MolecularCloning Expression Escherichia Dimethyl Sulfoxide ...The dimethyl sulfoxide (DMSO) reductase operon coding for a membrane-bound iron-sulfur, molyb doenzyme, whichfunctions

Vol. 170, No. 4JOURNAL OF BACTERIOLOGY, Apr. 1988, p. 1511-15180021-9193/88/041511-08$02.00/0Copyright © 1988, American Society for Microbiology

Molecular Cloning and Expression of theEscherichia coli Dimethyl Sulfoxide Reductase Operon

PETER T. BILOUS AND JOEL H. WEINER*Department of Biochemistry, University ofAlberta, Edmonton, Alberta, Canada T6G 2H7

Received 10 September 1987/Accepted 30 December 1987

The dimethyl sulfoxide (DMSO) reductase operon coding for a membrane-bound iron-sulfur, molyb-doenzyme, which functions as a terminal reductase in Escherichia coli, has been isolated and cloned from anE. coli gene bank. Two clones, MV12(pLC19-36) and MV12(pLC43-43), overexpressed both DMSO andtrimethylamine N-oxide (TMAO) reductase activites 13- to 15-fold compared with wild-type cells. Amplificationwas highest in cells grown anaerobically on fumarate, while cells grown on DMSO or TMAO displayed reducedlevels of enzyme amplification. Growth on nitrate or aerobic growth repressed expression of the enzyme. A6.5-kilobase-pair DNA restriction endonuclease fragment was subcloned from pLC19-36 into the vectorpBR322, yielding a recombinant DMSO reductase plasmid, pDMS159. Two polypeptides were amplified andidentified on sodium dodecyl sulfate-polyacrylamide gels of proteins from E. coli HB101 harboring pDMS159:a membrane-bound protein with molecular weight 82,600 and a soluble polypeptide with molecular weight23,600. Three plasmid-encoded polypeptides with molecular weights of 87,500, 23,300, and 22,600 weredetected by in vivo transcription/translation studies. The smallest subunit was poorly defined and not detectableby Coomassie blue staining. The DMSO reductase operon was localized to the 20.0-min position on the E. colilinkage map.

Anaerobic respiration by Escherichia coli on fumarate andnitrate is well established due to extensive biochemical andmolecular biological characterization of the respective ter-minal reductases (9, 13). However, alternate forms of anaer-obic respiration are known. Recent studies have focused ontrimethylamine N-oxide (TMAO) reduction, due to the wide-spread distribution of this compound in the natural environ-ment, and the demonstration of anaerobic respiration onTMAO by various bacteria (3, 24, 28). TMAO reduction isassociated with the marine genera, nonsulfur photosyntheticbacteria, and certain genera of intestinal bacteria includingE. coli. (3). Studies with E. coli have demonstrated thepresence of one constitutive and three or four inducibleforms of TMAO reductase in the cell (21, 23). The majorinducible form of TMAO reductase has been purified andcharacterized (30).Although bacterial reduction of dimethyl sulfoxide

(DMSO) has been known for some time (1), only recentlyhas its role in anaerobic respiration been determined (19, 25,31). Like TMAO, DMSO is associated with marine environ-ments as a by-product of phytoplankton activity (2) and isconsidered to be an intermediate in the global sulfur cycle(16). We recently demonstrated that E. coli is capable ofanaerobic respiration on DMSO (5). Anaerobic growth of E.coli on DMSO, TMAO, methionine sulfoxide, or fumarateresults in the induction of a membrane-bound molyb-doenzyme catalyzing the reduction of DMSO to dimethylsulfide (4).

Studies in other bacteria have suggested that TMAO andDMSO are reduced by the same enzyme system (20, 25). Ourinital studies suggested some similarities and differencesbetween DMSO and TMAO reduction in E. coli (4). Todefine the role of DMSO reductase in the anaerobic growthof E. coli, the structural genes for the enzyme were clonedand characterized. Cells harboring the DMSO reductase

* Corresponding author.

plasmid displayed amplified levels of DMSO, TMAO andmethionine sulfoxide reductase activities. Studies with thepurified enzyme indicate that DMSO reductase has a broadsubstrate specificity, reducing various sulfoxides and N-oxides (29). The enzyme has been designated DMSO reduc-tase due to the high affinity for this substrate.

MATERIALS AND METHODS

Bacterial strains and plasmids. The E. coli strains andplasmids used in this study are listed in Table 1. E. coliMV12 carries ColEl hybrid plasmids prepared by Clarke andCarbon (8).

