High-throughput screening to identify potent and ... - ACS Publications

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

High-throughput screening to identify potent and specific inhibitors of microbial sulfate reduction Hans K. Carlson, Mark Russell Mullan, Lorraine A. Mosqueda, Steven Chen, Michelle R. Arkin, and John D. Coates Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 14, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

Environmental Science & Technology

1

High-throughput screening to identify potent and specific inhibitors of microbial

2

sulfate reduction

3 4

Running title: Sulfate reduction high-throughput screen

5 6

Hans K. Carlson1,2, Mark R. Mullan1, Lorraine A. Mosqueda1, Steven Chen3, Michelle R.

7

Arkin3, John D. Coates1,4,5,*

8 9

1

Energy Biosciences Institute, UC Berkeley

10

2

Physical Biosciences Division, Lawrence Berkeley National Lab

11

3

Small Molecule Discovery Center, UC San Francisco

12

4

Earth Sciences Division, Lawrence Berkeley National Lab

13

5

Department of Plant and Microbial Biology, UC Berkeley

14 15 16

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

17

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

18

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

19 20 21

Keywords:

22

screening, hydrogen sulfide, souring

sulfate-reducing microorganisms, selective inhibitor, high-throughput

23

ACS Paragon Plus Environment

1


Page 2 of 25

24

Abstract

25

The selective perturbation of complex microbial ecosystems to predictably influence

26

outcomes in engineered and industrial environments remains a grand challenge for

27

geomicrobiology. In some industrial ecosystems, such as oil reservoirs, sulfate reducing

28

microorganisms (SRM) produce hydrogen sulfide which is toxic, explosive and

29

corrosive. Despite the economic cost of sulfidogenesis, there has been minimal

30

exploration of the chemical space of possible inhibitory compounds, and very little work

31

has quantitatively assessed the selectivity of putative souring treatments.

32

developed a high-throughput screening strategy to identify potent and selective inhibitors

33

of SRM, quantitatively ranked the selectivity and potency of hundreds of compounds and

34

identified previously unrecognized SRM selective inhibitors and synergistic interactions

35

between inhibitors. Zinc pyrithione is the most potent inhibitor of sulfidogenesis that we

36

identified, and is several orders of magnitude more potent than commonly used industrial

37

biocides. Both zinc and copper pyrithione are also moderately selective against SRM.

38

The high-throughput (HT) approach we present can be readily adapted to target SRM in

39

diverse environments and similar strategies could be used to quantify the potency and

40

selectivity of inhibitors of a variety of microbial metabolisms. Our findings and approach

41

are relevant to efforts to engineer environmental ecosystems and also to understand the

42

role of natural gradients in shaping microbial niche space.

We have

43


2

Page 3 of 25

44


Introduction

45

In the biomedical sphere, high-throughput screening has been successfully applied

46

to identify antimicrobial compounds for the treatment of infectious diseases in human

47

health and agriculture

48

have led to valuable therapeutic agents and strategies, but to our knowledge, no high-

49

throughput

50

microorganisms.

51

human or other host cell lines is often quantified, and selectivity screens towards specific

52

lifestyles of a pathogen have been established 3, very few screens systematically evaluate

53

the selectivity of a compound for sub-populations within a microbial community.

54

Selectivity is of importance as sub-lethal concentrations of an antibiotic can drive

55

resistance 4. Also, selective compounds will allow the establishment of an alternative

56

stable state for an environmental ecosystem which will be more resistant to invasion and

57

re-establishment of undesirable microbial sub-populations.

screen

1, 2

, Such screening efforts in industrial and academic laboratories

has

been

developed

to

target

subsurface

environmental

Furthermore, while selectivity against microbial pathogens versus

58

In environmental microbiology, selective inhibitors of respiratory processes are

59

valuable tools for studying the structure of microbial ecosystems and determining the

60

function of microbial subpopulations. Some compounds are known selective inhibitors

61

of respiratory metabolisms 5. For example, molybdate (MoO42-) inhibits sulfate reduction5

62

and 2-bromoethanesulfonic acid (BES) inhibits methanogenesis 5, but the inhibitory

63

potency and selectivity of these compounds is rarely quantitatively and systematically

64

evaluated for a given environmental system. Thus, in many cases, these compounds may

65

be used at concentrations at which they are not selective.

66

Biological sulfate reduction represents a major problem in the oil and gas

67

industry.

68

microbial sulfate respiration results in a variety of oil recovery problems, including oil

69

reservoir souring, contamination of crude oil, metal corrosion, and the precipitation of

70

metal sulfides which can subsequently plug pumping wells 6. Nitrate treatment is one

71

approach to souring control that has been widely championed

72

such as perchlorate

73

alternatives.

The generation of hydrogen sulfide (H2S) as a metabolic end-product of

10-12

or monofluorophosphate

13

6-9

, and other approaches,

, are emerging as promising


3


Page 4 of 25

74

However, while the oxyanion treatments are selective, they are less potent

75

inhibitors of microbial growth (millimolar IC50s) compared with biocides or other

76

antimicrobials (micromolar IC50s). Some previous work has provided insights into the

77

impact of common antibiotics and biocides on sulfidogenesis and SRM in natural systems

78

14-16

79

Desulfovibrio isolates, but the selectivity of small molecules against Desulfovibrio or

80

SRM in general is largely unknown

81

and selectivity of a panel of inorganic oxyanions against microbial sulfate reduction

82

and identified monofluorophosphate as a selective inhibitor of sulfidogenesis that has

83

been overlooked. In the present work, we have extended this approach to develop a high-

84

throughput screening strategy to screen small molecule libraries to identify specific

85

inhibitors of microbial sulfate reduction with low micromolar potency.

