www.sciencemag.

org/cgi/content/full/334/6058/986/DC1

Supporting Online Material for

H2S: A Universal Defense Against Antibiotics in Bacteria

Konstantin Shatalin, Elena Shatalina, Alexander Mironov, Evgeny Nudler*

*To whom correspondence should be addressed. E-mail: evgeny.nudler@nyumc.org

Published 18 November 2011, Science 334, 986 (2011)

DOI: 10.1126/science.1209855

This PDF file includes

Materials and Methods

SOM Text
Figs. S1 to S20
Table S1
References
 Supporting Online Material for

H2S: A Universal Defense against Antibiotics in Bacteria

Konstantin Shatalin, Elena Shatalina, Alexander Mironov, and Evgeny Nudler

correspondence to: evgeny.nudler@nyumc.org

This PDF file includes:

Materials and Methods

SOM Text
Figs. S1 to S20
Table S1

1
Materials and Methods

Strains and growth conditions

E.coli, S.aureus and P.aeruginosa strains were grown in Luria-Bertani (LB) broth or on LB plates
supplemented with 1.5% Bacto agar at 37°C. B.anthracis strains were grown in BHI media
supplemented with glycerol at 37°C. Construction of sseA deletion and sseA overexpression strains of
E.coli were produced according to the method of Datsenko and Wanner (20). Briefly, the attL-KmR-
attR cassette (21) was amplified with primers
5’atgcgtgagaatttacgttatgtaattcagtatcaccgctcaagttagtataaaaaagct3’ and
5’ttatttcactggctcaaccggtaaatctgcccgcgtgaagcctgcttttttatactaag3’ and transformed into a MG1655 strain
containing pKD46. A sseA mutant was selected on LB agar supplemented with Km and verified by
PCR with primers 5’attagcatgcaggcggctac3’ and 5’tgctggaaaggccgcgaa3’.
To construct the sseA overexpression strain we substituted the sseA promoter for PLtet-O1 (22).
Briefly, the attL-CmR-attR cassette (21) was amplified with primers 5’cgcagatcttgaagcctgcttttttatac3’
and 5’cgctcaagttagtataaaaaagct3’ and ligated with PLtet-O1 obtained with primers 5’-
cgcagatctcgagtccctatcagtg-3’ and 5’-aatttctcctctttccatgg-3’. The PLtet-O1- attL-CmR-attR cassette was
then amplified with primers 5’ccctgccacaatggcccgttagcaacgtcgaataacgctcaagttagtataaaaaagct3’ and
5’gtgatactgaattacataacgtaaattctcacgcatggtacctttctcctctttaatga3’. The first primer contains the upstream
region of sseA and sequence of attR, while the second contains the coding region of sseA and sequence
of PLtet-O1. The PCR fragment was transformed into MG1655 containing pKD46. CmR clones were
tested on the presence of the PLtet-O1- attL- CmR – attR cassette by PCR with primers 5’attagcatgcag-
gcggctac3’ and 5’gcaaaagaggctgatttggct3’.
E. coli metB, metC, malY, cysM, cysK, and tnaA deletion mutants were obtained from the E. coli
Keio Knockout Collection (Thermo Scientific).
The B.anthracis nos deletion strain Sterne 34F2 was described previously (7) and cbs/cse
deletion mutants were generated according to (23).
The conditional KO (CO) of cbs/cse in B.anthracis 34F2 was made using a protocol described in
(24). The 0.5 kb fragment of cbs from -20 to 480 with respect to the translational start site was
amplified from 34F2 genomic DNA by using primers designed for use in the Gateway cloning system
(Invitrogen). The resulting amplicon was transferred to pDONRtet using standard BP reaction
conditions (Invitrogen), followed by transformation of competent DH10B E.coli and selection on LB
plates containing 10 µg/ml of tetracycline. Cloned amplicons were transferred to pNFd13 (Ts pE194
ori) under the spac promoter using the standard LR reaction (Invitrogen), followed by transformation of
DH10B E. coli and selection on LB plates containing 50 µg/ml Km. Integration of the plasmid into the
targeted B. anthracis locus was done at 390C essentially as described in (24). The nos deletion in the
resulting 34F2 cbs/cse::pNFd13 strain was obtained as described previously (7), except that a
spectinomycin cassette was used instead of Km.
P. aeruginosa strains were from Dr. Newman’s collection of PA-14 mutants (MIT).
Generation of growth curves
Growth curves were obtained on a Bioscreen C automated growth analysis system. Subcultures of
specified strains were grown overnight at 30OC, diluted in fresh media 1:100 and grown to
OD600 ~0.8 at 37OC. Then, cultures were diluted 1:100 in LB or BHI media with appropriate antibiotics
or reagents as described in the text or figure legends. 300 µl of each mixture was inoculated into
honeycomb wells in triplicate and grown at 37OC with maximum shaking on the platform of the
Bioscreen C instrument. OD600 values determined were recorded automatically at different times and
the means of the triplicate cultures plotted.
Generation of survival curves
Overnight cultures were inoculated into LB supplemented with 0.5 mM Cys and grown at 37o C to ~
2
2x107 cells per ml. Then, cells were diluted 100 times in sterile 0.9% NaCl and treated by Gm (50
µg/ml) or Km (250 µg/ml) or Cm (70 µg/ml for P. aeruginosa strains and 35 µg/ml for other strains) at
room temperature in the presence or absence of 0.2 mM of NaHS. CBS/CSE inhibitors, 0.5 mM PAG
and 50 µM AOAA, were used in experiments with WT S. aureus and P. aeruginosa. At different times
samples of cells were placed on LB agar and incubated at 37oC for 16-18 hours. Cell survival was
determined by counting CFU and is shown as the mean ± SD from three independent
experiments.
H2S detection
To monitor H2S production in wild type (wt) and mutant cells we utilized a lead acetate detection
method (9). Paper strips saturated by 2% of Pb(Ac)2 were affixed to the inner wall of a cultural tube,
above the level of the liquid culture of wild type or mutant bacteria. Overnight cultures were diluted
1:50 in LB or BHI with or without Cys (25 – 500 µM; see figure legends) and incubated for 18-20
