Vol. 153, No. 3JOURNAL OF BACTERIOLOGY, Mar. 1983, P. 1513-15200021-9193/83/031513-08$02.00/0Copyright © 1983, American Society for Microbiology

chlC (nar) Operon of Escherichia coli Includes StructuralGenes for ox and a Subunits of Nitrate Reductase

ELLEN S. EDWARDS, SHEILA S. RONDEAU, AND JOHN A. DEMOSS*

Department ofBiochemistry and Molecular Biology, The University of Texas Medical School at Houston,Houston, Texas 77025

Received 30 July 1982/Accepted 5 November 1982

The synthesis of the ot and p subunits of nitrate reductase by 20 chiC::TnSinsertion mutants of Escherichia coli was determined by immune precipitation ofthe subunits from fractions of cell extracts. Only two of the mutants producedeither subunit in detectable amounts; these two accumulated the a subunit, but no

p subunit. In both cases the ot subunit was present in the cytosolic fraction, incontrast to wild-type cells, in which both subunits are present mainly in themembrane fraction. EcoRI restriction fragments containing the Tn5 inserts fromfive of the mutants were cloned into pBR322. The insertions were localized on twocontiguous EcoRI fragments spanning a 5.6-kilobase region that overlapped thecontiguous ends of the two fragments. An insertion that permitted subunitformation defined one end of the 5.6-kilobase region. The results indicated thatthe genes encoding the and p subunits of nitrate reductase were part of a chiC(nar) operon that is transcribed in the direction a

Nitrate reductase is a membrane-bound anaer-obic electron transport enzyme in Escherichiacoli that is repressed by oxygen and induced bynitrate. The isolated enzyme is composed ofthree distinct subunits (9, 11, 24). Two of thesesubunits, ax (155,000 daltons) and ,B (60,000 dal-tons), are present in all active preparations ofnitrate reductase, as assayed with artificial elec-tron donors (5, 22, 27). The y subunit (19,000daltons) is a cytochrome b1 which tends todissociate from the enzyme during purification(9), but appears to be required for associationwith the cell membrane (24, 26) as well as forfunctional association with physiological elec-tron donor systems, such as formate dehydroge-nase (10).The formation of nitrate reductase is affected

by mutations at several distinct genetic loci,designated chl; but only one of these, chiC, hasbeen directly implicated as a structural gene (8,25). From the biochemical properties of themutants arising from bacteriophage Mu inser-tions in the chlC region, Bonnefoy-Orth et al. (4)concluded that two closely linked genes, chiC(encoding nitrate reductase) and chlI (encodingcytochrome b1), are part of a multicistronicoperon. Stewart and MacGregor (32) reachedsimilar conclusions from studies with mutantsarising from transposon TnWO insertions andidentified several additional biochemically unde-fined mutant classes. No direct information onthe coding region for the P subunit was provided

by either of these studies, primarily because ofthe difficulty in determining the presence of thatsubunit in immunoprecipitation experiments.Since it is assumed (4, 23) that the ct subunit isthe catalytic subunit of nitrate reductase, itremained unclear whether the coding region forthe ,B subunit was part of the chlC region.To investigate the organization of the chiC

region that encodes the subunits of nitrate re-ductase, we determined the effects of transpo-son TnS insertions on the formation of ot and Psubunits. By cloning and mapping segments ofthe chromosome which contain the Tn5 inserts,we were able to assign promoter-proximal, ox, andP gene functions to specific regions of the clonedDNA. We conclude that the a and 3 subunits areboth encoded by the chiC region in a multicis-tronic operon which is transcribed in the ordera,-B*P.

MATERIALS AND METHODS

Strains and growth conditions. Wild-type E. coli K-12 strain PK27 (14) and all mutant strains were main-tained on L agar (20) and grown on L broth or minimalmedium (34) supplemented with 1% glucose and 1%potassium nitrate where indicated. The mutant strainswere maintained in the presence of 20 ,ug of kanamycinsulfate per ml.

