Vol. 170, No. 7

Construction of a Dihydrofolate Reductase-Deficient Mutant ofEscherichia coli by Gene Replacement

ELIZABETH EHRHARDT HOWELL,t* PAUL G. FOSTER, AND LISA M. FOSTERThe Agouron Institute, 505 Coast Boulevard South, La Jolla, California 92037

Received 13 October 1987/Accepted 10 April 1988

The dihydrofolate reductase (fol) gene in Escherichia coli has been deleted and replaced by a selectablemarker. Verification of the Afol::kan strain has been accomplished using genetic and biochemical criteria,including Southern analysis of the chromosomal DNA. The Afol::kan mutation is stable in E. coli K549 [thyApoUA12 (Ts)] and can be successfully transduced to other E. coli strains providing they have mutations in theirthymidylate synthetase (thyA) genes. A preliminary investigation of the relationship betweenfol and thyA geneexpression suggests that a Fol- cell (i.e., a dihydrofolate reductase deficiency phenotype) is not viable unlessthymidylate synthetase activity is concurrently eliminated. This observation indicates that either the nonpro-ductive accumulation of dihydrofolate or the depletion of tetrahydrofolate cofactor pools is lethal in a Fol-ThyA+ strain. Strains containing the thyA Afol::kan lesions require the presence of Fol end products forgrowth, and these lesions typically increase the doubling time of the strain by a factor of 2.5 in rich medium.

Dihydrofolate reductase (DHFR; EC 1.5.1.3) catalyzesthe reduction of dihydrofolate to tetrahydrofolate with theconcurrent oxidation ofNADPH to NADP+. This activity isnecessary in maintaining intracellular pools of tetrahydrofo-late cofactors which are essential for biosynthetic reac-tions involving the transfer of one-carbon units. Inhibition ofDHFR activity initially results in depletion of N5,N'0-meth-ylene tetrahydrofolate, followed by inhibition of DNA syn-thesis and ultimately cell death. Antifolate drugs which haveDHFR as their target include trimethoprim (antibacterial),methotrexate (anticancer), and pyrimethamine (antimalar-ial).

Genetic studies of the DHFR (fol) locus in Escherichia colihave typically described mutations which increase DHFRprotein production or decrease inhibitor binding to DHFR,or both (9, 26, 27, 30, 31). No temperature-sensitive muta-tions in the fol gene have been reported, and only recentlyhas one DHFR-deficient strain been isolated (29). TheDHFR-deficient strain is apparently devoid of DHFR activ-ity, and it requires the presence of Fol end products (thy-mine, adenine, glycine, methionine, pantothenate) forgrowth. The lesion (a likely point mutation) is uncharacter-ized and could be in the DHFR coding sequence or in acontrol region.Our interest in a true fol strain arose from needing to

express mutant E. coli DHFR genes in a Fol- (DHFR-deficient) background. Our mutant DHFR genes are ob-tained by site-directed mutagenesis techniques and arecloned in multicopy plasmids (14, 34). While expression ofthe chromosomal gene typically results in <1% contamina-tion of the purified mutant protein by wild-type DHFR, thislevel of contamination can cause significant kinetic effectswhen the mutant DHFR activity is low (14). Therefore, toexpress our mutant DHFR genes and minimize any possiblerecombination between chromosomal and plasmid E. coliDHFR genes, we have deleted the DHFR gene from the E.coli chromosome and studied the effects of this null allele onstrain viability. The relationship between expression of

* Corresponding author.t Present address: Biochemistry Department, University of Ten-

nessee, Walters Life Science Bldg., Knoxville, TN 37996-0840.

DHFR and thymidylate synthetase activities was addition-ally investigated when we found our 4fol::kan mutation wasnot viable in a ThyA+ background.

