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Of note, clindamycin, a structurally similar lincosamide, was not a selective. 262 inhibitor of sulfidogenesis (Dataset S1). Such divergent activity f...
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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

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Environmental Science & Technology

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

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sulfate reduction

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Running title: Sulfate reduction high-throughput screen

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

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

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1

Energy Biosciences Institute, UC Berkeley

10

2

Physical Biosciences Division, Lawrence Berkeley National Lab

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3

Small Molecule Discovery Center, UC San Francisco

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4

Earth Sciences Division, Lawrence Berkeley National Lab

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5

Department of Plant and Microbial Biology, UC Berkeley

14 15 16

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

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

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

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Keywords:

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screening, hydrogen sulfide, souring

sulfate-reducing microorganisms, selective inhibitor, high-throughput

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Abstract

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The selective perturbation of complex microbial ecosystems to predictably influence

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outcomes in engineered and industrial environments remains a grand challenge for

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

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

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

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

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has quantitatively assessed the selectivity of putative souring treatments.

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

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

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identified previously unrecognized SRM selective inhibitors and synergistic interactions

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

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

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

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

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

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

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

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role of natural gradients in shaping microbial niche space.

We have

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Environmental Science & Technology

Introduction

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

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

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health and agriculture

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

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throughput

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microorganisms.

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

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

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

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

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

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

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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

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

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function of microbial subpopulations. Some compounds are known selective inhibitors

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

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

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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

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be used at concentrations at which they are not selective.

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

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industry.

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

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

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

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approach to souring control that has been widely championed

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such as perchlorate

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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

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

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

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

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

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14-16

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

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SRM in general is largely unknown

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

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and identified monofluorophosphate as a selective inhibitor of sulfidogenesis that has

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

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throughput screening strategy to screen small molecule libraries to identify specific

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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

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

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

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

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and inhibitor cocktails are used in oil recovery systems

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

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

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

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inhibitors of sulfidogenesis than commonly used biocides such as THPS or

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

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sulfidogenesis in our marine enrichment culture and displays some selectivity against

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SRM.

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

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compared with other biocides.

8, 16

, but the relative potency and

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

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Experimental Section

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Media and cultivation conditions

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Desulfovibrio alaskensis G20 was cultivated in anoxic basal Tris-buffered

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

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

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

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

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

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

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

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microplates for screening and dose-response growth experiments.

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Marine enrichment cultures were passaged anoxic planktonic communities from

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continuous flow reactor columns inoculated from marine sediments collected from San

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Francisco Bay

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

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

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

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inoculation of cultures for experiments.

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High-throughput screen

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

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

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

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VT, USA).

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

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

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

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

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with potent antimicrobial activity and known modes of action.

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

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

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

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

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

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The ChemBridge

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

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

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

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

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

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

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

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

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

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

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

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(Sulfide, OD 660)

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

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

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

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

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

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

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absorbance at 660 nm.

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

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

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

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

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results were analysed using the UCSF SMDC web application called HiTS

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

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

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

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

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effects

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Bioconductor bioinformatics software package

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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

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

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

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

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serial dilutions starting from 100 µM.

All putative screen hits were

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

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

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

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

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

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

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

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

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inhibited cultures containing 100 µM metronidazole.

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

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

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

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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.

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Results and Discussion

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

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

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

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reservoir in Ventura County, CA

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

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

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

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

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

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

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

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(Table 1, Dataset S1).

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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

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

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

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

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

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Rex regulon which contains central enzymes involved in sulfate reduction including

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

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

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

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

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

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futile substrates of Sat such as molybdate or selenate 12.

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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

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(Figure 2A).

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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,

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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

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sulfate reduction pathway that only becomes detrimental for growth when chemical

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

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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

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

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

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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

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

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to the sulfate-reduction pathway outside of the Rex regulon

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

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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

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selective against sulfidogenesis in marine enrichments

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

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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

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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).

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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

.

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We identified several known antibacterial compounds in our primary screen

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

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

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

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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).

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

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community which are likely more resistant to fluoroquinolones and aminoglycosides.

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

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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

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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

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

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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

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respiratory enzymes

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electron transport proteins

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

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

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

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and dimetridazole displayed some selectivity against sulfide production in our enrichment

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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

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8, 34

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inhibit sulfidogenesis in oil reservoirs

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

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, 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-

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

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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

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compounds for Gram-negative bacteria.

Gram-positive fermentative bacteria in the

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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

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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

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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

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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.

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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

protein biosynthesis

1400 (860-2200)

280 (200-380)

5

kanamycin

aminoglycoside

protein biosynthesis

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

protein biosynthesis

>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

reactive electrophile

>3000

440 (350-560)

>6.8

biocide

releases NH4+ and Br-

3800 (1700-8900)

780 (580-1100)

4.9

biocide

reactive electrophile

2200 (1400-3400)

680 (590-790)

3.2

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

358 359

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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

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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.

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Figure 2 57x18mm (600 x 600 DPI)

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Figure 3 40x19mm (600 x 600 DPI)

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Figure 4 65x24mm (600 x 600 DPI)

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Figure 5 56x37mm (600 x 600 DPI)

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(Millions of potential targets)

Compound Libraries

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

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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)

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Specific inhibitors