Isolation of TnS insertion mutants. Strain PK27 wasgrown to mid-log phase in TYM broth (7) and muta-genized by infection with X b221 c1857 rex::Tn5 (fromP. Berget) at a multiplicity of infection of 50. Afteradsorption of A::TnS at 25°C for 45 min, the cells were

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1514 EDWARDS, RONDEAU, AND DEMOSS

grown for 60 min at 32°C and then plated on X agar (7)containing kanamycin (20 ,ug/ml), 5 mM sodium pyro-phosphate, 1% potassium nitrate, and triphenyl tetra-zolium chloride (TTC) (50 Vtg/ml) (12). Plates wereincubated at 42°C under anaerobic conditions. Accord-ing to Fimmel and Haddock (12), chlC colonies are adark red color on anaerobic TTC-nitrate plates, where-as other chl mutant and wild-type strain colonies arewhite. Kanamycin-resistant strains giving red colonieswere selected and tested for nitrate reductase activityby the overlay method of Glaser and DeMoss (14).Mutations were mapped by conjugation (14) and byP1-mediated cotransduction with the trp locus (14).Enzyme assays. For the assay of enzyme activities,

strains were grown on L broth supplemented with 1%potassium nitrate, 10 FM sodium selenite, and 10 F.Msodium molybdate under anaerobic conditions at 37°C.Cultures were harvested at a turbidity of 80 to 100Klett units (no. 54 filter). Cells were washed andsuspended in 50 mM potassium phosphate (pH 7.0).Methyl viologen-nitrate reductase activity was deter-mined by the procedure described by Showe andDeMoss (30). Phenazine methosulfate-formate dehy-drogenase activity was measured as described byRuiz-Herrera et al. (28). Protein concentration wasdetermined by the procedure of Lowry et al. (21).

Labeling and detection of nitrate reductase subunits.Strains were grown aerobically in minimal medium toa turbidity of 150 Klett units. The cultures were thenmade anaerobic by sparging with 95% N2-5% CO2 forseveral minutes. The medium was supplemented with1% potassium nitrate, 10 ,uM sodium selenite, 10 p.Msodium molybdate, and 0.5 to 5 mM L-[35S]methionine(1 to 2 mCi/mmol), and the cultures were incubated at37°C for 60 min. The cultures were harvested, washed,and suspended in 200 mM Tris-hydrochloride (pH 8.0).Crude extracts were prepared by passing the cellsuspension through a cold French pressure cell at10,000 Ib/in2. The supernatant fraction was preparedby ultracentrifugation at 200,000 x g for 90 min at 4°C.The membrane pellet was suspended in 50 mM potas-sium phosphate (pH 7.2), and proteins were solubi-lized by treatment with 2% Triton X-100 at 37°C for 30min in the presence of 0.1 mM tosyllysine chloro-methyl ketone. The solubilized membrane proteinswere clarified by ultracentrifugation at 200,000 x g for90 min at 4°C. 35S incorporation into the supernatantand membrane proteins was determined by precipitat-ing and washing each fraction with 5% ice-cold tri-chloroacetic acid and counting the precipitate in aliquid scintillation spectrophotometer.

Nitrate reductase subunits were precipitated fromsamples (approximately 500 ,ug of protein) of thesupernatant and solubilized membrane protein frac-tions with various dilutions of purified immunoglob-ulin G (IgG) from specific anti-nitrate reductase serum(9) by the procedure of Firestone and Heath (13). Insome cases the fractions were mixed with preimmuneserum or unfractionated antiserum and incubated at25°C for 1 h and 4°C for 16 h, and Formalin-treatedStaphylococcus aureus cells were used to precipitatethe immune complexes (9).The immunoprecipitates and samples of the mem-

brane and supernatant fractions were treated with 2%sodium dodecyl sulfate (SDS) (16). The samples weresubjected to electrophoresis on SDS-polyacrylamideslab gels as described by Keesey et al. (16), with a 7 to

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15% gradient. Radioactive molecular weight standardswere from New England Nuclear Corp., and theirsizes were as follows: phosphorylase b, 97,000; bovineserum albumin, 68,000; ovalbumin, 43,000; carbonicanhydrase, 29,000; and cytochrome c, 12,300. Autora-diography of the dried acrylamide gels was for 3 to 5days at room temperature.