.

The efficacy of small panels of common antibiotics has been determined against 17

. Recently, we quantitatively ranked the potency 13

86

The screening strategy we present is generic, can be applied to diverse microbial

87

systems, and could be adapted to target other respiratory metabolisms. The centerpiece

88

of our screening strategy is an assay that allows us to efficiently identify compounds that

89

are selective against SRM in the context of a microbial community. Dozens of biocides

90

and inhibitor cocktails are used in oil recovery systems

91

selectivity of these compounds against SRM is largely unknown. In our screen, most

92

biocides and antibiotics are non-selective, but we did identify several small molecules

93

with selectivity against sulfate reduction, and some compounds that were more potent

94

inhibitors of sulfidogenesis than commonly used biocides such as THPS or

95

glutaraldehyde. For example, zinc pyrithione is the most potent inhibitor of

96

sulfidogenesis in our marine enrichment culture and displays some selectivity against

97

SRM.

98

corrosion, but our findings suggest that it may be a more cost-effective treatment strategy

99

compared with other biocides.

8, 16

, but the relative potency and

In some industrial systems, zinc pyrithione is used as an inhibitor of SRM


4

Page 5 of 25


100

Experimental Section

101

Media and cultivation conditions

102

Desulfovibrio alaskensis G20 was cultivated in anoxic basal Tris-buffered

103

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

104

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

105

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

106

were added from stocks according to a recipe in 18, 19. Desulfovibrio alaskensis G20 were

107

recovered from 1 mL freezer stocks in 10 mL anoxic basal medium in Hungate type tubes

108

(Bellco, Vineland, NJ, USA) with 1 g/L yeast extract and 1 mM sodium sulfide and

109

washed in basal medium to remove residual yeast extract prior to inoculation of

110

microplates for screening and dose-response growth experiments.

111

Marine enrichment cultures were passaged anoxic planktonic communities from

112

continuous flow reactor columns inoculated from marine sediments collected from San

113

Francisco Bay

114

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

115

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

116

stored as -80 °C glycerol stocks, recovered in seawater medium, and washed before

117

inoculation of cultures for experiments.

118

High-throughput screen

11

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

119

384 well microplates (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) were pre-

120

filled with 40 µL water using a Biotek EL406 microplate dispenser (Biotek, Winooski,

121

VT, USA).

122

ChemBridge Premium Diversity (ChemBridge, San Diego, CA, USA) compound

123

libraries were added to pre-filled microplates from 10 mM DMSO stocks by 4 iterations

124

of a 50 nL pinning tool using a Biomek FxP liquid handling robot (Beckman Coulter,

125

Pasadena, CA USA). The Microsource Pharmakon collection contains many compounds

126

with potent antimicrobial activity and known modes of action.

127

Premium Diversity collection contains a diverse set of organic scaffolds and, as such, is

128

good source of lead compounds. Plates containing compounds in water were made

129

anoxic by storing the plates in an anaerobic chamber (COY, Grass Lake, MI, USA) for 48

130

hours prior to inoculation with cultures. Microbial cells were added in an anaerobic

Microsource Pharmakon (Microsource, Gaylordsville, CT, USA) and


The ChemBridge

5


Page 6 of 25

131

chamber using a 384-well aspiration manifold connected to a bottle-top dispenser VP

132

179A (V&P Scientific, Irvine, CA, USA) or a BioMek 1000 liquid handling robot

133

(Beckman Coulter, Pasadena, CA USA). 40 µL of 2x microbial cultures in 2x media was

134

added to compounds in 384 well plates to obtain a final optical density of OD 0.02, a

135

compound concentration of 25 µM and a final volume of 80 µL. Positive controls for

136

screens were metronidazole (100 µM) which completely inhibited growth in all cultures.

137

Negative controls were 0.25% DMSO in water. Microplates were sealed with PCR plate

138

seals (Thermo Fisher Scientific, Waltham, MA, USA) and kept in anoxic BD GasPak

139

anaerobic boxes (BD, Franklin Lakes, NJ, USA) and incubated at 30 °C. Growth was

140

monitored by measuring optical density at 600 nm (Growth, OD 600) and sulfide was

141

monitored using the Cline colorimetric assay and measuring optical density at 660 nm

142

(Sulfide, OD 660)

143

contained 10 mL sulfuric acid, 0.5 g zinc acetate and 0.5 g N,N dimethyl-p-phenylene

144

diamine sulfate per 500 mL of water. Cline reagent B contained 2.5 g FeCl3 • 6H2O in

145

500 mL water (all chemicals from Sigma-Aldrich, St. Louis, MO, USA). Assay reagents

146

were stored at 4 °C for up to 1 month.

147

transferred into 384 well plates containing 20 µL of Cline reagent A. 20 µL of Cline

148

reagent B was added and color was allowed to develop for 10 minutes before reading

149

absorbance at 660 nm.

150

sulfide. Plate absorbance reads were recorded using a Tecan M1000 Pro microplate

151

reader (Tecan Group Ltd., Männendorf, Switzerland). A 48-hour timepoint was chosen

152

for screening and dose-response as this timepoint represents early stationary phase for D.

