Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) inSalmonella enterica Serovar Typhimurium

Tamiko Oguri, Barbara Schneider, and Larry Reitzer

Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas, USA

Cysteine is potentially toxic and can affect diverse functions such as oxidative stress, antibiotic resistance, and swarming motil-ity. The contribution of cysteine catabolism in modulating responses to cysteine has not been examined, in part because thegenes have not been identified and mutants lacking these genes have not been isolated or characterized. We identified the genefor a previously described cysteine desulfhydrase, which we designated cdsH (formerly STM0458). We also identified a diver-gently transcribed gene that regulates cdsH expression, which we designated cutR (formerly ybaO, or STM0459). CdsH appearsto be the major cysteine-degrading and sulfide-producing enzyme aerobically but not anaerobically. Mutants with deletions ofcdsH and ybaO exhibited increased sensitivity to cysteine toxicity and altered swarming motility but unaltered cysteine-en-hanced antibiotic resistance and survival in macrophages.

Cysteine desulfhydrase (CDS) degrades cysteine to pyruvate,ammonia, and sulfide (19, 26, 43). The major CDS in Esche-

richia coli appears to be tryptophanase (TnaA), which has an ap-parent Km for cysteine of 11 mM (56). TnaA primarily catabolizestryptophan, which appears to be abundant in the intestinal tract(29). In E. coli, TnaA can become 10% of soluble protein, whichsuggests the potential to also degrade cysteine in vivo, despite anunfavorable Km (56). Several factors regulate TnaA expression.TnaA is repressed by glucose, pyruvate, and acetate (6, 9, 10, 31).Expression requires cyclic AMP and is induced by tryptophan,cysteine, indole, growth in alkaline broth, depletion of heme andphosphatidyl glycerol, and anaerobic growth with an alternateelectron acceptor (9, 51, 52). From these factors, it could be spec-ulated that TnaA contributes to energy generation by degradingtryptophan and possibly cysteine to pyruvate.

Salmonella enterica does not have TnaA, yet cysteine induces apowerful CDS (19, 26, 43). This CDS has been purified from S.enterica, and similarly to TnaA, it contains pyridoxal-5=-phos-phate (42). The kcat/Km ratio for L-cysteine, a measure of catalyticefficiency, is 10,340 mM�1 s�1 for S. enterica CDS (calculatedfrom values reported by Kredich et al. [42, 43]) and 6.7 mM�1 s�1

for E. coli TnaA (calculated from values reported by Snell [56]). Inother words, the S. enterica CDS appears to be more than threeorders of magnitude more efficient than TnaA. To explore thefunction of the S. enterica CDS and cysteine catabolism, we iden-tified the gene for the major CDS and characterized its regulationand the phenotype of mutants lacking this enzyme.

MATERIALS AND METHODSStrains and plasmids. Table 1 lists the strains and plasmids used in thisstudy. All gene deletions and replacements were performed originally intothe TT22971 background strain as described previously (21), followed byP22 transduction into the appropriate genetic background, and the prod-ucts were ensured to be phage free by cross-streaking against P22-H5 ongreen plate agar (15). The following codons were deleted: �cdsH, 8 to 321;�ybaO, 15 to 446; �metC, 14 to 386; �sbp, 11 to 317; �cysJ, 11 to 587;�asrA, 11 to 335; �phoP, 1 to 224; �cydD, 2 to 574; and �cysB, 11 to 315.

The cdsH gene and promoter region were amplified from genomic S.enterica TA1650 and cloned into the SmaI site of pUC18. The EcoRI-HindIII fragment of pUC18-cdsH containing cdsH was ligated into EcoRI-and HindIII-cleaved pMMB190. This places the cdsH gene under control

of the Ptaclac promoter in a low-copy vector. The cdsH promoter fusionswere constructed as described previously (27). A PCR product from 57bases downstream to 138 bases upstream of the cdsH start codon wascloned into translational fusion vector pBLS17 and transcriptional fusionvector pBLS18, a derivative of pBLS17 containing a trp terminator-deletedlacZ from pAH125 (36). The cdsH-lacZ fusion plasmids were integratedinto the � site of TT22971 and P22 transduced into TR10000 for study.Strains were verified with P2 and P3 CRIM primers (27) and S. enterica-specific primers P1 (5=-GGCATAATAGCAATGTACTGG-3=) and P4(5=-GCGTTCTGGCACGATAT-3=). For ybaO complementation, theASKA vector containing ybaO was used (38), and its corresponding emptyASKA vector was constructed by an SfiI cut/religation of the 4,510-bpbackbone of ASKA-cysE.

Growth of bacterial cells. Cells were grown in minimal medium con-taining W salts (10.5 g/liter K2HPO4, 4.5 g/liter K2HPO4, and 0.05 g/literMgSO4), 0.4% of a carbon source, and 0.2% (NH4)2SO4 as a nitrogensource, when appropriate. For determination of anaerobic growth rates,8.5 ml medium was placed in a 9-ml screw-cap tube, and cells were incu-bated without shaking in a 37°C water bath.

Enzyme assays. Detection of L-cysteine desulfhydrase was performedusing two assays. Assay 1 measured methylene blue formation at 670nm (58). Methylene blue forms when N=,N=-dimethyl-p-phenylenedi-amine reacts with sulfide in the presence of FeCl3. One unit is definedas the amount catalyzing the formation of 1 �mol sulfide min�1. Assay2 was an activity stain for proteins subjected to electrophoresis in anondenaturing gel (3). In this assay, bismuth reacts with sulfide toform a black precipitate at the site of a cysteine catabolic protein. Theassay for �-galactosidase has been previously described (36), with spe-cific activity units defined as nanomoles of product per minute permilligram of protein.

Sulfide detection assays. Sulfide detection was performed in fourways. Assay 1 involved stabbing cells into LB containing 0.6% agar, 0.1%FeSO4 · 7H2O, and 5 mM cysteine. Assay 2 was an anaerobic test with filterdiscs. Four-section plates contained the following layers from bottom to

Received 27 April 2012 Accepted 4 June 2012

Published ahead of print 8 June 2012

Address correspondence to Larry Reitzer, reitzer@utdallas.edu.

