JOURNAL OF BACTERIOLOGY, Apr. 1987, p. 1391-13970021-9193/87/041391-07$02.00/0Copyright © 1987, American Society for Microbiology

Vol. 169, No. 4

A New Methionine Locus, metR, That Encodes a trans-ActingProtein Required for Activation of metE and metH in Escherichia

coli and Salmonella typhimuriumMARK L. URBANOWSKI, LORRAINE T. STAUFFER, LYNDA S. PLAMANN, AND GEORGE V. STAUFFER*

Department of Microbiology, University of Iowa, Iowa City, Iowa 52242

Received 17 October 1986/Accepted 29 December 1986

We isolated an Escherichia coli methionine auxotroph that displays a growth phenotype similar to that ofknown metF mutants but has elevated levels of 5,10-methylenetetrahydrofolate reductase, the metF geneproduct. Transduction analysis indicates that (i) the mutant carries normal metE, metH, and metF genes; (ii)the phenotype is due to a single mutation, eliminating the possibility that the strain is a metE metH doublemutant; and (iii) the new mutation is linked to the metE gene by P1 transduction. Plasmids carrying theSalmonella typhimurium metE gene and flanking regions complement the mutation, even when the plasmid-borne metE gene is inactivated. Enzyme assays show that the mutation results in a dramatic decrease in metEgene expression, a moderate decrease in metH gene expression, and a disruption of the metH-mediated vitaminB12 repression of the metE and metF genes. Our evidence suggests that the methionine auxotrophy caused bythe new mutation is a result of insufficient production of both the vitamin B12-independent (metE) and vitaminB12-dependent (metH) transmethylase enzymes that are necessary for the synthesis of methionine fromhomocysteine. We propose that this mutation defines a positive regulatory gene, designated metR, whoseproduct acts in trans to activate the metE and metH genes.

The methylation of homocysteine to form methionine canbe carried out by either of two transmethylases in Salmo-nella typhimurium and Escherichia coli (for a review, seereference 15). The first is a vitamin B12-independent enzyme,the product of the metE gene; the second is a vitaminB12-dependent enzyme, the product of the metH gene. Themethyl donor for both enzymes is 5-methyltetrahydrofolate,produced by the metF gene product at a point of conver-gence of two major pathways, the methionine biosyntheticpathway and the C1 pathway (Fig. 1). The cell regulates theflow of C1 units through this convergence point on severallevels to balance the requirements for protein synthesis,methylation reactions, and nucleic acid synthesis.The genes in the nonfolate branch of the methionine

pathway (metA, metB, metC, and metK) and those in thefolate branch of the pathway (metF, metE, and, to a smallextent, metH) are all negatively controlled by the metJrepressor system. In addition, the metH gene product isinvolved in repression of the metE and metF genes when thecells are grown in medium containing vitamin B12. We reporthere the finding of a third regulatory mechanism at themethionine-C, convergence point, namely, the positive ac-tivation of the metE and metH genes.

MATERIALS AND METHODS

Bacterial strains, plasmids, and bacteriophages. All bacte-rial strains used are derivatives of E. coli K-12 and aredescribed in Table 1. Plasmids pGS47 and pGS69, and theirmetE::Tn5 derivatives have been described previously (16).Plasmid pMC1403 (4) was from M. Casadaban. Bacterio-phage Xgt2 (13) was from R. Davis. Plasmid pBR322 hasbeen described previously (9). Plasmid pGS191, the lacZ

* Corresponding author.

fusion plasmids, and lacZ fusion phage were isolated duringthis investigation.

Media. Luria broth (LB), Luria agar (L-agar) and glucoseminimal (GM) medium have been described previously (19).Supplements were added at the following concentrations:amino acids, 50 jig/ml, except for D-methionine (150 ,ug/ml);vitamins B1 and B12, 1 ,ug/ml; ampicillin, 100 ,ug/ml; tetracy-cline, 10 ,ug/ml; kanamycin, 25 ,ug/ml.P1 transductions. Transductions were performed with the

P1 cml clr-100 phage as described (10).Mutant isolation and classification. Strain GS162 was

mutagenized with phage Mu cts (3) and penicillin counterse-lected in GM medium supplemented with phenylalanine,vitamin B1, and penicillin G (20,000 U/ml). Surviving cellswere plated on GM plates supplemented with phenylalanine,vitamin B1, and methionine and then scored for their abilityto grow without the methionine supplement. Methionineauxotrophs were single-colony purified on L-agar plates andagain tested for the methionine requirement.Methionine auxotrophs were tentatively classified as de-

scribed previously (1). Cells were grown overnight in LB andwashed twice in GM medium, and 0.1-ml samples werespread on GM plates supplemented with phenylalanine andvitamin Bl. Crystals of cystathionine, homocysteine, vita-min B12, and methionine were then placed on sectors of theplates. Mutants that grew on cystathionine or homocysteinewere classified as carrying metA or metB mutations, thosethat grew on homocysteine were classified as carrying metCmutations, those that grew on vitamin B12 were classified ascarrying metE mutations, and those that grew only onmethionine were classified as carrying metF mutations.One mutant tested grew well only with a methionine

supplement, although slow growth occurred with a vitaminB12 supplement after 72 h of incubation. This mutant,designated strain GS190, was tentatively classified as a metF

