

Chlorite

17 (halogen)

2.8 (1-7.5)

1.17 (0.8-1.7)

2.4

BrO3-

Bromate

17 (halogen)

0.59 (0.40-0.86)

0.39 (0.12-1.2)

1.5

IO4-

Periodate

17 (halogen)

0.40 (0.27-0.60)

0.40 (0.27-0.59)

1

IO3-

Iodate

17 (halogen)

0.37 (0.19-0.72)

0.36 (0.13-0.31)

1

NO3-

Nitrate

15 (pnictogen)

46 (34-62)

8.0 (7.0-9.0)

5.75

NO2-

Nitrite

15 (pnictogen)

5.5 (3.3-9.3)

0.12 (0.1-0.4)

45.8

HPO32-

Phosphite

15 (pnictogen)

>100

>100

Not inhibitory

SPO32-

Thiophosphate

15 (pnictogen)

>100

>100

Not inhibitory

FPO32-

Monofluorophosphate

15 (pnictogen)

>100

1.9 (0.29-4.4)

53

AsO42-

Arsenate

15 (pnictogen)

0.41 (0.21-0.76

0.36 (0.055-2.4)

1.1

AsO32-

Arsenite

15 (pnictogen)

0.011 (0.008-0.014)

~0.008

1.4

VO43-

Vanadate

5 (Transition metal)

2.7 (0.89-8.2)

0.17 (0.12-0.26)

16

CrO43-

Chromate


0.027 (0.020-0.036)

0.031 (0.026-0.037)

0.9

MoO42-

Molybdate


0.69 (0.24-1.9)

0.0069 (0.0032-0.015)

100

WO42-

Tungstate


1.3 (0.57-3.0)

0.0024 (00075-0.0079)

542

0.9 >26.3 >17.5 >83

675 676 677


23


678 679

Page 24 of 29

Table 2. Inhibition by monomeric oxyanions of Desulfovibrio alaskensis G20 wild-type and Rex mutant IC50 (95% CI) against Desulfovibrio alaskensis strains (mM)

Oxyanion attributes Molecular formula

Group

Wild-type (sulfate)

tn5::rex (sulfate)

(CH3)SO2

16 (chalcogen)

>100

>100

CH3SO3-

16 (chalcogen)

>100

>100

110 2.4-470 0.022 (0.018-0.028) 0.45 (0.32-0.63) 0.11 (0.028-0.40)

110 (2.4-470) 0.025 (0.012-0.060) 0.011 (0.016-0.79) 0.077 (0.061-0.091)

NH2SO3-

16 (chalcogen)

SeO42-

16 (chalcogen)

SeO32-

16 (chalcogen)

TeO42-

16 (chalcogen)

TeO32-

16 (chalcogen)

0.022 (0.023-0.13)

0.039 (0.032-0.047)

ClO4-

17 (halogen)

24 (20-32)

51 (37-70)

ClO3-

17 (halogen)

6.4 (4.9-8.0)

25 (30-31)

ClO2-

17 (halogen)

4.7 (0.5-41)

9.6 (6.7-13)

BrO3-

17 (halogen)

2 (1.3-3.2)

7.2 (1.8-28)

IO4-

17 (halogen)

5.6 (2.0-16)

2.3 (1.5-3.8)

IO3-

17 (halogen)

15 (0.61-370)

1.6 (1.1-2.3)

NO3-

15 (pnictogen)

51 (40-65)

250 (90-300)

NO2-

15 (pnictogen)

0.42 (0.32-0.56)

0.1 (0.09-1.2)

HPO32-

15 (pnictogen)

>100

>100

SPO32-

15 (pnictogen)

>100

>100

FPO32-

15 (pnictogen)

1.2 (0.88-1.6)

0.46 (0.33-0.66)

AsO42-

15 (pnictogen)

22 (17-29)

2.2 (1.5-3.1)

AsO32-

15 (pnictogen)

0.93 (0.94-1.6)

0.73 (0.6-2.8)

VO43-


>10

>10

CrO43-


>10

>10

MoO42-


0.089 (0.043-0.19)

0.092 (0.021-0.24)

WO42-


1.6 (0.71-3.4)

0.027 (0.019-0.39)

680 681 682


24

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683 684


Table 3. Inhibition by monomeric oxyanions of Desulfovibrio alaskensis G20 wild-type under different growth conditions Molecular formula

IC50 (95% CI) against Desulfovibrio alaskensis G20 growth (electron acceptor) (mM)

Putative target

Wild-type (sulfate)

Wild-type (pyruvate)

Wild-type (sulfite)

ClO4-

24 (20-32)

170 (62-450)

>100

Competitive Sat inhibitor

ClO3-

6.4 (4.9-8.0)

32 (13-78)

33 (12-92)


NO3-

51 (40-65)

>100

>100


NO2-

0.42 (0.32-0.56)

1.5 (0.31-7.2)

0.015 (0.0094-0.023)

Competitive Dsr substrate

FPO32-

1.2 (0.88-1.6)

2.2 (0.68-7.4)

4.3 (2.1-9.1)

Futile Sat substrate, F-

MoO42-

0.089 (0.043-0.19)

0.092 (0.021-0.41)

1.2 (0.46-2.9)

Futile Sat substrate

685 686 687 688


25



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35x14mm (600 x 600 DPI)


Monofluorophosphate Is a Selective Inhibitor of Respiratory Sulfate

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