MATERIALS AND METHODS

Bacteria and plasmids. The E. coli (K-12) strains andplasmids used in this research are listed in Table 1. P1 virwas the generous gift of Doug Smith (University of Califor-nia, San Diego), and plasmids pAGts and M14) were the giftsof Ernest Villafranca (Agouron Pharmaceuticals, Inc.) andChris Morris (California Institute of Technology), respec-tively. pBR328 and pUC4K were obtained from Boehringer-Mannheim and Pharmacia, respectively.To grow E. coli thyA Afol::kan strains on Bonner-Vogel

minimal plates (35) it was necessary to supplement themedium with thymine (200 p.g/ml), adenine (20 p.g/ml), pan-tothenate (1 ,ug/ml), and glycine and methionine (50 ,ug/mleach) (29). Any E. coli thyA strains grown on Bonner-Vogelminimal plates were supplemented with 200 ,ug of thymineper ml. Cells were typically grown in L broth. Whenrequired, trimethoprim, kanamycin, ampicillin, and chlor-amphenicol were added at concentrations of 10 to 20, 25 to50, 100 to 200, and 30 ,ug/ml, respectively.

Strain manipulations. Deletion of the DHFR gene wasperformed as described by Russel and Model (25) except thatthe deletion was performed and verified in E. coli K549[polA12(Ts)] and not immediately transduced to alternatestrains. Subsequent P1 vir transductions were performed bythe method of Miller (20) using K549 (Afol::kan) as the hoststrain. Selection of transductants at 37°C used LB platescontaining 50 ,ug of kanamycin per ml.DNA techniques. BamHI digests of 1 ,ug of chromosomal

DNA and 0.25 .g of plasmid markers were size fractionatedon a 0.6% agarose gel and blotted onto a Nytran filter by themethods of Maniatis et al. (16). Southern hybridizationswere performed by the method of Maniatis et al. (16) withthe following exceptions: the prehybridization solution con-tained tRNA (0.1 U of type X per ml; Sigma) instead ofsalmon sperm DNA; hybridizations were done at 65°C; and75°C washes contained 6x SSC (1 x SSC is 0.15 M NaCl plus0.015 M sodium citrate)-0.1% sodium dodecyl sulfate. Nick-

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TABLE 1. E. coli K-12 strains and plasmids used

Designation Relevant properties" Reference or source

E. coli strainsED8654 supE supF met hsdR (8)ED8654 (thyA) Same as ED8654 but thyA This studyJM107 A(lac proAB) endAl gyrA96 thi-J hsdRI7 supE44 relAl X- F' traD36 Bethesda Research Laboratories; (36)

proAB+ lac(Vq-ZZAM5)+K549 thyA rha lac str polA12(Ts) F' (25)K549 (Afol::kan) Same as K549 but Afol::kan This studyNM522 Alac-pro supE hsdRS F' lac(lq-ZAM15)+ pro' (10)NM522 (thyA) Same as NM522 but thyA This studyLH18 NM522 thyA Afol::kan This study

PlasmidsM14 M13mp8 with a HincII-PstI fragment containing the promoterless E. coli Chris Morris, unpublished work

DHFR gene sequence; the HindlIl site was obtained by site-directed mu-tagenesis

P700 pUC8 with R67 DHFR gene insert; Ampr Tmpr Lynn ElwellpACYC177 Ampr Kanr (6)pAG101 pUC8 with E. coli DHFR gene on a 1.1-kb fragment; Ampr Tmpr (34)pAGts pUC8 with DHFR promoter controlling expression of E. coli thymidylate Ernest Villafranca

synthetase gene insert; AmprpBR328 Tetr Clmr Ampr (32)pCV29 pBR322 with 8.3-kb BamHI insert containing E. coli DHFR gene; Ampr (31)

TmprpLMF20 pACYC177 with 8.3-kb BamHI insert containing the DHFR gene; Ampr This study

Kanr TmprpLMF21 pLMF20 with kanamycin resistance gene (from pACYC sequence) inacti- This study

vated; Ampr TmprpLMF22-3 pLMF21 with kanamycin resistance gene from pUC4K inserted in Sall site This study

of BamHI fragment; Tmpr Ampr KanrpLMF22-3 B-4 pLMF22-3 with 1.2 kb deleted, encompassing the entire DHFR (fol) gene; This study

