JOURNAL OF BACTERIOLOGY, Nov. 1982, p. 650-6600021-9193/82/110650-11$02.00/0Copyright © 1982, American Society for Microbiology

Vol. 152, No. 2

Escherichia coli Phenylalanyl-tRNA Synthetase Operon:Characterization of Mutations Isolated on Multicopy Plasmids

JACQUELINE A. PLUMBRIDGE* AND MATHIAS SPRINGERInstitut de Biologie Physico-Chimique, 75005 Paris, France

Received 2 April 1982/Accepted 26 July 1982

Plasmid pBl carries the genes for threonyl-tRNA synthetase, phenylalanyl-tRNA synthetase, and translation initiation factor IF3. Strains carrying thisplasmid overproduce phenylalanyl-tRNA synthetase about 100-fold. Spontaneousmutant plasmids were obtained which no longer caused the overproduction of theenzyme. Three classes of mutations were found. (i) Deletion mutations werefound, some of which had the interesting property of fusing different genestogether, e.g., putting phenylalanyl-tRNA synthetase under the control of thethreonyl-tRNA synthetase promoter. (ii) Insertion mutations were found; oneinsertion in particular was studied. This insertion is located in front of thestructural gene for phenylalanyl-tRNA synthetase and is shown to interrupt a cis-acting regulatory region. (iii) Mutations that showed no major change in DNAstructure were found. One of these mutations is apparently purely structural, as itproduces a small subunit of phenylalanyl-tRNA synthetase with a reducedmolecular weight. This protein is less stable than the wild-type enzyme. Thesemutations represent useful tools to investigate how the phenylalanyl-tRNAsynthetase operon is regulated.

Phenylalanyl-tRNA synthetase is a tetramericenzyme with two different subunits (a2A2 struc-ture). The genes corresponding to the smallsubunit (pheS) and to the large subunit (pheT)have been shown to be adjacent on the Esche-richia coli chromosome at 38 min (10, 23). A Atransducing phage was isolated which carries,besides pheS and pheT, the structural genes forthreonyl-tRNA synthetase (thrS) and for transla-tion initiation factor IF3 (infC) (12, 13, 26). Theorder of the genes is thrS infC pheS pheT (23,25). These four genes are transcribed in thedirection from thrS to pheT (i.e., counter-clock-wise on the standard E. coli map [2]), and pheSand pheT are transcribed from the same promot-er, located after infC (21, 27).The expression of the pheS, Toperon is consti-

tutive, as expected, since phenylalanyl-tRNAsynthetase is an essential enzyme. However,this synthetase, like several other aminoacyl-tRNA synthetases, is subject to metabolic regu-lation (20), i.e., its synthesis rate increases withthe growth rate of the cell. It has also beenshown that withdrawing phenylalanine from aphenylalanine bradytroph provokes an in-creased synthesis of phenylalanyl-tRNA synthe-tase (19). The biosynthesis of multimeric proteinsystems presents a special problem, namely, thestoichiometric synthesis of the various compo-nents. The cotranscription of the pheS, T operoncauses the equimolar synthesis of the mRNA ofthe two genes; this, however, does not necessar-

ily ensure the equimolar synthesis of the geneproducts.One way to investigate the mechanisms regu-

lating the expression of the pheS,T operon is tostudy mutants with an altered expression. Thiswork describes the isolation and characteriza-tion of spontaneous mutations directly on plas-mids. The fact that the mutations are isolated onplasmids has several advantages: (i) mutationscan be isolated that would be lethal on thechromosome; (ii) the analysis of the mutantplasmid DNA makes it easy to classify themutations as deletions, insertions, or point mu-tations; (iii) as wild-type pheS,T mRNA isstrongly expressed from the type of multicopyplasmid employed, significant mRNA measure-ments can be made even with "down" muta-tions.A variety of information has been derived

directly from the mutants described here, e.g.,relative strength of promoters and physical loca-tion of regulatory and structural regions of thepheS,T operon. Some of the mutants character-ized in this paper are the object of the transcrip-tion studies reported in the accompanying paper(22) and provide information about how theoperon is regulated.

MATERIALS AND METHODSBacterial strains and plasmids. IBPC1365 is F- and

thi-I argE3 his4 proA2 lacYl gaIK2 mtl-l xyl-5 tsx-29supE44 A- (27); IBPC2001 has the same markers asIBPC1365, except it is recAl and rpoB (27); IBPC1671

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E. COLI PHENYLALANYL-tRNA SYNTHETASE 651

has the same markers as IBPC1365, except it is rpsL,recAl pheS5 (23); IBPC1772 has the same markers asIBPC1365, except that it is rpsL recAl pheT3S4 (23).IBPC4771 is identical to IBPC1365, except that it isrpsL thrS1029 (27). Plasmid pBl (23; with revised sizesof the DNA restriction fragments, G. Fayat et al.,personal communication) is shown in Fig. 1, togetherwith the structures of the mutant plasmids derivedfrom it. The isolation of plasmids pGC87 and pG6-7has been described elsewhere (21). They carry, re-spectively, the PstI2 to PstI3 and PstI2 to PstI4fragments of pBl cloned into plasmid pGA39 (1).Plasmid pG6-7 carries the whole structural gene forpheS, whereas pGC87 carries only the promoter proxi-mal part. Both plasmids confer resistance to chloram-phenicol on the host bacteria.