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HOMOSERINE

I metA

O-SUCCINYLHONOSERINE

_tBK CYSTEINE 3-PHOSPHOGLYCERATE

CYSTATHIONINE

I etC

HOMOCYSTEINESERINE

metE or metH .tF5-methylTHF_-- 5,10-methyieneTHF1 glyA

NETHIONINE GLYCINE

I etKS-ADENOSYLETHIONINE

FIG. 1. The methionine pathway in E. coli and S. typhimurium.The gene designations and gene products are as follows: metA,homoserine succinyltransferase; metB, cystathionine -y-synthetase;metC, ,B-cystathionase; metF, 5,10-methylenetetrahydrofolatereductase; metE, homocysteine transmethylase; metH, homocyst-eine transmethylase (vitamin B12-dependent); metK, S-adenosylmethionine synthetase; glyA, serine hydroxymethyltrans-ferase; THF, tetrahydrofolate.

mutant. To stabilize the mutation in strain GS190, cells weregrown in LB at 30°C, and then 0.1-ml samples were spreadon L-agar plates and incubated overnight at 42°C (3). Tem-perature-resistant survivors were single-colony purified andtested for the methionine requirement. One temperature-resistant survivor with a phenotype identical to that of strainGS190 was saved and designated as strain GS244.Enzyme assays. Cells were grown in the appropriate media

and harvested in the mid-log phase of growth. The vitaminB12-dependent homocysteine transmethylase was assayed aspreviously described (21). The 5,10-methylenetetrahydrofo-late reductase was assayed as described by Kutzbach andStokstad (7). Protein determinations were made by themethod of Lowry et al. (8). ,-Galactosidase levels weremeasured as described by Miller (10) by using the chloro-form-sodium dodecyl sulfate lysis procedure.

Construction of the met-lacZ fusion phages. The ABlacfusion phage was described previously (20). The construc-tion of the AElac, XFlac, and XHlac fusion phages wassimilar to that of ABlac and will be described in detailelsewhere (manuscript in preparation). Briefly, a DNA frag-ment encoding the amino terminus of the respective S.typhimurium methionine structural gene plus the upstreamtranscriptional and translational control regions was fused toamino acid codon 8 of the lacZ gene in the plasmid fusionvector pMC1403. These constructs encode chimeric pro-teins, each consisting of an enzymatically active p-galactosidase moiety having an amino terminus derived fromthe respective met structural gene. The fused gene systems,located on approximately 7-kilobase-pair (kbp) EcoRI-Sallfragments on the fusion plasmids, were ligated into phageXgt2 by using the methods described previously for the XBlacphage (20). The resulting hybrid phages were used tolysogenize various strains in this study. In phage XElac, themetE-lacZ fusion occurred at codon 22 of the metE gene andincluded approximately 280 base pairs (bp) of upstreamDNA. In phage XFlac, the metF-lacZ fusion occurred atcodon 13 of the metF gene and included approximately 600bp of upstream DNA. The precise point of translationinitiation has not yet been determined for the metH gene,and so, in the XHlac phage, the codon numbering has notbeen assigned in the open reading frame in which the

TABLE 1. List of strains used in this investigationStrain Genotype Source

GS38 ili, This laboratoryGS162 pheA905 thi AlacUl69 araD129 rpsL G. ZurawskiGS190 pheA905 thi AlacU169 araD129 rpsL This laboratory

metR::Mu ctsGS232 metF63 pro-22 B. BachmannGS243 pheA905 thi AlacU169 araD129 rpsL This laboratory

AmetE: :MuGS244 pheA90S thi AlacUl69 araD129 rpsL This laboratory

AmetR: :MuGS470 trpR lacZ metE70 This laboratoryGS472 trpR lacZ metE70 metH This laboratoryGS597 pheA905 thi AlacUl69 araDJ29 rpsL This laboratory

metJ97

metH-lacZ fusion occurs. The XHlac phage includes at least400 bp of upstream DNA.

Construction of the X lysogens. Appropriate strains werelysogenized with XHlac, AElac, XFlac, and ABlac fusionphages by the procedure described previously (20). Afterpurification, lysogens were tested for the presence of only asingle copy of the A phage by comparing the relative levels of,-galactosidase enzyme produced in several isolates and byinfecting the isolates with X c190 c17 phage (17).