Preparation of colicin El. Colicin El was prepared from E.coli W3110(ColE1) by the procedure of Schwartz and Hel-inski (22), except that cells were disrupted by two passagesthrough a French pressure cell (American Instrument Co.,Silver Spring, Md.) at 110 MPa. The crude lysate wascentrifuged at 150,000 x g for 1 h before ammonium sulfateprecipitation of the soluble material according to the pub-lished procedure. The final preparation was stored in 50 mMpotassium phosphate buffer (pH 7.5) containing 50% glyceroland assayed as described previously (12).Growth of cells and preparation of everted membrane

vesicles. For enzyme expression studies, cells were routinelygrown in glycerol minimal medium (4) supplemented withthe appropriate antibiotics (100 ,ug ampicillin or streptomy-cin sulfate per ml), amino acids (0.003%), and terminalelectron acceptor (nitrate, 100 mM; fumarate, 40 mM;TMAO, 100 mM; or DMSO, 70 mM). Cultures were grownfor 36 h at 37°C, harvested, and then lysed by Frenchpressure cell treatment. Membranes were prepared from thecrude lysate material as described previously (4).Enzyme assay. Reductase activity was assayed by moni-

toring the substrate-dependent oxidation of reduced benzylviologen at 570 nm (4). One unit of activity corresponds to 1,umol of benzyl viologen oxidized per min at 23°C. Specific

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1512 BILOUS AND WEINER

TABLE 1. Bacterial strains and plasmids

Strain or Description Sourceplasmid

E. coli strainHB101 F- hsdR hsdM pro leu lac gal thi recA rpsL Lab collectionK38 HfrC (X) S. TaboraMV12 F+ recA Atrp thr leu thi Lab collectionTG1 A(lac-pro) supE thi hsdDSIFl' traD36 proA+B+ la, lacZAM15 W. ParanchychbW3110 F- X- Lab collection

PlasmidColEl ColElr, coding for colicin El Lab collectionpBR322 Apr Tcr Boehringer MannheimpDMS159 Apr dms+ This studypDMS201 Apr dms, derivative of pDMS159 This studypDMS216 Apr Kmr dms+, derivative of pMS159 This studypDMS219 Apr dms, derivative of pDMSt1iS This studypDMS222 Aprdms+, pTZ18R derivative of pDMS2l6 This studypDMS229 Apr dms+, pTZ18R derivative qo pDMS216 This studypGP1-2 Kmr clts857, coding for T7 RNA polymerase under X PL control S. TaborpLC19-36 ColElrdms+ This studypLC43-43 ColElr dms+ This studypTZ18R Apr lacZ' Pharmacia

"Harvard Medical School, Boston, Mass.b University of Alberta, Edmonton, Alberta, Canada.

activity is expressed as units of reductase activity per mil-ligram of protein.

Screening of the Clarke and Carbon colony bank. Each ofthe 2,112 clones from the Clarke and Carbon colony bank (8)was growp aq erpbically at 37°C for 36 h in screw-cap testtubes (13 by "#'mm) containing 8.5 ml of complex medium(glucose, 0.1 Bacto-Peptone [Difco Laboratories, Detroit,Mich.], 0.4%; yeast extract, 0.4%; 70 mM potassium phos-phate buffer,; pEH 6.8) supplemented with 40 mM sodiumfumarate (pP 7 4hiamine (0.003%), and colicin El at 1U/ml. Cultures" Werd mixed continuously during growth bygently rocking horizontally on a platform shaker. Cells wereharvested at 4,400 x g for 5 min (IEC clinical centrifuge,model CL), washed once with 5 ml of 50 mM sodiumphosphate buffer, pH 6.8, and then suspended in 0.5 ml ofthe same buffer. A 50-,ul portion of each cell suspension wasadded to individual wells on microtiter plates. Enzymeassays were initiated by the rapid addition of 200 p.l of assaymixture (50 mM sodium phosphate buffer, pH 6.8, 0.5 mMdithiothreitol, 0.2 mM benzyl viologen, 1.0 mM sodiumdithionite, 10 mM either DMSO or TMAO). The rate ofoxidation of reduced benzyl viologen in each well (purple tocolorless transition) was monitored visually, and the approx-imate time for complete oxidation was recorded.