153

alaskensis G20 and for the bulk growth of the marine enrichment culture. Primary screen

154

results were analysed using the UCSF SMDC web application called HiTS

155

(http://smdc.ucsf.edu/hits/). This web-based analysis platform provides a data analysis

156

environment for viewing primary screen and dose-response results. Screen hits represent

157

compounds that gave B-scores > 3 standard deviations from the mean of B-score values.

158

B-scores are a measure of inhibition that takes into account “within plate” systematic

159

effects

160

Bioconductor bioinformatics software package

161

compounds were further clustered by Tanimoto similarity coefficient using Dotmatics

21

20

.

Cline assays were conducted as follows:

Cline reagent A

For each assay, 2.5 µL of cultures were

Control marine enrichments reproducibly produced 15 mM

and software to calculate this can be accessed through the open-source 22

.

The Microsource Pharmakon


6

Page 7 of 25


162

Vortex software (Dotmatics, San Diego, CA, USA).

163

confirmed in dose-response secondary screens against D. alaskensis G20 and the marine

164

enrichment culture. Dose-response plates for screen hits were prepared with eight 2-fold

165

serial dilutions starting from 100 µM.

All putative screen hits were

166

Data analysis for dose-response inhibition experiments was carried out in

167

GraphPad Prism 6 (GraphPad, San Diego, CA, USA) and curves were fit to a standard

168

inhibition log dose-response curve to generate an IC50 value. 95% Confidence intervals

169

are reported and all IC50s are the mean of at least three biological replicates. All dose-

170

response experiments are based on measurement of growth or sulfide at 48 hours for

171

microplate cultures inoculated at an initial OD 600 of 0.02. The IC50 values are the

172

concentration at which 50% of the microbial process (e.g. growth, sulfide production) is

173

inhibited relative to uninhibited control cultures containing 0.25% DMSO and fully

174

inhibited cultures containing 100 µM metronidazole.

175

counterscreens were confirmed by ANOVA comparison of IC50s. Synergy was assessed

176

using the equation for Fractional Inhibitory Concentration Index (FICI) based on the IC50

177

for each inhibitor A and B in the absence (IC50(A), IC50(B)) or presence of the other

178

inhibitor (IC50(AB), IC50(BA)):

Selective compounds in

179 180

FICI = FICA + FICB = IC50(AB)/IC50(A) + IC50(BA)/IC50(B).

181 182 183

A FICI score < 0.5 implies synergism, whereas a FICI score > 2 implies antagonism. An FICI between 1 and 2 implies indifference 23.

184


7


185

Page 8 of 25

Results and Discussion

186

We developed a high-throughput screening strategy to identify potent and specific

187

inhibitors of SRM (Figure 1A). The strategy begins with a primary screen against

188

Desulfovibrio alaskensis G20, a model oil field SRM isolated from a producing oil

189

reservoir in Ventura County, CA

190

screened the Microsource Pharmakon Collection (Figure 1B) and ChemBridge Premium

191

Diversity collection (Figure 1C) obtaining a hit rate of 2.86% (55 hits from 1,920

192

compounds) and 0.4% (111 hits from 30,000 compounds) respectively. Screen hits were

193

filtered using two separate counter-screens based on dose-response to confirm hits and

194

quantify potency and selectivity against SRM. We also quantified the potency and

195

selectivity for several additional common biocides and antibiotics not identified in the

196

primary screen, because of their importance and widespread use in industrial systems

197

(Table 1, Dataset S1).

198

24

, at a compound concentration of 25 µM.

We

Counter-screens against a cell line that overproduces the presumed target of an 25

199

inhibitor candidate are often used to confirm the inhibitor:target interaction

200

counter-screen A (Figure 1A) we compare the inhibitory potency of screen hits against a

201

parental D. alaskensis G20 strain and a D. alaskensis G20 strain with a transposon

202

insertion in a transcriptional repressor of the central pathway of sulfate reduction

203

(tn5::rex, tn5::dde_2702, Rex mutant)

204

Rex regulon which contains central enzymes involved in sulfate reduction including

205

qmoABCD (Dde_1111:Dde_1114), ATP sulfurylase (sat) (Dde_2265), adenylate kinase

206

(Dde_2028), pyrophosphatase (Dde_1178), a sulfate transporter (Dde_2406), an ATP

207

synthase, atpFFHAGD (Dde_0990:Dde_0984) and atpIIBE (Dde_2698:Dde_2701)

208

We and others have demonstrated that tn5::rex strains are resistant to competitive

209

inhibitors of sulfate reduction including nitrate 12, 26, chlorate 12 and perchlorate 12, but not

210

futile substrates of Sat such as molybdate or selenate 12.

211

26, 27

.

In

. The tn5::rex strain overproduces the core

27

.

We did not identify any screening hits to which tn5::rex strains were resistant

212

(Figure 2A).

213

inhibitors of Rex mutants compared to wild-type G20 cells (Figure 2A). While the exact

214

basis for this frequent sensitivity of tn5::rex rex strains to chemical stressors is unknown,

215

it is plausible that there is a subtle energetic penalty associated with overexpression of the

Rather, many antibiotics, biocides and screen hits were more potent


8

Page 9 of 25


216

sulfate reduction pathway that only becomes detrimental for growth when chemical

217

stressors which require active efflux or repair to damaged cellular components are

218

introduced. Importantly, this observation suggests that spontaneous mutations in rex

219

during nitrate or perchlorate treatment are unlikely to be competitive in the environment

220

in the presence of other inhibitory small molecules. Another important consideration is

221

that cell permeable competitive inhibitors of the sulfate reduction pathway analogous to

222

the oxyanions nitrate, chlorate and perchlorate

223

and therefore compounds to which tn5::rex strains are resistant will be rare. Current

224

work in our laboratory is aimed at developing high-throughput screening strategies to

225

identify potent inhibitors of the Sat enzyme. These screens are likely to yield competitive

226

inhibitors of Sat to which tn5::rex strains will be resistant.