Construction of plasmid pGS191. The general proceduresfor plasmid DNA isolation, cloning, restriction enzymecleavage, and transformation have been described previ-ously (9). Plasmid pGS47, which contains the S. typhi-murium chromosomal fragment carrying the metE and metRgenes, was digested with restriction endonucleases HindIIIand Sall (Fig. 2). The digestion products were run on a 1%low-melting-temperature agarose gel, and the approximately

1-3 kb7pGS47 | PGS6 \. SacilECoRI Hhdil Pstl Pstt

Ap t Hind III + SallT4 DNA LigaseTransform GS244 (metR)

pBR322 Select Met+ Apt transformants

met

pstiAP

pGS 191

FIG. 2. Construction of the metR plasmid pGS191. Details of theconstruction are given in Materials and Methods. A comparison ofthe physical maps of pGS47 (metR+) and pGS69 (metR) suggeststhat the region of DNA on pGS47 most likely to encode thetrans-acting metR product includes an approximately 700-bp seg-ment (hatched box) that is not present in pGS69. Plasmid pGS47 alsocontains a spontaneous 1.3-kbp internal deletion (V) (16). The SalIand HindlIl sites in pGS47 that were used to construct pGS191 arein bold type. The solid square (-) in pGS191 represents the end ofthe truncated metE gene.

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2-kbp fragment deduced to carry the metR gene was iso-lated. This fragment was then ligated into the HindIlI andSall sites of plasmid pBR322. The ligation mixture was usedto transform strain GS244, and the cells were plated ontoGM plates containing phenylalanine, vitamin B1, andampicillin and incubated at 37°C. Several Met' transform-ants were single-colony purified, and plasmid DNA wasprepared. The resulting plasmids were checked by restric-tion enzyme analysis (data not shown) to confirm the pres-ence of the 2-kbp HindIII-SalI fragment. One of theseplasmids was again used to transform strain GS244 tomethionine prototrophy on GM plates containing phenylal-anine, vitamin B1, and ampicillin to confirm the presence ofthe metR gene. The plasmid was designated pGS191.

RESULTS

Transduction analysis. During a routine screening proce-dure for methionine auxotrophs, we isolated one mutantwith an unusual phenotype. This mutant, strain GS244, grewwell only with a methionine supplement, although slowgrowth occurred over 72 h with a vitamin B12 supplement.This phenotype is characteristic of either a metF mutant or ametE metH double mutant (1). Thus, an initial series oftransductions was carried out to determine the status of themetE, metF, and metH genes in strain GS244. P1 cml clr-100phage was grown on strains GS232 (metF63), GS470(metE70), and GS472 (metE70 metH) and used in transduc-tion with strain GS244 as the recipient. Met+ transductantswere selected on GM plates supplemented with phenylala-nine and vitamin B1 with strains GS232 and GS470 as donorsand on GM plates supplemented with phenylalanine, vitaminB1, and vitamin B12 with strain GS472 as the donor. Trans-ductants and abortive transductants were observed in allthree transductions, suggesting that the mutation in strainGS244 is not in metF, metE, or metH. Since it is possiblethat the inability of strain GS244 to grow on either GMmedium or GM medium plus vitamin B12 is due to more thanone mutation, transductants selected on the vitamin B12-supplemented plates with GS472 as the donor were scoredon GM plates supplemented with phenylalanine and vitaminB1. All transductants selected on the vitamin B12-supplemented plates could grow on GM plates without thevitamin B12 supplement (50 of 50 scored), suggesting that asingle mutation is responsible for the phenotype of strainGS244. We have designated this mutation as metR.

Reciprocal transductions were also performed with P1 cmlclr-100 phage grown on strain GS244 and with strains GS232(metF63), GS470 (metE70), and GS472 (metE70 metH) as

recipients. Met+ transductants were selected on GM platessupplemented with proline with GS232 as the recipient, GMplates with GS470 as the recipient, and GM plates supple-mented with vitamin B12 with GS472 as the recipient. Trans-ductants and abortive transductants were observed with allthree recipients. Since either metE+ or metH+ transductantscan grow on GM plates supplemented with vitamin B12 whenstrain GS472 was used as the recipient, the transductantswere scored on GM plates without the vitamin B12 supple-ment. metH+ transductants can be distinguished frommetE+ transductants since the former would still requiremethionine for growth in the absence of vitamin B12. Over90% of the transductants (97 of 100 scored) still required amethionine supplement, verifying that they were metH+transductants. All of these results indicate the presence offunctional metF, metE, and metH genes in strain GS244.Furthermore, when GS472 was used as the recipient, we

TABLE 2. Results of transductions demonstrating that themutation in strain GS244 is cotransducible with ilv

Scoring on mediumb:Recipienta ~~~~~~~~%Cotrans-Recipient GMGMGM + Leu + ductioneGM ~~Vat + Ile

GS470 (metE70) 45 64 30GS243 (metE) 24 29 17GS244 (metR) 28 46 39

a Only the relevant markers are given. The mutation in strain GS244 wasdesignated as metR. See Table 1 for complete genotypes. In each case, thedonor was strain GS38 (ilv).