Preparation and analysis of plasmid DNA. Plasmid DNAwas isolated from cells grown in M9CA medium by chlor-amphenicol amplification and sodium dodecyl sulfate (SDS)lysis as described by Maniatis et al. (17). The isolatedplasmid DNA was purified by equilibrium centrifugation oncesium chloride-ethidium bromide gradients. Isolation ofplasmid DNA on a smaller scale was performed by thealkaline-SDS procedure of Birnboim and Doly (6).

Electrophoresis. SDS-polyacrylamide gel electrophoresiswas performed on vertical slab gels of 12.5% (wt/vol)acrylamide-0.33% bisacrylamide, with a stacking gel of 3%acrylamide-0.08% bisacrylamide. The discontinuous SDSbuffer system of'Laemmli (14) was used. Gels were stainedand destained as described previously (15). Gels containing35S-labeled proteins were dried and autoradiographed di-rectly without further treatment.

Protein determination. Protein was estimated by an SDSmodification of the Lowry procedure (18), using crystallinebovine serum albumin (Bio-Rad Laboratories, Richmond,Calif.) as the protein standard.

Construction of recombinant pTZ18R plasmids. For in vivopolypeptide expression studies, recombinant plasmidspDMS222 and pDMS229 were constructed from pDMS159and pTZ18R (Pharmacia) as follows. A Kmr cartridge (Gen-Block, Pharmacia) was ligated into the unique EcoRI site ofpDMS159 to proviol an additional SalI site for convenientisolation and subsequent ligation of the 6.5-kilobase (kb)chromosomal insert. The resulting plasmid, pDMS2'16, wasdigested with SalI to yield a 6.5-kb SalI-SalI fragment ofchromosomal DNA, which was subsequently purified fromagarose gels by electroelution (D-gel; KONTES, Vineland,N.J.). The fragment was ligated into the SalI site of pTZ18Rand then used to transform TG1 host cells. Plasmid DNAwas isolated from transformed cells, and the orientation ofthe insert was determined by restriction endonuclease m'ap-ping. pDMS222 and pDMS229 contained the chromosomalinsert from pDMS159 in opposite orientations with respectto the T7 RNA polymerase promoter region of pTZ18R.

Labeling of plasmid-encoded polypeptides. E. coli K38(pGP1-2), coding for T7 RNA polymerase under c1857, APLcontrol, was transformed with either pDMS222 orpDMS229. Cell proteins were labeled in these strains with[35S]methionine 'as outlined by Tabor and Richardson (26)with minor modifications. Transformed cells were grownovernight at 30°C in LB medium (17) containing kanamycin(40 ,ug/ml) and ampicillin (100 ,ug/ml) and then diluted 1:50 infresh LB medium. Cells were grown to an A600 of 0.5, and200-,ul samples were removed and washed twice with 1.0 mlof M9 medium (17) before suspension in 1.0 ml of M9medium plus amino acids (0.1%, minus methionine andcysteine), thiamine, and antibiotics. Cells were grown for 60min at 30°C and shifted to 42°C for 15 min, and then rifampin(200 ,ug/ml) was added followed by a further 10-min incuba-tion at 42°C. Cells were then shifted to 30°C for 20 min, atwhich time 6 p.Ci of L-[35S]methionine (1,330 Ci/mmol) wasadded. At the appropriate time intervals, samples were

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CLONING OF DMSO REDUCTASE 1513

removed, added to cold trichloroacetic acid (10% final), andthen incubated on ice for 30 min. Samples were washedtwice with 10% trichloroacetic acid, suspended in Laemmlisolubilization buffer (14) modified to contain 0.2 M Trizmabase (Sigma Chemical Co., St. Louis, Mo.), electrophoresedon SDS-polyacrylamide gels, and then autoradiographed.For pulse-chase experiments, 1.0 ml of cells was pulsed for1 min with 24 pLCi of [35S]methionine, followed by a chasewith 0.01% methionine.

Reagents. All chemicals used in this study were of analyt-ical grade and obtained commercially. L-[35S]methioninewas purchased from Amersham Canada Ltd., Oakville,Ontario.