Isolation of mutant pBl plasmids. The mutant plas-mids described here all arose spontaneously. System-atic screening was carried out as follows. Independentisolates of IBPC1365 and IBPC2001 carrying pBl weregrown in 3 ml of LB supplemented with ampicillin (50,ug ml-') and tetracycline (10 ,ug ml-'). After beingaged at room temperature for 1 to several weeks, thecultures were diluted and regrown to saturation. Afterbeing streaked out on LB plates containing 50 ±g ofampicillin per ml and 10 ,ug of tetracycline per ml,single colonies were taken and grown in LB containingampicillin and tetracycline, and extracts were ana-lyzed by sodium dodecyl sulfate-12.5% polyacryl-amide gel electrophoresis as described previously (15).Plasmid DNA was extracted from non-overproducingmutant strains by the rapid alkaline extraction method(4) and analyzed with restriction enzymes as describedpreviously (23). Plasmid DNA was introduced intocells made competent by treatment with CaCl2 (23).Strains were cured of plasmids by many generations ofgrowth in the absence of antibiotic selection.

Assay of aminoacyl-tRNA synthetases. IBPC2001 car-rying pBl, pBlT, pB1S, or pBR322 was grown inmorpholinepropanesulfonic acid (MOPS) medium sup-plemented with all of the amino acids (20), 50 gig ofampicillin per ml, and 10 Fig of tetracycline per ml.Samples of exponentially growing cells (absorbance at650 nm [A650] = 0.4) were spun down in small polyeth-ylene tubes and frozen at -80°C. Just before beingassayed, S30 extracts were made by suspending thecells in 300 tl of 10 mM Tris-hydrochloride (pH 6.8)-0.1 mM EDTA-O1o glycerol-10 mM mercaptoethanoland then sonicating 4 x 15 s, with 15 s cooling in ice inbetween. The sonicated extracts were centrifuged for4 min in a microfuge to give a clear supernatant, whichwas decanted and assayed immediately for synthetaseactivity.

Synthetases were assayed in the following buffer: 50mM Tris-hydrochloride (pH 7.5), 10 mM MgCl2, 10mM KCI, 10 mM 3-mercaptoethanol, 1 mM ATP, 4 mgof tRNA per ml, and 50 ,uM 14C-labeled threonine orphenylalanine (specific activity, -20 mCi mmol-1) or"5S-labeled methionine (specific activity, -50 mCimmol-1). Incubation was at 37°C in 100 ,ul. The S30swere diluted as necessary into the sonication buffer togive a linear response in 15 min (less than 20%omaximum tRNA charging capacity). Reactions werequenched with ice-cold 7% trichloroacetic acid con-taining 0.5% Casamino Acids. Precipitates were col-lected on Whatman GF/C filters, extensively washedwith the trichloroacetic acid containing Casamino Ac-

ids, rinsed with 70% ethanol, and dried. Protein con-centration in the S30s was estimated by the Coomassieblue method (6), using bovine serum albumin as astandard.

Labeling of cels with [3SJmethionine and immuno-precipitation. IBPC2001 carrying plasmids pBl, pB1T,pBlS, or pBR322 was grown as described for the testfor synthetases. At A650 = 0.4, the cells were spundown, washed, and suspended in the same mediumminus methionine. After 10 min of growth to starve formethionine, the cultures were pulse-labeled with[35S]methionine at 40 ,Ci ml-' (specific activity, 200Ci mmol-1). After 2 min, unlabeled methionine wasadded to a final concentration of 1 mM. Samples (1 ml)of the labeled cultures were removed at 2, 10, 30, anm120 min and, when the cultures were saturated, werechilled in ice, collected by centrifugation, washed with10 mM Tris-hydrochloride (pH 6.8)-i mM EDTA andfrozen at --80°C. The frozen cells were suspended in10 mM Tris-hydrochloride (pH 6.8)-1.0 mM EDTA,lysed by three cycles of freezing and thawing, treatedwith 100 pLg of DNase per ml, and suspended in sodiumdodecyl sulfate sample buffer (14). Samples wereimmune precipitated with antisera to phenylalanyl-tRNA synthetase in 10 mM sodium phosphate (pH7.0)-150 mM NaCI-0.5% Triton X-100-0.1% sodiumdodecyl sulfate as described previously (27). Theimmunoglobulins with bound antigens were separatedfrom the bulk of the radioactive material by absorbingto protein A-Sepharose (Pharmacia Fine Chemicals,Uppsala, Sweden). After being washed the antigen-antibody-protein A complex was disrupted by theaddition of sodium dodecyl sulfate sample buffer andthen analyzed on 2-mm-thick 10o sodium dodecylsulfate-polyacrylamide gel. The gel was treated forfluorography (17) and exposed to RP X-Omat film at-700C.Construction of AB1S and AB1T. The structures of

the starting phages X512 (14), an EcoRI substitutionvector, and XNM540 (5), a Hindlll insertion vector areshown in Fig. 2. The principle of the cloning is to insertthe EcoRI-HindIII fragment of pBlS and pBlT be-tween the left-hand arm of X512, ending with an EcoRIsite, and the right-hand arm of ANM540, ending with aHindIII site (Fig. 2). Phages X512 and XNM540 weredigested, respectively, with EcoRl or HindlIl, andpBlS and pBlT were digested with both enzymes.Each phage DNA (0.6 jLg) was ligated with 0.2 ,g ofpBlS or pBlT and used to transfect IBPC2001 madecompetent with CaCl2 (23). Several hundred plaques(over four plates) were obtained, which were pooled toproduce a low-titer lysate. Phages capable of transduc-ing IBPC4771 (a thrS mutant auxotrophic for threo-nine) to prototrophy were selected by plating thelysate on IBPC4771 and picking lysogens from thecenter of the plaques. Lysogens capable of growing inthe absence of threonine and exhibiting the imm2lcharacter of XNM540 were purified. UV induction ofthese lysogens gave lysates with titers of 109 PFUml-'. These lysates were tested for their ability totransduce the thermosensitive pheS and pheT recom-bination-deficient (IBPC1671 and IBPC1772) strains tothermoresistance.