hours at 370C with aeration. Stained paper strips were scanned and quantified with an AlphaImager
(Imgen Technologies). The results were normalized per ODs.
Assay of chromosomal DNA damage
The assay was performed as described previously (6). Cells were grown to OD600 = 1.0 in LB media
and treated with H2O2 (1 mM – E. coli; 2 mM - B. anthracis; 2.5 mM – P. aeruginosa) or antibiotics
Em (1 µg/ml – B. anthracis); Ap (10 µg/ml – E. coli; 125 µg/ml - P. aeruginosa) for 4 hours. Cells
were equalized to OD600=1.0 by addition of fresh media and the cells from 2 ml of the equalized
cultures were harvested by centrifugation. Total genomic DNA was isolated from the bacteria pellets
with the Qiagen Kit or according to the GenElute Bacterial Genomic DNA Kit protocol for Gram
Positive Bacteria (Sigma). DNA was extracted with phenol/chloroform and quantified with the
PicoGreen dsDNA quantitation reagent (Molecular Probes) using λ phage DNA as a standard.
Arbitrarily chosen 10-kb, 6-kb, and 5.8-kb fragments of E.coli, B. anthracis and P. aeruginose
genomes, respectively, were used for PCR. Primer sequences were as follows: E.coli 5’-
TTCCATTGGGATGTAGATGCTG-3’ (forward) and 5’-GGTAAAAGAGTCAAGGGAAGAACC-3’
(reverse), B.anthracis 5'-ACGCTTTGACTTCTCTCACTTCGGT-3' (forward) and 5'-
AAACATTTGCTCTTGATGTCCTGGA-3' (reverse), P.aeruginose 5'-
CTTTGCAACCTGTACATGCCTTG-3' (forward) and 5'-CATCGTAGTAGTTGATCGGATGGAC-3'
(reverse). PCR was performed with Phusion DNA polymerase (Finnenzymes). The 50-µl PCR mixture
contained 0.5 ng of genomic DNA as a template, 1.5 µM primers, 200 µM dNTPs (Fermentas), 5µl
Phusion GC PCR buffer, and 0.5 µl of DNA polymerase. DNA was subjected to 29 cycles of PCR
(98°C for 30 s, 53°C for 30 s, 72°C for 3 or 9 min). PCR products were separated by electrophoresis in
a 0.8% or 1% agarose gel, stained with ethidium bromide, scanned, and quantified with an
AlphaImager (Imgen Technologies).
Assay of DNA damage in vitro (Fenton reaction)
The nicking reaction mixture contained 0.1 µg pBR322 plasmid DNA in 20 mM Tris HCl buffer, pH
8.0, 30 µM FeCl3, 4 mM Cys and 4 mM H2O2, in the presence or absence of 0.2 mM NaHS. After
incubating the mixture at room temperature for 15 min, the DNA samples were applied to an 0.8%
agarose gel in a TAE buffer system, and electrophoresis was performed at 80 V for 30 min. Following
electrophoresis, gels were stained with ethidium bromide for 30 min. After washing, the bands were
visualized in a UV transilluminator. The modification of the fluorescence intensity of the bands is due
to DNA strand breakage that leads to a decrease in the proportion of the supercoiled form and to an
increase in the relaxed form produced by a nick in one strand.
Pulsed-field gel electrophoresis (PFGE)
E. coli cells were grown to OD600 ~0.8 at 320C. 200 µM NaHS, 10 µg/ml Ap, or 1mM peroxide were
added as indicated and cells were allowed to grow at 370C for 4 more hours. The agarose plugs were
3
prepared using 3x108 cells/plug and treated with lysozyme and Proteinase K according to the BioRad
CHEF protocol. Linearized 4.6 Mb E. coli chromosomal DNA marker was made by digesting the
genomic DNA plug of strain SMR8476 with I-SceI endonuclease. DNA fragments were separated in a
1% agarose gel in 0.5x TBE at 140C for 24 hours at 6 V/cm using a BioRad CHEF-DR II angle system
with a 2.8-26.3 second linear switch time ramp. Gels were stained with EtBr and visualized with UV-
trans-illumination. DNA was quantified (integrated density of the linear products) using ImageJ
software.
CBS/CSE expression assay. B. anthracic Stern 34F2 cbs/cse::pNFd13 cells (see above) carrying the
lacZ reporter under the native cbs promoter were used. To monitor expression of cbs-lacZ fusions,
bacteria were grown overnight at 37 °C in LB medium, washed and diluted 1:25 in fresh medium
containing erythromycin or H2O2 at the specified concentrations. Cultures were incubated for 2-2.5 hr
at 37oC (OD600=0.5-0.6) before measurement of β-galactosidase activity. Shown β-galactosidase
activities correspond to mean values from at least three independent experiments. Error bars
correspond to the standard deviation.
Assays of the SOS response
Overnight cultures of strain 10973 harboring a recA’-gfp chromosomal fusion (25) was grown in Luria
broth with kanamycin (20 µg/ml). The cultures were diluted in LB to OD600 = 0.1 and grown at 37OC to
an OD600 ~1.0. Asp (3 mM), Gm (5 µg/ml) and NaHS (200 µM) were added at OD600 = 0.5.
Fluorescence was measured (Ex. wave length: 480 nm and Em. wavelength: 520 nm) in a Perkin Elmer
LS55 spectrofluorimeter. Fluorescence values were normalized to OD600 values and plotted.
Catalase activity Assay
Degradation of H2O2 was monitored in real time as a decrease in absorbance at 240 nm (26). Aliquots
of extracts to be monitored or of pure catalase were mixed with 50 mM phosphate buffer (pH 7.0) and
placed into a 1 ml quartz cuvette. 40 mM H2O2 solution was added and the kinetics of its degradation
recorded. Total H2O2 degrading activity was measured as the decrease of H2O2 concentration per
milligram of total protein per second. OD240 was converted to the concentration of H2O2 according to a
calibration curve (10 mM H2O2 = 0.36 OD240).
Superoxide dismutase activity assay
SOD activity of cell extracts was determined using a Cayman Chemical Company Superoxide
Dismutase Assay Kit (Cat#706002) according to the manufacture’s recommendations.
Chemicals and reagents
All chemicals were from Sigma, except PYO and the Superoxide Dismutase Assay Kit, which were
purchased from Cayman. The CuFL NO sensor was provided by Stephen Lippard (MIT).