RESULTSIsolation of DMSO reductase plasmids. To determine the

mechanism of DMSO reduction and its relationship to otherwell-defined anaerobic respiration pathways, cloning of theDMSO reductase operon was carried out. Our initial ap-proach to cloning DMSO reductase was by the mutantcomplementation procedure, using a group of mutants wepreviously characterized to be defective in DMSO reductaseactivity (4). The mutants were complemented with an E. coligene bank prepared by ligating HindIlI-digested chromo-somal DNA into plasmid vectors pBR322 and pUC13. Themutants fell into two complementation groups, and the DNAfragments complementing each of the two mutant classeswere cloned and characterized. Expression studies indicatedthat neither fragment coded for the DMSO reductase struc-tural gene. A preliminary report of these findings has beenpresented (P. T. Bilous, and J. H. Weiner, Abstr. Annu.Meet. Am. Soc. Microbiol. 1987, K119, p. 222), and work isin progress to characterize the gene products.

Since the mutagenesis approach did not result in theisolation of the structural gene for DMSO reductase, theClarke and Carbon E. coli gene bank (8) was screened forclones which expressed amplified levels ofDMSO or TMAOreductase activity. Elevated expression was expected inappropriate clones, due to the multicopy nature of the ColElvector. Each of the approximately 2,100 clones harboringrecombinant ColEl plasmids was grown anaerobically on aglucose-peptone medium. The cells were harvested as de-scribed in Materials and Methods and then used directly forassay of enzyme activity by following the DMSO- or TMAO-dependent oxidation of reduced benzyl viologen. Previousstudies have indicated that both substrates have readyaccess to the enzyme in whole cells or crude lysates (data

PLASMID

pLCI9- 36

RESTRICTION MAP

Sc CPB B BPA A C SE

not shown). Two clones were identified, E. coli MV12(pLC19-36) and MV12(pLC43-43), both of which displayed atwo- to fourfold-faster DMSO- and TMAO-dependent oxi-dation of reduced benzyl viologen than the average E. coliMV12 clone. Air oxidation of the reduced benzyl viologenwas at least twofold slower than the average substrate-dependent oxidation reaction. No clones were found by thisscreening procedure, which amplified DMSO or TMAOreductase individually.

Restriction mapping and subcloning. The recombinantColEl plasmids were isolated from clones MV12(pLC19-36)and MV12(pLC43-43), and restriction endonuclease mapswere determined relative to a unique EcoRI site (Fig. 1). Thetwo plasmids were found to contain a similar chromosomalDNA fragment with an overlapping region of approximately15 kb. To identify the DNA region coding for DMSOreductase, various restriction fragments were generated andsubcloned into vector pBR322, yielding plasmids pDMS159,pDMS201, and pDMS219 (Fig. 1). As shown, only E. coliHB101 cells transformed with plasmid pDMS159 expressedamplified levels of DMSO reductase activity comparable tothe levels seen with MV12(pLC19-36) or MV12(pLC43-43).Chromosomal mapping of DMSO reductase operon. The

precise location of the coding region for DMSO reductasewas determined by comparison of the restriction endonucle-ase sites on pLC19-36 to a BamHI-EcoRI-HindIII restrictionmap of the E. coli chromosome (10). The location, kind, andnumber of endonuclease sites on pLC19-36 agree perfectlyover a 14-kb region with the restriction sites determined fora region of the 210-kb NotI chromosomal DNA fragment(Fig. 2). The NotI fragment was shown by restriction sitecomparison (D. Daniels and F. Blattner, unpublished data)to contain the rpsA and ompF genes which are located on thelinkage map at 20.5 and 20.8 min, respectively. DNA se-quencing analysis (P. T. Bilous, S. T. Cole, W. F. Andersonand J. H. Weiner, manuscript in preparation) has demon-strated the presence of three open reading frames associatedwith DMSO reductase, organized as an operon. The DMSOreductase operon dms was localized to the region of pLC19-36 shown in Fig. 2. The dms operon is therefore situated at20.0 min on the E. coli linkage map.Growth and enzyme expression. E. coli MV12(pLC19-36)

and HB101(pDMS159) cells were grown anaerobically onglycerol-fumarate medium, and the enzyme activities in themembranes and soluble fractions of the cells were deter-mined. We have previously shown that growth on fumarateresults in the expression of DMSO reductase at levels

DMSO REDUCTASE(Specific Activity, U/mg)

9.9Pu Sm

pLC43-43

pDMS159

pDMS201

pDMS219

Sc cPB 8 BPA A C SE

CPS B BPA AC S A... . - . . ' ' J1.