b Met+ transductants were selected on GM plates supplemented withisoleucine, leucine, and valine and then spotted on the scoring media. Allplates were also supplemented with phenylalanine and vitamin B1, sincestrains GS243 and GS244 carry the pheA9OS and thi markers.

c Percent cotransduction refers to the percentage of the Met + transductantsthat received the ilv marker from the donor.

expected about 50% of the transductants to grow on GMplates without vitamin B12, since the metE and metH genesare not linked by P1 transduction (2). One explanation forthe low yield of metE+ transductants is that the new muta-tion leading to methionine auxotrophy is closely linked to themetE gene by P1 transduction.To further test the possibility that metR is linked to metE,

we mapped the new mutation with respect to anothermarker, ilv, known to be cotransducible with metE (2). P1cml clr-100 phage was grown on strain GS38 (ilv) and used totransduce strains GS470, GS243, and GS244 to methionineindependence on GM plates supplemented with phenylala-nine, vitamin B1, isoleucine, leucine, and valine. Met+transductants were then scored on GM plates supplementedwith phenylalanine and vitamin B1. Of the Met+ transduc-tants with strain GS244 as the recipient, 39% are ilv, sug-gesting that the metR mutation in GS244 is linked to ilv andcould lie near the metE gene (Table 2).Measurement of metF, metE, and metH gene expression. As

mentioned above, either a metF mutant or a metE metHdouble mutant has a nutritional requirement satisfied only bya methionine supplement. Although the transduction analy-sis indicated that all three genes are intact, we tested thepossibility that the mutation in strain GS244 somehow pre-vents the expression of these genes and is therefore respon-sible for the phenotype of strain GS244. We measured theexpression of these three genes in strain GS244 and theparent strain GS162 grown under conditions that derepress(D-methionine) the metF, metE, and metH genes. Since thesubstrate for the vitamin B12-independent transmethylasewas unavailable, we measured metE gene expression indi-rectly by using a metE-lacZ gene fusion. In this system, alambda phage carrying the metE-lacZ fusion (XElac) wasused to lysogenize strains GS244 and GS162. The productionof P-galactosidase activity in these lysogens is directed bythe S. typhimurium metE gene control region. Expression ofthe metF gene was actually higher in strain GS244 than in theparent strain GS162 (Table 3). However, expression of boththe metH gene and the metE-lacZ fusion was reduced instrain GS244 compared with the parent strain GS162. Theseresults suggest that the new locus defined by the metRmutation is involved with activation in trans of the metE andmetH genes and that the methionine auxotrophy of strainGS244 is a result of insufficient production of bothtransmethylase enzymes.Complementation in trans of the GS244 mutation. Since the

transduction analysis suggested that the metR mutation inGS244 lies near the metE gene, it was possible that one or

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TABLE 3. Comparison of activities of the enzymes in the folatebranch of the methionine pathway in the parent strain GS162 and

the mutant strain GS244

Enzyme assayed Strain Sp acta

5,10-Methylenetetrahydrofolate reductase (metF) GS162 0.29GS244 0.41

B12-homocysteine transmethylase (metH)

metE-lacZ fusedb ,3-galactosidase (metE)

GS162 0.67GS244 0.12

GS162 217GS244 19

a The growth media contained phenylalanine, vitamin B1, and D-methionine(vitamin B12 was added to cultures for the metH assay). Units of specificactivities are, for reductase, nanomoles of HCHO formed per minute per

milligram of protein at 30°C; for B12-transmethylase, nanomoles of methionineformed per minute per milligram of protein at 37°C; and for P-galactosidase,nanomoles of O-nitrophenol formed per minute per milligram of protein at28°C.

b Substrate for the vitamin B12-independent transmethylase (metE) was

unavailable. Thus, metE gene expression was determined by measuringl-galactosidase enzyme levels in XElac lysogens of GS162 and GS244 carryingthe metE-lacZ gene fusion.

more of the plasmids previously constructed carrying the S.typhimurium metE gene (16) also carries the metR gene. Totest this possibility, we transformed GS244 with four metEplasmids. Two of these plasmids, pGS47 and pGS69, carry a