4
SOM Text

3MST is the major source of H2S in E.coli

In addition to 3MST (sseA), the E.coli genome encodes several other enzymes that could potentially
generate H2S, including cystathionine-γ-synthase MetB, cystathionine-β-synthases MetC and MalY,
and CysM, CysK, and TnaA, which are all cysteine desulfurases (27). Pb(Ac)2 analysis of individual
strains harboring knockouts of each of these genes indicates that 3MST is the major source of H2S
production under specified growth conditions (fig. S19).

Anaerobic conditions diminish the effectiveness of antibiotics and H2S-mediated protection

against them.
Fig. 2B and fig. S8 demonstrate that nalidixic acids (NA) and pyocyanin, respectively, are considerably
less potent in anaerobic growth conditions than in aerobic conditions. Such a substantial difference
supports the notion that these antibiotics kill bacteria in large part by causing oxidative stress (8, 10,
15). Because H2S completely fails to protect against NA in anaerobic conditions, we conclude that, at
least in the case of this quinolone, all the protective effect of H2S can be attributed to its ability to
mitigate the oxidative damage caused by the antibiotic. These results also suggest that the effectiveness
of many antibiotics whose potencies have been associated with oxidative stress (8, 10) would be
compromised during infections caused by anaerobic bacteria such as Bacteroides or Clostridium. It
remains to be determined to what extent H2S production by bacteria during aerobic and anaerobic
infection contributes to antibiotic resistance. However, considering the parallels between NO and H2S
production by bacteria (Fig. 3) and the essential role of bacterial NO in survival of germinating B.
anthracis in macrophages (7), we believe that H2S-mediated protection against oxidative stress
contributes to bacterial infection and antibiotic resistance as well.

H2S prevents antibiotic-inflicted chromosomal damage.

The extent of genomic DSBs can be assessed by PCR of an arbitrarily chosen segment of the bacterial
chromosome (6). In this assay, equal amounts of genomic DNA from wt and mutant cells grown with
or without antibiotics or H2O2 were isolated for PCR. The number of reaction cycles was selected so
that DSBs influenced the yield of the final PCR product. The yield of the PCR product from
exponentially growing, H2S-deficient B. anthracis, E. coli, or P. aeruginosa treated with H2O2 or
antibiotics was substantially lower than that from similarly treated wt cells (fig. S12).
To further implicate H2S in genome maintenance we examined its effect on the SOS response
using a chromosomal recA-gfp gene fusion (25). Addition of a sub-lethal amount of gentamicin
induced significant GFP fluorescence (fig. S13). However, induction of RecA by the antibiotic was
more pronounced in aspartate-treated (3MST-compromised) cells than in untreated cells (fig. S13).
NaHS completely suppressed the hyper-induction of SOS by gentamicin (fig. S13).

The mechanism of H2S-mediated cytoprotection

Our results suggest several non-mutually exclusive mechanisms that could explain the H2S-mediated
defense against oxidative stress (fig. S14): (i) stimulation of the major antioxidative enzymes, catalase
and SOD (Fig. 2E); (ii) direct neutralization of peroxide (H2S + H2O2 → S + 2H2O); (iii) transient
depletion of free cysteine, a reducing agent that fuels the Fenton reaction (28), and (iv) depletion of
ferrous iron, a catalytic agent of the Fenton reaction.
We think that the sequestration of ferrous iron by H2S is the largest contributor to H2S-mediated
protection against oxidative stress (although other mechanisms play an important role as well, see
below). To test this hypothesis we measured the effect of NaHS on cell survival after treatment with a
5
lethal dose of H2O2. Within one minute of pretreatment with NaHS, cells became >20 times more
resistant to peroxide (fig. S10). This rules out any protective mechanism that depends on gene
expression. Note, that the amount of NaHS was ten times smaller than that of peroxide, i.e. the
protective effect could not be explained by direct reaction of NaHS with peroxide in this case.
Furthermore, pretreatment of cells with the Fe chelator dipyridyl also protected them rapidly against
peroxide (fig. S10). Because NaHS does not provide extra protection beyond that of dipyridyl (fig.
S10) we conclude that, like dipyridyl, NaHS protected cells from the Fenton reaction by eliminating
Fe++.
To further support this conclusion we examined the effect of H2S on the Fenton reaction in vitro
(fig. S15). Hydrogen peroxide alone or together with Fe3+ did not produce significant strand brakes in
pBR322 DNA (fig. S15), whereas addition of Cys along with Fe3+ caused dramatically accelerated
DNA damage. Addition of NaHS (NaHS:Fe3+ - 6:1) completely inhibited DNA damage under
physiological pH (pH=8). Note that NaHS was 20 times less abundant than H2O2. This result shows
that H2S can effectively interfere with Fenton chemistry by scavenging cellular iron.
To examine the role of free Cys in oxidative stress associated with antibiotics, we studied its
effect on gentamicin (Gm) toxicity in wt and 3MST-deficient cells. WT E.coli became more resistant to
the antibiotic in Cys enriched media (0.5 mM L-cysteine) than in regular LB (<20 µM L-cysteine) (fig.
S20). In contrast, 3MST-deficient cells became more sensitive to Gm in Cys rich media. This stronger
sensitivity of the 3MST mutant cells to Gm killing in the presence of Cys is attributed to the higher
level of intercellular pro-oxidative Cys that could not be efficiently converted to protective H2S.
Indeed, addition of the thiol-depleting diamide protected 3MST mutant cells against Gm (fig. S20).

6
Supporting figures

Fig. S1. Schematic representation of basic metabolic pathways of H2S synthesis. CBS and CSE
generate H2S, pyruvate and ammonia directly from Cys. 3MST generates H2S and pyruvate from 3-
mercaptopyruvate (3MP), which is synthesized along with glutamate by CAT from Cys and α-
ketoglutarate. The orthologs of corresponding mammalian genes are present in the majority of bacterial
species whose genomes have been fully sequenced (fig. S1). Chemicals that specifically inhibit CBS,
CSE, and 3MST are indicated. A sample of clinically relevant pathogens carrying genes that encode
3MST or CBS/CSE is shown on the right.