Pu Sm

c

CPS BP A A C S A cMI I --+ .-

PB B BP E P_ _ _ II_ ----- ------

6.4

10.5

.8

1.3i_ kb

FIG. 1. Partial restriction endonuclease maps of chromosomal DNA from various plasmids containing the DMSO reductase gene andactivities in membranes prepared from cells harboring each of the plasmids. DMSO reductase specific activities were determined in themembrane fractions ofE. coli MV12 (for ColEl plasmids) and E. coli HB101 (for pBR322 plasmids) grown anaerobically on Glycerol-fumaratemedium. A, AvaI; B, BamHI; C, ClaI; E, EcoRI; P, PstI; Pu, PvuII; S, Sall; Sc, SacII; Sm, SmaI. Vectors: ColEl (-); pBR322 (---).

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E cOOiGENOME

20.0' 20.5S 2Q8'

N dnw rp*A ompF

iH 6lV B E H B BH H

pLC19-36H11 H A IB

NHH

A A H

kbH

N 10kbAI

IkbH

FIG. 2. Location of DMSO reductase operon on the E. coli chromosome. The DMSO reductase operon dms was localized on the E. colichromosome by comparison of the BamHI (B), EcoRI (E), and HindIll (H) endonuclease restriction sites on plasmid pLC19-36 to the kind,number, and position of these restriction sites on a 210-kb Notl (N) fragment of the E. coli chromosome. The positions of several genes onthe Notl fragment are indicated. The restriction map of the Notl chromosomal fragment and data on the location of the genes shown werekindly provided by D. Daniels (personal communication). The operon coding for DMSO reductase, dms, was localized on the pLC19-36chromosomal fragment from DNA sequence data. (Bilous et al., in preparation).

comparable to or better than those obtained with DMSO aselectron acceptor in the growth medium (4; unpublishedobservations). The results of a typical expression study areshown in Table 2.The E. coli MV12(pLC19-36) clone isolated from the

Clarke and Carbon colony bank displayed a 13- to 15-foldamplification of the membrane-bound DMSO and TMAOreductase activity when compared with a typical E. coliMV12 clone harboring a random DNA insert. Approxi-mately 90% of the DMSO or TMAO activity in these cellswas associated with the membrane fraction of the cell, inagreement with the nitrate and fumarate reductase activities.Interestingly, the ratio of TMAO/DMSO reductase activitywas constant at about 4:1 both in wild type and in E. coliMV12(pLC19-36). This suggested that one enzyme wasresponsible for both TMAO and DMSO reductase activitiesunder these growth conditions. A similar amplification anddistribution of activity was observed with E. coli MV12(pLC43-43) (data not shown). Neither clone overexpressedfumarate or nitrate reductase activity.

E. coli HB101 cells harboring the dms plasmid pDMS159expressed elevated levels of TMAO and DMSO reductaseactivity eight- to ninefold greater than those observed for E.coli HB101 (Table 2). Approximately 70 to 80% of theamplified activity was membrane associated, comparable to

that observed with E. coli MV12(pLC19-36) and in agree-ment with previous observations with wild-type cells (4).However, the level of enzyme expression with pDMS159 inE. coli HB101 was not as high as observed with pLC19-36 inE. coli MV12. The results may reflect strain differences.Previous growth studies with E. coli HB101 have shown thatmethionine sulfoxide, an analog of DMSO, could substitutefor DMSO in both the growth medium and the benzylviologen assay (4). Amplification of activity (as shown byDMSO and TMAO) was also observed with methioninesulfoxide as substrate (Table 2). The results suggest that oneenzyme is responsible for all three activities.

Identification of DMSO reductase gene product. The pro-tein electrophoretic pattern of membranes and soluble frac-tions from E. coli HB101 harboring pDMS159 is shown inFig. 3. An amplified protein band with molecular weight of82,600 + 2,000 (mean standard deviation, eight determi-nations) was evident in the membrane fraction of E. coliHB1O1(pDMS159) (lane 2) when compared with the wild-type control (lane 1). A polypeptide with identical molecularweight was present in the soluble fraction of the cell (lane 5),but the intensity of this band varied from preparation topreparation. It is probably identical to the membrane-boundpolypeptide of the same size. A membrane-bound polypep-tide with molecular weight of approximately 72,000 is evi-

TABLE 2. Expression and localization of reductase activities in E. coli MV12 and HB101 harboring DMSO reductase plasmidsa

Sp act (U/mg)E. coli strain Cell fraction

Nitrate Fumarate TMAO DMS0 MetSO

MV12(pLC22-12)b Membranes 1.3 (>%)c 6.3 (98) 2.6 (90) 0.75 (93) dSoluble


1 2 3 4 5

-.

