Supplementary Methods
Cultivation conditions in microplates
For cultivation of Desulfovibrio in microplates, plates were inoculated in an anaerobic
chamber (Coy). Desulfovibrio were resuspended in 2x concentrated anoxic basal media
containing 2 mM sodium sulfide and added at a 2x dilution to microplates containing
water or compounds. Nitrate, nitrite, perchlorate, and chlorate were sodium salts (Sigma).
DETANONOate (Cayman) is a nitric oxide donor with a 56 hour half life at 22-25 C at
pH 7.4, but stable in base. Stocks in 0.1 M NaOH were added to plates or Hungate tubes
and serial dilutions made immediately prior to inoculation. Microplates were filled with
compounds aerobically using a Biomek FxP liquid handling robot (Beckman instruments)
and allowed to degas in Coy anaerobic chambers for 48 hours prior to inoculation. All
microplates were inoculated at an initial OD 600 of 0.02 and the growth rate determined
for timepoints between 16 and 48 hours. Microplates were sealed with PCR plate seals
(VWR) and kept in anoxic BD GasPak anaerobic boxes except when timepoints were
being recorded. Growth IC50 values were determined using fine dilutions of each stressor
over an order of magnitude in 96 and 384 well plates. Growth was monitored by
measuring optical density at 600 nm (OD 600) and sulfide (OD 660) was monitored using
the Cline colorimetric assay (Cline 1969).
Dose-response data analysis
Data analysis for inhibition experiments was carried out in GraphPad Prism 6 and
curves were fit to a standard inhibition log dose-response curve to generate an IC50 value.
95% Confidence intervals are reported and all IC 50s are the mean of at least three
biological replicates.
Except for pre-induction experiments, all dose-response
experiments are based on measurement of growth and sulfide at 48 hours for microplate
cultures inoculated at an initial OD 600 of 0.02. Synergy was assessed using the equation
for Fractional Inhibitory Concentration Index (FICI) based on the IC50 for each inhibitor
A and B in the absence (IC50(A), IC50(B)) or presence of the other inhibitor (IC50(AB),
IC50(BA))
FICI = FICA + FICB = IC50(AB)/IC50(A) + IC50(BA)/IC50(B). FICI < 0.5
implies synergism. FICI > 2 implies antagonism. An FICI between 1 and 2 implies
indifference (European Committee for Antimicrobial Susceptibility Testing of the
European Society of Clinical and Infectious 2000).
Cross-induction of resistance
Desulfovibrio alaskensis G20 was grown to an OD of 0.2-0.3, centrifuged and resuspended in triplicate microplates containing 2x dilutions of stressors and 1 mM sodium
sulfide at an initial OD 600 of 0.02. The initial growth rates were determined between 0
and 6 hours and used to calculate a growth IC50 for nitrate and (per)chlorate against pregrown cells.
16S rRNA gene amplicon sequencing of marine enrichment cultures
For 16S rRNA gene amplicon sequencing, marine enrichment cultures were
grown in 96-well plates in the presence of 2-fold serial dilutions of nitrate or perchlorate
(gradient plates). The gradient plate cultures were inoculated at an initial OD 600 of 0.02
in a volume of 200 L. After 48 hours (OD 600 ~ 0.3-0.4), cultures were harvested by
centrifugation, 180 L supernatant was removed, and genomic DNA was extracted from
the remaining pellet and supernatant using the Wizard SV 96 Genomic DNA Purification
System (Promega). Following quantification of genomic DNA (Quant-IT BR, Life
Technologies), the V3V4 variable region of the 16S rRNA gene was amplified from ~5
ng of each template in 48 separate PCR reactions. Each PCR reaction mix contained: 1X
Phusion buffer, 200 M of each deoxyribonucleotide triphosphate, 0.05M of each
primer, and 1 unit of Phusion DNA polymerase (Thermo Scientific) in a final volume of
20 L. Cycling conditions were 98°C for 30 s followed by 30 cycles of 98°C for 10 s,
50°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 7 min. Primers used to
amplify the V3V4 region are a unique combination of dual-indexed primers with attached
Ilumina adapters (Nicholas Justice, manuscript in preparation), and are generally similar
to previously published designs (Fadrosh et al 2014, Kozich et al 2013). The genespecific regions of the primers are 341F (5’- CCTACGGGAGGCAGCAG) and 806R
(5’- GGACTACHVGGGTWTCTAAT).
PCR products from each reaction were
normalized to 0.25 ng/L and pooled and concentrated using a Promega DNA Clean-up
kit. The concentration of the final sample was confirmed by qPCR and Qubit (Life
Technologies). The final yield of pooled PCR products was 20 L at a concentration of
31 nM. 10 L of 2 nM pooled PCR product was used for Illumina MiSeq sequencing.