functional S. typhimurium metE gene. PlasmidspGS47metE::TnS and pGS69metE::TnS are derivatives ofpGS47 and pGS69, respectively, in which the metE gene onthe plasmid has been inactivated by insertion of the trans-posable element TnS. Transformants were selected on L-agar plates containing the appropriate antibiotics and werethen scored on GM plates supplemented with phenylalanineand vitamin B1. The metE strain GS243 was also trans-formed with these plasmids as a control. Both of the metE+plasmids, pGS47 and pGS69, complement the metR muta-tion in strain GS244 (Table 4). In addition, one metEplasmid, pGS47metE::TnS, also complements strain GS244,supporting the transduction data indicating that the metEgene is not the site of the mutation in strain GS244. How-ever, the other metE plasmid, pGS69metE::TnS, fails tocomplement strain GS244, suggesting that the metE gene onthis plasmid is involved in the ability of the parent plasmidpGS69 to complement strain GS244. Therefore, we testedplasmids pGS47 and pGS69 for the presence of the trans-acting factor by measuring metE-lacZ gene expression.When lysogen 244XElac was transformed with plasmidpGS69, the P-galactosidase levels (reflecting metE geneexpression) were 25-fold lower than when the same lysogenwas transformed with plasmid pGS47 (Table 5). Thus, plas-mid pGS47 carries the metR gene necessary for the activa-tion in trans of the metE-lacZ fusion. We have recentlyshown that the metR gene encodes a polypeptide of Mr31,000 (L. Plamann and G. Stauffer, unpublished results).Plasmid pGS69 does not carry the metR gene but probablycomplements the methionine auxotrophy of GS244 on thebasis of the high copy number of the metE gene in thetransformant. In support of this view, high-copy plasmidswhich carry the metH gene are also capable of complement-ing GS244 when grown in the presence of vitamin B12 (datanot shown). As expected, plasmid pGS47metE::TnS is ableto activate the expression of the metE-lacZ fusion, indicatingthat this plasmid carries a functional copy of the metR gene.

In addition, since the expression of the metH gene isreduced in GS244 (Table 3), we tested whether the metR

TABLE 4. Complementation of strains GS244 and GS243 bymetE plasmids

Growth on mediumb:Strain

metE statusaof plasmid GM GM + GM +

B12 Met

GS244 - + + + +GS244(pGS47) metE+ + + + +++ + + +GS244(pGS47 metE::TnS) metE + + + + + + + + +GS244(pGS69) metE+ + + + +++ + + +GS244(pGS69 metE::TnS) metE - + + + +GS244(pGS191) metE + + + +++ + + +

GS243 - + + + + + +GS243(pGS47) metE+ + + + +++ + + +GS243(pGS47 metE::TnS) metE - + + + + + +GS243(pGS69) metE+ + + + + + + + + +GS243(pGS69 metE::Tn5) metE - + + + + + +GS243(pGS191) metE - + + + + + +

a The status of the metE gene on the plasmid was determined by comple-mentation tests with known metE mutants (16).

b Test plates were GM supplemented with phenylalanine, vitamin B1, and,when indicated, vitamin B12 or L-methionine. Symbols: -, no growth after72 hr; +, slight growth after 72 h;++ + , good growth after 24 h. Incubationwas at 37°C.

gene carried on the plasmids could increase expression ofthe metH gene. We used a metH-lacZ fusion system carriedon a lambda phage (XHlac) in which P-galactosidase produc-tion is directed by the S. typhimurium metH gene controlregion. Strain GS244 was lysogenized with XHlac, and thelysogen was transformed with the same plasmids as above.All plasmids except pGS69 activate the expression of themetH-lacZ fusion (Table 5).

Location of the trans-acting factor on pGS47. A comparisonof the physical maps of plasmids pGS47 and pGS69 (Fig. 2)(16) suggests that the region ofDNA on pGS47 most likely toencode the trans-acting metR product includes an approxi-mately 700-bp segment that is not present in pGS69. Wetherefore isolated a 2-kbp HindIII-SaIl DNA fragment frompGS47 containing the 700-bp segment and an inactive seg-ment of the metE gene and ligated it into the HindIII-SalIsites of the plasmid vector pBR322. Transformation ofGS244 with this ligation mixture resulted in Apr transform-ants that grew on GM plates supplemented with phenylala-nine and vitamin B1 without a methionine supplement. Oneof these transformants was purified, and plasmid DNA was

TABLE 5. Activation in trans of the A metE-lacZ and the XmetH-1acZ GS244 lysogens transformed with possible

metR plasmids

Strain 13-Galactosidasesp acta

244XElac(pGS69). 8244Elac(pGS47).201244XElac(pGS47 metE: :TnS).565244XElac(pGS191).581

244XHlac(pGS69).33244XHlac(pGS47).333244XHlac(pGS47 metE: :Tn5).333244XHlac(pGS191) 321

a Units of specific activity are nanomoles of O-nitrophenol produced perminute per milligram or protein at 28°C. The growth medium was GMsupplemented with phenylalanine and vitamin B1 and with D-methionine forthe 244XElac lysogens or with D-methionine plus vitamin B12 for the 244XHlaclysogens.

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isolated. This new plasmid, designated pGS191, was used toagain transform GS244 to methionine independence to con-firm that the metR gene is present on the cloned fragment(Table 4). Since GS244 is an E. coli K-12 strain, the ability ofthe metR gene on plasmid pGS191 (which was derived fromS. typhimurium chromosomal DNA) to complement the E.coli mutation indicates that both organisms have a similaractivation system for the metE and metH genes.