RESULTSIsolation of the mutants. Plasmid pBl, a deriv-

ative ofpBR322 with an E. coli EcoRI to HindlIl

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insert (Fig. 1), carries thrS, infC, pheS, andpheT (21). Cells carrying this plasmid overpro-duced phenylalanyl-tRNA synthetase, transla-tion initiation factor IF3 (IF3) and, to a lesserextent, threonyl-tRNA synthetase. Enzyme ac-tivity measurements (Table 1) showed thatstrains carrying pBl overproduce phenylalanyl-tRNA synthetase on the order of 100-fold andthreonyl-tRNA synthetase on the order of 10-fold compared with pBR322-carrying strains.Similar levels of overproduction of the twosynthetases have been measured by the ATP-pyrophosphate exchange assay (A. Ducruix, G.Fayat, S. Blanquet, personal communication).Sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis of extracts from pBl-carryingstrains shows an overproduction of both sub-units of phenylalanyl-tRNA synthetase and alsoof IF3, but no clearly visible overproduction ofthreonyl-tRNA synthetase (Fig. 3, lane 2).The overproduction of proteins caused by the

presence of pBl in the cell might be somehowdetrimental to the cell and could explain whyspontaneous mutants, which no longer causephenylalanyl-tRNA synthetase overproduction,are easily isolated. The frequency of these muta-tions is such that they can be screened for by thesystematic polyacrylamide gel electrophoresisof extracts from clones isolated from pBl-carry-ing strains. In all cases, the loss of proteinoverproduction was found to be associated withthe plasmid. A strain that no longer overpro-duced phenylalanyl-tRNA synthetase and hadbeen cured of its plasmid gave, upon retransfor-mation with wild-type pBl DNA, a strain over-producing both phenylalanyl-tRNA synthetase

and IF3. Plasmid DNA extracted from the non-overproducing strain, on introduction into an-other strain, exhibited the mutant pattern ofprotein synthesis.

Polyacrylamide gel analysis of several suchnon-overproducing mutants is shown in Fig. 3.Several of these mutants overproduced neitherIF3 nor phenylalanyl-tRNA synthetase, somestill overproduced IF3 but not the synthetase,and one still overproduced the synthetase butnot IF3. All of the mutants, except pBlTZ, wereisolated from pBl in a single step. As well asbeing analyzed for protein overproduction insaturated cultures, exponentially growing cellswere pulse-labeled with [35S]methionine, andextracts were examined on denaturing poly-

TABLE 1. Aminoacyl-tRNA synthetase activities inIBPC2001 carrying different plasmidsa

Aminoacyl-tRNA synthetasePlasmid

Thr Phe Met

pBR322 7 ± 3 20 ± 8 2 ± 2pBl 55 ± 25 1,500 ± 500 0.8 ± 0.5pBlS 100 t 20 70 ± 30 1.1 ± 0.1pBiT 110 ± 60 20 ± 10 1.2 ± 0.3pBlM1 5 ± 2 1,020 ± 150 1.6 ± 0.1pBlM5 33 19 1.8

a Activities are expressed in picomoles of tRNAaminoacylated per microgram of extract per 15 min.Error limits indicate the level of reproducibility ofextracts made from cultures grown at different times.For pBl and pBR322, these are the average of at leastfive experiments. For pBlS, pB1T, and pBlMl, theyare the mean of two experiments; for pBlM5, theresult of a single experiment is shown.

FIG. 1. Structure of plasmid pB1 and mutants derived from it. The positions of the four genes thrS (_),infC (=), pheS (a=), and pheT (_) are taken from data in references 23, 24, and 27. The positions of therestriction sites are taken from the revised map of pBl (G. Fayat et al., personal communication). Deletions areindicated by blocked-in regions. The serrated edges indicate that the exact end of the in vivo deletions is notknown.

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654 PLUMBRIDGE AND SPRINGER

PheRS__

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FIG. 3. Proteins synthesized by bacteria carryingpBl and derived plasmids. Plasmid-carrying strainswere grown to saturation in LB medium containing 50,ug of ampicillin per ml and 10 .g of tetracycline perml. Cells were collected by centrifugation and lysed bysuspending in sodium dodecyl sulfate sample buffer(15) and heating in a boiling water bath for 4 min.Samples equivalent to 0.1 A650 U were analyzed on a12.5% polyacrylamide gel. Proteins were visualizedwith Coomassie blue. The positions of the small andlarge subunits of phenylalanyl-tRNA synthetase(aPheRS and PPheRS, respectively) and translationalinitiation factor IF3 are indicated.

acrylamide gels. Comparing strains carrying mu-tant plasmids with a strain carrying pBR322, wefound that pulse-labeling sometimes reveals thesynthesis of plasmid-coded proteins that are notvisible as overproduced proteins when saturatedcells are examined. This would suggest that thestability of the gene products has somehow beenaffected by the mutation. Plasmids which showthis phenomenon are pBlT, pBlT2, pBlE4, andpBlS4. To test the complementation propertiesof the mutant plasmids, we used their DNA totransform pheS and pheT thermosensitive re-combination-deficient (recA) strains. The trans-formed strains were subsequently tested forgrowth at the restrictive temperature. Table 2summarizes the properties of a representativesample of the mutants found.The level ofprotein overproduction in bacteri-

al strains due to the presence of genes carried onmulticopy plasmids is critically dependent onthe copy number of the plasmids studied. pBl isa derivative of pBR322, a relaxed copy numberplasmid, and several observations make us thinkthat the mutations, isolated within the E. coliDNA insert of pBl, do not affect the copynumber of the plasmid. When a wild-type genewas carried by a plasmid, the overproductionwas constant, e.g., when seen on denaturinggels, the level of IF3 was constant (Fig. 2).