Supplemental material for this article may be found at http://jb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.00729-12

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top: 1 ml LB containing 0.6% agar, a 6-mm Whatman no. 1 paper filterdisc with 15 �l of a chemical solution affixed with 200 �l soft agar, 0.5 mlovernight culture cells mixed with 1 ml LB containing 0.6% agar, and 6 mlLB containing 1.5% agar to the top of the plate. Discs contained 5 �l 1 Mcysteine, 5 �l 1 M NaNO3, 5 �l 40% glucose, or 5 �l 10% FeSO4 · 7H2Owhen indicated. The assay was specific for cysteine, since methionine,glutathione, cystine, and dithiothreitol (DTT) did not generate sulfide.Only FeSO4, and not FeCl3 or FeCl2, generated sulfide. Sulfide did notresult from sulfate reduction, since mutants blocked in the reductionpathways (TO5 [�asrA], TO6 [�cysJ], and TO7 [�sbp]) still generated

sulfide. Assay 3 detects sulfide from silver nitrate-impregnated strips (45).Microcentrifuge tubes (1.5 ml) contained 1.3 ml LB, 0.1% Na2SO4 ·10H2O, and 5 mM cysteine. The strips were sealed within the caps abovethe liquid cultures. A small hole was pierced in the caps to allow air toescape, and tubes were incubated at 37°C without shaking. Assay 4 usedless-reactive lead acetate strips (Fisher Scientific). Silver nitrate and leadacetate strips produce a black stain directly proportional to the amount ofvolatile sulfide formed. Photographs were taken with the Multi-Doc-Itdigital imaging system using Launch Doc-It LS image acquisition soft-ware.

TABLE 1 Bacterial strains and plasmids

Strain or plasmid Relevant genotype or phenotypeSource and/orreference

StrainsTR10000 Wild-type S. enterica serovar Typhimurium LT2 47TO1 TR10000 �cdsH This studyTO2 TR10000 �cdsH �metC::cat This studyTO3 TR10000 �ybaO This studyTO4 TR10000 �cysB::cat This studyTO5 TR10000 �asrA::cat This studyTO6 TR10000 �cysJ::cat This studyTO7 TR10000 �sbp::cat This studyTO9 TR10000 �metC::cat This studyTO10 TR10000 �cydD::cat This studyTO11 TR10000 [� (cdsH-lacZ) Kanr] This studyTO12 TR10000 �cdsH [� (cdsH-lacZ) Kanr] This studyTO13 TR10000 �ybaO [� (cdsH-lacZ) Kanr] This studyTT22971 metA22 metE551 trpD2 ilv-452 leu-pro-(leaky) hsdLT6 hsdSA29 hsdB strA120/pDK46 4714028s Wild-type virulent strain; B serotype 8TO15 14028s �cdsH This studyTO16 14028s �ybaO This studyTO17 14028s �phoP::cat This studySL1433 S. enterica wild-type virulent strain; B serotype 61TO18 SL1433 �cdsH::cat This studyTO19 SL1433 �ybaO::cat This studyW3110 E. coli K-12 lacL8 lacIq Laboratory stockDH5� E. coli K-12 F� endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG �80dlacZ�M15

�(lacZYA-argF)U169 hsdR17(rK� mK

�) ��

Laboratory stock

BW23474 E. coli K-12 �(lacZYA-argF)U169 rpoS(Am) robA1 creC510 hsdR514 �uidA(MluI)::pir-116 endA(BT333) recA1

Genetic stock center

PlasmidspMMB190 Broad-host-range vector, Ampr, lacUV5 promoter, lacZ� 44pMMB-cdsH pMMB190 containing cdsH (low copy) This studypUC18 Cloning vector Laboratory stockpUC18-cdsH pUC18 containing cdsH (high copy) This studypKD46 araC bla oriR101 repA101(Ts) � red (gam� bet � exo�) 21pCP20 FLP (FRT-specific) recombinase, Ampr Camr 21pKD13 oriR bla with the Kanr cassette flanked by FRT sites 21pDK13-Cat pKD13 with the Kanr replaced by Camr This studypKD4 oriR bla with the Kanr cassette flanked by FRT sites 21pINT-s � integrase, Ampr, pSC101 origin 27pAH125 lacZ transcriptional fusion vector, Kanr Genetic stock centerpBLS17 lacZ translational fusion vector (from pAH125) Laboratory stockpBLS18 lacZ transcriptional fusion vector (from pBLS17) This studyEmpty ASKA SfiI-cut and religated backbone of JW3582 This studyJW3582 pCA24N carrying cysE ASKA library (38)JW0437 pCA24N carrying ybaO ASKA library (38)JW2514 pCA24N carrying iscS ASKA library (38)JW1670 pCA24N carrying sufS ASKA library (38)JW2414 pCA24N carrying cysM ASKA library (38)JW2407 pCA24N carrying cysK ASKA library (38)

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Swarming. Swarming agar was prepared as described previously (37)(1% Fisher tryptone, 0.25% NaCl, 0.5% Bacto agar, and 0.5% glucose orxylose). One microliter of an overnight culture grown in LB was spotted inthe center of each plate section and was dried for 5 min in a flow hood withthe lids removed. To ensure consistent inoculum densities, cell countswere determined by the drop plate method (16) and swarm plates wereaccepted only if the inoculum size started within 4 mm in diameter. Plateswere placed on a tray covered in Saran Wrap to maintain humidity andincubated at 37°C before time point photos were taken with the sameimaging system.

Antibiotic resistance. MICs were determined by the broth microdi-lution method (63) in LB with 0.1% Na2SO4 · 10H2O. Plates were sealedwith AeraSeal breathable film covers and incubated at 37°C. After 14 h ofincubation, final optical densities at 600 nm (OD600) were measured usingan Infinite M200 Tecan reader and iControl version 1.2.7.0 software.

Survival in macrophages. The bacterial survival assay was modifiedfrom previous methods (13, 39, 53); details are provided in the supple-mental material. At various time points, macrophages were washed 3times with phosphate-buffered saline (PBS) and lysed with 1 ml 0.5%deoxycholate–PBS, and surviving bacteria were enumerated by serial di-lutions using the drop plate method (16).

RESULTSBioinformatics of the cysteine desulfhydrase gene. The majorCDS in S. enterica has been purified and characterized (42). From thepublished amino acid composition, we used the AACompIdent toolof the ExPASy Bioinformatics Resource Portal and the TrEMBLdatabase to identify candidate genes. The tool provides a least-squares score using the differences in moles percent for eachamino acid. The website indicates a likely identification if (i) thesame protein has the lowest score on three different lists, includingone which compares all proteins in all species, (ii) the score isunder 30, and (iii) there is a large difference (e.g., 2-fold) betweenproteins in the same species with the lowest two scores. STM0458has the lowest score on all three lists (see Fig. S1 in the supplemen-tal material). The lowest-scoring proteins in S. enterica areSTM0458 and the phosphate-starvation inducible PhnW (2-ami-noethylphosphonate aminotransferase), which have scores of 5and 12, respectively (see Fig. S1 in the supplemental material). Theonly proteins in Salmonella species with a score of less than 30 thatare involved in cysteine metabolism are MetC (a cystathioninelyase) and IscS (a cysteine desulfurase), which have scores of 21and 26, respectively.