AN* ~ *s

_~ ~

FIG. 3. SDS-polyacrylamide gel electrophoresis of membrane-bound and soluble proteins from E. ccli HB101 and HB101(pDMS159) grown on Glycerol-fumarate minimal medium. Cellswere grown for 36 h on glycerol-fumarate minimal medium, and

membranes and soluble fractions were prepared as outlined in

Materials and Methods. Samples (75 p.g of protein per lane) were run

on a 12.5% SDS-polyacrylamide gel and then stained for proteinwith Coomassie brilliant blue. Lane 1, E. coli HB101 membranefraction; lane 2, E. coli HB101(pDMS159) membrane fraction; lane3, protein molecular weight standards; lane 4, E. ccli HB101 solublefraction; lane 5, E. ccli HB101(pDMS159) soluble fraction. Themolecular weights of protein standards shown in lane 3 are as

follows: phosphorylase b, 97,400; bovine albumin, 66,000; eggalbumin, 45.000; carbonic anhydrase, 29,000; trypsin inhibitor,

20,100. Arrowheads mark amplified protein bands in cells harboringthe DM80 reductase plasmid pDMS159.

dent in lane 2 and is believed to be a proteolytic fragment ofthe 82,600-molecular-weight polypeptide as its intensity in-creased during storage of the samples. An additional ampli-fied protein band with molecular weight of 23,600 ± 800

(mean ± standard deviation, eight determinations) was

present in the soluble fraction of E. coli HB101(pDMS159)(lane 5). DNA sequence analysis of pDM.159 has indicated

the presence of three open reading frames organized in an

operon, suggesting a three-subunit structure for DM80

reductase with calculated molecular weights of 87,350,

23,070, and 30,789 (Bilous et al., in preparation). The 23,600-

molecular-weight polypeptide present in the soluble fraction

of the cell during isolation is probably equivalent to the

23,070-molecular-weight polypeptide coded by the dms op-eron and is loosely associated with the 82,600-molecular-

weight membrane-bound subunit. The two subunits have

been shown to copurify during purification of DM80 reduc-

tase (29). It would appear that the 30,789-molecular-weight

polypeptide, which has a very hydrophobic amino acid

composition, is not detected by Coomassie blue staining of

crude cellular preparations.Effect of growth conditions on DM80 reductase activity. It

was previously shown that optimal levels of DM80 reduc-

tase were induced by anaerobic growth on fumarate (4). Theglycerol-fumarate medium was the preferred medium forgrowth studies because the products of both DMSO andTMAO reduction are volatile and have unpleasant odors.However, it was of interest to determine the activity levelsof DMSO reductase in E. coli HB1O1(pDMS159) whengrown with various terminal electron acceptors. Cells weregrown for 48 h (early stationary phase) on nitrate, fumarate,TMAO, and DMSO minimal media. Crude membranes wereprepared from E. coli HB1O1(pDMS159) and then assayedfor reductase activity levels. The results are shown in Table3. The presence of the DMSO reductase plasmid resulted inmaximal DMSO and TMAO reductase activities only whengrown anaerobically on fumarate. Surprisingly, unelevatedlevels of activity were obtained when cells were grown onDMSO. Growth on TMAO resulted in a similar low level ofexpression when compared with growth on fumarate, but afive- to sevenfold-higher level of expression when comparedwith E. coli HB101 grown on glycerol-TMAO. Anaerobicgrowth on nitrate or aerobic growth (data not shown) com-pletely repressed the synthesis of DMSO, TMAO, andfumarate reductase activities. A similar pattern of enzymeexpression was demonstrated with E. coli MV12(pLC19-36)grown under the conditions described above (unpublishedobservations). In agreement with these results, examinationof the protein electrophoretic pattern on SDS-polyacryl-amide gels revealed an amplified protein band with molecu-lar weight 82,600 present only in the membranes of glycerol-fumarate-grown cells (data not shown).