Sequencing was done using the 600 bp V3 MiSeq kit (Illumina). Overlapping reads were
first merged using PEAR v0.9.1 (Zhang et al 2014), then quality-filtered, keeping reads
with fewer than two expected errors by summation of phred scores (94.85% of reads
passed filter). Quality reads were then demultiplexed, matching 5’ and 3’ barcodes in a
greedy fashion by allowing at most two mismatches. Demultiplexed reads then served as
input to the pick_de_novo_otus.py script of QIIME v1.7.0 (Caporaso et al 2010) to
quantify OTU relative abundances. Python code to perform this analysis is available on
GitHub at https://github.com/polyatail/arkin.
qPCR assay for quantifying dsrA
DNA was pooled from 4 replicate 96-well gradient plates (~800 L of culture)
and extracted using the Wizard SV Genomic DNA Purification System (Promega) as
described above. Quantification of genomic DNA was accomplished using a Quant-IT,
BR kit (Life Technologies) and used to normalize all samples to approximately 2ng/l.
PCR
amplification
was
performed
using
primers
DSR1-F+
(5’-
ACSCACTGGAAGCACGGCGG-3’) and DSR-R (5’-GTGGMRCCGTGCAKRTTGG3’) (Bourne et al 2011, Leloup et al 2007) with cycling conditions and primer
concentrations as described by Bourne et al. (Bourne et al 2011). The amplicon (221 bp)
was cloned (TOPO TA Kit, Invitrogen) and sequenced. All sequences were 90% identical
to the partial dsrA sequence of an uncultured sulfate-reducing bacterium (GenBank:
EF052879.1) and 90% identical to the Desulfovibrio desulfuricans ND132 dsrA partial
sequence. Taqman qPCR according to Bourne et al. (Bourne et al 2011) was used to
quantify dsrA using primers DSR1-F+ and DSR-R and Taqman probe DSRtaq: (HEX)-50
-CCGATAACRCYGCCGCCGTAACCGA-30 -(TAMRA). The cloned amplicon was
used to generate a standard curve, ranging from 3,000,000 to 300 copies of dsrA. All
samples and standards were run in triplicate on a StepOnePlus Real-Time PCR System
(Applied Biosystems) with primer/probe concentrations and cycling conditions as
described by Bourne et al. (Bourne et al 2011). The size of the qPCR products was
confirmed via gel electrophoresis. No non-specific amplification was observed.
Calculated r2 values for each run were 0.998. Negative controls were also run in
triplicate. Data analysis was done using the StepOnePlus Applied Biosystems software,
version 2.2. DNA concentrations in each sample were again quantified (Qubit, HS kits)
and dsrA copy number was normalized to the ng of total DNA added to each reaction.
The values were further normalized to % control: all uninhibited samples (12 biological
replicates) were averaged and set at 100%. The fully inhibited treatments were also
averaged and set to 0%. The normalized values were plotted in GraphPad Prism 6 and fit
to an inhibition dose-response curve to generate IC50 values. 95% confidence intervals
for the fits are reported as error.
Proteomics and data analysis
Cells for proteomics were harvested from 50 mL mid-log phase cultures (OD 0.30.5). Cells were pelleted anaerobically at 4000 rcf, supernatant was decanted and cells
were resuspended in 100 mM ammonium bicarbonate, pH 7.4, sonicated and digested
with trypsin according to the following procedure. 50 L of a 1 g/L lysate sample was
placed in a capped low-rentention microcentrifuge tube. 10 L of 100 mM NH4HCO3,
pH 7.5 was added along with 25 L of a 0.2% Rapigest SF solution. The sample was
placed in a block heater set to 80 C and heated for 15 minutes. The sample was
removed from the block heater, centrifuged and 10 L of 100 mM dithiothreitol was
added and the samples were heated to 60 C for 30 minutes. 10 L of 100 mM
iodoacetamide was added and the samples kept in the dark for 30 minutes at room
temperature. Samples were digested overnight at 37 C by addition of 10 L of 0.5
g/L Trypsin Gold porcine protease (Promega). The following morning, 10 L of 0.5%
trifluoroacetic acid was added to quench digests and samples were incubated at 37 C for
30 minutes. Precipitated cell debris was pelleted by centrifugation at 14,000 RPM, 6 C
for 30 minutes and the supernatant containing soluble peptides was transferred into a new
low-rentention microcentrifuge tube. Samples were filtered through 0.2 m cellulose
syringe filters (National Scientific F2404-16) and kept in autosampler vials at 4 C until
analysis. LC-MS/MS methods and conditions were as described below.
Peptide counts were normalized by dividing the number of peptides observed for
a given protein in a sample by the total number of peptides observed in that sample.
Normalized peptide counts were log2 transformed and Rex regulated genes were plotted
in Graph-pad Prism 6.
LC-MS/MS Methods
Trypsin-digested proteins were analyzed using a Thermo Dionex UltiMate3000
RSLCnano liquid chromatograph (LC) that was connected in-line with an LTQ Orbitrap
XL mass spectrometer equipped with a nanoelectrospray ionization (nanoESI) source
(Thermo Fisher Scientific, Waltham, MA). The LC was equipped with a C18 analytical
column (Acclaim® PepMap 100, 150 mm length × 0.075 mm inner diameter, 3 µm
particles, 100 Å pores, Thermo) and a 1 µL sample loop. Acetonitrile (Fisher Optima
grade, 99.9%), formic acid (1 mL ampules, 99+%, Thermo Pierce), and water purified to
a resistivity of 18.2 MΩ·cm (at 25 °C) using a Milli-Q Gradient ultrapure water
purification system (Millipore, Billerica, MA) were used to prepare mobile phase
solvents for liquid chromatography-tandem mass spectrometry (LC-MS/MS). Solvent A
was 99.9% water/0.1% formic acid and solvent B was 99.9% acetonitrile/0.1% formic
acid (v/v).