We also compared other properties of the purified CDS withseveral proteins that could conceivably generate sulfide: MetC,IscS, and SufS (cysteine desulfurases) and CysK and CysM (cys-teine synthases A and B, respectively). The amino-terminal resi-due of purified CDS is serine. Of the potential sulfide-producingenzymes considered, only STM0458 and CysK have a serine afterthe amino-terminal methionines. The reported mass of the puri-fied CDS subunit (37,000 Da) is closer to the deduced mass ofSTM0458 (38,889 Da) than to the mass of CysK (34,535 Da),CysM (32,645 Da), or any cysteine desulfurase (�44,500 Da) (25,32, 42, 68). These findings strongly suggest that the purified CDSfrom S. enterica is the product of STM0458, and biochemical evi-dence presented below confirmed this conclusion. We designatethis gene cdsH (cysteine desulfhydrase).

The BLAST algorithm indicates the presence of four S. entericaproteins with �20% amino acid identity with STM0458: CysM(cysteine synthase B, 28%), CysK (cysteine synthase A, 24%),STM1002 (a putative diaminopropionate ammonia lyase, 24%),and IlvA (threonine dehydratase, 23%). Due to this homology,

STM0458 (cdsH) is annotated as a putative cysteine synthase/cys-tathionine �-lyase.

Biochemical evidence that CdsH is the predominant cys-teine-inducible CDS. Deletion of cdsH reduced CDS activity by10-fold (Fig. 1A). This result was observed for cells grown aerobi-cally or anaerobically, in broth or minimal medium (data notshown). The major CDS activity is eliminated from an extract of a�cdsH strain subjected to electrophoresis in a nondenaturing gel(Fig. 1B, lane 3, and C, lanes 2 and 5). Plasmid pMMB-cdsH re-stored this activity in a �cdsH strain (Fig. 1D, lane 1). To furtherconfirm that cdsH specifies a CDS, we transformed plasmidpMMB-cdsH into E. coli W3110, which lacks a prominent cys-teine-inducible CDS. These cells acquired CDS activity that was100-fold over the background level and 17-fold over the inducedlevel in S. enterica (Fig. 1A and B, lanes 6 to 8).

A distinctive feature of the cysteine-inducible CDS activity in S.enterica is its high catalytic activity. We observed 600 mU/mg pro-tein of inducible activity in a crude extract (Fig. 1A), which iscomparable to the previously reported 260 mU/mg protein (42).The purified S. enterica CDS has a specific activity of about450,000 mU/mg protein (42). In contrast, purified NifS, a cysteinedesulfurase, has a specific activity of 90 mU/mg, which is less thanthe CDS activity in a crude extract (69).

A second distinguishing feature of the major CDS in S. entericais its induction by cysteine (26). We confirmed this result fromassays from a crude extract (Fig. 1A) and a cell extract subjected tonative gel electrophoresis (Fig. 1B, lanes 2 and 4, and C, lane 4). Toinvestigate whether expression of cdsH matched the enzyme activ-ities, we constructed strains with cdsH-lacZ transcriptional andtranslational fusions. Cysteine induced transcriptional activity inbroth (Fig. 2A) and glucose minimal medium (Fig. 2B). The trans-lational fusion showed the same trend (data not shown). Induc-tion of cdsH occurred with cysteine concentrations greater than200 �M. Expression appeared sooner and was consistently higherin TO12 (�cdsH), which might suggest a higher intracellular levelof cysteine without CdsH (Fig. 2B).

Other sulfide-producing enzymes. Native gels revealed afaster-migrating CDS activity, which generated sulfide hours afterthe predominant activity. We suspected that this other proteinwas MetC, which can degrade cystine as a side reaction (22). Thelag before sulfide detection presumably results from the time re-quired to generate cystine from cysteine oxidation. Strains with adeletion of �metC did not have the lower band (Fig. 1B, lanes 4and 5).

Ten percent residual CDS activity was found in TO1 (�cdsH)and TO2 (�cdsH �metC::cat) (not shown), suggesting the pres-ence of other enzymes with CDS activity that were not cysteineinducible. We did not genetically analyze the enzymes that con-tribute to this basal activity. Instead, we examined the ability oftwo cysteine desulfurases, SufS and IscS, and both cysteine syn-thases, CysK and CysM, to generate sulfide by our assays. Thesefour genes were expressed from high-copy ASKA plasmids. Quan-titative assays indicated activity 2- to 4-fold above background forall four enzymes in a �cdsH strain (Fig. 1D). However, this activitywas at least 20-fold less than that in a strain with pMMB-cdsH,which is a low-copy-number plasmid (Fig. 1D). These results wereexpected, since CysM and CysK can catalyze the cleavage of cys-teine but at only about 1% of the level for the forward reaction(25). The same samples subjected to electrophoresis in a nonde-naturing gel detected IscS (just below MetC) but not SufS. The two

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cysteine synthases, CysK and CysM, generated sulfide, althoughwith substantially less activity and at entirely different positionsthan the predominant CDS. These results were also expected,since the cysteine synthases are dimeric, while the previously char-acterized CDS is hexameric (40, 42). We did not further examinewhether these enzymes contribute to the basal CDS activity in a�cdsH strain.

ybaO regulates cdsH. The ybaO gene, which is just upstream ofcdsH, specifies a putative Lrp/AsnC family transcriptional regula-tor (67). Lrp (leucine-responsive regulatory protein) binds severalamino acids, e.g., leucine and alanine (50). Therefore, we exam-ined whether ybaO controls cdsH expression. A ybaO deletionreduced cysteine-inducible CDS activity 10-fold (Fig. 1A and C)and essentially eliminated �-galactosidase activity from a tran-

FIG 1 L-Cysteine desulfhydrase activity of crude extracts. (A) Cells were grown in LB to stationary phase at 37°C and assayed for CDS activity by the methyleneblue assay. **, empty pMMB190 vector; *, empty ASKA plasmid. (B and C) Activity staining of cysteine desulfhydrase on a nondenaturing 10% polyacrylamidegel. (B) Lanes 1 to 5, activity from strains in an S. enterica TR10000 background: lane 1, TR10000 (wild type); lane 2, TR10000; lane 3, lane TO1 (�cdsH); lane 4,TO9 (�metC); lane 5, TO2 (�cdsH �metC). Lanes 6 to 8, activity from E. coli W3110: lane 6, W3110; lane 7, W3110 containing pMMB190 (empty vector); lane8, W3110 containing pMMB190-cdsH. (C) Extracts from derivatives of S. enterica TR10000: lane 1, TR10000; lane 2, TO1 (�cdsH); lane 3, TO3 (�ybaO); lane 4,TR10000; lane 5, TO1 (�cdsH); lane 6, TO3 (�ybaO); lane 7, TO3 (�ybaO) with empty ASKA vector; lane 8, TO3 (�ybaO) with ASKA-ybaO; lane 9, TO3(�ybaO) containing pMMB190; and lane 10, TO3 (�ybaO) with pMMB190-cdsH. (D) Extracts from derivatives of S. enterica TR10000 �cdsH, with vectorscarrying genes indicated above each lane and respective CDS activity units measured by the methylene blue assay below each lane.