J. BACTERIOL.

Extracts of [35S]methionine-labeled culturesshowed similar amounts of pulse-labeledthreonyl-tRNA synthetase. We also measuredthe level of P-lactamase inside plasmid-carryingstrains. Samples of exponentially growing cellswere washed three times and sonicated, and 1B-lactamase was measured by the ability of peni-cilloic acid (the product of the action of 1-lactamase on penicillin) to reduce iodine (M.Trudel, M. Graffe, and M. Springer, manuscriptin preparation). These experiments (data notshown) suggest that the copy number of pBl ison the order of one-fifth that of pBR322. Thelevels of ,B-lactamase in pBl- and mutated-pBl-carrying strains varied by a maximum of factortwo. This again suggests that the pattern ofprotein overproduction seen with pBl- and mu-tated-pBl-bearing strains is not a function ofplasmid copy number.

Classiication of the mutant plasmids. The mu-tant plasmid DNAs were analyzed with restric-tion endonucleases. The structure of these plas-mids (Fig. 1) permits a classification of themutations into three categories: (i) large dele-tions, such as pB1S3, pBlS4, pBlM1, andpBlM5; (ii) insertions, such as pB1S, pB1T2,and pBlTZ; (iii) point mutations where no grosseffect on the DNA restriction pattern is ob-served; pBlT and pB1E4 belong in this catego-ry. Initially, we describe the properties of thesethree classes of mutants and subsequently de-scribe experiments with two of the mutants toexplore the regulation of the pheS,T operon.

(i) Deletions which caused fusions with thepheS,T operon. All of the characterized genescarried on pB1 are transcribed in the samedirection (21); creating deletions in pBl thusyields gene or protein fusions. As all four genesare precisely located on a restriction map of pBl(27), it is possible to identify the fusions by theanalysis of mutant plasmid DNA. Several fu-sions between thrS and pheS,T were character-ized. The deletion of pBlS4 starts within thethrS structural gene and ends to the right ofPstI3 (Fig. 1). This plasmid expresses pheT, asshown by the complementation and pulse-label-ing of pBlS4-carrying strains (Table 2). Thelarge subunit was not overproduced alone (atleast not at the level of pBl-carrying strains).The pB1S3 plasmid carries a similar deletion,also starting within the thrS structural gene andending to the right of PstI3. This plasmid did notexpress pheT in a pulse, but did weakly comple-ment pheT mutants (Table 2), suggesting that theexpression of the pheT gene has been affected bythe deletion. A possible explanation for theweak complementation is that the deletion re-moves the NH2-terminal part of the structuralpheT gene, creating a protein fusion betweenthrS and pheT. This hypothetical hybrid protein

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656 PLUMBRIDGE AND SPRINGER

would be too unstable to be seen in a pulse-labeling experiment but still sufficiently active toweakly complement pheT mutants.A different type of fusion is shown by the

plasmid pBlM5. It caused the overproduction ofa 22-kilodalton (kd) protein (Fig. 3, lane 9) whichwas shown by immune precipitation to carry IF3antigenic determinants (data not shown). Thelocation of the deletion would strongly suggest ahybrid between infC and pheS genes. However,antisera against the small subunit of phenyl-alanyl-tRNA synthetase failed to precipitate the22-kd protein. This might be expected if thepheS part of the hybrid gene is small. However,the size of the pB1M5 deletion and the molecularweight of the overproduced protein do not fitwith a hybrid protein made of the NH2-terminalpart of IF3 and the COOH-terminal part of thesynthetase small subunit. DNA sequence analy-sis (G. Fayat, C. Sacerdot, S. Blanquet, person-al communication) shows that the distance be-tween the beginning of infC and the end ofpheS

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FIG. 4. Immunoprecipitation with anti-phenyla-lanyl-tRNA synthetase of IBPC2001 carrying eitherpBl, pB1T, pBlS, or pBR322. Exponentially growingcultures were pulse-labeled with ["Simethionine for 2min and then chased with nonradioactive methionineas described in the text. Samples of the labeled cells,equivalent to 5 x 105 cpm, were immune precipitatedwith antiserum as described in the text and thenanalyzed on a 10%o sodium dodecyl sulfate-polyacryl-amide gel (15). After electrophoresis, the gel wastreated for fluorography (17) and exposed to R.P. X-Omat film at -70°C for 3 days. We performed theimmune precipitation of IBPC2001 pB1, exponentiallylabeled (lane 1) and then chased for 8 min (lane 2), 28min (lane 3), 118 min (lane 4), and allowed to saturate(lane 5). Lanes 6 to 10, As for lanes 1 to 5, but with aculture of IBPC2001 pB1T. Lanes 11 and 12, Immuneprecipitate of exponentially labeled IBPC2001 pBlSand IBPC2001 pBR322. Lane 13, Total extract ofexponentially labeled IBPC2001 pBl (50,000 cpm).

is 2.5 kilobases (kb). The pBlM5 deletion isabout 1.3 kb, leaving 1.2 kb for the hybrid gene.A DNA fragment of 1.2 kb should code for aprotein of molecular weight greater than 40 kd.As the hybrid protein is of 22 kd, it seems likelythat the fusion starting with infC is not in thecorrect reading frame of pheS, causing transla-tion of the pheS part of the hybrid gene toterminate quickly at a normally out-of-phasetermination codon. The weak complementationof pheS mutants with pBlM5 would, in thiscase, be due to some residual expression ofpheSdue to, for instance, an internal translation initi-ation, giving a protein lacking its NH2-terminalend.

Plasmid pBlM1 caused the overproduction ofphenylalanyl-tRNA synthetase but not IF3.Analysis of the DNA showed a large deletionstarting within thrS and ending after the AvaI3site. As discussed later, this eliminates thepheS, T promoter, so that the expression ofphenylalanyl-tRNA synthetase is presumablyfrom the thrS promoter. The overproduction bypBlM1 of the two subunits of the synthetasewith wild-type molecular weights demonstratestwo facts. It shows, firstly, that the intact struc-tural genes of pheS, T are located to the right ofthe AvaI3 site and, secondly, that the overpro-duction of phenylalanyl-tRNA synthetase is nota function of the pheS,T promoter. The thrSpromoter can cause about the same level ofphenylalanyl-tRNA synthetase as the pheS,Tpromoter (Table 1), whereas its natural product,threonyl-tRNA synthetase, is only overpro-duced about 10-fold. This anomaly is still underinvestigation.