7
A
HS 1 MASPQ--------LCRALVSAQWVAEALRAPRAGQPLQLLDASWYLPKL---GRDARREFEERHIPGAAFFDIDQCSDRT 69
EC 1 MSTTW------------FVGADWLAEHIDDPE----IQIIDARMASPGQ--EDRNVAQEYLNGHIPGAVFFDIEALSDHT 62
YP 1 ---------MN---SDFLVTPQWLAAHANDAN----IVILDARMSPPGVVPKR-NIQAEFEQGHIPGAVYFDIDAIADHS 63
ST 1 MTTAF------------FVAADWLAEHIDDPE----IQILDARMAPPGQ--EHRDMAGEYRAGHIPGALFFDIEALSDRA 62
BS 1 MTEKS----------AFVVSRDWLKERLHKPG----LAIVDASWYLPAA---GRNGQEEYEKAHIPGAVFFDQDKIADKE 63
ER 1 MSASSSDLPVS---HERFVSADWLANHLNDSS----ITLIDARMLPPGN--DTRDIHAEYRAAHLPGAVFFDIENLSDHS 71
PL 1 ---------MN---NAYFVTPQWLKDHLDDKN----LVILDATAPPP---PQQIDCHKLWLNTHIPGAQFLDLDKIANHQ 61
RP 1 MTAHT-D---------PLVSTDWLAERLGDPS----VKIIDASFKMPGV--LPL-PADDYLAAHIPGAVFFDVDAVSDHA 63
NW 1 MSLMM-DIAMNPTLDDPLVSTEWLAAHLGE------VKAIDASFKMPGV--LPL-AVDDFYAAHIPGAVFFDVDAVSDRA 70
SB 1 MSTTW------------FVGADWLAEHIDDPE----IQIIDARMASPGQ--EDRNVAQEYLNGHIPGAVFFDIEALSDHT 62
JS 1 MAEVI-----TGDDPQTLVSTDWLAAHFNDPD----LRIIDASYYLAEM---NRDAKAEYDAGHIPGARFFDIDDISDAR 68

HS 70 SPYDHMLPGAEHFAEYAGRLGVGAATHVVIYDASDQGLYSAPRVWWMFRAFGHHAVSLLDGGLRHWLRQNLPLSSGKSQP 149
EC 63 SPLPHMLPRPETFAVAMRELGVNQDKHLIVYDEG--NLFSAPRAWWMLRTFGVEKVSILGGGLAGWQRDDLLLEEGAVEL 140
YP 64 TGLPHMLPSPQLFSEMAGQLGITEQHTVVIYDEG--NLFSAPRVWWTFRTFGAKNVRILASGLSGWQQAGYKLESGPAHP 141
ST 63 SPLPHMMPRPEAFAVAMRELGVRQDKHLVIYDEG--NLFSAPRAWWMLRTFGAEKVSILAGGLAGWQRDEWLLREGEEAH 140
BS 64 SGLPHTLPSPEFFAQQVGTLGITADETVVVYDGP--GMFSAPRVWWMFRVMGVKNVYVLDGGFDGWKKAGYPVTDEVTKI 141
ER 72 TDLPHMMPTCENFARAMGELGIDNQQHLVIYDEG--NLFSAPRAWWMLHTFGATSISILSGGLAGWKAQNLPLEQGYVTR 149
PL 62 SGLPHMLPDPQTFSQAVGAMGISENHLVVIYDQG--NMFSAPRAWWTFKIFGSHNVRILDGGLQGWQQAGFPTASGEVKR 139
RP 64 SSLPHMYPSADQFARDVEALGISSGDTVVAYDAG--GWVAAPRAWWMFLSFGHANIRILDGGLKKWVAEGLPTEAGKPTI 141
NW 71 SPLPHMYPDAAQFGRDVGALGISSKDTVVVYDNG--GWLAGPRAWWMFLSFGHAGVRVLDGGLKKWRAEGRPVESGKVSP 148
SB 63 SPLPHMLPRPKTFAVAMRELGVNQDKHLIVYDEG--NLFSAPRAWWMLRTFGVEKVSILGGGLAGWQRDDLLLEEGAVEL 140
JS 69 SELPHMVAPVEKFMSRMRAMGVGDGHQVVVYDGR--GVFSAARVWWNFRLMGKTDVAVLDGGLPKWVAEGRPVEDMPPII 146

HS 150 APAEFRAQLDPAFIKTYEDIKENLESRRFQVVDSRATGRFRGTEPEPRDGIEPGHIPGTVNIPFTDFLS-QEGLEKSPEE 228

EC 141 PEGEFNAAFNPEAVVKVTDVLLASHENTAQIIDARPAARFNAEVDEPRPGLRRGHIPGALNVPWTELV--REGELKTTDE 218
YP 142 TPQTFNVTFNAAAVSSVDEVLAVLGNNEVQILDARPSARYRAQEPEPRPGLRLGRIPGSINIPWGTMVE--NGHLKSPQA 219
ST 141 EGGEFEAKFAPQAVVRLTDVLLASHEKTAQIVDARPAARFNAQADEPRPGLRRGHIPGALNVPWTELV--YEGELKTTDE 218
BS 142 AATFFKPSFNKDAVVDFQEMRKIVDEKRSQIADARGAGRFTGRDAEPRAEMRSGHMPGARNVPVTTLSE--NGELKDLES 219
ER 150 KPVTFHATLDENAIRSRDDVLSISRDKSEQIVDARPASRFHAEVDEPRPGLHRGHIPGSLNVPWTDLVN--NGALKPNAE 227
PL 140 NPQIFNTDFNADKVKSLEQILGALNDQQIQIVDARATDRFQAKAPEPRPGLRMGHIPGSKNIPWTMLVE--NGHFKSETE 217
RP 142 APGRFSAKLDPSFIRSRDQLVANLDSGAEQVIDARAAPRFEGSVAEPRPGLRAGHIPGSRNLPYNELFDAATGTMKPLAE 221
NW 149 EPGHFTATFDPLFVRDKAQLVSNLSSCREQLVDARAAARFTGAVMEPRQGLRSGHIPGSRNLSYAELFDPGTGVMKPLDD 228
SB 141 PEGEFNAAFNPEAVVKVTDVLLASHENTAQIIDARPAARFNAEVDEPRPGLRRGHIPGALNVPWTELV--REGELKTTDE 218
JS 147 RDRHMTVQRQAHLVKDVTQVASASKLGDWQIVDARAPARFRGEEPETRPGLRAGRIPNSKNVHYASLFK-PDGTMKEGDA 225