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scriptional cdsH-lacZ strain (Fig. 2B). Finally, all deficiencies in aybaO mutant were restored by a plasmid carrying ybaO (Fig. 1Aand C [lanes 7 and 8] and 2B). We conclude that YbaO mediatesinduction by cysteine.

Growth defects of �cdsH and �ybaO mutants. In E. coli, ex-ogenous cysteine transiently causes amino acid starvation, whichis overcome by addition of leucine, isoleucine, valine, and threo-nine (20, 33). The toxicity is only transient, possibly because ofinduction of cysteine catabolic enzymes (2, 57). If CdsH degradescysteine in vivo, then mutants with defects in cysteine catabolismmight respond poorly to exogenous cysteine. We examined theeffect of cysteine in different media. First, 1 mM cysteine in glu-cose-ammonia minimal medium increased the lag phase beforerapid growth for both TO1 (�cdsH) and TO3 (�ybaO) (Fig. 3A).(TO1 and TO3 are here designated the �cdsH and �ybaO mu-tants, respectively.) Cells were grown anaerobically to minimizethe complication of cysteine oxidation to cystine. Second, we grewcells with cysteine as the sole nitrogen source, which requires cys-teine catabolism. If the nitrogen from 1 mM cysteine can be uti-lized, then we would expect growth equivalent to that supportedby 1 mM NH4Cl (A600 of 0.6) (65). However, TR10000 (wildtype [WT]) did not grow with cysteine as the sole nitrogen source,possibly due to toxicity (33). To overcome this inhibition, either0.2 mM (NH4)2SO4 or 0.02% Casamino Acids was added witheither glucose or xylose, respectively, as the carbon source. Cys-teine utilization as a nitrogen source with 0.2 mM (NH4)2SO4 wasapparent for the wild-type strain but was severely diminished forboth the �cdsH and �ybaO mutants (Fig. 3B and C). Since othersulfide-producing enzymes, and possibly cysteine catabolic en-zymes, exhibit glucose repression (18), cells were also grown withxylose as a carbon source. Similar results were seen for cells grownwith xylose (Fig. 3D and E). This growth defect was reversed bycomplementation with plasmid pMMB-cdsH (Fig. 3D). Finally, inglucose-ammonia minimal medium with 5 mM cysteine, cells ofthe �cdsH and �ybaO strains, but not the wild type, turned theculture yellow (spectral peak at around 380 nm) and aggregated.The basis for these properties is unknown.

Sulfide generation in vivo. Impaired growth with cysteine asthe major nitrogen source suggests that �cdsH and �ybaO mu-

tants might have diminished sulfide production in vivo. Leadacetate strips sealed above a culture with cysteine as the majornitrogen source were used to detect sulfide. The mutants had di-minished sulfide generation with cysteine as the major nitrogensource compared to the wild-type strain, which was restored bycdsH complementation (Fig. 3D). For extended incubations, bothmutants eventually generated sulfide (Fig. 3E), which suggests thepresence of an additional cysteine catabolic, sulfide-producing en-zyme(s).

Sulfide production distinguishes S. enterica from related en-teric bacteria and is often detected using sulfide-indole motility(SIM) agar (1). SIM medium contains hydrolyzed peptides,iron, and thiosulfate (23). Cells are stabbed into this medium,and sulfide production results in FeS, which forms a black pre-cipitate (14). In modified SIM medium without thiosulfate(cysteine becomes the only source of sulfide), the �cdsH mu-tant and TO2 (�cdsH �metC) produced as much sulfide asTR10000 (wild type), whereas the �ybaO mutant producedlittle sulfide (see Fig. S2A in the supplemental material). Theseresults suggest YbaO-dependent, CdsH-independent sulfideproduction from whole cells. This is consistent with the possi-bility of a second YbaO-regulated cysteine catabolic enzyme.Further qualitative observations are consistent with this possi-bility. Wild-type and �cdsH strains, unlike a �ybaO mutant,produced a detectable sulfide odor and a grayish-green cellpellet (see Fig. S2B in the supplemental material). A plausibleexplanation for this observation is that sulfide precipitatestrace metals in the medium. Finally, to ensure that YbaO-reg-ulated cysteine catabolism was not an artifact of a specific lab-oratory strain, we tested for and observed YbaO-dependentsulfide generation from cysteine in three different S. entericastrains (see Fig. S2C in the supplemental material).

To confirm CdsH-independent sulfide production and tomore easily control the sulfide source, the assay for sulfide wasperformed in a different way. We grew cells on LB plates contain-ing ferrous sulfate and with a potential sulfide source on a filterdisc. Iron sulfide (FeS) will form a black ring around the disc ifsufficient sulfide is produced. These assays had to be performedanaerobically (under a thick layer of agar), since there was no

FIG 2 Cysteine-inducible regulation of cdsH using cdsH-lacZ transcriptional fusions. (A) TR10000 (wild type) was grown in LB. The arrow indicates additionof 10 mM cysteine. The dotted line represents cell growth in Klett units (100 units is an A600 of 0.6), and the solid line represents �-galactosidase activity froma strain with a transcriptional cdsH-lacZ fusion. (B) Strains with a transcriptional cdsH-lacZ fusion, i.e., TO11 (WT), TO12 (�cdsH), TO13 (�ybaO), and TO13with pYbaO (JW0437), were grown in glucose-ammonia minimal medium with increasing concentrations of cysteine. For TO11, number of determinations(n) � 5; for TO12 and TO13 with pYbaO, n � 2; and for TO13, n � 3.

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detectable precipitate if the discs were affixed to the aerobic side(Fig. 4A). When cysteine was added to the disc, the wild-type and�cdsH strains produced sulfide, but again, a �ybaO strain did not(Fig. 4B). This phenotype was not due to growth differences (seeFig. S3 in the supplemental material). Well-characterized enzymesgenerate sulfide from thiosulfate and tetrathionate independent ofcysteine catabolism (5, 17, 54). When thiosulfate or tetrathionatereplaced cysteine on the disc, the �cdsH and �ybaO mutants pro-

duced as much sulfide as the wild type (Fig. 4B). These resultsshow that YbaO regulates sulfide generation from cysteine but notfrom thiosulfate or tetrathionate. Glucose and nitrate repress sul-fide generation from thiosulfate and tetrathionate (5, 18). Glucoseand nitrate also repressed anaerobic sulfide generation from cys-teine (Fig. 4B).

We examined sulfide production from cells with a plasmid thatoverexpressed CdsH. Overproduction increased CdsH activity

FIG 3 Cysteine toxicity and sulfide generation. (A) Anaerobic growth of cells with or without cysteine in glucose-ammonia minimal medium at 37°C. Note thatthe WT and �cdsH curves overlap. (B to E) Growth measurements (A600) were recorded for cells grown with cysteine as the major nitrogen source. (B and C) Withglucose as the carbon source and 0.003% (NH4)2SO4 as a trace nitrogen source, growth was measured for 18 h (B) and 24 h (C) without cysteine (black bars) andwith 2 mM cysteine (gray bars). (D and E) With xylose as a carbon source and 0.02% Casamino Acids as a trace nitrogen source, growth was measured at 18 h(D) and 24 h (E) without cysteine (black bars) and with 2 mM cysteine (gray bars). Lead acetate strips were affixed to the inner wall of tubes and are shown abovethe graphs. Blackening of the strip is proportional to the sulfide generated.