(ui) Insertion mutants. Plasmid pBlS carries aninsertion of about 1.3 kb located between theHpaI5 and AvaI3 sites (Fig. 2) to the left ofphenylalanyl-tRNA synthetase structural genes.This insertion has been given the designationfl(pheSp::1.3 kb) 1001. Indicative of the fact thatthis is a regulatory mutant, no synthesis of eitherpheS or pheT gene products was detected bypulse-labeling (Table 2 and Fig. 4, lane 11).There was, however, some residual synthesis ofphenylalanyl-tRNA synthetase detected by theactivity measurements and complementationdata (Table 1). As shown later, the insertioninterrupts a cis-acting regulatory region.

Plasmid pB1T2 carries an insertion of about0.2 kb between the PstI3 and BamHI sites ofpBl (Fig. 2). This fragment contains the end ofpheS and the start of pheT. In a pulse, pheS isexpressed normally, indicating that the insertioninterferes with pheT expression only. The inser-tion is thus located either within the intercis-tronic region or within pheT.

Plasmid pBlTZ is a double mutant. It wasisolated as an IF3 non-overproducer of pBlT,

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E. COLI PHENYLALANYL-tRNA SYNTHETASE 657

already a phenylalanyl-tRNA synthetase non-overproducer. An insertion, about 0.6 kb, waslocated between HpaI4 and SacII2. This couldbe within the infC structural gene or a regulatoryregion. The insertions, which appear at a fairlyhigh frequency, are apparently stable DNAstructures, and no spontaneous revertants werefound.

(ill) Point mutations of the pheS,T operon.Initially classified as a point mutation, pBlT wassubsequently shown to be a very small deletion(20 to 30 bp) between AvaI3 and PstI3 (Fig. 2).This deletion has been given the designationApheS1002. Although genetically a deletion, it isquite different from the large deletions coveringseveral kb of DNA and which were found withmuch greater frequency. Only one other pointmutation, called pBlE4, has been detected. De-tailed analysis of this latter plasmid has not yetbeen undertaken.The deletion in pBlT (ApheS1002) is within

the pheS structural gene, and the pheS geneproduct of this plasmid is slightly smaller thanthe wild type (approximately 1 kd smaller, corre-sponding to 25 to 30 bp). As DNA sequenceanalysis (G. Fayat, C. Sacerdot, S. Blanquet,personal communication) shows that the pheSgene ends 216 bp to the right of the PstI3 site, itseems reasonable that the ApheSO002 deletion(located to the left of PstI3) creates an in-phasefusion within pheS. (This point is further dis-cussed in the accompanying paper [22].)

Further analysis of the pBlS and pBlT muta-tions. (i) Genetic complementation. Both plas-mids complemented thermosensitive mutationsin pheS and pheT (Table 2). However, comple-mentation with multicopy plasmids has not thesame significance as classical one-copy comple-mentation with F' episomes of X transducingphages. To test whether a single copy of themutations, present on pBlS and pB1T, wassufficient to complement chromosomal muta-tions in pheS and pheT, we transferred themutations to an integration-proficient A vector.The isolation of AB1S and AB1T (Fig. 2) frompBlS and pBlT, respectively, has been de-scribed above. Lysogens of these phages withIBPC1671 (pheS) and IBPC1772 (pheT) thermo-sensitive recombination-deficient strains wereobtained. Both sets of lysogens were thermore-sistant. Hence, a single copy (or perhaps twocopies if double lysogens were formed) of themutations complemented pheS and pheT de-fects. The fact that the fQpheSpl001 mutation isnot a null mutation is suggested by the activitymeasurement of Table 1; strains carrying plas-mid pBlS showed a level of phenylalanyl-tRNAsynthetase activity somewhat higher than that ofa pBR322-bearing control strain. The ApheS1002mutation is a structural mutation resulting in a

less stable protein (see below) and low phenyla-lanyl-tRNA synthetase activities in vitro (Table1). However, within the cell the protein wassufficiently stable to complement a pheS muta-tion.

(ii) Proteins synthesized by pBlS and pB1T.Mutations which still allow complementationbut not overproduction could be of two conceiv-able types: those which have a diminished levelof expression of the operon (regulatory muta-tions) and those which show a normal level ofexpression of an unstable enzyme (structuralmutations). To investigate whether the muta-tions carried by pBlS and pBlT (fQpheSpl001and ApheS1002, respectively) are structural orregulatory, strains carrying these plasmids werepulse-labeled with [35S]methionine and chasedwith an excess of nonradioactive methionine forincreasing times before lysis. The extracts of thelabeled and chased strains were immunoprecipi-tated with anti-phenylalanyl-tRNA synthetaseimmune serum, and the immunoprecipitateswere analyzed by polyacrylamide gel electro-phoresis. The fluorogram of such a gel, madefrom extracts of pBl-, pBlT-, pBlS-, andpBR322-carrying strains, is shown in Fig. 4. Theimmunoprecipitate of a pulse from a pBl-carry-ing strain shows the synthesis of the two wild-type subunits (Fig. 4, lane 1); for a pBlT-carrying strain the pulse shows a somewhatreduced synthesis of an enzyme whose smallsubunit is smaller than the wild-type one (Fig. 4,lane 6). With a pulse, no more phenylalanyl-tRNA synthetase synthesis was detected withpBlS (Fig. 4, lane 11) than with the controlstrain carrying pBR322 (Fig. 4, lane 12). ForpBlT-carrying strains, the mutated subunit wascompletely degraded between 30 and 120 min ofchase (Fig. 4, lanes 9 and 10). At these samechase times, both subunits of phenylalanyl-tRNA synthetase were detectable with pBl-carrying strains (Fig. 4, lanes 4 and 5). Thesedata show that pBlT synthesized a less stablephenylalanyl-tRNA synthetase, with its smallsubunit smaller than the wild type, and thusseems to be a typical structural mutant. On theother hand, pBlS synthesized only a low, resid-ual quantity of the synthetase, not detectable byimmunoprecipitation, but which must still besufficient to complement pheS and pheT mu-tants. This latter mutant is, as discussed later, atrue regulatory mutant. The other minor bandsprecipitated by the anti-phenylalanyl-tRNA syn-thetase serum are presumably due to antibodiesof different specificities contaminating the anti-sera, since the same bands, at the same intensi-ties, are precipitated from pBR322- and pBlS-carrying strains (Fig. 4, lanes 11 and 12).