Identification of plasmid-encoded polypeptides. To identifyall gene products which were plasmid encoded, an in vivotranscription/translation expression study was performed.Recombinant plasmids pDMS222 and pDMS229 were con-structed as described in Materials and Methods and asshown in Fig. 4. Plasmids pDMS222 and pDMS229 containthe chromosomal insert in opposite orientations with respectto the T7 promoter site of pTZ18R. E. coli K38, containingplasmid pGP1-2 which expresses a T7 RNA polymeraseunder temperature control, was transformed with pDMS222or pDMS229. Cells under the appropriate expression condi-tions were pulsed for 3 min with [35S]methionine. Totalcellular protein was precipitated with trichloroacetic acid,separated by SDS-polyacrylamide gel electrophoresis, andthen visualized by autoradiography. The resulting autoradio-gram is shown in Fig. SA.

TABLE 3. Reductase activity levels in membranes of E. coliHB101 and HB101(pDMS159) grown on glycerol minimal medium

with various terminal electron acceptorsa

Growth Sp act (U/mg)Snmedium Nitrate Fumarate TMAO DMSO

HB101 GLY-nitrate 26.3 NDb ND NDGLY-FUM 4.5 3.7 3.2 1.4GLY-TMAO 4.1 1.0 1.0 0.46GLY-DMSO 5.0 3.1 6.8 1.1

HB101(pDMS159) GLY-nitrate 14.6 ND ND NDGLY-FUM 0.6 3.2 29.0 7.8GLY-TMAO 0.9 1.3 7.0 2.5GLY-DMSO 0.7 1.4 5.6 1.9

Cells were grown anaerobically for 48 h at 37°C in 250 ml of glycerol(GLY) minimal medium with nitrate, fumarate (FUM), TMAO, or DMSO aselectron acceptor. Membranes were prepared and reductase activities wereassayed as described in Materials and Methods.

b ND, Not detected.

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preparation of DMSO reductase on SDS-polyacrylamidegels (29).

DISCUSSION

In this paper we report on the isolation and cloning of ananaerobically induced, membrane-bound, terminal reduc-tase of E. coli (4, 5). The isolation of the operon wasfacilitated by the use of a whole-cell enzyme assay to screena gene bank of potential clones expressing higher levels ofDMSO or TMAO reductase activity. Two clones wereidentified which amplified both DMSO and TMAO reductaseactivity. No clone was discovered which amplified only oneof the two substrates tested. The results suggest that oneenzyme is responsible for the reduction of both substrates.We have designated the cloned enzyme as DMSO reductasefor the following reasons. (i) The enzyme is geneticallydistinct from the genetic loci reported for TMAO reductase.(ii) The purified enzyme displays a higher affinity for DMSOthan methionine sulfoxide, TMAO, or other N-oxide com-pounds (29). (iii) Whole cells challenged with both substratesreduced DMSO at a faster rate than TMAO (P. T. Bilous,B. D. Sykes, and J. H. Weiner, unpublished observations).

Analysis of protein electrophoretic patterns on SDS-poly-acrylamide gel electrophoresis suggested that a membrane-bound polypeptide with molecular weight of 82,600 waslikely associated with DMSO reductase activity. In vivopolypeptide expression studies identified three polypeptideswith molecular weights of 87,500, 23,300, and 22,600 asso-

AB12 3 4 5

FIG. 4. Construction of recombinant pTZ18R plasmids carryingthe DMSO reductase gene. Recombinant plasmids pDMS222 andpDMS229 containing the chromosomal insert from pDMS159 in bothorientations with respect to the T7 promoter region were con-structed from DMSO reductase plasmid pDMS159 and pTZ18R asdescribed in Materials and Methods. The direction of transcriptionof the dms operon and its location on the chromosomal DNA insertwere determined from DNA sequencing data (Bilous et al., inpreparation). Apr, Ampicillin resistance; dms, DMSO reductaseoperon; E, EcoRI; H, HindIII; Kmr, kanamycin resistance; T7, T7RNA polymerase promoter; S, SaIl.

Two polypeptides with molecular weights of 87,500 ± 900(mean standard deviation, three determinations) and23,300 300 (three determinations) were clearly expressedby pDMS222 (lane 3), but not by pDMS229 (lane 4) or in theappropriate controls (lanes 1 and 2). An additional polypep-tide with molecular weight of 22,600 ± 400 (three determi-nations) was evident as a fuzzy band in lane 3. These resultsestablish the direction of transcription of the DMSO reduc-tase operon. To determine whether proteolytic activity dur-ing the 3-min pulse experiment was perhaps responsible forthe ill-defined 22,600-molecular-weight protein band, apulse-chase experiment was performed with a 1-min pulsefollowed by 2-, 5-, and 10-min chases with cold methionine.The results shown in Fig. SB display an identical pattern andrelative intensity to that seen with the pulse experiment (Fig.5A, lane 3). These results demonstrate the absence of anyfurther modification of the polypeptides during the course ofthese experiments and suggest a three-subunit structure forDMSO reductase. A polypeptide pattern identical to thatshown in Fig. 5A, lane 3, has been observed with a purified