HS 229 IRHLFQEKKVDLSKPLVATCGSGVTACHVALGAYLCGKPDVPIYDGSWVEWYMRARPEDVISEGRGKTH 297

EC 219 LDAIFFGRGVSYDKPIIVSCGSGVTAAVVLLALATLDVPNVKLYDGAWSEWGARADLPVE--PVK---- 281
YP 220 LAEIFAAQGVDLTKPIITSCGSGVTAAVVVLGLAAVNARSVSLYDGSWAEWGASNSLPIDATPLA---- 284
ST 219 LNEVFFSHGVSFDRPIIASCGSGVTAAVVVLALATLDVPDVTLYDGAWSEWGARTDLPVE--PA----- 280
BS 220 LRRIFDEAGIDLSGPVVTSCGSGVTAAVITLALTSLGHKDNRLYDGSWSEWGSRQDTPVVTGEAE---- 284
ER 228 LATILHKHGVDFTRPIVASCGSGVTASVVVLALTQLNVPNVTLYDGSWSDWGSRDDVPIARD------- 289
PL 218 ITDIFHKQGVDLNKPVITSCGSGMTAAVLVLGLDIIGKKDVYLYDGSWAEWGADEALPLEK-------- 278
RP 222 LRQAFERAGLDLGRPVVTSCGSGVSAAVLTLALYRLGVRGSALYDGSWSEWGLQDGPPVATGPAA---- 286
NW 229 IRAAFSRAGVDLAKPVVTTCGSGVSAAVLTLALYRLGARGSALYDGSWSEWGLVEGPPIATGPA----- 292
SB 219 LDAIFFGRGVSYDKPIIVSCGSGVTAAVVLLALATLDVPNVKLYDGAWSEWGARADLPVE--PVK---- 281
JS 226 LRAAFEAGGVDLDKRIITTCGSGVTAAILMLGLTRLGHQDVSLYDGSWSEWGQFEQLKVETG------- 287

B
HS 81 LKKIGDTPMVRINKIGKKFGLKCELLAKCEFFNAGGSVKDRISLRMIEDAERDGTLKPGDTIIEPTSGNTGIGLALAAAV 160
BA 8 HELIGHTPIVEITRFSLPEGVR--LFAKLEFYNPGGSVKDRLGRELIEDALEKGLVTEGGTIIEPTAGNTGIGLALAALQ 85
PA 10 LDLIGNTPLVRVTRFD---TGPCTLYLKLESQNPGGSIKDRIGVAMIEAAERDGRLRPGGTIVEATAGNTGLGLALVGRA 86
SA 4 YDLIGNTPLVLLEHYS-DDKVK--IYAKLEQWNPGGSVKDRLGKYLVEKAIQEGRVRAGQTIVEATAGNTGIGLAIAANR 80

HS 161 RGYRCIIVMPEKMSSEKVDVLRALGAEIVRTPTNARFDSPESHVGVAWRLKNEIPNSHILDQYRNASNPLAHYDTTADEI 240

BA 86 HDLRVIVCVPEKFSIEKQELMKALGATVVHTPTEQGMTG---AIAKAKELVNEIPNSYSPSQFANEANPRAYFKTLGPEL 162
PA 87 KGYRVVLVVPDKMSTEKVLHLRAMGAEVHITRSDVGKGHPEYYQDVAARLAQDIPGAFFADQFNNPANPLAHECGTGPEL 166
SA 81 HHLKCKIFAPYGFSEEKINIMIALGADVSRTSQSEGMHG---A-QLAARSYAEKYGAVYMNQFESEHNPDTYFHTLGPEL 156

HS 241 LQQCDGKLDMLVASVGTGGTITGIARKLKEKCPGCRIIGVDPEGSILAEPEELNQ-TEQTTYEVEGIGYDFIPTVLDRTV 319

BA 163 WSALNGEINIFVAGAGTGGTFMGTASYLKEKNIDIKTVIVEPEGSIL------NG-GKAGSHETEGIGLEFIPPFLKTSY 235
PA 167 WAQTGHDLDAIVVGVGSSGTLTGLTRFFQKVQPELEMVLADPEGSIMAEYSRSGTLGTPGSWAVEGIGEDFVPAIADLSS 246
SA 157 TSALQ-QIDYFVAGIGSGGTFTGTARYLKQHHV--QCYAVEPEGSVL------NG-GPAHAHDTEGIGSEKWPIFLERRL 226

HS 320 VDKWFKSNDEEAFTFARMLIAQEGLLCGGSAGSTVAVAVKAAQELQEGQRCVVILPDSVRNYMTKFLSDR----WMLQKG 395

BA 236 FDEIHTISDRNAFLRVKELAQKEGLLVGSSSGAAFHASLLEAEKAAPGTNIVTIFPDSSERYLSKDIYK----GWE---- 307
PA 247 VRHAYSISDEESFAMARELLRVEGIPGGSSTGTLLAAALRFCREQKEPKRVVSFVCDTGTRYLSKIYNDQ----WMTDQG 322
SA 227 VDGIFTIKDQDAFRNVKSLAINEGLLVGSSSGAALQGALNLKAQLSEGT-IVVVFPDGSDRYMSKQIFNYEENDYE---- 301