(iii) Insertion of pBlS interrupts a cis-actingsequence. The insertion of pBlS is situated in

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658 PLUMBRIDGE AND SPRINGER

front of the structural genes (discussed later). Itis thus possible that it exerts its effect either byinterrupting a trans-acting regulatory gene or acis-acting sequence. To distinguish these twopossibilities, a cis-trans test was devised basedon the overproduction of phenylalanyl-tRNAsynthetase. Two plasmids have been described(21) with parts of the wild-type pheS,T operoninserted into a plasmid vector (pGA39 [1]) com-patible with pBR322 (and thus with pBlS orpB1T). One plasmid, pGC87, carried the PstI2-PstI3 fragment of pBl (Fig. 2). This fragmentcarried the end of thrS, infC, part of the pheSstructural gene, and all of the regulatory regionof the pheS,T operon. It did not complementpheS mutants. The second plasmid, pG6-7, car-ried the PstI2-PstI4 fragment (Fig. 3) and didcomplement pheS mutants without causing theoverproduction of the small subunit of phenyla-lanyl-tRNA synthetase. Both plasmids (confer-ring resistance to chloramphenicol) were intro-duced into strains carrying pBlT and pBlS(conferring resistance to ampicillin and tetracy-cline). The strains were grown in LB containingthe three antibiotics, and samples of saturatedcultures were analyzed on denaturing polyacryl-amide gels (Fig. 5).

Neither the presence of pGC87 nor that of

N

IN

PheRS_..

_,..... .. ...

iPheRSMAsot j

CmAc _ _ _@i

FIG. 5. Cis-trans test for overproduction ofphenylalanyl-tRNA synthetase. IBPC2001, carryingthe plasmids indicated, was grown to saturation in LBmedium containing the appropriate antibiotics: ampi-cillin (50 ,ug ml-1) and tetracycline (10 Fg ml-') forpBl, pBlS, pB1T, and pBR322 and chloramphenicol(25 pg ml-') for pGC87 and pG6-7. Samples wereprepared as described in the legend to Fig. 3 andelectrophoresed on an 11% sodium dodecyl sulfate-polyacrylamide gel (17). Proteins were stained withCoomassie blue. The positions of the small and largesubunits of phenylalanyl-tRNA synthetase (aPheRSand ,IPheRS, respectively) and chloramphenicol acet-ylase (CmAc), coded by pGC87 and pG6-7, are indi-cated.

pG6-7 affected the level of phenylalanyl-tRNAsynthetase synthesis from pBlS (Fig. 5, lanes 7and 8). With pB1T, there was no effect withpGC87 (Fig. 5, lane 4), but the presence of pG6-7(Fig. 5, lane 5) caused the overproduction ofboth subunits of the synthetase. The fact thatsupplying a whole pheS gene in trans to a pheSstructural mutation causes overproduction vali-dates the test as a method of detecting trans-acting products. The overproduction ofphenyla-lanyl-tRNA synthetase with the two plasmids isless than with pBl. This could be due to thelower copy number of the pG6-7 plasmid (carry-ing the P15A replicon [8]).

Since no increased synthesis of phenylalanyl-tRNA synthetase was detectable from supplyingpBlS with the wild-type region, equivalent towhere the insertion is located (carried by pCG87and pG6-7) in trans, we concluded that thisinsertion exerts its effect in cis either by inter-rupting a cis-acting regulatory region or by sepa-rating a cis-acting regulatory region froni thestructural genes.

DISCUSSIONIdentification of a regulatory region for the

pheS,T operon. The mutations described hereare of such a variety that they give informationabout many different aspects of phenylalanyl-tRNA synthetase biosynthesis. The most im-portant result is that the pBlS insertion(fQpheSpl001) has permitted the identification ofa cis-acting regulatory sequence for the pheS,Toperon. Other examples of cis-acting regulatorymutations for aminoacyl-tRNA synthetase havebeen described, but this is the first where thenature and approximate position of the mutationis known. Based on the isolation of thermoresis-tant revertants of thermosensitive aminoacyl-tRNA synthetases, three other cis-acting muta-tions, serO (9), leuX (16), and vaiX (3), closelylinked to their corresponding aminoacyl-tRNAsynthetase genes, have been defined. Thesemutations act by producing an increased amountof the thermosensitive enzyme. In addition, mu-tations conferring resistance to the antibioticborrelidin cause an overproduction of wild-typethreonyl-tRNA synthetase. These mutations,linked to thrS, are cis-dominant (11). The natureof the mutations is unknown in all cases, butthey have been tentatively identified as opera-tor-promoter mutations.The fQpheSpl001 mutation also defines a regu-

latory region since it reduces considerably theamount of phenylalanyl-tRNA synthetase syn-thesized. The exact point of the insertion has yetto be determined, but it is situated in the HpaI5-AvaI3 fragment (Fig. 1), implying that it is manybp on the 5' side of the structural pheS,T genes.Sequence data (G. Fayat, C. Sacerdot, S. Blan-

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quet, personal communication) show that thepheS structural gene starts 85 bp to the right ofAvaI3. However, the functional promoter forpheS,T is situated to the left of this site. Springeret al. (25) described an in vivo-deleted derivativeof Xp2 (a transducing phage which carries all ofthe E. coli genes for pBl); this phage, Xppl-16,does not express any of the genes of pBl.However, DNA restriction analysis shows thatthe deletion is to the left of AvaI3, leaving theAvaI3 site and all of the DNA to the right of itintact (i.e., the entire structural genes of pheSand pheTare present). This observation predictsthat the natural promoter for pheS, T is beforethe AvaI3 site.