12, 13

are likely rare in compound libraries

227

While tn5::rex strains are a useful counter screen to identify competitive

228

inhibitors of the sulfate reduction pathway, SRM have other conserved enzymes unique

229

to the sulfate-reduction pathway outside of the Rex regulon

230

candidates must retain potency and selectivity against SRM in the context of a complex

231

microbial ecosystem. Also, tn5::rex strains are not resistant to inorganic oxyanions that

232

are futile substrates of Sat (i.e. molybdate) even though these compounds are very

233

selective against sulfidogenesis in marine enrichments

234

compounds against D. alaskensis G20 pure cultures are not always well correlated with

235

inhibitory potency against sulfidogenesis (Figure 2B).

13

28

, and, ultimately, inhibitor

. Finally, inhibitory potency of

236

In counter screen B, we compare the IC50s against growth of a marine enrichment

237

culture as measured by OD 600 with IC50s against sulfide production as measured using

238

the colorimetric Cline assay (Expermental Section for details) (Figure 1A, Figure 2C).

239

Previously, we found that several inorganic oxyanions including molybdate, nitrate,

240

perchlorate and monofluorophosphate are selective inhibitors of sulfide production

241

We quantify selectivity as a selectivity index which is the ratio of the IC50s against

242

growth and sulfide production (SI = growth IC50/sulfide IC50). For example, molybdate

243

has a SI of 100, indicating that sulfidogenesis is 100-fold more sensitive to MoO4=

244

relative to general microbial growth in our marine enrichment culture 13.

13

.

245

We identified several known antibacterial compounds in our primary screen

246

against D. alaskensis G20 in a diversity of compound classes (Figure 3, Dataset S1).


9


Page 10 of 25

247

These compounds have a range of potencies and selectivities for inhibition of D.

248

alaskensis G20 and for inhibition of growth and sulfidogenesis in the marine enrichment

249

culture (Figure 2, Figure 3). Furthermore, different compound classes displayed differing

250

potencies against D. alaskensis G20 (Figure 3A) and sulfidogenesis (Figure 3B) in the

251

marine enrichment (Figure 3, Figure 2B). Perhaps because our primary screen was

252

carried out against a Gram-negative bacterium (D. alaskensis G20) some hit compounds

253

are antibiotics with known selectivity towards Gram-negative bacteria 29, 30. These screen

254

hits include several fluoroquinolones

255

clinafloxacin and nalidixic acid and the aminoglycosides

256

(Table 1, Figure 4).

257

Desulfovibrio, and there are Gram-positive bacteria (i.e. Clostridiales)

258

community which are likely more resistant to fluoroquinolones and aminoglycosides.

259

The most selective inhibitor of sulfate reduction was lincomycin, a lincosamide

260

antibiotic. The basis for this selectivity is unclear, as lincosamides, like macrolides,

261

target the ribosome

262

32

positives . Of note, clindamycin, a structurally similar lincosamide, was not a selective

263

inhibitor of sulfidogenesis (Dataset S1). Such divergent activity for clindamycin and

264

lincomycin has been previously observed, but is not well understood

265

compounds differ only by a single R group substitution of a chlorine atom (clindamycin)

266

for a hydroxyl (lincomycin) 32.

29

including lomefloxacin, ciprofloxacin, 30

gentimicin and kanamycin

The dominant SRM in the marine microbial enrichment is a

31, 32

12

in the

and have been reported to be selective inhibitors of Gram-

32

as these

267

Several nitroimidazoles were among the most potent inhibitors of D. alaskensis

268

G20 and sulfidogenesis in the marine enrichment (Figure 3, Table 1, Dataset S1). These

269

compounds are known to have selectivity against anaerobic bacteria and eukaryotes

270

and become antimicrobial through reduction of the nitro group by anaerobic redox active

271

respiratory enzymes

272

electron transport proteins

273

through substrate level phosphorylation and do not require periplasmic electron flow.

274

Thus, anaerobic sulfate respirers may be more susceptible to nitroimidazoles or other

275

compounds that require redox activation. Consistent with this hypothesis, secnidazole

276

and dimetridazole displayed some selectivity against sulfide production in our enrichment

277

cultures (Table 1, Figure 4D). Nitroimidazoles have been considered as strategies to

33

.

33, 34

Sulfate-reducing bacteria contain a number of periplasmic 28, 35

, whereas fermentative bacteria primarily generate energy


10

Page 11 of 25


8, 34

278

inhibit sulfidogenesis in oil reservoirs

279

inhibition of sulfate reduction versus fermentation in a natural system has been lacking.

280

, but until now evidence of their selectivity for

Also of note, some compounds displayed selectivity in favor of sulfide production

281

in our microbial enrichment cultures.

282

clinafloxacin (Dataset S1) inhibited growth to less than 50% of controls while allowing

283

sulfide production to persist.

284

industrial systems. This observation underscores the importance of a dose-response assay

285

such as counterscreen B. An uninformed choice of a biocide could result in a treatment

286

strategy that drives an industrial system towards sulfidogenesis.