8
C
HS 1 MQEKDASSQGFLPHFQHFATQAIHVGQDPEQWTSRAVVPPISLSTTFKQGAPGQ-HSGFEYSRSGNPTRNCLEKAVAALD 79
BA 1 MRAK---------------TKLIHGIRIGEP-STGSVNVPIYQTSTYKQEAVGK-HQGYEYSRTGNPTRAALEEMIAVLE 63
PA 1 MSQHDQHPDA---PAQAFATRVIHAGQAPDP-STGAIMPPIYANSTYIQESPGV-HKGLDYGRSHNPTRWALERCVADLE 75
SA 1 MNKK---------------TKLIHGGHTTDD-YTGAVTTPIYQTSTYLQDDIGDLRQGYEYSRTANPTRSSVESVIAALE 64
*
HS 80 GAKYCLAFASGLAATVTITHLLKAGDQIICMDDVYGGTNRYFRQVASE-FGLKISFVDCSKIKLLEAAITPETKLVWIET 158
BA 64 NGHAGFAFGSGMAAITATIMLFSKGDHVILTDDVYGGTYRVITKVLNR-FGIEHTFVDTTNLEEVEEAIRPNTKAIYVET 142
PA 76 GGTQAFAFASGLAAISSVLELLDAGSHIVSGNDLYGGTFRLFERVRRRSAGHRFSFVDPTDLQAFEAALTPETRMVWVET 155
SA 65 NGKHGFAFSSGVAAISAVVMLLDKGDHIILNSDVYGGTYRALTKVFTR-FGIEVDFVDTTHTDSIVQAIRPTTKMLFIET 143
** * **
HS 159 PTNPTQKVIDIEGCAHIVHKHGDIILVVDNTFMSPYFQRPLALGADISMYSATKYMNGHSDVVMGLVSVNC-ESLHNRLR 237
BA 143 PTNPLLKITDIKKISTLAKEKG-LLTIIDNTFMTPYWQSPISLGADIVLHSATKYLGGHSDVVAGLVVVNS-PQLAEDLH 220
PA 156 PSNPLLRLTDLRAIAQLCRARG-IISVADNTFASPYIQRPLELGFDVVVHSTTKYLNGHSDVIGGIAIVGDNPDLRERLG 234
SA 144 PSNPLLRVTDIKKSAEIAKEHG-LISVVDNTFMTPYYQNPLDLGIDIVLHSATKYLGGHSDVVAGLVATSD-DKLAERLA 221

HS 238 FLQNSLGAVPSPIDCYLCNRGLKTLHVRMEKHFKNGMAVAQFLESNPWVEKVIYPGLPSHPQHELVKRQCTGCTGMVTFY 317

BA 221 FVQNSTGGILGPQDSFLLLRGLKTLGIRMEEHETNSRAIAEFLNNHPKVNKVYYPGLASHQNHELATEQANGFGAIISFD 300
PA 235 FLQNSVGAISGPFDAFLTLRGVKTLALRMERHCSNALALAQWLERQPQVARVYYPGLASHPQHELAKRQMRGFGGMISLD 314
SA 222 FISNSTGGILGPQDSYLLVRGIKTLGLRMEQINRSVIEIIKMLQAHPAVQQVFHPSIESHLNHDVHMAQADGHTGVIAFE 301
* * *
HS 318 IKGTLQHAEIFLKNLKLFTLAESLGGFESLAELPAIMTHASVLKNDRDVLGISDTLIRLSVGLEDEEDLLEDLDQALKAA 397
BA 301 VDSE-ETLNKVLERLQYFTLAESLGAVESLISIPSQMTHASIPADRRKELGITDTLIRISVGIEDGEDLIEDLAQAL--A 377
PA 315 LRCDLAGARRFLENVRIFSLAESLGGVESLIEHPAIMTHASIPAETRADLGIGDSLIRLSVGVEALEDLQADLAQALAKI 394
SA 302 VKNT-ESAKQLIKATSYYTLAESLGAVESLISVPALMTHASIPADIRAKEGITDGLVRISVGIEDTEDLVDDLKQALDTL 380

Fig. S2. Sample sequence alignments of H2S-producing enzymes

(A) 3MST from Homo sapiens (HS) and its orthologs from E.coli (EC), Y.pestis CO92 (YP),
S.typhimurium LT2 (ST), B.suis 23445 (BS), E.pyrifoliae Ep1/96 (ER), P.luminescens laumondii
TTO1(PL), R.palustris CGA009 (RP), N.winogradskyi Nb-255 (NW), S.boydii Sb227 (SB), and
Jannaschia sp. CCS1 (JS) (29,30).
(B) and (C) CBS and CSE from Homo sapiens (HS) and their homologs from B.anthracis Sterne (BA),
P.aeruginose PA01 (PA) and S.aureus N315 (SA). Conserved amino acids are highlighted.
Active-site loop residues of HS 3MST and CBS are shown in red (31-33). Asterisks indicate key
active-site residues of CSE (34).

9
A

Fig. S3. Genome organization of 3MST and CBS/CSE in selected bacteria. (A) Localization of
sseA (3MST) orthologs (red) in selected genomes of pathogenic bacterial. Genes of the same color are
from the same ortholog group. Light yellow - no COG assignment; white - pseudo gene. (B) cbs (red)
and cse (yellow) orthologs in B.anthacis, S.aureus and P.aeruginose genomes.

10
Fig. S4. The results of Phenotype MicroArray (PMA) as provided by Biolog, Inc. The growth
curves for wt E.coli are shown in red, for ΔsseA in green and overlay in yellow. Data are shown as the
means from two experiments. To perform the MicroArray a standardized cell suspension was
inoculated into the wells of a multiwell plate. The plate was then incubated for 24 hours at 370C under
aerobic conditions. PMA uses Biolog's proprietary redox chemistry that detects cell respiration as a
universal reporter. To compare the phenotypes of two cell lines, one is recorded as a red tracing and
one as green. Dedicated software is used to overlay the graphs and detect differences. Areas of overlap
(i.e. no change) are colored yellow; any differences are highlighted as patches of red or green. The
MicroArray was repeated in two replicates and bioinformatics software was used to select the wells
with reproducible results. The average values of growth inhibition (negative numbers) are presented in
Table S1.

Fig. S5. Growth of wt and mutant E.coli, B.anthracis, and P.aeruginose in LB.

11
Fig. S6. Endogenous H2S protects various bacteria against diverse antibiotics
(A) Representative optical density (OD) growth curves of E. coli (MG1655), B. anthracis (Sterne) or P.
aeruginosa (PA14) (black curves) and their H2S-deficient counterparts (red curves) in the presence of
spectinomycin (Sp, 60 µg/ml), nalidixic acid (NA, 2.5 µg/ml), erythromycin (Em, 1 µg/ml), or
ampicillin (Amp, 125 µg/ml). Green curves show the growth of E. coli that overexpress 3MST. Cells
were grown in triplicate at 37°C with aeration using a Bioscreen C automated growth analysis system.
The curves represent averaged values from three parallel experiments with a margin of error of less
than 5%.
(B) Inhibitors of CSE/CSB and 3MST increase antibiotic sensitivity. Panels show representative
growth curves of methicillin-resistant S. aureus (MW2), E. coli, or P. aeruginosa (black) in the
presence of chloramphenicol (Cm, 0.5 µg/ml), vancomycin (Van, 0.5 µg/ml), acriflavine (Acr, 8
µg/ml), or pyocyanin (Pyo, 50 µM). Red and blue curves represent cellular growth in the presence of
CSE/CSB and 3MST inhibitors (inh) with or without antibiotics, respectively.
(C) Exogenous H2S restores antibiotic resistance in H2S-deficient bacteria. Panels show representative
growth curves of wt E. coli or B. anthracis (black) or H2S-defcient strains (red) in the presence of
gentamicin (Gm, 1 µg/ml), NA (2.5 µg/ml), cefuroxime (Cef, 20 µg/ml), and Pyo (50 µM). Green
curves show growth of cells pretreated with the H2S donor (NaHS) in the presence of the indicated
antibiotic.