Ampr KanrpLH30-2 pBR328 with an 8.6-kb BamHI insert from pLMF22-3 B-4; Ampr Kanr Clmr This studypUC4K pUC8 with kanamycin resistance gene; Ampr Kanr (23)a Antibiotic resistance phenotypes: Amp, ampicillin; Tmp, trimethoprim; Kan, kanamycin; Tet, tetracycline; CIm, chloramphenicol.

translation of plasmid DNA with [a-32P]dCTP (New EnglandNuclear Corp.) and DNA polymerase I (New England Nu-clear Corp.) was performed per McCarter and Silverman(18).Other general DNA manipulations such as polyacrylamide

and agarose gel electrophoresis, restriction enzyme diges-tions, ligations, transformations, cesium chloride plasmidDNA preparations, and chromosomal DNA preparationswere performed as described by Maniatis et al. (16) andSilhavy et al. (28). Restriction endonucleases were obtainedfrom 3oehringer Mannheim. BAL 31 was obtained fromNew England Biolabs. BAL 31 digestions were quenched bythe addition of ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and treated with pro-teinase K to remove the nuclease.

Protein analysis. Purification ofDHFR followed a standardprocedure in which cellular lysates were passed over amethotrexate affinity column (2). High-salt and higb-pHrinses were followed by elution ofDHFR with 1 mM folate-1 M KCl-10 mM EDTA-10 mM P-mercaptoethanol in 0.2 Msodium borate buffer (pH 9.0). Salt and folate were subse-quently removed by dialysis. DHFR activity was monitoredby spectrophotometric assays (14). Activity stains of cellularlysates run on nondenaturing electrophoretic gels were alsoused to evaluate the level of DHFR activity (13).

RESULTS AND DISCUSSION

Vector construction. The vector used to delete the E. colichromosomal DHFR gene was designed (i) to have a largeDNA insert so that recombination between vector and

chromosome would readily occur, (ii) to concurrently inserta selectable marker (kanamycin resistance) upon deletion ofthe chromosomal gene for DHFR, and (iii) to utilize a system[e.g., ColEl plasmid in a polA(Ts) E. coli strain] such thatplasmid integration and segregation into the chromosomecould be easily selected (11, 25). Figure 1 summarizes thesteps utilized in the construction of such a vector.The first step in constructing a vector for DHFR gene

deletion involved cloning the 8.3-kilobase (kb) BamHI frag-ment containing the -0.5-kb E. coli DHFR structural gene(methionine start point to TAA stop) from pCV29 (31) intopACYC177 (6). The resulting plasmid was designatedpLMF20 and it conferred resistance to trimethoprim, ampi-cillin, and kanamycin. The kanamycin resistance gene ofpLMF20, associated with the pACYC portion of the plas-mid, was then inactivated by a unique HindIII cut, followedby BAL 31 nuclease digestion and blunt-end ligation. Thisplasmid, pLMF21, conferred only trimethoprim and ampi-cillin resistance. Next, a 1.5-kb Sall fragment encoding thekanamycin resistance gene from pUC4K (23) was clonedinto the Sall site of the 8.3-kb BamHI fragment, resulting inthe plasmid pLMF22-3 (conferring kanamycin, ampicillin,and trimethoprim resistances). In this construction the ka-namycin resistance gene is contained in the BamHI frag-ment. The orientation of the 1.5-kb kanamycin resistance-conferring insert was determined by DNA fragment sizesfrom double restriction enzyme digests (XhoI and SaII) (23;Pharmacia technical information). Transcription of the Ka-namycin resistance-conferring (aminoglucoside 3'-phospho-transferase) gene occurred in the same direction as DHFR