Meclocycline (Figure 4E, Dataset S1) and

Such compounds may favor the growth of SRM in

287

From the ChemBridge Premium collection, we identified a number of 1,2,4-

288

oxadiazoles that are potent inhibitors of D. alaskensis G20 (Supplemental Dataset S1,

289

Figure 3A). Many oxadiazoles are known antimicrobials

290

low micromolar inhibitors of G20 may warrant further investigation. However, we did

291

not observe correspondingly potent inhibition of either growth or sulfide production in

292

the counter screen against the marine enrichment culture. This result highlights the

293

importance of counter screens that evaluate a compound in the context of a microbial

294

community.

36

, and their identification as

295

In the oil industry, a number of biocides are used to control sulfide production and

296

SRM growth in pipelines. We tested a panel of common biocides to rank their potency

297

against sulfide production (Dataset S1). We observed that glutaraldehyde, methenamine,

298

2,2-dibromo-cyanoacetamide (DBNPA), 2-methyl-4-iosthiazolin-3-one (MIT) and zinc

299

pyrithione were selective inhibitors of sulfide production (Table 1, Dataset S1, Figure 4)

300

whereas formaldehyde and tetrakis(hydroxymethyl)phosphonium sulfate (THPS) were

301

not (Dataset S1). Some of this selectivity may be explained by selectivity of these

302

compounds for Gram-negative bacteria.

Gram-positive fermentative bacteria in the

303

marine enrichment culture (e.g. Clostridia)

12, 13

304

some advantage in resistance to less cell permeable inhibitors.

305

glutaraldehyde forms oligomers that more slowly transit cell envelopes

306

formaldehyde. Only two compounds in our primary screening hits are commonly used as

307

biocides in the oil and gas industry, methenamine and zinc pyrithione 8 (Dataset S1). All

with a thicker cell envelope may have


For example, 37

compared to

11


Page 12 of 25

308

of the other biocides we tested were less potent relative to these two compounds, with

309

IC50s above our primary screening concentration of 25 µM (Dataset S1).

310

Zinc pyrithione was the most potent inhibitor of sulfidogenesis in our marine

311

microbial community and is moderately selective. Pyrithione is a multifaceted compound

312

that functions as a both protonophore 38 and a chelator that can also carry metals such as

313

copper into the cytoplasm of microbial cells

314

abundance in Desulfovibrio species 41

40

39

.

Zinc and copper are both in low

, and these and other chalcophile elements can 39

315

deplete glutathione

316

important Fe-S containing proteins. We hypothesize that both the metal and ligand are

317

important for the activity of zinc pyrithione in our marine enrichment culture. Consistent

318

with this hypothesis, when applied as a sodium salt, pyrithione is synergistic with zinc

319

and copper (Figure 5A, B) for inhibition of sulfidogenesis. Interestingly, copper displays

320

some selectivity against sulfidogenesis (Figure 5C), but zinc does not (Figure 5D). The

321

precise basis for the selectivity of zinc pyrithione remains obscure, but it may be a

322

general SRM selective biocide.

323

. Copper can damage Fe-S clusters

, and SRM have abundant

Our results demonstrate that a high-throughput screening strategy can be applied

324

to target a specific microbial sub-population in a natural community.

325

compounds display little or no selectivity against sulfidogenesis, some compounds are

326

selective. The context of our marine microbial enrichment may determine the efficacy

327

and selectivity of a given compound. For example, the sulfate reducer in this system is a

328

Gram-negative Desulfovibrio, and as such, antimicrobials that have selectivity against

329

Gram-negatives are likely enriched in our screening hits.

330

methodology can be adapted to other microbial enrichment cultures. For example, higher

331

temperatures, salinities, pH values, alternative carbon sources, or alternative electron

332

donors can be used in place of our conditions. Additionally, other microbial inocula,

333

such as those present in wastewater treatment facilities or oil reservoirs can be used to

334

identify selective treatment strategies tailored to these environments. Furthermore, the

335

approach of assaying the selectivity of compounds for a microbial sub-population with a

336

counter-screen against respiratory end-products or respiratory enzymes can be extended

337

to other respiratory metabolisms. Identifying new metabolism specific inhibitors from

338

small molecule libraries or panels of naturally occurring compounds could provide


While many

However, our screening

12

Page 13 of 25


339

insights into how biogeochemical context structures microbial communities. Finally,

340

while our screen hits represent promising leads for field application, evaluating

341

compound biogeoavailability in column studies, mesocosms, and pilot studies remains

342

important for validating any treatment strategy for an industrial ecosystem

343 344

Acknowledgements

345

We would like to thank members of the Coates Laboratory and the UC Berkeley Energy

346

Biosciences Institute for critical comments and suggestions. We particularly like to thank

347

John Pierce, BP (currently Devenir consulting) for early support and encouragement.

348

Work in the laboratory of JDC on biosouring is supported by the UC Berkeley Energy

349

Biosciences Institute.