12
Fig. S7. H2S protects Gram(+) bacteria against chloramphenicol-mediated killing. Survival curves
show the effect of CBS/CSE (B. anthracis and P. aeruginosa) and 3MST (E.coli) deletions or
CBS/CSE inhibition (S. aureus) by AOAA/PAG (inh) on chloramphenicol (Cm, 50 µg/ml)-mediated
killing. This “bacteriostatic” antibiotic kills Gram(+) bacteria (S. aureus and B. anthracis) within a
relatively short time period, but not Gram(-) E.coli and P. aeruginosa. Our data suggest that Cm, and
perhaps other bacteriostatic antibiotics, exert oxidative damage in Gram(+) bacteria, against which H2S
provides protection (see below), thereby improving growth in the presence of these antibiotics in liquid
culture (Fig. S6B).

Fig. S8. Endogenous H2S renders B. anthracis more resistant to pyocyanin in aerobic conditions,
but fails to do so in anaerobic conditions. A paper disk saturated with 50 µM pyocyanin was placed
on wt or CBS/SCE-deficient B. anthracis lawns that were grown aerobically or anaerobically for the
next 20 hours. Zone borders are marked with dashed lines.

13
Fig. S9. 3MST-deficent cells exhibit a much greater sensitivity to hydrogen peroxide than other
cysteine desulfurase mutants. Overnight cultures of indicated E.coli strains were diluted with fresh
LB 1:50 and grown to OD600 ~1. H2O2 was added to 2.5 mM for 10 min. Cell survival was determined
by counting CFU and is shown as the mean ±SD from three independent experiments.

Fig. S10. Rapid protective effect of H2S against oxidative stress. Wt E.coli cells were grown in LB
to OD600 ~1.0, treated with NaHS (200 µM), or the iron chelator dipyridyl (0.5 mM), or both for 1 min,
followed by the addition of H2O2 (2 mM) for 10 min. Cell survival was determined by counting CFU
and is shown as the mean ±SD from three independent experiments.

Fig. S11. Inhibitors of CBS/CSE render S. aureus sensitive to hydrogen peroxide, whereas an H2S
donor reverses this sensitivity. The panel shows growth curves of methicillin-resistant S. aureus
(MW2) pretreated with PAG and AOAA for 3 min, followed by the addition of H2O2 (0.5 mM) (red).
NaSH was added to 100 µM prior to challenge with H2O2 (green).

14
Fig. S12. H2S protects against H2O2- and antibiotic-inflicted DNA damage. Sub-lethal amounts of
H2O2 and ampicillin (Amp) inflicted greater chromosome damage in CBS/CSE-deficient B. anthracis
and P. aeruginosa and 3MST-deficient E. coli cells than in wt cells. The integrity of chromosomal
DNA was monitored by PCR. Representative agarose gels show chromosomal fragments amplified
from equal amounts of genomic DNA isolated from wild type (wt) or mutant (Δ) cells. M, 1 kb DNA
marker. “%” indicates the fraction of the full-length PCR products. Values are the averages from three
experiments with a margin of error of less than 10%.

15
Fig. S13. H2S suppresses antibiotic-induced SOS response. Gentamicin (Gm, 20 µg/ml) induces
fluorescence in recA’::gfp E. coli (wt). SOS caused by Gm was greater in Asp-treated cells, but fully
suppressed by exogenous NaHS. The genotoxic agent mitomycin (MMC, 0.2 µg/ml) was used as a
positive control. In each case, the basal level of fluorescence before induction was assigned a value of
1. Values are the mean ±SD from three experiments. (*) p<0.05, (**) p<0.01.

Fig. S14. The proposed mechanism of H2S-mediated defense against antibiotics. Despite having
different primary targets, many antibiotics kill bacteria by generating ROS (S11,12). The ability of
H2S-producing enzymes (CBS, CSE, and 3MST) to alleviate oxidative stress is achieved by several
mechanisms: (i) depletion of free cysteine (Cys), which fuels the Fenton reaction; (ii) inhibition of the
Fenton reaction by H2S, which reacts with H2O2 and diminishes free Fe2+; (iii) stimulation of catalase
and superoxide dismutase activities. Antibiotics stimulate the activities of CBS, CSE, and 3MST,
thereby ensuring their specific defensive responses.

16
Fig. S15. Effect of Cys and NaHS on Fenton-mediated DNA damage in vitro. As indicated, the
supercoiled pBR322 plasmid (0.5 µg) was treated with 30 µM FeCl3, 4 mM Cys, 200 µM NaHS, or 4
mM H2O2 in 20 mM Tris-HCl buffer (pH 8). After a 30-min incubation at room temperature, the
reaction was stopped and separated in a 1% agarose gel. RF, relaxed form; SF, supercoiled form.

Fig. S16. H2S-generating CBS/CSE becomes essential in the absence of bNOS. B. anthracis (34F2
Δnos cbs/sce::Pspac) cells carrying the chromosomal copy of cbs/cse under the IPTG-inducible spac
promoter were plated on LBA plates with or without 0.1 mM IPTG for overnight incubation at 370C.

17
 Fig. S17. H2S induction in response to erythromycin or H2O2 challenge. E.coli cells were grown
in 96 wells plates in LB+Cys (25 or 200 µM) covered with lead acetate soaked paper at 37°C with
aeration using a Bioscreen C automated growth analysis system. Em (2.5 µg/ml) or H2O2 (1 mM)
were added at OD600 ~0.6 for 12 hours. PbS brown/black stain is proportional to the amount of H2S.