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rc= isr

-

I 1I

DHFR

coding

seuence

77 Barn Hi Ban HI digest

digest ligaseBam

Bam HI

Bam HI

Bam HI

I

ItBam HI

Mnt

Barn HI

FIG. 1. Construction of the vector used to delete the DHFR genefrom E. coli. The 8.3-kb BamHI DNA fragment containing the E.coli DHFR gene (see top of figure) was obtained from plasmidpCV29 (30). The HindIII site located between the DHFR promoter(HinclI site) and coding sequence is present only in the M1+plasmidand was obtained by oligonucleotide-directed mutagenesis (ChrisMorris, personal communication). The presence of a functionalDHFR gene confers resistance to trimethoprim. Pertinent restrictionenzyme sites are noted. The plasmids were transformed into E. coliJM107. Antibiotic resistance genes: KAN, kanamycin; AMP, ampi-cillin; CLM, chloramphenicol; TET, tetracycline.

transcription (i.e., towards the PstI site noted at the top ofFig. 1).To delete the DHFR gene from the BamHI fragment, the

pLMF22-3 plasmid was cut at the unique EcoRI site in theDHFR-coding sequence, and the DNA was digested withBAL 31 for 10 min. After blunt-end ligation and transforma-tion of the DNA, kanamycin-resistant colonies were ob-tained and screened for trimethoprim sensitivity. One plas-mid conferring these properties (pLMF22-3 B-4) wasanalyzed by restriction enzyme mapping to determine thesize of the deletion. Approximately 1.2 kb was found to bedeleted from the BamHI fragment by the BAL 31 digestion.As determined by restriction enzyme mapping, the HincII(promoter region) and PstI (approximately 540 base pairsafter the end of the DHFR gene) sites were both lost (see topof Fig. 1). However, the SalI site on the 3' end of thekanamycin resistance gene was still present. Clearly, theentire DHFR structural gene and its promoter sequencewere deleted from the plasmid vector. Note that insertion ofa 1.5-kb kanamycin resistance fragment and deletion of 1.2kb around the DHFR gene slightly increased the BamHIfragment size to 8.6 kb.Gene deletion. Since our selection procedure for the gene

deletion process relies on the replication of ColEl plasmidsin temperature-sensitive polA strains (11, 25), the BamHIfragmnent from pLMF22-3 B-4 was cloned into the ColElvector pBR328. This vector was named pLH30-2 and itconferred kanamycin, chloramphenicol, and ampicillin resis-tances on the host cell.

Transformation of pLH30-2 into E. coli K549 poIA12(Ts)allows expression of the ColEl replicon by host DNApolymerase A at permissive temperatures (.30'C) (21). Atrestrictive temperatures (.370C), growth only occurs whenthe plasmid integrates into the chromosome (11, 25). WhenK549 cells transformed with pLH30-2 were grown at 42°C,the number of kanamycin and chloramphenicol-resistantcolonies obtained was decreased 50-fold as compared with30°C growth. Those colonies that grew at 42°C were pre-sumed to have the plasmid integrated in the bacterial chro-mosome at the DHFR gene locus (fol maps at 1 min [3]).Integration of plasmids into the chromosome is proposed tooccur by homologous recombination (primarily from a singlecross-over event between the vector and the DHFR chro-mosomal gene) which produces a nontandem duplicationseparated by the plasmid DNA (11).Subsequent growth of the strain (containing the presumed

integrated plasmid) at 30°C allows the plasmids to segregatefrom the chromosome. Segregation of a nontandem duplica-tion from the chromosome will result , deletion of either theplasmid DNA containing the chromouomal DHFR gene orthe plasmid DNA containing the kanamycin resistance frag-ment (11, 25). Loss of the latter would restore the cell to itsoriginal genotype, whereas loss of the former would deletethe DHFR chromosomal gene.The segregated plasmid (carrying the chromosomal DHFR

gene) in the Afol cell may then be lost if antibiotic pressureis not maintained and if deletion of the DHFR gene results ina viable cell. Cells still containing plasmid (integrated orsegregated) can be identified by their ability to grow in thepresence of kanamycin, chloramphenicol, and ampicillin. Incontrast, Afol::kan cells which have lost their plasmid canconfer resistance to kanamycin only. Several kanamycin-resistant and chloramphenicol-sensitive cells were identifiedand were analyzed to determine whether the DHFR chro-mosomal gene had been deleted.