Cloning procedures. DNA was isolated (6) fromchiC: :Tn5 mutants and digested to completion with therestriction endonuclease EcoRI. With T4 DNA ligase(7), fragments of mutant DNA were cloned into theunique EcoRI site of plasmid pBR322. Hybrid plas-mids containing a fragment of DNA carrying TnS wereselected by transforming the wild-type strain andselecting for resistance to kanamycin and tetracycline(or ampicillin). Transformation was carried out by aprocedure of A. R. Poteete (personal communication).Cells were grown to 50 Klett units (no. 54 filter) in 10ml of L broth, harvested, and suspended in 5 ml of 0.1M CaCI2-10 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 6.8) for 15 min on ice, thencentrifuged and suspended again in the same buffer onice. DNA was diluted to a concentration of 0.2 ,ug/mlwith 10 mM PIPES, 10 mM CaC12, and 10 mM MgCl2.Cells (0.3 ml) and DNA (0.2 ml) were mixed andincubated for 30 min at 0°C, 2 min at 37°C, and then 10min at 25°C. L broth (1 ml) was added, and the mixturewas incubated at 37°C for 30 min before being spreadon L agar containing kanamycin (20 ,ug/ml) and tetra-cycline (25 ,ug/ml) (or ampicillin [50 ,ug/ml]). Isolateswere retested for drug resistance. Plasmid DNA wasisolated (19) from the drug-resistant transformants andanalyzed by restriction endonuclease mapping (7). Therestriction endonucleases were purchased from Be-thesda Research Laboratories, Inc., or Boehringer-Mannheim Biochemicals (Cial). Products were ana-lyzed on 0.8% agarose and 5% polyacrylamide gels.

Hybridization of plasmid DNA with mutant DNAfragments. Chromosomal DNA was isolated fromstrain PK27 and the chIC::Tn5 mutants by a rapidisolation procedure (6). A [32P]dCTP-labeled DNAprobe was prepared from plasmid pSR201S with anick-translation kit and protocol obtained from Amer-sham Corp. Chromosomal DNA was digested withEcoRI and subjected to electrophoresis on a 0.8%agarose gel. Included in the gels were radioactivemolecular weight standards prepared by end-labeling ADNA fragments generated by ClaI digestion (29). TheDNA fragments were transferred to diazobenzyloxy-methyl paper and hybridized with the labeled probe(35). Autoradiography was for I to 2 days at -80°Cwith intensifying screens.

RESULTS

Isolation of chlC::TnS mutants. To determinewhether both the a and I subunits are encodedby the chlC region and are part of a multicis-tronic operon, we examined the effects of tran-sposon Tn5 insertions into the chiC gene on theformation of each subunit. This approach takesadvantage of the observation that, when insertedin a multicistronic operon, Tn5 inhibits the tran-scription and expression of cistrons within the

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FIG. 1. Induction and labeling of nitrate reductase.Strain PK27 was grown aerobically on minimal medi-um. At time A, [35S]methionine and nitrate were addedas described in the text and the culture was shifted toanaerobic conditions by sparging with 95% N2-5%cCO2. Units of nitrate reductase are given as micro-moles of nitrite produced per min per milligram ofprotein.

operon that are promoter distal from the inser-tion site (1, 2, 17, 18).Twenty chlC::Tn5 mutants were isolated, and

their mutations were mapped by the proceduresdescribed above. The wild-type strain was in-fected with A: :Tn5, and mutants were selected at42°C on TTC agar with kanamycin after induc-tion of the TnS5 insertion as described above.Mutants that produced no nitrate reductase(NR-) when scored by the overlay technique ofGlaser and DeMoss (14) had their mutationsmapped by conjugation and by P1-mediated co-transduction with trp. Mutants that cotrans-duced the NR- phenotype with trp at a frequen-cy of approximately 50% were taken to be chiCmutants. Those that transferred Kanr obligatori-ly with the NR- phenotype were assumed toresult from Tn5 insertions.None of the 20 selected chlC::Tn5 mutants

produced significant levels of nitrate reductasewhen grown under conditions which fully inducethe wild-type strain, PK27. Furthermore, eachof the mutants expressed wild-type levels ofphenazine methosulfate-linked formate dehy-drogenase, indicating that the Tn5 insertionsproduced none of the pleiotropic effects that

characterize all of the other chl gene mutations,as well as certain chiC point mutations (8, 14).