Since several of the non-overproducing mu-tant plasmids still complement mutations inpheS and pheT, complementation is not a suffi-cient criterion for the presence of a completewild-type gene. A better criterion for the wild-type pheS,T operon on a plasmid would appearto be the overproduction of phenylalanyl-tRNAsynthetase. By employing a cis-trans test foroverproduction of the synthetase, the insertion,flpheSp1001, was shown to act in cis. SupplyingpBlS in trans with wild-type DNA of the region,where the insertion is located (via the compati-ble plasmids pGC87 or pG6-7), did not stimulatean increased synthesis of phenylalanyl-tRNAsynthetase. Thus, the insertion is not affecting atrans-acting gene but is affecting the pheS, Tpromoter or is situated between the promoterand structural genes. Although the size of thisinsertion (1.3 kb) is similar to that of manyinsertion elements (7), we have not identified it.Only minimal expression of the pheS,T genes

seems to be necessary for complementation tobe observed. This is particularly apparent forpBlS. No phenylalanyl-tRNA synthetase pro-tein synthesis was observed by pulse-labeling ofIBPC2001 carrying pBlS, and activity measure-ments showed only a small enhancement ofamino acid charging activity compared withpBR322. However, when transferred to a Xphage, a single copy of the mutated operon stillcomplemented perfectly. The expression ofpheS,T from the lysogen can only be a fractionof the normal wild-type expression. This couldmean that phenylalanyl-tRNA synthetase is nor-mally synthesized in excess.

Structural mutations of the pheS,T operon. ThepBlT mutation (ApheSJ002) was studied as anexample of a purely structural mutation. The 20-to 30-bp deletion within the pheS gene produceda smaller protein whose molecular weight iscompatible with the size of the deletion. Thisprotein was much less stable than the wild-typesubunit (Fig. 4), and this is, presumably, themajor reason why phenylalanyl-tRNA synthe-tase was not overproduced in pBlT-carrying

strains. However, a 2-min pulse-labeling ofpBlT with [35S]methionine apparently labeledless phenylalanyl-tRNA synthetase than did asimilar pulse with pBl (cf. Fig. 4, lanes 1 and 6),suggesting that there is a reduction in the synthe-sis of the mutated enzyme. This might be indica-tive of some role of the wild-type protein incontrolling its own synthesis. This point is con-sidered in detail in the accompanying paper (22).Supplying pBlT with a wild-type pheS gene intrans restored the ability to overproduce phenyl-alanyl-tRNA synthetase. Thus, although nor-mally cotranscribed from the single pheS, Tpromoter (21), neither cotranscription nor co-translation is necessary for phenylalanyl-tRNAsynthetase overproduction. This is again sugges-tive of the importance of the wild-type tetramer-ic enzyme in its own regulation. Finally, we notethat pBlM1, deleted for the regulatory regionsof pheS,T up to, and including, the AvaI3 site,still considerably overproduced phenylalanyl-tRNA synthetase, although transcription is fromthe thrS promoter.

In summary, we have described the isolationand preliminary characterization of several mu-tated plasmids which are shown to be useful instudies of the expression of the pheS,T operon.Plasmid pBlM1 localizes the structural genes tothe right ofAvaI3, whereas plasmid pBlS showsthat DNA to the left of AvaI3 is essential for thenormal expression of the pheS,T operon. Themutation in plasmid pBlT caused a structuralchange in the small subunit of phenylalanyl-tRNA synthetase and produced a less stableenzyme. This mutated enzyme is useful for bothin vivo experiments to see the effect of analtered protein on phenylalanyl-tRNA synthe-tase biosynthesis and also, potentially, for invitro analysis of the biochemistry of the enzyme.Some of the mutants seem to reveal specificfeatures of the pheS,T operon, e.g., the weakpheS complementation and strong pheT expres-sion of pBlM5 could be explained by a transla-tional restart within pheS, and the weak pheTcomplementation with pBlS3 could be due to anunstable hybrid protein exhibiting some residualpheT activity. The fact that these mutations areisolated on multicopy plasmids makes them easi-er to analyze and eliminates any problems ofessential genes not being susceptible to mosttypes of mutation described here.

ACKNOWLEDGMENTSWe acknowledge the continued enthusiasm and support of

Marianne Grunberg-Manago in this work. We thank GuyFayat, Christine Sacerdot, Sylvain Blanquet, and ArnaudDucruix for communicating results before publication anduseful discussion and August Bock for the gift of anti-phenyla-lanyl-tRNA synthetase serum.

This work was supported by grants from the Centre Nation-al de la Recherche Scientifique (Groupe de Recherche 18,

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A.T.P. "Biologie Mol6culaire du Gene," and "Microbiologie1979") and the D6l6gation G6nerale a la Recherche Scientifi-que et Technique (convention 80.E.0872).

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3. Baer, M., K. B. Low, and D. S611. 1979. Regulation of thebiosynthesis of aminoacyl-transfer ribonucleic acid syn-thetases and of transfer ribonucleic acid in Escherichiacoli. V. Mutants with increased levels of valyl-transferribonucleic acid synthetase. J. Bacteriol. 139:165-175.