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12-fold (from 0.6 U to 7.5 U) and contributed to aerobic sulfidegeneration with cysteine as a nitrogen source (Fig. 3D). However,overproduction did not result in detectable sulfide in vivo by theanaerobic disc assay (Fig. 4C). S. enterica containing the wild-typecdsH gene and a cdsH-lacZ transcriptional fusion grown anaero-bically with cysteine induced �-galactosidase and CdsH as indi-cated in activity staining in a native gel (not shown). These resultssuggest that CdsH is inactive in vivo in either S. enterica or E. coligrown anaerobically.

Swarming. Cysteine, but not other amino acids, is importantfor swarming motility. Swarming is a form of motility on semi-solid surfaces (34). Cysteine auxotrophs do not swarm on a low-cysteine-containing medium, but additional exogenous cysteinerestores swarming (30, 48, 60).

Mutants with defects in cysteine catabolism consistently exhib-ited a decreased lag phase before swarming (Fig. 5A and B). Oncethey were swarming, it appeared that both mutants swarmed atthe same speed as the wild type. Such a property has been called

precocious swarming (7, 35). Swarming lags are poorly under-stood, but they can be overcome by increasing inoculum den-sity, certain mutations, or hyperflagellation (34). To rule outthe first factor, colony counts of the initial inoculum densitywere measured for each swarm assay to ensure that all strainsstarted with an average of 5 � 109 CFU/ml (Fig. 5C). As nega-tive controls, we constructed TO10 (�cydD) and TO4 (�cysB),which exhibit defective swarming (30, 48, 60), and verifiedtheir impaired swarming (data not shown). To ensure that themutant phenotype is specific to swarming motility, we inocu-lated the strains on swimming motility plates containing 0.25%agar. There was no difference in swimming motility betweenwild-type, �cdsH, and �ybaO strains (Fig. 5D). We also ob-served precocious swarming on xylose swarm plates (data notshown). We conclude that defective cysteine catabolism affectsswarming-specific motility.

Antibiotic resistance. It has been previously shown that cys-teine synthesis is required for antibiotic resistance of swarming

FIG 4 Anaerobic sulfide detection using cysteine as the sulfur source. (A) Filter discs containing a sulfur source were placed on top of (aerobic) or under(anaerobic) LB agar for S. enterica TR10000. (B) Anaerobic disc assay for various strains with the indicated additions. WT, S. enterica TR 10000; �cdsH, TO1;�ybaO, TO3; E. coli, W3110. (C) Assay of sulfide generation when cdsH is overproduced from a plasmid in S. enterica TO3 (�ybaO) or E. coli W3110. IPTG,isopropyl-�-D-thiogalactopyranoside.

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cells (60). Therefore, we examined the effect of cysteine catabo-lism on antibiotic resistance. We measured the MICs of variousantibiotics in LB medium (63). High exogenous cysteine increasedantibiotic resistance (Fig. 6). This effect is specifically seen for

aminoglycosides (streptomycin, spectinomycin, gentamicin, andkanamycin) but not for other antibiotic classes (chloramphenicoland tetracycline) (data not shown). However, a minor effect isseen with ampicillin (not shown), which may be consistent with aprevious report that cysteine increases resistance to penicillin(49). Defective cysteine catabolism did not affect antibiotic sus-ceptibility with or without cysteine (Fig. 6).

Virulence. Macrophages are monocyte-derived cells that at-tack phagocytosed bacteria with acidification, reactive oxygenand nitrogen species, antimicrobial peptides, and lysosomalenzymes (62). S. enterica survives and replicates within macro-phages. Monocytes increase cysteine uptake when activated byflagellar interaction with pathogen-associated molecular pat-terns (PAMPS) (24). Gene profiling has shown that both cdsHand ybaO are upregulated at 4 and 8 h postinfection of macro-phages (28). Also, cdsH is upregulated in response to reactive ni-trogen species (11). It is possible that cysteine contributes to theinteraction between bacteria and macrophages. However, assay ofS. enterica survival in a macrophage line showed no differencebetween wild-type and mutant strains after several hours (see Fig.S4 in the supplemental material).

FIG 5 Swarming motility. (A) Swarming motility on 0.5% agar captured at various time points for which the diameters are represented graphically. (B) Swarmdiameters. (C) Serial dilutions of the overnight cultures used to inoculate swarm medium to ensure the same initial starting densities. (D) Swimming motility on0.25% agar.

FIG 6 Cysteine-enhanced antibiotic resistance. Growth with the indicatedconcentration of gentamicin, with or without 5 mM cysteine, is shown.

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DISCUSSIONCdsH, CutR/YbaO, and cysteine catabolism. We identified thegene for the major CDS in S. enterica as STM0458 (cdsH) usingbioinformatic and biochemical evidence. The evidence includedthe lowest least-squares score when comparing the amino acidcomposition of the previously purified CDS to that of all proteins,subunit size, the amino-terminal amino acid, high catalytic activ-ity, loss of activity in a deletion strain, acquisition of activity by aplasmid that contains cdsH in E. coli (which lacks a major CDS),induction of the gene and protein by cysteine, and the observationthat several other known sulfide-producing enzymes could notaccount for significant CDS activity. We proposed the redesigna-tion of STM0458 as cdsH (cysteine desulfhydrase).

The gene adjacent to (but divergently transcribed from) cdsHspecifies the major regulator of cdsH expression. Its current des-ignation is ybaO (STM0459). Since this regulator controls multi-ple enzymes that catabolize cysteine, we propose the designationcutR (cysteine utilization regulator). (The identification and char-acterization of a second cysteine catabolic enzyme will be the sub-ject of another communication.) CutR is homologous to Lrp, theleucine-responsive regulatory protein, which binds leucine andseveral other amino acids. It is plausible that CutR binds cysteineand activates transcription. The primary sequence of YbaO fromS. enterica has been aligned with those of the three Lrp homo-logues from E. coli, Lrp, AsnC, and YbaO (66). It has been previ-ously shown that CysB indirectly affects cysteine-inducible CDSactivity by affecting transport (4). However, induction of CDSactivity, which is presumably CdsH, is normal in various cysBmutants (41). Mutants with a deletion of either cdsH or cutR havedistinct phenotypes that distinguish them from each other and theparental strains. Both mutants were more sensitive to cysteinetoxicity, had altered sulfide production aerobically, and exhibitedprecocious swarming. However, only the cutR mutants had re-duced sulfide production anaerobically.