Production of at and , subunits by mutants.The effect of each insertion on the production ofthe individual a and 1B subunits was determinedby growing the strains under conditions in whichlarge amounts of newly formed enzyme wereproduced in the wild-type strain (Fig. 1). Thewild-type strain, PK27, was grown aerobicallyon minimal medium in the absence of nitrate to aturbidity of approximately 150 Klett units. Ni-trate and [35S]methionine were added, and theculture was shifted to anaerobic conditions bysparging with 95% N2-5% CO2. Sufficient[35S]methionine was added to sustain rapidincorporation into protein for approximately 60min, a period that encompassed the rapid phaseof nitrate reductase formation but was assumedto be short enough to minimize turnover of thecomponent subunits. The labeled cells werefractionated into the supernatant and detergent-solubilized membrane fractions. These fractionswere then immunoprecipitated with antiserumprepared against purified, heat-released nitratereductase. This antiserum contains antibodiesspecific for both c- and 13 subunits (R. H. Scottand J. A. DeMoss, unpublished data).

Autoradiography of SDS-polyacrylamide gelsof the immunoprecipitated fractions from thewild-type strain showed that the 35S-labeled oX,1, and y subunits were precipitated mainly fromthe membrane fraction (Fig. 2). Comparison ofthe immunoprecipitated fraction with the samefraction before precipitation showed the speci-ficity of this procedure. Several proteins wereprecipitated by both the preimmune serum andthe specific anti-nitrate reductase serum, butthese did not interfere with visualization of the ot

and 1 bands. There were some a. and 1 subunitsspecifically precipitated from the supernatantfraction, but at greatly reduced levels comparedwith those from the membrane fraction. Theseresults established that the rapid labeling proce-dure permitted identification of both the a and 1subunits. In addition, it was also assumed thatthe relatively short labeling period helped avoidartifacts caused by the possible turnover ofunassociated subunits.The 20 chiC::Tn5 mutants were analyzed for

their ability to form intact (x and 13 subunits bythe labeling and immunoprecipitation proce-dures (Fig. 1 and 2). All fell into one of the twoclasses represented by the two mutants shown inFig. 3. With mutant EE1, no detectable amountsof intact a or 13 subunit were precipitated fromeither the supernatant or membrane fraction,although significant labeling of the membraneand supernatant proteins occurred, as shown bythe lanes containing the unprecipitated fractions(Fig. 3, lanes B and E). With mutant EE201, two

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FIG. 2. Autoradiogram of SDS-polyacrylamide gelprepared from immunoprecipitates of wild-type frac-tion. Cells were induced and labeled and cell fractionswere prepared and precipitated with unfractionatedanti-nitrate reductase serum as described in the text.Immunoprecipitates were subjected to SDS-polyacryl-amide gel electrophoresis. Except where indicated,fractions contained 500 ,ug of protein. Lanes: A,supematant fraction, unprecipitated (150 jig of pro-tein); B, supernatant fraction precipitated with 20 ,ul ofpreimmune serum; C, supernatant precipitated with 20,ul of antiserum; D, molecular weight standards; E,solubilized membrane proteins, unprecipitated (150 jigof protein); F, solubilized membrane proteins precip-itated with 20 jil of preimmune serum; G through I,solubilized membrane proteins precipitated with 10(G), 20 (H), or 50 jl (I) of serum; and J, 1,000 jig ofsolubilized membrane protein precipitated with 20 jilof serum.