350 351 352 353 354 355


13


356 357

Page 14 of 25

Table 1. Selective inhibitors of growth and sulfidogenesis in marine enrichment cultures identified in this study. Compound attributes Name lincomycin hydrochloride clinafloxacin hydrochloride

IC50 (95% CI) against marine enrichment cultures (µ µM)

Compound class

Putative target

Growth OD 600

Sulfide OD 660

Selectivity index (Growth IC50: Sulfide IC50)

lincosamide

protein biosynthesis

77 (54-109)

5.89 (3.8-9.3)

13.1 7.4

fluoroquinolone

DNA gyrase

103 (88-121)

13.96 (5.51435.37)

lomefloxacin

fluoroquinolone

DNA gyrase

410 (240-720)

120 (76-200)

3.4

ciprofloxacin

fluoroquinolone

DNA gyrase

360 (270-480)

83 (63-110)

4.3

nalidixic acid

fluoroquinolone

DNA gyrase

1700 (1000-2600)

260 (180-360)

6.5

gentimycin

aminoglycoside


1400 (860-2200)

280 (200-380)

5

kanamycin

aminoglycoside


850 (570-1300)

230 (150-350)

3.7

secnidazole

nitroimidazole

releases nitro radical

12 (9-15)

6.211 (5.4837.036)

1.9

dimetridazole

nitroimidazole

releases nitro radical

40 (31-52)

11 (7.4-17)

3.6

allopurinol

purine analog

unknown

620 (440-870)

110 (75-160)

5.6

mupirocin

pseudomonic acid


>100

24.66 (14.4442.11)

>4

zinc pyrithione

biocide

unknown

0.905 (0.901-1.2)

0.4 (0.32-0.5)

2.3

methenamine

biocide

reactive electrophile

>100

64 (19-213)

>1.5

glutaraldehyde

biocide


>3000

440 (350-560)

>6.8

biocide

releases NH4+ and Br-

3800 (1700-8900)

780 (580-1100)

4.9

biocide


2200 (1400-3400)

680 (590-790)

3.2

2,2,-dibromo-2cyanoacetamide (DBNPA) 2-methyl-4isothiazolin-3-one (MIT)

358 359


14

Page 15 of 25

360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401


Figure Captions: Figure 1. (A) Screening strategy to identify sulfate reduction specific inhibitors. (B-C) Results of primary screen against (B) the Microsource Pharmakon Collection and (C) the ChemBridge Premium Diversity collection. Lines representing 1, 2 and 3 standard deviations from the average b-score are shown in green, blue and red respectively. Screen hits are compounds with b-scores greater than 3 standard deviations from the mean (Materials and Methods). The Microsource Pharmakon collection is clustered by Tanimoto similarity coefficient (Materials and Methods). Figure 2. IC50ss from dose-response counterscreens. Triangles represent oxyanions and circles represent screening hits, selected biocides and antibiotics. (A) IC50s for D. alaskensis G20 (WT) and tn5::rex mutant (Rex) plotted against each other. Solid symbols represent non-selective compounds and open symbols represent selective compounds for which WT and Rex IC50s are significantly different (ANOVA). (B) IC50s for D. alaskensis G20 (WT) and sulfidogenesis in a marine enrichment culture plotted against each other. C. IC50s for inhibition of growth (growth) and sulfidogenesis (sulfide) in a marine enrichment culture plotted against each other. Solid symbols represent non-selective compounds and open symbols represent selective compounds for which growth and sulfide IC50s are significantly different (ANOVA). Figure 3. Comparison of IC50s by compound class for screening hits. (A) IC50s for D. alaskensis G20. (B) IC50s for inhibition of sulfidogenesis in a marine enrichment culture. Figure 4. Selected dose-response curves for screening hits, selected biocides and antibiotics. Open symbols represent sulfide production and closed symbols represent growth relative to control cultures in which no inhibitor was added. Figure 5. (A-B) Isobolograms showing synergistic inhibition of sulfidogenesis in marine enrichment cultures by combinations of (A) zinc and pyrithione, FICI = 0.49 and (B) copper and pyrithione, FICI = 0.23. (C-D) Dose-response curves for (C) copper and (D) zinc for inhibition of sulfide production (open symbols) and growth (closed symbols) relative to control cultures in which no inhibitor was added. Table 1. Selective inhibitors of growth and sulfidogenesis in marine enrichment cultures identified in this study. Dataset S1. Compound information and dose-response data from counterscreens for screening hits, selected antibiotics and biocides.


15


Page 16 of 25

402 403 404

References 1. Tietjen, K.; Drewes, M.; Stenzel, K., High throughput screening in agrochemical research. CCHTS 2005, 8 (7), 589-94.

405 406 407

2. Arkin, M. R.; Ang, K. K.; Chen, S.; Davies, J.; Merron, C.; Tang, Y.; Wilson, C. G.; Renslo, A. R., UCSF Small Molecule Discovery Center: innovation, collaboration and chemical biology in the Bay Area. CCHTS 2014, 17 (4), 333-42.

408 409

3. Derbyshire, E. R.; Prudencio, M.; Mota, M. M.; Clardy, J., Liver-stage malaria parasites vulnerable to diverse chemical scaffolds. PNAS 2012, 109 (22), 8511-6.

410 411

4. Hughes, D.; Andersson, D. I., Selection of resistance at lethal and non-lethal antibiotic concentrations. Curr. Op. microbiol. 2012, 15 (5), 555-60.

412 413

5. Oremland, R. S.; Capone, D. G., Use of specific inhibitors in biogeochemistry and microbial ecology. Adv. Microb. Ecol. 1988, 10, 285-383.

414 415

6. Gieg, L. M.; Jack, T. R.; Foght, J. M., Biological souring and mitigation in oil reservoirs. Appl. Microbiol. Biotech. 2011, 92 (2), 263-282.