The cdsH gene is one of several genes whose products metab-olize sulfur-containing compounds in S. enterica but not E. coli.This group includes ttrABC (tetrathionate reductase), phsABC(thiosulfate reductase), and asrABC (sulfite reductase). The prod-ucts of these operons can accept electrons for anaerobic respira-tion. The known properties of CdsH do not suggest an obviousrelation to anaerobic respiration. Phylogenetic considerationsalso suggest that cdsH is not linked to these particular genes. Forexample, within the family Enterobacteriaceae, some genera lackboth cdsH and ttrA (Erwinia), some have both (Salmonella, Kleb-siella, Serratia, and Yersinia), and some have one but not the other(Citrobacter and Proteus).

The cdsH gene is present in Salmonella, Klebsiella, Serratia, Yer-sinia, and Citrobacter but not Escherichia, Shigella, Erwinia, andProteus. In contrast, all genera have cutR (ybaO). It seems likelythat CutR/YbaO regulates more genes than cdsH. The widespreaddistribution of cutR (ybaO) and cdsH suggests that they are notrecent acquisitions within the Enterbacteriaceae.

Cysteine toxicity. High exogenous cysteine is toxic to bacteriaand eukaryotes. Several mechanisms have been proposed, includ-ing inhibition of anabolic enzymes, reduction of Fe(III) to Fe(II)and subsequent stimulation of the Fenton reaction and hydroxylradical production, and inhibition of electron transport (46, 49).The greater lag before exponential growth indicated that cdsH andcutR mutants were more sensitive to exogenous cysteine. The cutR

mutant has a longer lag than the cdsH mutant, which suggests thata cutR-regulated enzyme(s) contributed to degradation of cyto-toxic cysteine.

Sulfide generation in vivo and cysteine catabolism. A recentstudy shows that hydrogen sulfide protects a variety of bacteriafrom antibiotics (55). The basic mechanism appears to involvelowering the concentration of reactive oxygen species. Endog-enous sulfide generation is therefore an important cellularreaction. CdsH generates sulfide with cysteine as the majornitrogen source for cells grown aerobically. In contrast, foranaerobic growth in broth with exogenous cysteine, CdsH wasnot a major contributor to sulfide generation. Instead, a secondcysteine catabolic enzyme generated most of the sulfide. Thisenzyme, like CdsH, was controlled by CutR. Even when CdsHwas overproduced 12-fold, CdsH did not detectably generatesulfide in vivo. We propose that some factor inside cells inhibitsCdsH activity. A possible candidate is sulfide itself. The Ki forCdsH for sulfide is 10 �M (43). If the second enzyme has ahigher Ki, then CdsH will be inactive when sufficient sulfideaccumulates.

Our results suggest that at least two enzymes in S. enterica candegrade cysteine and generate sulfide. We suggest that there areseveral significant sulfide-generating reactions and that which isactive will depend on what compounds are available (e.g., cys-teine) and the physical environment (the presence of oxygen). Inaddition to sulfide-generating reactions of cysteine catabolism, S.enterica also possesses enzymes that can generate sulfide fromthiosulfate and tetrathionate. Both of the latter compounds arereadily available in the intestinal tract during inflammation (64).Their utilization is controlled by glucose or alternate electron ac-ceptors, such as nitrate (5). Such regulation was also apparent forthe CdsH-independent, CutR-regulated cysteine catabolic en-zyme (Fig. 4B). Figure 7 summarizes the various reactions andpathways that generate sulfide.

FIG 7 Sulfide-producing pathways. There are several sulfide-generating path-ways and enzymes. The assimilatory reduction of sulfite generates sulfide forcysteine synthesis. The dissimilatory pathway of sulfite reduction uses sulfite asan electron acceptor for anaerobic respiration. CdsH is a cysteine catabolicenzyme that is controlled by YbaO. YbaO also controls a second enzyme,indicated by “?,” which will be the topic of another communication. The Sbpprotein is the sulfate-binding protein that transports sulfate into the cell. Inaddition to these enzymes, the cysteine desulfurases and CysK and CysM pro-teins might provide background sulfide as side reactions.

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Cysteine catabolism, swarming motility, and antibiotic re-sistance. Mutants impaired in cysteine synthesis had defectiveswarming, even though cells were tested in a broth that allowedcysteine auxotrophs to grow. It was suggested that swarming re-quires a higher-than-normal level of intracelluar cysteine and analtered metabolism (30, 49, 60). Our results support this conclu-sion. Mutants defective in cysteine catabolism, which would bepredicted to have higher intracellular cysteine, exhibited preco-cious swarming. A possible mechanistic link is that high intracel-lular cysteine inhibits certain enzymes, which may be sufficient toalter metabolism in a way that promotes swarming.

Turnbull and Surette have also shown that swarming cells haveelevated resistance to antibiotics and that cysteine enhances anti-biotic resistance of swarming cells (60). We confirmed the effect ofcysteine on antibiotic resistance, but we observed a cysteine effectfor planktonic cells. Loss of cysteine catabolic enzymes did notaffect antibiotic susceptibility. Because of the different effects ofloss of cysteine catabolism on swarming and antibiotic resistance,we propose that the effect of cysteine occurs by two differentmechanisms. A previous study revealed that increasing exogenouscysteine decreases reduced periplasmic cytochrome c levels (49).Since aminoglycoside antibiotic uptake relies on anionic trans-porters energized mainly by the respiratory chain, impaired elec-tron transport could explain the observed increase in aminogly-coside resistance with cysteine (12, 59). Defects in cysteinecatabolism may have little effect on exogenous cysteine underthese conditions, and this could explain the lack of effect on anti-biotic susceptibility.

ACKNOWLEDGMENTS

This work was supported in part by grants MCB-0323931 from the Na-tional Science Foundation and GM085536 from the National Institutes ofHealth.

We thank Helene Andrews-Polymenis, D. J. Kopecko, and LoraHooper for strains and the macrophage cell line, Eric Hansen and his labfor training in the use of the macrophage line, Piyush B. Lal for construc-tion of the S. enterica metC and cydD mutants, Juan González and his labfor training and use of equipment, and Jeff DeJong and Santosh D’Mellofor the use of tissue culture facilities.

REFERENCES1. Appelbaum PC, et al. 1982. Comparison of three methods for identifi-

cation of Enterobacteriaceae. Eur. J. Clin. Microbiol. 1:76 – 81.2. Awano N, Wada M, Mori H, Nakamori S, Takagi H. 2005. Identification

and functional analysis of Escherichia coli cysteine desulfhydrases. Appl.Environ. Microbiol. 71:4149 – 4152.

3. Awano N, et al. 2003. Effect of cysteine desulfhydrase gene disruption onL-cysteine overproduction in Escherichia coli. Appl. Microbiol. Biotech-nol. 62:239 –243.

4. Baptist EW, Kredich NM. 1977. Regulation of L-cystine transport inSalmonella typhimurium. J. Bacteriol. 131:111–118.

5. Barrett EL, Clark MA. 1987. Tetrathionate reduction and production ofhydrogen sulfide from thiosulfate. Microbiol. Rev. 51:192–205.