proteins that migrated similarly to the a subunitwere precipitated from the supernatant but notthe membrane fraction (lanes K and J, respec-tively). One band appeared to be identical to thea subunit, whereas the other migrated slightlyfaster.Two mutants, EE201 and EE104, produced

the a subunit but no ,B subunit, and the other 18mutants produced neither a nor p subunits.Mutant EE104 gave results identical to those forEE201 (Fig. 3); i.e., the a subunit was precipitat-ed from the supernatant fraction only, alongwith a slightly faster migrating band. It wasestablished for each mutant that the membraneand supernatant proteins were significantly la-beled during the induction period on the basis ofboth total 35S incorporation into trichloroaceticacid-precipitable material and incorporation intoprotein bands separated from the unprecipitatedfractions by SDS-polyacrylamide gel eldctro-phoresis.

Cloning and restriction mapping of the chiCregion. To delineate the proximity of the variousTn5 insertions to each other within the chiC

region, we cloned the segments of the E. colichromosome containing the TnS inserts fromseveral of the mutants into the unique EcoRI siteof plasmid pBR322. Mixtures of EcoRI-digestedDNA from pBR322 and from each of fivechiC: :Tn5 mutants were ligated and used totransform the wild-type strain. Transformantsresistant to both kanamycin and tetracyclinewere found to harbor hybrid plasmids withEcoRI inserts that included Tn5. Plasmids pSR1,pSR10, pSR105, pSR201, and pSR207 were de-rived from EE1, EE10, EE105, EE201, andEE207, respectively.

Digestion of each hybrid plasmid with EcoRIrevealed that the TnO inserts occurred in twodistinct EcoRI fragments (Fig. 4). Hybrid plas-mids pSR1, pSR207, and pSR201 contained a12.3-kilobase (kb) insert, whereas pSR10 andpSR105 contained a 21-kb insert.The orientation of the cloned fragment in each

hybrid plasmid and the specific site and orienta-tion of TnO in each fragment were determinedfrom restriction enzyme analysis and the known

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FIG. 3. Autoradiogram of SDS-polyacrylamide gelprepared from immunoprecipitates of mutant frac-tions. Cell induction and labeling, preparation of frac-tions, precipitation with IgG purified from anti-nitratereductase serum, and gel electrophoresis were carriedout as described in the text. Mutant fractions (contain-ing 500 jig of protein) were precipitated with 100 jLl ofpurified IgG. Unprecipitated fractions contained 150jig of protein. Lanes: A and L, a and ,B subunitsprecipitated from solubilized membrane fraction of thewild-type strain, PK27; B through G, mutant EEl:solubilized membrane fraction (B) precipitated withpreimmune (C) and anti-nitrate reductase (D) sera, andsupernatant fraction (E) precipitated by preimmune(F) and anti-nitrate reductase (G) sera; H through K,mutant EE201: solubilized membrane fraction (H)precipitated by anti-nitrate reductase serum (J), andsupernatant fraction (I) precipitated by anti-nitratereductase serum (K).

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restrictionas shownorientatiorEcoRI sitedistancesSall site inthe insertedistances casymmetriA sumn

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shown by taking advantage of the orientation ofthe Tn5 insertion in mutant EE201, as deter-mined from plasmid pSR201 (Fig. 5). In thisstrain the kanamycin resistance gene was locat-ed between the unique Sall restriction site in theTn5 insert and the region of the 6.6-kb fragmentthat contained the other insertions (Fig. 5 and 6).By cloning DNA from mutant EE201 that hadbeen digested with Sall into the unique Sall siteof pBR322 and selecting for resistance to bothkanamycin and ampicillin in transformed E. coli

A B c D E F cells, hybrid plasmid pSR201S was isolated (Fig.6B). Restriction enzyme analysis revealed that

Agarose gel electrophoresis of EcoRI-di- pSR201S contained a 15-kb Sall restriction frag-,mids. Lanes: A, pBR322; B, pSR1; C, ment with a single EcoRI restriction site (Fig.pSR207; E, pSR201; F, pSR105. The upper 6B). The 8.4- and 7.0-kb fragments produced in ae A iS assumed to be undigested pBR322 SalI-EcoRI digest corresponded precisely to the

sizes expected if the two EcoRI fragments (Fig.6B) are contiguous on the E. coli chromosome.