Confirmation of DHFR gene deletion. The two primary

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A B

8.3

3.7

2.7

8 2 ,

FIG. 2. A Southem blot of equivalent amounts of chromosomal

DNA from E. coli strains K549 and K549 4fol::kan digested with

BamHl, size fractionated oni a 0.6% agarose gel, and blotted onto a

Nytran filter. (A) Autoradiogram of a filter which was probed with

32P-labeled M14o plasmid DNA to identify the DHFR gene. (B)

Comparable filter probed with 32P-labeled pUC4K plasmid DNA to

identify the kanamycin resistance gene. The 32P label was incorpo-

rated into the plasmid DNA by nick-translation. Lanes 1 and 3,

K549 DNA; lanes 2 and 4, K549 Afo1::kan DNA. Sizes in kilobases

(KB), from plasmid markers, are noted on the left.

methods used to assess whether the fol gene deletion oc-

curred were Southern hybridizations of the chromosomal

DNA, using a DHFR-specific probe, and determination of

DHFR activity loss from cellular lysates eluted from a

methotrexate affinity column. Additional screens which are

useful in assessing the DHFR deficiency phenotype include

(i) the requirement of Fol end product auxotrophy, (ii)

restoration of Fol end product prototrophy by transforma-

tion of the Afol: :kan cell with a cloned DHFR gene, and (iii)

activity stains of cellular lysates run on nondenaturing

electrophoretic gels.Southern blots were performed on BamHl digests of

chromosomal DNA from the putative Afol: :kan strains. A

nick-translated probe of M14~DNA (a HindIII-PstI fragment

of the DHFR gene carried in M13mp8; see top of Fig. 1) was

used to probe for the presence of any DHFR gene se-

quences. The Ml1) probe was found to hybridize only to

DNA from K549 (Fig. 2). In contrast, when a nick-translated

probe from pUC4K (the kanamycin resistance-conferring

plasmid) was utilized, hybridization with the K549 Afol: :kan

strain, but not with K549 itself, 'Was observed. As expected,

the size of the pUC4K-hybridizing fragment from K549

4fol: :kan was -8.6 kb, corresponding to the BamHl frag-

ment size in pLH30-2. These results clearly substantiate our

hypothesis that the DHFR gene has been deleted from the

chromosome of E. coli K549 Afol: :kan.

To assess the amount ofDHFR activity present in both the

K549 and K549 Afol: :kan strains, any DHFR present was

purified from cellular lysates by using a methotrexate affinity

column. While the K549 strain yielded 0.18 U of activity

from 1 liter of cells, the K549 Afol: :kan strain yielded no

detectable activity from an equivalent number of cells (the

lower limit for the assay is --0.004 U). This result also

supports the conclusion that the DHFR gene has been

deleted from K549 Afol::kan.Relationship between thyA and Afol::kan alleles. Initial

attempts to transduce the Afol: :kan lesion from K549

tafol::kan into E. coli JM1o7 (and other strains) by using P1

vir were unsuccessful unless a plasmid containing either the

E. coli DHFR gene or the nonhomologous R67 plasmid-

encoded DHFR gene (P700; 5) was present in trans. This

DHFR

NADP+

IDIHYDROFOLTI o

dTMP

THYMIDYLATE SYNTHETASE

dUMP

ITETRAHYDROFOLATE I~NS,N10METHYLENE _TETR HYROFOLATE

SERINEHYDROXYMETHYLTRANSFERASE

SERINE GLYCINE

FIG. 3. The three-enzyme cycle in pyrimidine metabolism com-posed of DHFR, thymidylate synthetase, and serine hydroxy-methyltransferase activities.