6. Beggs WH, Lichstein HC. 1965. Repression of tryptophanase synthesis inEscherichia coli. J. Bacteriol. 89:996 –1004.

7. Belas R, Schneider R, Melch M. 1998. Characterization of Proteus mira-bilis precocious swarming mutants: identification of rsbA, encoding a reg-ulator of swarming behavior. J. Bacteriol. 180:6126 – 6139.

8. Bogomolnaya LM, Santiviago CA, Yang HJ, Baumler AJ, Andrews-Polymenis HL. 2008. ‘Form variation’ of the O12 antigen is critical forpersistence of Salmonella typhimurium in the murine intestine. Mol. Mi-crobiol. 70:1105–1119.

9. Botsford JL. 1975. Metabolism of cyclic adenosine 3=,5=-monophosphateand induction of tryptophanase in Escherichia coli. J. Bacteriol. 124:380 –390.

10. Botsford JL, DeMoss RD. 1971. Catabolite repression of tryptophanase inEscherichia coli. J. Bacteriol. 105:303–312.

11. Bourret TJ, et al. 2008. Nitric oxide antagonizes the acid tolerance re-sponse that protects Salmonella against innate gastric defenses. PLoS One3:e1833. doi:10.1371/journal.pone.0001833.

12. Bryan LE, Nicas T, Holloway BW, Crowther C. 1980. Aminoglycoside-resistant mutation of Pseudomonas aeruginosa defective in cytochromec552 and nitrate reductase. Antimicrob. Agents Chemother. 17:71–79.

13. Buchmeier NA, Heffron F. 1989. Intracellular survival of wild-type Sal-monella typhimurium and macrophage-sensitive mutants in diverse pop-ulations of macrophages. Infect. Immun. 57:1–7.

14. Carrington GO, Cleveland P, Jr, von Graevenitz A, Rupp WD. 1975.Biochemically aberrant Salmonella enteritidis ser. Newington from humansources in Connecticut. Yale J. Biol. Med. 48:83– 89.

15. Chan RK, Botstein D. 1976. Specialized transduction by bacteriophageP22 in Salmonella typhimurium: genetic and physical structure of thetransducing genomes and the prophage attachment site. Genetics 83:433–458.

16. Chen CY, Nace GW, Irwin PL. 2003. A 6 � 6 drop plate method forsimultaneous colony counting and MPN enumeration of Campylobacterjejuni, Listeria monocytogenes, and Escherichia coli. J. Microbiol. Methods55:475– 479.

17. Clark MA, Barrett EL. 1987. The phs gene and hydrogen sulfide produc-tion by Salmonella typhimurium. J. Bacteriol. 169:2391–2397.

18. Clark MA, Barrett EL. 1987. Catabolite repression of thiosulfate reduc-tion by Salmonella typhimurium. Curr. Microbiol. 16:27–31.

19. Collins JM, Monty KJ. 1973. The cysteine desulfhydrase of Salmonellatyphimurium. Kinetic and catalytic properties. J. Biol. Chem. 248:5943–5949.

20. Cowman RA, Baron SS, Fitzgerald RJ. 1983. Cysteine toxicity for oralstreptococci and effect of branched-chain amino acids. Infect. Immun.39:1107–1113.

21. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomalgenes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci.U. S. A. 97:6640 – 6645.

22. Dwivedi CM, Ragin RC, Uren JR. 1982. Cloning, purification, andcharacterization of �-cystathionase from Escherichia coli. Biochemistry21:3064 –3069.

23. Ederer GM, Lund ME, Blazevic DJ, Reller LB, Mirrett S. 1975. Motility-indole-lysine-sulfide medium. J. Clin. Microbiol. 2:266 –267.

24. Edinger AL, Thompson CB. 2002. Antigen-presenting cells control T cellproliferation by regulating amino acid availability. Proc. Natl. Acad. Sci.U. S. A. 99:1107–1109.

25. Flint DH, Tuminello JF, Miller TJ. 1996. Studies on the synthesis of theFe-S cluster of dihydroxy-acid dehydratase in Escherichia coli crude ex-tract. Isolation of O-acetylserine sulfhydrylases A and B and �-cystathio-nase based on their ability to mobilize sulfur from cysteine and to partic-ipate in Fe-S cluster synthesis. J. Biol. Chem. 271:16053–16067.

26. Guarneros G, Ortega MV. 1970. Cysteine desulfhydrase activities of Sal-monella typhimurium and Escherichia coli. Biochim. Biophys. Acta 198:132–142.

27. Haldimann A, Wanner BL. 2001. Conditional-replication, integration,excision, and retrieval plasmid-host systems for gene structure-functionstudies of bacteria. J. Bacteriol. 183:6384 – 6393.

28. Hautefort I, et al. 2008. During infection of epithelial cells Salmonellaenterica serovar Typhimurium undergoes a time-dependent transcrip-tional adaptation that results in simultaneous expression of three type 3secretion systems. Cell. Microbiol. 10:958 –984.

29. Hirakawa H, Kodama T, Takumi-Kobayashi A, Honda T, YamaguchiA. 2009. Secreted indole serves as a signal for expression of type III secre-tion system translocators in enterohaemorrhagic Escherichia coli O157:H7. Microbiology 155:541–550.

30. Inoue T, et al. 2007. Genome-wide screening of genes required forswarming motility in Escherichia coli K-12. J. Bacteriol. 189:950 –957.

31. Isaacs H, Jr, Chao D, Yanofsky C, Saier MH, Jr. 1994. Mechanism ofcatabolite repression of tryptophanase synthesis in Escherichia coli. Micro-biology 140:2125–2134.

32. Kambampati R, Lauhon CT. 1999. IscS is a sulfurtransferase for the invitro biosynthesis of 4-thiouridine in Escherichia coli tRNA. Biochemistry38:16561–16568.

33. Kari C, Nagy Z, Kovacs P, Hernadi F. 1971. Mechanism of the growthinhibitory effect of cysteine on Escherichia coli. J. Gen. Microbiol. 68:349 –356.

Cysteine Catabolism in S. enterica

August 2012 Volume 194 Number 16 jb.asm.org 4375

on August 27, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

34. Kearns DB. 2010. A field guide to bacterial swarming motility. Nat. Rev.Microbiol. 8:634 – 644.

35. Kim DJ, Boylan B, George N, Forst S. 2003. Inactivation of ompRpromotes precocious swarming and flhDC expression in Xenorhabdusnematophila. J. Bacteriol. 185:5290 –5294.

36. Kim SH, Schneider BL, Reitzer L. 2010. Genetics and regulation of themajor enzymes of alanine synthesis in Escherichia coli. J. Bacteriol. 192:5304 –5311.