maps of Tn5 (16) and pBR322 (3, 33), Determination of Tn5 insertion sites by hybrid-for pSR201 and pSR10 (Fig. 5). The ization with labeled plasmid. Plasmid pSR201Si of the cloned fragment in the unique was labeled with [32P]dCTP by nick translationof pBR322 was determined by the and used as a probe in a Southern analysis (35)

of restriction sites from the unique to identify homologous sequences in EcoRI di-pBR322. Similarly, the orientation of gests of DNA from the wild-type and severald transposon was determined by the mutant strains (Fig. 7). In the wild-type digest,)f restriction sites from the unique and only the expected 6.6- and 15-kb fragmentsic Sall and Sma sites within TnS. hybridized with the probe. For each of thenary of the restriction maps deter- mutants, the size of one of the two hybridizingthe two EcoRI fragments containing fragments was larger than the correspondingnsertions is shown in Fig. 6A. The fragment in the wild type. In mutant EE1 theze of the fragments without the insert- 6.6-kb fragment was replaced by a 12.3-kb frag-ere 15 and 6.6 kb, respectively. The ment, and in mutant EE103 the 15-kb fragmentin mutants EE1O and EE105 occurred was replaced by a 21-kb fragment, as werekb at one end of the 15-kb fragment. expected for insertion of 5.7 kb of Tn5 DNA intoinsertions were distributed throughout each fragment. The third fragment observed infragment. EE1 (Fig. 7) was incompletely digested DNA.zse two EcoRI fragments represent a These results established that the Sall fragments region of the chromosome was cloned into pSR201S contained only sequences

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FIG. 6. Summary of restriction enzyme maps of the chiC region. (A) TnS insertion sites deduced from therestriction maps of five cloned chlC::TnS insertion fragments. The triangle represents the TnS insertion, with theorientation indicated by the position of the kanamycin resistance gene (represented by the heavy bar within thetriangle). (B) Restriction enzyme map of hybrid plasmid pSR201S. The half-triangle representing the fragment ofTn5 included in the 7.0-kb fragment is not drawn in direct proportion to the chromosomal DNA, but represents2.7 kb of Tn5-derived DNA.

from the two EcoRI fragments that containedthe five TnS insertions. The EcoRI fragmentswere arranged contiguously on the E. colichromosome so that the TnS insertions wereclustered within a 5.6-kb region, with the ,B geneinsertion in mutant EE201 being located at oneend of the cluster (Fig. 6).

DISCUSSION

Transposon insertions into the chiC region ofthe chromosome of E. coli affect the formationof both the at and subunits of nitrate reductase.Because Tn5 is known to terminate transcriptionand prevent the expression of genes distal to thesite of insertion, the pattern of expression of thetwo subunits in Tn5 insertion mutants reflectsthe organization of the genes within the chiCregion. If the at and ,B subunits are encoded byseparate transcriptional units, a TnS insertion inone gene should result in the loss of one subunitonly, with the other subunit still being produced.If both subunits are encoded by a single tran-scriptional unit, then many insertions, includingthose in the promoter and the promoter-proxi-mal gene, should lead to the loss of bothsubunits, whereas insertion within the promoter-distal gene would lead to loss of expression ofonly that gene.The fact that 18 of 20 chiC: :Tn5 mutants

produced neither subunit indicates that bothsubunits are produced from the same transcrip-tional unit. The formation of the a subunit aloneby two mutants further indicates that the direc-tion of transcription is a-*. Thus, TnS inser-tions in either the a gene or the region of theoperon that is promoter proximal to the ax geneleads to failure to produce both the a and a

subunits, whereas Tn5 insertions in the : genepermit expression of the a gene but preventexpression of any other distal genes of theoperon. We assume that the large proportion ofmutants producing neither subunit resulted fromthe relatively large size of the a gene and pro-moter region compared with the size of thegene.By cloning DNA restriction fragments con-

taining the Tn5 inserts from five of the mutants,we showed that the two types of mutants result-ed from insertions into a relatively restrictedregion of the chromosome. Although the fivemutants cloned contained TnS inserts in twodistinct EcoRI fragments, the Sall fragmentcloned from mutant EE201 contained the ends ofthe two EcoRI fragments arranged contiguously.