Samples contained in polypropylene autosampler vials with septa caps
(Wheaton, Millville, NJ) were loaded into the autosampler compartment prior to analysis.
The autosampler compartment was maintained at 4 ºC. The elution program consisted of
isocratic flow at 5% B for 4 min, a linear gradient to 30% B over 159 min, isocratic flow
at 95% B for 5 min, and isocratic flow at 5% B for 12 min, at a flow rate of 300 nL/min.
The column exit was connected to the nanoESI emitter in the ion source of the
mass spectrometer using polyimide-coated, fused-silica tubing (20 µm inner diameter ×
280 µm outer diameter, Thermo). Full-scan mass spectra were acquired in the positive
ion mode over the range m/z = 350 to 1500 using the Orbitrap mass analyzer, in profile
format, with a mass resolution setting of 60,000 (at m/z = 400, measured at full width at
half-maximum peak height). In the data-dependent mode, the eight most intense ions
exceeding an intensity threshold of 30,000 counts were selected from each full-scan mass
spectrum for tandem mass spectrometry (MS/MS) analysis using collision-induced
dissociation (CID). MS/MS spectra were acquired using the linear ion trap, in centroid
format, with the following parameters: isolation width 3 m/z units, normalized collision
energy 28%, default charge state 2+, activation Q 0.25, and activation time 30 ms. Realtime charge state screening was enabled to exclude singly charged ions and unassigned
charge states from MS/MS analysis. To avoid the occurrence of redundant MS/MS
measurements, real-time dynamic exclusion was enabled to preclude re-selection of
previously analyzed precursor ions, with the following parameters: repeat count 2, repeat
duration 10 s, exclusion list size 500, exclusion duration 60 s, and exclusion mass width
20 ppm. Data acquisition was controlled using Xcalibur software (version 2.0.7 SP1,
Thermo). Raw data files were searched against the Desulfovibrio alaskensis strain G20
protein database using Proteome Discoverer software (version 1.3, SEQUEST algorithm,
Thermo) for tryptic peptides (i.e., peptides resulting from cleavage at the C-terminal end
of
lysine
and
arginine
residues)
with
up
to
three
missed
cleavages,
carbamidomethylcysteine as a static post-translational modification, and methionine
sulfoxide as a variable post-translational modification. A decoy database was used to
characterize the false discovery rate and the target false discovery rate was 0.01 (i.e.,
1%).
Extraction and Measurement of Intracellular NADH and NAD+
NADH and NAD+ were extracted from Desulfovibrio alaskensis G20 according to
a protocol modified from (Sporty et al 2008).
50 mL mid-log phase cultures of
Desulfovibrio alaskensis G20 were pelleted under anoxic conditions.
Pellets were
resuspended in 1 mL anoxic 100 mM ammonium acetate, pH 7.4 and lysed by 3 x 30
seconds sonication with a microtip using a Misonix ultrasonic sonicator S-4000 and
centrifuged at 14,000 rpm to pellet cell debris. The clarified lysate was added to 10 mL
chloroform and shaken vigorously to mix the layers. 1 mL anoxic 50:50 100 mM
ammonium acetate in acetonitrile was added to the cell debris pellet, vortexed and
centrifuged at 14,000 rpm for 1 minute and the supernatant added to the clarified lysate in
chloroform.
After vigorous shaking, the extraction was kept on ice for 30 minutes to
allow the aqueous layer to separate from the chloroform. The aqueous extracts were
removed and frozen at -80 C, lyophilized and resuspended in 150 L of 20 mM
ammonium acetate, pH 7.7. 80 L injections were used for quantification of intracellular
nucleotides.
Agilent Eclipse Plus reversed-phase C-18 column (5 M ID, 9.4 x 250mm) was
used to separate intracellular nucleotides on an Agilent 1100 series HPLC with diodearray (DAD) and fluorescence (FLD) detectors.
NADH was quantified by integrating
the fluorescence emission signal at 460 nm for a 340 nm excitation, PMT gain = 16.
NAD+ was quantified by integrating the absorbance at 260 nm for the NAD+ peak.
Stocks of NADH and NAD+ in water were used to generate standard curves for
quantification. Buffer A was 20 mM ammonium acetate, pH 7.7 and Buffer B was
methanol. The following program was used to separate and elute NAD+ and NADH: 0
min: 100%A, 1min: 100% A, 16 min: 85% A, 16.1 min: 0% A, 18 min:100% A, 28min:
100% A. Post-run time: 4 min. DAD 250, 260, 340 nm, FLD excitation 340 nm:emission
460 nm, PMT gain = 16.
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