result suggested that the Afol::kan construction was stable inK549 because of a possible suppressor mutation in thisstrain. An example of compensatory mutations in a secondgene is the rescue of DNA topoisomerase I (Atop) mutantcells by mutations in either of the DNA gyrase genes (gyrA,gyrB) (7). The increased growth rates associated with thedouble-mutant cells suggest that a balance of the topoisom-erase and gyrase activities is necessary for cell viability.One possible suppressor in our K549 Afol::kan strain was

the thyA lesion, as thymidylate synthetase (thyA), dihydro-folate reductase, and serine hydroxymethyltransferase com-pose a three-enzyme cycle (Fig. 3) in pyrimidine metabo-lism, and it has been shown that inhibition of intracellularDHFR activity decreases or abolishes expression of thy-midylate synthetase (15, 22). When DHFR activity is low oreliminated and thymidylate synthetase activity is unaffected,the intracellular concentration of dihydrofolate will increaseand the N5,Nl0-methylene tetrahydrofolate pool will bedepleted. Depletion of the N5,N10-methylene tetrahydrofo-late pool negatively affects one-carbon biosynthetic pathwayreactions. In this situation, a concurrent decrease or loss inthymidylate synthetase activity would allow the N5,N10-methylene tetrahydrofolate pool to be maintained (15). Thisconnection between DHFR and thymidylate synthetase ac-tivities may explain our inability to transduce the Afol::kanmutation into ThyA+ strains.Another possible interpretation of our inability to trans-

duce Afol::kan into a ThyA+ strain has been suggested bythe recent results of Allegra et al. (1). They hypothesize thatan increased dihydrofolate concentration in a Fol- cell(obtained by inhibiting DHFR with methotrexate or trimeth-oprim) inhibits other critical enzymic activities. 5-Aminoim-idazole carboxamide ribotide transformylase, methylene te-trahydrofolate reductase, and thymidylate synthetase are allinhibited by polyglutamylated forms of dihydrofolate (Ki -5x 10-8 M) (1). Buildup of dihydrofolate, followed by inhi-bition of the above enzyme activities, could inhibit cellgrowth. Again, if thymidylate synthetase activity were de-creased or eliminated in a Fol- cell, buildup of intracellulardihydrofolate would not occur, increasing cell viability.Clearly dihydrofolate accumulation is linked to tetrahydro-folate cofactor depletion, but whether one or both conditionscause the lethal effect of the Fol- ThyA+ condition is not yetknown.

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The connection between DHFR and thymidylate synthe-tase activities is further illustrated by the current method ofselecting thyA strains of E. coli, i.e., inhibiting intracellularDHFR activity with trimethoprim in the presence of thymine(20, 33). Another illustration of this relationship is seen inprotozoa, where DHFR and thymidylate synthetase are

genetically linked and a bifunctional protein is produced(19).To investigate the relationship between the thyA and

Afol::kan alleles in our strain, we transformed an E. colithymidylate synthetase gene carried in pUC8 (pAGts, underconstitutive DHFR promoter control; E. Villafranca, per-sonal communication) into K549 and K549 Afol::kan. Whilewe have never observed any transformants of K549&fol::kan by pAGts, transformation of this plasmid intoK549 and JM107 typically produces >500 colonies. Controltransformations of pUC8 into all three strains give equiva-lent numbers of colonies. Therefore we hypothesize thatFol- ThyA' is probably a lethal condition (at least when thethymidylate synthetase gene is carried on a multicopy plas-mid).Based on the above observations, several strains of E. coli

were made thyA (20) for use as recipient strains in transduc-tion experiments. P1 vir transductions, performed with E.coli strains NM522 (10) and NM522 thyA, produced large,stable colonies for the NM522 thyA strain and an equivalentnumber of very small, unstable colonies for the NM522strain. The transduced NM522 strains grew initially (albeitslowly) but then lost their ability to grow, even in richmedium. Similar results were obtained with transductants ofE. coli strains ED8654 (8) and ED8654 thyA. These prelim-inary results suggest that a single functional copy of thethymidylate synthetase gene may be lethal in a Fol- strain,even in rich media. This result is surprising, as the presenceof Fol end products, supplied by rich medium apparently donot rescue the Fol- ThyA+ phenotype. Perhaps the rate oftransport of one (or more) of the Fol end products into thecell is less than the rate of depletion of this end product in thecell.