Nature of regulation of methionine biosynthesis by metR.The plasmid complementation tests described above suggestthat the metR locus encodes a protein that is involved in theactivation of the metE and metH genes. As a first step indiscerning the nature of this trans-activation by the metRgene product, the effects of the metR mutation in GS244were analyzed by comparing the expression of several metgenes in this strain with those in the Met+ parent strainGS162 and the metE mutant strain GS243. These compari-sons were facilitated by lysogenizing these strains withAElac, XHlac, XFlac, and XBlac fusion phages (see Materialsand Methods), allowing 3-galactosidase levels to be assayedas an indication of expression of the respective met gene

being examined. The XBlac phage was used as a represen-

tative gene from the nonfolate branch of the methioninebiosynthetic pathway (Fig. 1). The results of these compar-

isons are shown in Table 6.The expression of the metE-1acZ, metH-lacZ, metF-lacZ,

and metB-lacZ fusions in response to methionine limitation(D-methionine) or methionine excess (L-methionine) in theGS162 lysogens (Met+) and GS243 lysogens (metE) gener-

ally followed the patterns reported previously for the respec-tive met genes in E. coli (1, 6). The addition of L-methionineto the GM growth media substantially repressed the expres-sion of the metE, metF, and metB genes, especially whencompared with the derepressed levels in the methionineauxotroph GS243. The addition of L-methionine marginallyrepressed the expression of the metH gene. In addition, themetE and metF genes were repressed by the addition ofvitamin B12 to the GM media.

In contrast, regulation of the metH-lacZ, metF-lacZ, and,particularly, the metE-lacZ fusions is altered in GS244.P-Galactosidase levels in lysogen 244XElac grown undermethionine-limiting conditions was 200-fold lower than inlysogen 243XElac. Since lysogens 244XFlac and 244XBlacboth showed derepressed P-galactosidase levels under thesegrowth conditions, it is clear that GS244 is methioninelimited in the D-methionine-supplemented GM media, andthus the low P-galactosidase levels seen in 244XElac and244XHlac lysogens cannot be due to repression by adequateinternal pools of methionine.The metR mutation in GS244 also disrupted the normal

vitamin B12-dependent repression of the metE-lacZ andmetF-lacZ fusions, although the effect was more apparentfor the metF gene. The vitamin B12 repression seen in243XFlac was nearly abolished in 244XFlac, and the I-

galactosidase activity approached the level seen for244XFlac grown under derepressing conditions (Table 6).Interestingly, the release of the vitamin B12-dependent re-

pression in lysogens 244XElac and 244XFlac was paralleledby lower expression of the metH gene in 244XHlac undersimilar growth conditions. Since the metH gene product hasbeen shown to be involved in the vitamin B12-dependentrepression of the metE and metF genes (6, 11, 22), it isplausible that the decreased production of metH gene prod-uct in GS244 is responsible for the disruption of the vitaminB12-dependent repression mechanism. In contrast, it is clearthat the addition of L-methionine to the growth medium

TABLE 6. Effects of GS244 mutation on expression of met-lacZgene fusions in XElac, XHlac, XFlac, and X13lac lysogens

3-Galactosidase sp act withaStrain

D-Met L-Met D-Met + B12

162XElac (Met') 240 45 27243XElac (metE) 4,496 61 37244XElac (metR) 19 6 14

162XHlac 97 56 116243AHlac 116 66 143244XHlac 41 35 39

162XFlac 590 170 215243XFlac 1,845 240 288244XFlac 1,034 97 800

162XBlac 227 95 NDb243XBlac 2,940 194 ND244XBlac 1,920 188 ND

a Units of specific activity are nanomoles of O-nitrophenol produced perminute per milligram of protein at 28°C. The growth medium was GM mediumsupplemented with phenylalanine, vitamin B1, and, when indicated, D-methionine, L-methionine, or D-methionine plus vitamin B12.

b ND, Not done.

results in significant repression of all the met-lacZ fusionstested, including the GS244 derivatives. This suggests thatthe metJ-mediated repression system functions indepen-dently of metR and can override the stimulatory effect ofmetR.

DISCUSSION

We have provided evidence for the existence of a newregulatory element, designated metR, that encodes a trans-acting protein required for expression of the metE and metHgenes of E. coli and S. typhimurium. Consistent with thisinterpretation, the metR mutation shares a number of char-acteristics with mutations in other positive activator genes(14): (i) the mutation occurs outside the target genes (metEand metH) and thus acts in trans; (ii) the mutation isrecessive to the wild-type allele; and (iii) the mutation affectsthe synthesis of gene products rather than their activity. Thislast point would be crucial in the assignment of the metRgene product as an activator. Since the two genes it controls(metE and metH) code for gene products having identicaltransmethylase functions, it is possible that the metR geneproduct functions as a subunit necessary for the formation ofan active transmethylase complex. However, our resultsshow that the metR gene product increases the levels ofactivity of the unrelated enzyme P-galactosidase in metE-lacZ and metH-lacZ fusion strains (Table 6), demonstratingthat it must function either directly or indirectly by increas-ing transcription or translation of the metE and metH genes.Furthermore, the metE and metH genes in high copy cancomplement the metR mutant, suggesting that the metR geneproduct is not a necessary subunit for transmethylase activ-ity.Smith and Childs (18) previously reported that the metE

locus in S. typhimurium could be divided into two comple-mentation groups and suggested that the vitamin B12-independent transmethylase might consist of two differentpolypeptide subunits. The group I metE mutants were iso-lated seven times more frequently than were the group IImutants, suggesting a larger target site for the group I locus.