37. Kim W, Surette MG. 2003. Swarming populations of Salmonella repre-sent a unique physiological state coupled to multiple mechanisms of an-tibiotic resistance. Biol. Proc. 5:189 –196.

38. Kitagawa M, et al. 2005. Complete set of ORF clones of Escherichia coliASKA library (a complete set of E. coli K-12 ORF archive): unique re-sources for biological research. DNA Res. 12:291–299.

39. Klumpp J, Fuchs TM. 2007. Identification of novel genes in genomicislands that contribute to Salmonella typhimurium replication in macro-phages. Microbiology 153:1207–1220.

40. Kredich NM. 1996. Biosynthesis of cysteine, p 514 –527. In Neidhardt FC,et al (ed), Escherichia coli and Salmonella: cellular and molecular biology,2nd ed. ASM Press, Washington, DC.

41. Kredich NM. 1971. Regulation of L-cysteine biosynthesis in Salmonellatyphimurium. I. Effects of growth of varying sulfur sources and O-acetyl-L-serine on gene expression. J. Biol. Chem. 246:3474 –3484.

42. Kredich NM, Keenan BS, Foote LJ. 1972. The purification and subunitstructure of cysteine desulfhydrase from Salmonella typhimurium. J. Biol.Chem. 247:7157–7162.

43. Kredich NM, Foote LJ, Keenan BS. 1973. The stoichiometry and kineticsof the inducible cysteine desulfhydrase from Salmonella typhimurium. J.Biol. Chem. 248:6187– 6196.

44. Morales VM, Backman A, Bagdasarian M. 1991. A series of wide-host-range low-copy-number vectors that allow direct screening for recombi-nants. Gene 97:39 – 47.

45. Natusch DF, Sewell JR, Tanner RL. 1974. Determination of hydrogensulfide in air—an assessment of impregnated paper tape methods. Anal.Chem. 46:410 – 415.

46. Park S, Imlay JA. 2003. High levels of intracellular cysteine promoteoxidative DNA damage by driving the Fenton reaction. J. Bacteriol. 185:1942–1950.

47. Penrod JT, Roth JR. 2006. Conserving a volatile metabolite: a role forcarboxysome-like organelles in Salmonella enterica. J. Bacteriol. 188:2865–2874.

48. Pittman MS, Robinson HC, Poole RK. 2005. A bacterial glutathionetransporter (Escherichia coli CydDC) exports reductant to the periplasm. J.Biol. Chem. 280:32254 –32261.

49. Pittman MS, et al. 2002. Cysteine is exported from the Escherichia colicytoplasm by CydDC, an ATP-binding cassette-type transporter requiredfor cytochrome assembly. J. Biol. Chem. 277:49841– 49849.

50. Roesch PL, Blomfield IC. 1998. Leucine alters the interaction of theleucine-responsive regulatory protein (Lrp) with the Fim switch to stim-ulate site-specific recombination in Escherichia coli. Mol. Microbiol. 27:751–761.

51. Rompf A, Schmid R, Jahn D. 1998. Changes in protein synthesis as aconsequence of heme depletion in Escherichia coli. Curr. Microbiol. 37:226 –230.

52. Saito H, Kobayashi H. 2003. Bacterial responses to alkaline stress. Sci.Prog. 86:271–282.

53. Schwan WR, Huang XZ, Hu L, Kopecko DJ. 2000. Differential bacterialsurvival, replication, and apoptosis-inducing ability of Salmonella sero-vars within human and murine macrophages. Infect. Immun. 68:1005–1013.

54. Sekowska A, Kung HF, Danchin A. 2000. Sulfur metabolism in Esche-richia coli and related bacteria: facts and fiction. J. Mol. Microbiol. Bio-technol. 2:145–177.

55. Shatalin K, Shatalina E, Mironov A, Nudler E. 2011. H2S: a universaldefense against antibiotics in bacteria. Science 334:986 –990.

56. Snell EE. 1975. Tryptophanase: structure, catalytic activities, and mecha-nism of action. Adv. Enzymol. Relat. Areas Mol. Biol. 42:287–333.

57. Sorensen MA, Pedersen S. 1991. Cysteine, even in low concentrations,induces transient amino acid starvation in Escherichia coli. J. Bacteriol.173:5244 –5246.

58. Soutourina J. 2001. Role of D-cysteine desulfhydrase in the adaptation ofEscherichia coli to D-Cysteine. J. Biol. Chem. 276:40864 – 40872.

59. Taber HW, Mueller JP, Miller PF, Arrow AS. 1987. Bacterial uptake ofaminoglycoside antibiotics. Microbiol. Rev. 51:439 – 457.

60. Turnbull AL, Surette MG. 2008. L-Cysteine is required for induced anti-biotic resistance in actively swarming Salmonella enterica serovar Typhi-murium. Microbiology 154:3410 –3419.

61. Vaishnava S, Behrendt CL, Ismail AS, Eckmann L, Hooper LV. 2008.Paneth cells directly sense gut commensals and maintain homeostasis atthe intestinal host-microbial interface. Proc. Natl. Acad. Sci. U. S. A. 105:20858 –20863.

62. von Loewenich FD, Scorpio DG, Reischl U, Dumler JS, Bogdan C. 2004.Frontline: control of Anaplasma phagocytophilum, an obligate intracellu-lar pathogen, in the absence of inducible nitric oxide synthase, phagocyteNADPH oxidase, tumor necrosis factor, Toll-like receptor (TLR) 2 andTLR4, or the TLR adaptor molecule MyD88. Eur. J. Immunol. 34:1789 –1797.

63. Wiegand I, Hilpert K, Hancock RE. 2008. Agar and broth dilutionmethods to determine the minimal inhibitory concentration (MIC) ofantimicrobial substances. Nat. Protoc. 3:163–175.

64. Winter SE, et al. 2010. Gut inflammation provides a respiratory electronacceptor for Salmonella. Nature 467:426 – 429.

65. Xi H, Schneider BL, Reitzer L. 2000. Purine catabolism in Escherichia coliand function of xanthine dehydrogenase in purine salvage. J. Bacteriol.182:5332–5341.

66. Yokoyama K, Suzuki M. 2005. Orthologous and paralogous FFRPs in E.coli and related proteobacteria. Proc. Jpn. Acad. Ser. B 81:129 –139.

67. Yokoyama K, et al. 2006. Feast/famine regulatory proteins (FFRPs): Esch-erichia coli Lrp, AsnC and related archaeal transcription factors. FEMSMicrobiol. Rev. 30:89 –108.

68. Zhao C, Kumada Y, Imanaka H, Imamura K, Nakanishi K. 2006.Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli. Protein Expr. Purif. 47:607– 613.

69. Zheng L, White RH, Cash VL, Jack RF, Dean DR. 1993. Cysteinedesulfurase activity indicates a role for NIFS in metallocluster biosynthe-sis. Proc. Natl. Acad. Sci. U. S. A. 90:2754 –2758.

Oguri et al.

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