416 417 418

7. Sunde, E.; Torsvik, T., Microbial control of hydrogen sulfide production in oil reservoirs. In Petroleum Microbiology, Ollivier, B.; Magot, M., Eds. ASM Press: Washington D.C., 2005.

419 420

8. Fink, J. K., Petroleum Engineer's Guide to Oil Field Chemicals and Fluids. Petroleum Engineer's Guide to Oil Field Chemicals and Fluids 2012, 1-785.

421 422 423

9. Vance, I.; Thrasher, D. R., Reservoir souring: mechanisms and prevention. In Petroleum Microbiology, Ollivier, B.; Magot, M., Eds. ASM Press: Washington, DC, 2005.

424 425 426 427

10. Gregoire, P.; Engelbrektson, A.; Hubbard, C. G.; Metlagel, Z.; Csencsits, R.; Auer, M.; Conrad, M. E.; Thieme, J.; Northrup, P.; Coates, J. D., Control of sulfidogenesis through bio-oxidation of H2S coupled to (per)chlorate reduction. Environ. Microbiol. Rep. 2014, 6 (6), 558-564.

428 429 430 431

11. Engelbrektson, A.; Hubbard, C. G.; Tom, L. M.; Boussina, A.; Jin, Y. T.; Wong, H.; Piceno, Y. M.; Carlson, H. K.; Conrad, M. E.; Anderson, G.; Coates, J. D., Inhibition of microbial sulfate reduction in a flow-through column system by (per)chlorate treatment. Front. Microbiol. 2014, 5, 315.

432 433 434 435 436

12. Carlson, H. K.; Kuehl, J. V.; Hazra, A. B.; Justice, N. B.; Stoeva, M. K.; Sczesnak, A.; Mullan, M. R.; Iavarone, A. T.; Engelbrektson, A.; Price, M. N.; Deutschbauer, A. M.; Arkin, A. P.; Coates, J. D., Mechanisms of direct inhibition of the respiratory sulfate-reduction pathway by (per)chlorate and nitrate. ISME J. 2014, DOI 10.1038/ismej.2014.216.


16

Page 17 of 25


437 438 439 440

13. Carlson, H. K.; Stoeva, M. K.; Justice, N. B.; Sczesnak, A.; Mullan, M. R.; Mosqueda, L. A.; Kuehl, J. V.; Deutschbauer, A. M.; Arkin, A. P.; Coates, J. D., Monofluorophosphate is a selective inhibitor of respiratory sulfate-reducing microorganisms. Environ. Sci. Technol. 2015, 49 (6), 3727-36.

441 442

14. Saleh, A. M., Differences in the Resistance of Sulphate-Reducing Bacteria to Inhibitors. J Gen. Microbiol. 1964, 37, 113-21.

443 444

15. Cordova-Kreylos, A. L.; Scow, K. M., Effects of ciprofloxacin on salt marsh sediment microbial communities. ISME J 2007, 1 (7), 585-95.

445 446

16. Kelland, M. A., Production chemicals for the oil and gas industry. Second edition. ed.; CRC Press: Boca Raton, 2014; p xvii, 436 pages.

447 448

17. Lozniewski, A.; Labia, R.; Haristoy, X.; Mory, F., Antimicrobial susceptibilities of clinical Desulfovibrio isolates. Antimicrob Agents Chemother 2001, 45 (10), 2933-5.

449 450 451

18. Price, M. N.; Deutschbauer, A. M.; Skerker, J. M.; Wetmore, K. M.; Ruths, T.; Mar, J. S.; Kuehl, J. V.; Shao, W.; Arkin, A. P., Indirect and suboptimal control of gene expression is widespread in bacteria. Mol. Syst. Biol. 2013, 9, 660.

452 453 454 455 456

19. Mukhopadhyay, A.; He, Z.; Alm, E. J.; Arkin, A. P.; Baidoo, E. E.; Borglin, S. C.; Chen, W.; Hazen, T. C.; He, Q.; Holman, H. Y.; Huang, K.; Huang, R.; Joyner, D. C.; Katz, N.; Keller, M.; Oeller, P.; Redding, A.; Sun, J.; Wall, J.; Wei, J.; Yang, Z.; Yen, H. C.; Zhou, J.; Keasling, J. D., Salt stress in Desulfovibrio vulgaris Hildenborough: an integrated genomics approach. J. Bacteriol. 2006, 188 (11), 4068-78.

457 458

20. Cline, J. D., Spectrophotometric Determination of Hydrogen Sulfide in Natural Waters. Limnol. Oceanogr. 1969, 14 (3), 454-&.

459 460 461 462

21. Birmingham, A.; Selfors, L. M.; Forster, T.; Wrobel, D.; Kennedy, C. J.; Shanks, E.; Santoyo-Lopez, J.; Dunican, D. J.; Long, A.; Kelleher, D.; Smith, Q.; Beijersbergen, R. L.; Ghazal, P.; Shamu, C. E., Statistical methods for analysis of high-throughput RNA interference screens. Nat. Meth. 2009, 6 (8), 569-75.

463 464

22. Boutros, M.; Bras, L. P.; Huber, W., Analysis of cell-based RNAi screens. Gen. Biol. 2006, 7 (7), R66.

465 466 467 468

23. European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical, M.; Infectious, D., EUCAST Definitive Document E.Def 1.2, May 2000: Terminology relating to methods for the determination of susceptibility of bacteria to antimicrobial agents. Clin. Microbiol. Infect. 2000, 6 (9), 503-8.