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1396 URBANOWSKI ET AL.

The metE gene of S. typhimurium encodes a polypeptide ofapproximate Mr 92,500 (16), and the metR gene encodes apolypeptide of only Mr 31,000 (Plamann and Stauffer, un-published results). In addition, the group II mutants had anextended growth lag compared with group I mutants whenthe strains were grown in GM medium supplemented withvitamin B12. On the basis of our results, we predicated thatgroup I nmutations inactivate the metE gene coding for thetransmethylase, whereas group II mutations inactivate themetR gene coding for the activator. We tested S.typhimurium strains carrying group I mutations (metE205,metE230, and metE237) and strains carrying group II muta-tions (metE197, metE387, and metE396) in complementationtests with the metE metR+ plasmid pGS191. Consistent withthe above interpretation, pGS191 complements group II butnot group I mutations.The level of P-galactosidase produced in 244XElac

(pGS191) is significantly higher than in 244XElac(pGS47)(Table 5). This elevation is probably not due simply to ahigher copy of the metR gene in the pGS191 transformant,since a similar elevation was seen in 244XEiac(pGS47metE::TnS). It is unlikely that the TnS insertion into pGS47would raise the production of the metR gene product theexact amount necessary to compensate for a difference incopy number between pGS47 and pGS191. A more likelyexplanation is that inactivation of the plasmid-borne metEgene is responsible for the higher P-galactosidase levels seenin the pGS191 and pGS47metE::TnS transformants. ThemetE gene product could act negatively in its own regulationin two ways. First, it could function as an antagonist of themetR gene prQduct, either by interfering directly in theactivation mechanism or by repressing metR gene expres-sion. Alternatively, activation probably depends not only onthe presence of a functional metR gene product but also ona coactivator, e.g., an intermediate in the methioninebiosynthetic pathway. Methionine intermediates have beenimplicated in the regulation of the metE gene in E. coli (5)and the metH and metF genes in S. typhimurium (22). It wasconcluded that cystathionine may function as an inducer forthe metE gene and that O-succinylhomoserine may functionas an inducer for the metH and metF genes. In addition, in E.coli, a functional metF gene is required for vitamin B12-mediated repression of the metF gene, ahd 5-methyltetrahy-drofolate may be involved in this repression (12). Inactiva-tion of the plasmid-borne metE gene in 244XElac(pGS191)and 244XElac(pGS47 metE: :TnS) could allow a greater accu-mulation of the methionine intermediates O-succinylhomo-serine, cystathionine, homocysteine, and 5-methyltetrahy-drofolate than would 244XElac(pGS47). The pGS47transformant has multiple copies of the metE gene and thusmight drain off the intermediates more quickly, preventingtheir accumulation.The level of p-galactosidase produced in 244XHlac(pGS47)

was not significantly lower than in 244XHlac(pGS191) or244XHlac(pGS47 metE::TnS) (Table 5). Thus, multiple cop-ies of the metE gene did not decrease expression from themetH gene, suggesting that different coactivators are re-quired for expression of the metE and metH genes via themetR gene product. We are currently examining the effectsof combinations of mutations in other met genes to directlycontrol the concentrations of methiohine intermediates,thereby allowing identification of the coactivator(s).A requirement for the metR gene product thus far is

limited to the metE and metH genes. We have testedwhether the metR gene product affects the third gene in thefolate branch of the pathway, metF, and a gene in the

nonfolate branch, metB. Only a moderate decrease in theexpression of the metF-lacZ fusion or the metB-lacZ fusionin strain GS244 was seen compared with the decrease seenafter the addition of L-methionine to the growth medium(Table 6).The metE gene is activated to a much greater extent than

is metH. In E. coli cells grown under derepressing condi-tions, the metE gene product represents as much as 5% ofthe total cellular protein (23). However, when cells weregrown in media containing vitamin B12, synthesis of thenon-B12 transmethylase (metE gene product) was repressedover 100-fold, and there was a more moderate repression ofthe metF gene product (Table 6). It was suggested that thevitamin B12-dependent transmethylase is a more efficientenzyme and thus the cell finds it more economical to use theB12-dependent pathway when vitamin B12 is available (5). Itis possible that the availability of vitamin B12 to the organismis fairly reliable in its natural habitat, so that the B12-dependent (metH) pathway is normally used and inductionof the synthesis of the less efficient metE gene product isnecessary only when vitamin B12 is unavailable. This hypo-thesis predicts that the metH gene would be partially con-stitutive and would require only a low level of activation bymetR, whereas the metE gene would require a high level ofactivation to obtain sufficient expression. It is interesting inthis respect that GS244 reverts much more frequently whengrown on vitamih B12-containing medium than on non-B12-containing medium (unpublished results). Although we havenot yet characterized these revertants, it appears that themetH gene can overcome its dependence on metR activationmuch more easily than can metE.