FIG. 5. In vivo expression of plasmid-encoded polypeptides byT7 RNA polymerase-promoter expression system. For pulse-la-beling experiments (A), E. coli K38(pGP1-2) cells alone (lane 1) orcontaining pTZ18R (lane 2), pDMS222 (lane 3), or pDMS229 (lane 4)were pulse-labeled for 30 min with [35S]methionine as described inMaterials and Methods. Trichloroacetic acid-precipitated proteinswere electrophoresed on 12.5% SDS-polyacrylamide gels, fixed,dried, and then autoradiographed. For pulse-chase studies (B), E.coli K38(pGP1-2) cells transformed with pDMS222 were pulsed for1 min with [35S]methionine (lane 2) and then chased with unlabeledmethionine for 2 (lane 3), 5 (lane 4), and 10 (lane 5) min. Molecularweight standards shown in lanes 1 (B) and 5 (A) are identical to thosedescribed in the legend to Fig. 3, with the addition of ot-lactalbumin,14,200 molecular weight.

ES Km E

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ciated with the cloned DMSO reductase, which agrees withthe subunits associated with purified DMSO reductase (29).A consistent difference in molecular weight of the largersubunit was noted between growth and expression studiesand the values obtained from in vivo labeling experiments.The possibility of posttranslational modification of this sub-unit is under investigation.

It was perhaps fortuitous that the Clarke and Carboncolony bank was screened in a complex medium supple-mented with fumarate. Growth studies with E. coliHB101(pDMS159) or MV12(pLC19-36) have shown thatgrowth in the presence of DMSO or TMAO result in nearwild-type levels of DMSO reductase activity. In agreementwith the activity results, the 82,600-molecular-weight sub-unit is barely visible in the membrane fraction of DMSO- andTMAO-grown cells. Due to the different generation timesassociated with growth on the various terminal electronacceptors, cultures were grown to stationary phase. Allcultures were harvested at the same time and treated in anidentical fashion. It is therefore unlikely that proteolyticdigestion could be responsible for the observed results. It ispossible that the end products of reduction are toxic-to thecells and a repression mechanism exists when cells aregrown on Glycerol-DMSO or Glycerol-TMAO.

Studies of TMAO and DMSO reduction in Rhodobactercapsulatus and Proteus vulgaris have concluded that onlyone enzyme is responsible for both activities (20, 25).However, in E. coli multiple forms ofTMAO reductase havebeen reported (23). The major inducible form has beenpurified and characterized (30). Genetic studies have local-ized two inducible E. coli TMAO reductase genes to the 28.3(21)- and 77- to 84 (27)-min region of the chromosome. It isnot clear if the major inducible form of the enzyme which hasbeen purified by Yamamoto et al. (30) is coded by either ofthese genes.

In the present study, we have cloned a membrane-boundterminal reductase from E. coli which is situated at 20.0 minon the linkage map. The enzyme is induced by anaerobiosis,but does not require the presence of any added sulfoxide orN-oxide substrates for expression. The enzyme is thusanaerobically constitutive. The enzyme can use DMSO,TMAO, and methionine sulfoxide as substrates, but is ge-netically distinct from the reported TMAO reductases. Twomethionine sulfoxide reductases have been identified in E.coli, one reducing free methionine sulfoxide (11) and theother reducing protein-bound residues (7). Both have beenpurified and have molecular weights of 21,000 and 18,000 to20,000, respectively. Although the cloned DMSO reductaseis able to reduce methionine sulfoxide, it appears to bedistinct from the reported methionine sulfoxide reductasesbased on physical properties.

ACKNOWLEDGMENTS

We thank Stan Tabor for stain K38 and plasmid pGP1-2 andNancy Chung for construction of pDMS201. We are especiallyindebted to Donna Daniels and Frederick Blattner for providing theE. coli restriction map data.

This work was supported by a grant (MT5838) from the MedicalResearch Council of Canada. P.T.B. is a postdoctoral fellow of theAlberta Heritage Foundation for Medical Research.

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