Therefore, all five TnS inserts, including boththe (x and P gene insertions, mapped within a5.6-kb region that overlapped two specific

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FIG. 7. Southern blot of EcoRI-digested mutantand wild-type DNAs, with 32P-labeled pSR201S usedas the probe. Lanes: A, mutant EE103 DNA; B,mutant EE3 DNA; C, mutant EE1 DNA; D, wild-typestrain PK27 DNA.

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EcoRI restriction fragments of the E. coli chro-mosome. The presumed 1 gene insertion inmutant EE201 delineated one end of the region,and the insertions that led to the loss of produc-tion of both subunits were distributed over theremainder of the region. The latter insertionsmay be within the promoter and (x gene regionsor within any other genes which are promoterproximal to the ax gene in the operon. The 5.6-kbregion spanned by the five Tn5 insertions issufficient to code for over 200,000 daltons ofprotein. Since the total size of the ax and 1subunits is approximately 210,000 daltons, andsince it is unlikely that inserts into both extrem-ities of the two genes would be selected in fiverandom Tn5 insertions, the most promoter-prox-imal insertion in mutant EE105 is most likelylocated in the region preceding the ot gene in theoperon. This interpretation is supported by therecent demonstration that mutant EE1 accumu-lated a fragment of the ox subunit with an estimat-ed size of 70,000 daltons (J. K. Keesey and J. A.DeMoss, unpublished data). The distance be-tween the EE1 and EE105 insertion sites was 2.8kb, whereas only approximately 2 kb of DNA isrequired to encode a 70,000-dalton peptide. Thissuggests that the EE105 insertion site is outsidethe ax subunit coding region in the promoter-proximal region.Bonnefoy-Orth et al. (4) reported that certain

mutants resulting from Mu phage insertions inthe chiC region produce a soluble form of nitratereductase containing only cx and 1 subunits, butfail to produce demonstrable levels of the cyto-chrome b, associated with nitrate reductase.Since these mutations map closely to mutationsthat prevent the formation of detectableamounts of all three subunits, they proposedthat an additional gene, chlI, is part of a multicis-tronic operon which encodes at least the a. and y(cytochrome b1) subunits. Stewart and MacGre-gor (32) showed that at least five distinct pheno-typic classes, based on indicator plate respons-es, occur among mutants resulting from TnlOinsertions in the chiC region. Although theirimmunoprecipitation results permitted them toconclude only that the ot and -y subunits areencoded by the affected operon, they suggestedthat all three subunits, oa, 1, and -y, as well assome possible regulatory proteins are producedby this operon. Our results provide direct evi-dence for that suggestion and, together with theabove studies, indicate that the a-, 13, and -ysubunits are encoded by a multicistronic operonwhich is transcribed in the order ox--->P-y.We concur with the proposal of Stewart and

MacGregor (32) that the chiC region should beredesignated nar in recognition of its apparentrole in encoding the subunits of nitrate reduc-tase. As previously shown, chlorate resistance is

a constant feature of the pleiotropic mutations atchiA, B, D, E, and G, but it is not an invariableproperty of mutations in the chlC region (15, 32).The individual genes within this region should bedesignated narG, H, I, K, and L (31), corre-sponding to the number of genes in the operon,with narG, H, and I designating the genes for theOc, 1, and -y subunits, respectively, of nitratereductase.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grantGM 19511 from the National Institute of General MedicalSciences. E.S.E. was a predoctoral trainee under PublicHealth Service grant GM 07542 from the National Institutes ofHealth.We thank Peter Berget, Joseph Keesey, and Randolph Scott

for their helpful advice and stimulating discussions.

LITERATURE CITED

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