4. Birnbolm, H. D., and J. Doly. 1979. A rapid alkalineextraction procedure for screening recombinant plasmidDNA. Nucleic Acids Res. 7:1513-1523.

5. Borck, K., J. D. Beggs, W. J. Brammar, A. S. Hopkins,and N. E. Murray. 1976. The construction in vitro oftransducing derivatives of phage lambda. Mol. Gen. Gen-et. 146:199-207.

6. Bradford, M. M. 1976. A rapid and sensitive method forthe quantitation of microgram quantities of protein utiliz-ing the principle of protein-dye binding. Anal. Biochem.72:248-254.

7. Calos, M. P., and J. H. Miller. 1980. Transposable ele-ments. Cell 20:579-595.

8. Chang, A. C. Y., and S. N. Cohen. 1978. Construction andcharacterization of amplifiable multicopy DNA cloningvehicles derived from the P15A cryptic miniplasmid. J.Bacteriol. 134:1141-1156.

9. Clark, S. J., B. Low, and W. H. Konigsberg. 1973.Isolation and characterization of a regulatory mutant of anaminoacyl-tRNA synthetase in Escherichia coli K12. J.Bacteriol. 113:1096-1103.

10. Comer, M. M., and A. B6ck. 1976. Genes for the a and 1Bsubunits of the phenylalanyl transfer ribonucleicacid synthetase of Escherichia coli. J. Bacteriol.127:923-933.

11. Frohler, J., A. Rechenmacher, J. Thomake, G. Nass, andA. B6ck. 1980. Genetic analysis of mutations causingborrelidin resistance by overproduction of threonyl-trans-fer ribonucleic acid synthetase. J. Bacteriol. 143:1135-1141.

12. Henneck, H., A. B6ck, J. Thomale, and G. Nasa. 1977.Threonyl-transfer ribonucleic acid synthetase from Esch-erichia coli: subunit structure and genetic analysis of thestructural gene by means of a mutated enzyme and of aspecialized transducing A bacteriophage. J. Bacteriol.131:943-950.

13. Hennecke, H., M. Spriger, and A. B6ck. 1977. A special-ized transducing A phage carrying the E. coli genes forphenylalanyl-tRNA synthetase. Mol. Gen. Genet.152:205-210.

14. Kourilsky, P., M. Perricaudet, D. Gros, A. Garapin, M.Gottesman, A. Fritsh, and P. Trolais. 1978. Descriptionand properties of bacteriophage lambda vectors useful forthe cloning of EcoRI fragments. Biochimie 60:183-187.

15. Laemmll, U. K. 1970. Cleavage of structural proteinduring the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

16. La Rossa, R., G. VWgel, K. B. Low, and D. S6l1. 1977.Regulation of biosynthesis of aminoacyl-tRNA synthe-tases and of tRNA in Escherichia coli. II. Isolation ofregulatory mutants affecting leucyl-tRNA synthetase lev-els. J. Mol. Biol. 117:1033-1048.

17. Laskey, R. A., and A. D. MIDls. 1975. Quantitative filmdetection of tritium and (4C) in polyacrylamide gels byfluorography. Eur. J. Biochem. 56:335-341.

18. Lugtenberger, B., J. MeUers, R. Peters, P. v.d. Hoek, andL. v. Alphen. 1975. Electrophoretic resolution of the"major outer membrane protein" of Escherichia coli K12into four bands. FEBS Lett. 58:254-258.

19. Nasa, G., and F. C. Neidhardt. 1967. Regulation of forma-tion of aminoacyl-transfer ribonucleic acid synthetases inE. coli. Biochem. Biophys. Acta 134:347-359.

20. Neldhardt, F. C., P. L. Bloch, S. Pedersen, and S. Reeh.1977. Chemical measurements of steady-state levels oftenaminoacyl-transfer ribonucleic acid synthetases in Esche-richia coli. J. Bacteriol. 129:378-387.

21. Plumbrldge, J. A., and M. Springer. 1980. Genes for thetwo subunits of phenylalanyl-tRNA synthetase of E. coliare transcribed from the same promoter. J. Mol. Biol.144:595-600.

22. Plumbridge, J. A., and M. Springer. 1982. Escherichia coliphenylalanyl-tRNA synthetase operon: transcriptionstudies of wild-type and mutated operons on multicopyplasmids. J. Bacteriol. 152:661-668.

23. Plumbridge, J. A., M. Springer, M. Grafe, R. Goursot,and M. Grunberg-Manago. 1980. Physical localisation andcloning of the structural gene for E. coli initiation factorIF3 from a group of genes concerned with translation.Gene 11:33-42.

24. Sacerdot, C., G. Fayat, P. Dessen, M. Springer, J. A.Plumbridge, M. Grunberg-Manago, and S. Blanquet 1982.Sequence of a 1.26 kb DNA fragment containing thestructural gene for E. coli initiation factor IF3: presence ofan AUU initiator codon. Eur. Mol. Biol. J. 1:311-315.

25. Springer, M., M. Grafe, and M. Granberg-Manago. 1979.Genetic organization of the E. coli chromosome aroundthe structural gene for initiation factor IF3 (i4oC). Mol.Gen. Genet. 169:337-343.

26. Springer, M., M. Grate, and H. Hennecke. 1977. Special-ized transducing phage for the initiation factor IF3 gene inE. coli. Proc. Natl. Acad. Sci. U.S.A. 74:3970-3974.

27. Springer, M., J. A. Plumbridge, M. Trudel, M. Graffe, andM. Grunberg-Manago. 1982. Transcription units aroundthe gene for E. coli translation initiation factor IF3 (iqfC).Mol. Gen. Genet. 186:247-252.

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