ACKN)WLEDGMENTSThis investigation was supported by Public Health Service grant

GM26878 from the National Institute of General Medical Sciences.

LITERATURE CITED1. Ahmed, A. 1973. Mechanism of repression of inethionine bio-

synthesis in Escherichia coli. Mol. Gen. Genet. 123:299-324.2. Bachmann, B. J. 1983. Linkage map of Escherichia coli K-12,

edition 7. Microbiol. Rev. 47:180-230.3. Casadaban, M. J. 1975. Fusion of the Escherichia coli lac genes

to the ara promoter: a general technique using bacteriophageMu-i insertions. Proc. Natl. Acad. Sci. USA 72:809-813.

4. Casadaban, M. J., J. Chou, and S. N. Cohen. 1980. In vitro genefusions that join an enzymatically active 3-galactosidase seg-ment to amino-terminal fragments of exogenous proteins: Esch-erichia coli plasmid vectors for the detection and cloning oftranslational initiation signals. J. Bacteriol. 143:971-980.

5. Dawes, J., and M. A. Foster. 1971. Vitamin B12 and methioninesynthesis in Escherichia coli. Biochim. Biophys. Acta 237:455-464.

6. Kung, H.-F., C. Spears, R. C. Greene, and H. Weissbach. 1972.Regulation of the terminal reactions in mnethionine biosynthesisby vitamin B12 and methionine. Arch. Biochem. Biophys. 150:23-31.

7. Kutzbach, C., and E. L. R. Stokstad. 1971. Mammalianmethylenetetrahydrofolate reductase: partial purification, prop-erties, and inhibition by S-adenosylmethionine. Biochim. Bio-phys. Acta 250:459-477.

8. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

9. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

10. Miller, J. H. 1972. Experiments in molecular genetics, p.352-355. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

J. BACTERIOL.

on May 2, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

metR GENE 1397

11. Milner, L., C. Whitfield, and H. Weissbach. 1969. Effect ofL-methionine and vitamin B12 on methionine biosynthesis inEscherichia coli. Arch. Biochem. Biophys. 133:413-419.

12. Mulligan, J. T., W. Margolin, J. H. Krueger, and G. C. Walker.1982. Mutations affecting regulation of methionine biosyntheticgenes isolated by use of met-lac fusions. J. Bacteriol. 151:609-619.

13. Panasenko, S. M., J. R. Cameron, R. W. Davis, and I. R.Lehman. 1977. Five hundred-fold overproduction ofDNA ligaseafter induction of a hybrid lambda lysogen constructed in vitro.Science 196:188-189.

14. Raibaud, O., and M. Schwartz. 1984. Positive control of tran-scription initiation in bacteria. Annu. Rev. Genet. 18:173.-206.

15. Rowbury, R. J. 1983. Methionine biosynthesis and its regula-tion, p. 191-211. In K. M. lierrmann and R. L. Somerville (ed.),Amino acids: biosynthesis and genetic regulation. Addison-Wesley Publishing Co., Reading Mass.

16. Schulte, L. L., L. T. Stauffer, and G. V. Stauffer. 1984. Cloningand characterization of the Salmonella typhimurium metE gene.J. Bacteriol. 158:928-933.

17. Shimada, K., R. A. Weisherg, and M. E. Gottesman. 1972.

Prophage k at unusual chromosomal locations. I. Location ofthe secondary attachment sites and properties of the lysogens.J. Mol. Biol. 63:483-503.

18. Smith, D. A., and J. D. Childs. 1966. Methiorine genes andenzymes of Salmonella typhimurium. Heredity 21:265-286.

19. Stauffer, G. V., M. D. Plamann, and L. T. Stauffer. 1981.Construction and expression of hybrid plasmids containing theEscherichia coli glyA gene. Gene 14:63-72.

20. Urbanowski, M. L., and G. V. Stauffer. 1986. Autoregulation bytandem promoters of the Salmonella typhimurium LT2 metJgene. J. Bacteriol. 165:740-745.

21. Urbanowski, M. L., and G. V. Stauffer. 1986. The metH genefrom Salmonella typhimurium LT2: cloning and initial charac-terization. Gene 44:211-217.

22. Vhitehouse, J. M., and D. A. Smith. 1973. Methionine andvitamin B12 repression and precursor induction in the regulationof homocysteine methylation in Salmonella typhimurium. Mol.Gen. Genet. 120:341-353.

23. Whitfield, C. D., E. J. Steers, Jr., and H. Weissbach. 1970.Purification and properties of 5-methyltetrhydropteroyltrigluta-mate-homocysteine transmethylase. J. Biol. Chem. 245:390-401.

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