469 470 471

24. Wall, J. D.; Rapp-Giles, B. J.; Rousset, M., Characterization of a small plasmid from Desulfovibrio desulfuricans and its use for shuttle vector construction. J. Bacteriol. 1993, 175 (13), 4121-8.


17


Page 18 of 25

472 473

25. Silver, L. L., Challenges of antibacterial discovery. Clin. Microbiol. Rev. 2011, 24 (1), 71-109.

474 475 476

26. 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.

477 478 479 480

27. 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.

481 482 483

28. Pereira, I. A.; Ramos, A. R.; Grein, F.; Marques, M. C.; da Silva, S. M.; Venceslau, S. S., A comparative genomic analysis of energy metabolism in sulfate reducing bacteria and archaea. Front Microbiol 2011, 2, 69.

484 485

29. Oliphant, C. M.; Green, G. M., Quinolones: a comprehensive review. Am. Fam. Phys. 2002, 65 (3), 455-64.

486 487

30. Gonzalez, L. S., 3rd; Spencer, J. P., Aminoglycosides: a practical review. Am. Fam. Phys. 1998, 58 (8), 1811-20.

488 489 490

31. Leclercq, R., Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 2002, 34 (4), 482-92.

491 492

32. Spizek, J.; Rezanka, T., Lincomycin, clindamycin and their applications. Appl Microbiol. Biotechnol. 2004, 64 (4), 455-64.

493 494

33. Edwards, D. I., Nitroimidazole drugs--action and resistance mechanisms. I. Mechanisms of action. J Antimicrob. Chemother. 1993, 31 (1), 9-20.

495 496

34. Wiatr, C. L.; Fedyniak, O. X., Development of an obligate anaerobe specific biocide. J Ind Microbiol 1991, 7 (1), 7-13.

497 498 499

35. Price, M. N.; Ray, J.; Wetmore, K. M.; Kuehl, J. V.; Bauer, S.; Deutschbauer, A. M.; Arkin, A. P., The genetic basis of energy conservation in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. Front Microbiol 2014, 5, 577.

500 501 502 503 504 505

36. O'Daniel, P. I.; Peng, Z.; Pi, H.; Testero, S. A.; Ding, D.; Spink, E.; Leemans, E.; Boudreau, M. A.; Yamaguchi, T.; Schroeder, V. A.; Wolter, W. R.; Llarrull, L. I.; Song, W.; Lastochkin, E.; Kumarasiri, M.; Antunes, N. T.; Espahbodi, M.; Lichtenwalter, K.; Suckow, M. A.; Vakulenko, S.; Mobashery, S.; Chang, M., Discovery of a new class of non-beta-lactam inhibitors of penicillin-binding proteins with Gram-positive antibacterial activity. J. Am. Chem. Soc. 2014, 136 (9), 3664-72.

506 507

37. Monsan, P.; Puzo, G.; Mazarguil, H., Mechanism of glutaraldehyde-protein bond formation. Biochimie 1975, 57 (11-12), 1281-92.


18

Page 19 of 25


508 509 510

38. Chandler, C. J.; Segel, I. H., Mechanism of the antimicrobial action of pyrithione: effects on membrane transport, ATP levels, and protein synthesis. Antimicrob Agents Chemother 1978, 14 (1), 60-8.

511 512 513 514 515

39. Reeder, N. L.; Kaplan, J.; Xu, J.; Youngquist, R. S.; Wallace, J.; Hu, P.; Juhlin, K. D.; Schwartz, J. R.; Grant, R. A.; Fieno, A.; Nemeth, S.; Reichling, T.; Tiesman, J. P.; Mills, T.; Steinke, M.; Wang, S. L.; Saunders, C. W., Zinc pyrithione inhibits yeast growth through copper influx and inactivation of iron-sulfur proteins. Antimicrob Agents Chemother 2011, 55 (12), 5753-60.

516 517 518 519 520

40. Lancaster, W. A.; Menon, A. L.; Scott, I.; Poole, F. L.; Vaccaro, B. J.; Thorgersen, M. P.; Geller, J.; Hazen, T. C.; Hurt, R. A., Jr.; Brown, S. D.; Elias, D. A.; Adams, M. W., Metallomics of two microorganisms relevant to heavy metal bioremediation reveal fundamental differences in metal assimilation and utilization. Metallomics : integrated biometal science 2014, 6 (5), 1004-13.

521 522 523

41. Kushkevych, I. V.; Hnatush, S. O.; Mutenko, H. V., Glutathione level of Desulfovibrio desulfuricans IMV K-6 under the influence of heavy metal salts. Ukrains'kyi biokhimichnyi zhurnal 2011, 83 (6), 104-9.

524


19



Page 20 of 25

Page 21 of 25


Figure 2 57x18mm (600 x 600 DPI)





Page 22 of 25

Page 23 of 25







Page 24 of 25

Environmental Science & Technology Sulfidogenic High Throughput Screen

(Millions of potential targets)

Compound Libraries

384-well Primary Screen (Inhibitor ID) Candidate inhibitors % maximum OD 600 at 48 hours

Page 25 of 25

Dose response

100 80

Perchlorate

60 40

(Potency)

20 0

-4

-3

-2

-1

0

log[Inhibitor], M

384-well Secondary Screen

Growth IC50

Sulfide IC50 (Growth IC50/Sulfide IC50 = Selectivity)


Specific inhibitors

High-throughput screening to identify potent and ... - ACS Publications

Recommend Documents