Fig. S18. Wild type E. coli cells protect 3MST-deficient cells from antibiotic toxicity. WT and
ΔsseA cells were grown in LB+Cys (0.5 mM) to OD600 ~0.6. Then aliquots containing equal amounts of
cells were mixed together, washed, resuspended in M9 minimal media supplemented with Gm (5
µg/ml) and incubated for 30 min at 370C. Cell survival was determined by counting CFU and is shown
as the mean ±SD from three independent experiments.

18
Fig. S19. H2S production by E. coli depends primarily on 3MST.
Lead acetate soaked paper strips show a PbS brown/black stain as a result of reaction with H2S. Strips
were affixed to the inner wall of a cultural tube above the level of the liquid culture of wt or mutant
bacteria grown in LB supplemented with 0.5 mM Cys for 18 hours. Numbers (%) show the relative
change in H2S production.

Fig. S20. Protection of 3MST-deficient cells from Cys-mediated gentamicin toxicity by the thiol
oxidizer diamide and NaHS. Cells grown in LB or LB+Cys (0.5 mM) to OD600 ~1.0 were washed in
M9 minimal media supplemented with the thiol oxidizer diamide (25 µM) or NaHS (0.2 mM) or Cys
(0.5 mM). After 3 min of incubation gentamicin (50 µg/ml) was added for 5 min. Cell survival was
determined by counting CFU and is shown as the mean ±SD from three independent experiments.

19
Supporting table

Table S1. List of chemicals that selectively inhibit the growth of E.coli ΔsseA (3MST-deficient)
strain. The results are provided by Phenotype MicroArray (Biolog, Inc). The relative values of growth
inhibition (negative numbers) are presented in the table (see fig. S4 for raw data and explanation).

ΔsseA inhibition
Map position of growth Chemical
PM12B D09,D10,D12 -265 Novobiocin
PM16A B01,B02 -212 Norfloxacin
PM11C E09,E10 -183 Nalidixic acid
PM13B B11 -117 Oxolinic acid
PM14A A01,A02,A03 -295 Acriflavine
PM14A B02,B03 -142 9-Aminoacridine
PM20B D03 -93 Proflavine
PM13B G09,G10,G12 -372 Trifluoperazine
PM14A H05,H06 -262 Promethazine
PM17A D09,D10 -251 Chlorpromazine
PM16A E01,E02,E03,E04 -421 Streptomycin
PM20B A05,A06 -179 Apramycin
PM13B H09,H10,H11,H12 -520 Tylosin
PM15B F05,F06,F07 -498 Oleandomycin
PM11C F05,F06,F07,F08 -468 Erythromycin
PM19 A01,A02 -294 Josamycin
PM12B H01,H02 -235 Spiramycin
PM20B H09,H10 -203 Troleandomycin
PM11C F01,F02 -136 Chloramphenicol
PM15B C05,C06 -271 Fusidic acid
PM16A E09,E10,E11,E12 -352 Rifamycin SV
PM16A B09,B10 -212 Trimethoprim
2,4-Diamino-6,7-
PM12B E01,E02,E03 -429 diisopropylpteridine
PM12B B09,B10,B11 -231 Polymyxin B
PM11C C06 -102 Colistin
PM12B C05,C06 -198 Vancomycin
PM17A H01,H02,H03,H04 -201 Cefsulodin
PM11C H01,H02 -160 Cephalothin
PM17A G09,G10 -159 Cefoperazone
PM12B B01,B02 -346 Oxacillin
PM11C D09,D10 -227 Nafcillin
PM19 F01,F02 -216 Phenethicillin
PM12B A01,A02 -186 Penicillin G
PM11C B05,B06 -182 Cloxacillin
PM13B H06,H07 -171 Moxalactam
PM12B A09,A10 -164 Carbenicillin
PM20B A10,A11,A12 -336 Benserazide

20
PM17A G02,G03,G04 -177 Chlorambucil
PM20B B01,B02 -345 Orphenadrine
PM20B F09,F10 -206 Pridinol
PM20B B05,B06 -263 Propranolol
PM18C A01,A02,A03,A04 -468 Ketoprofen
PM17A H06,H07 -173 Caffeine
PM19 G09,G10 -150 Hydroxylamine
PM19 A09,A10,A11 -243 Coumarin
PM17A A01,A02,A03 -398 D-Serine
PM18C D09,D10 -269 Lidocaine
PM18C C01,C02,C03 -377 Poly-L-lysine
Methyltrioctylammonium
PM19 B01,B02 -326 chloride
PM16A C09,C10 -269 Cetylpyridinium chloride
PM12B E09,E10 -255 Benzethonium chloride
Dodecyltrimethyl ammonium
PM12B H09,H10 -225 bromide
PM15B D05,D06 -250 Domiphen bromide
PM15B E01,E02 -216 Alexidine
PM20B A01,A02,A03 -422 Amitriptyline
PM20B G01,G02,G03 -357 Captan
PM18C F06,F07 -223 Tinidazole
PM20B C09,C10 -203 Ornidazole
PM14A A05,A06 -162 Furaltadone
PM18C B09,B10,B11,B12 -341 Azathioprine
PM13B E01,E02,E03 -281 Cytosine arabinoside
PM19 D01,D02 -246 Disulfiram
PM09 G01,G02,G03,G04 -497 200mM Sodium Phosphate pH 7
PM16A D01,D02 -221 1-Chloro-2,4-dinitrobenzene
PM19 E09,E10,E11,E12 -330 Lawsone
Phenyl-methylsulfonyl-
PM19 D09,D10 -194 fluoride (PMSF)
PM19 F06,F07 -185 Blasticidin S
PM19 D05,D06 -215 Iodonitro tetrazolium violet
PM19 E01,E02,E03 -460 FCCP
PM15B G01,G02,G03,G04 -422 CCCP
PM19 F09,F10,F11,F12 -346 Sodium caprylate
PM19 B09,B10,B11 -296 2,4-Dinitrophenol
PM18C C09,C10,C11 -287 Pentachlorophenol
PM20B D09,D10,D11 -212 18-Crown-6 ether
PM19 C09,C10 -204 Cinnamic acid
PM13B E09,E10,E11 -268 Ruthenium red
PM20B E01,E02,E03,E04 -576 Crystal Violet
PM20B C01,C02,C03 -313 Thioridazine
PM20B B09,B10 -293 Tetrazolium violet
PM14A C09,C10,C11 -238 Sodium cyanate

21
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