Screening of the NM522 thyA Afol::kan strains to confirmtheir Fol- phenotype included DHFR activity stains ofcellular lysates run on nondenaturing electrophoretic gels(13). No DHFR activity was observed for the cellular lysatesfrom the Afol::kan strains, whereas activity was noted forthe NM522 thyA strain. Additionally, these /fol::kan strainsgrew on Bonner-Vogel medium supplemented with thymine,glycine, pantothenate, methionine, and adenine, but did notgrow on nonsupplemented medium (29). This result illus-trates Fol end product auxotrophy. As expected, theAfcl::kan strains could be rescued from Fol end productauxotrophy by transforming them with a plasmid carryingeither the E. coli DHFR gene (pAglOl; E. coli DHFR genecarried on a 1.1-kb fragment) (34) or the nonhomologousR-plasmid-encoded R67 DHFR (P700) (5). The ability of theR67 DHFR to rescue strain LH18 from Fol end productauxotrophy indicates that only the DHFR activity is neces-sary for cell viability. Any possible requirement for a specificprotein structure is eliminated, as the E. coli DHFR se-

quence and structure are totally different from those of theR67 DHFR (5, 17, 24). Finally, Southern blot analysis of thechromosomal DNA (not shown) verified the deletion of theDHFR gene. These various results indicate that the&fol: :kan lesion can be successfully transduced to thyAstrains of E. coli.

Finally, as the DHFR coding region is only 0.5 kb longwhereas the deletion that removed the DHFR gene was 1.2

kb long, the possibility exists that additional deletions inneighboring genes may have occurred. However, the likeli-hood that any additional deletions (or any polar effects)associated with the /fol::kan deletion are the source of thethyA requirement appears minimal. This interpretation issupported by the fact that the Afol::kan deletion can betransduced into a ThyA+ strain if a DHFR gene (either theE. coli or the R67 DHFR) is present on a plasmid (in trans).Since the nonhomologous R67 DHFR gene can "rescue" theE. coli Afol::kan strain from a thyA requirement and sincethe likelihood that the neighboring DNA sequences for theR67 DHFR gene are homologous with the neighboring E.coli DHFR gene sequences in the Afol::kan strain is ex-tremely remote, we conclude that any effects on neighboringgenes due to the Afol::kan deletion are extremely unlikely tocreate the thyA requirement.

Preliminary characterization of the thyA Afol::kan strain.The NM522 thyA Afol::kan strains were infected with P1 virto verify they were not P1 lysogens. One nonlysogenicstrain, named LH18, was further characterized. Our ob-served doubling times for NM522, NM522 thyA, and LH18in L broth at 37°C were 22, 22, and 51 min. The thyA/fol: :kan combination decreased the growth rate by a factorof approximately 2.5. This decrease in growth rate is pre-sumably due to the inability of rich medium to supply the Folend product, fMet-tRNA Met A previous study of E. coligrown in the presence of high concentrations of trimetho-prim (50 to 100 ,ug/ml) has shown that protein synthesis canbe initiated with unformylated Met-tRNAfMet, but cellgrowth is slowed by a factor of 3 (4, 12).Our observations indicate that deletion of the DHFR gene

in E. coli results in a viable cell, as long as thymidylatesynthetase activity in the cell is concurrently decreased oreliminated. This result contrasts with previous observationsby Singer et al. (29); e.g., their DHFR-deficient strain of E.coli apparently displays normal levels of thymidylate syn-thetase activity. However, since their DHFR activity assayswere performed on cellular lysates and their antibody com-petition experiments to identify any inactive DHFR proteinhad a lower limit of <12%, it seems most likely that theirstrain was not totally deficient in DHFR activity. An ex-tremely low level of DHFR activity could potentially rescuethe cell by balancing any effects due to the observed thy-midylate synthetase activity.

ACKNOWLEDGMENTS

This research was supported by Public Health Service grantGM35308 from the National Institutes of Health (to E.E.H.).We thank Mike Silverman, Linda McCarter, and Bob Belas for

their generous help and advice; Carol L. J. Booth for her excellenttechnical assistance; and Lewis N. Booth, Jr., for drafting Fig. 1.We additionally thank Terence Jones, Joseph Kraut, David Mat-thews, and Ernest Villafranca for helpful discussions.

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