Vol. 145, No. 1JouRNAL OF BACTERIOLOGY, Jan. 1981, p. 299-3050021-9193/81/010299-07$02.00/0

Infection of Salmonella typhimurium with Coliphage Mu dl(Apr lac): Construction ofpyr::lac Gene Fusions

LASZLO N. CSONKA,' MARTHA M. HOWE,2 JOHN L. INGRAHAM,3 LELAND S. PIERSON III,3 ANDCHARLES L. TURNBOUGH, JR.3*t

Plant Growth Laboratory' and Department of Bacteriology,3 University of California, Davis, California95616, and Department ofBacteriology, University of Wisconsin, Madison, Wisconsin 537062

A procedure was developed for introducing the coliphage Mu dl (Apr lac) intoSalmonella typhimurium in order to construct gene fusions that place thestructural genes of the lac operon under the control of the promoter-regulatoryregion of other genes. To introduce Mu dl from Escherichia coli K-12 into S.typhimurium, which is normally not a host for Mu, we first constructed an E. colidouble lysogen carrying the defective Mu dl phage and a Mu-Pl hybrid helperphage (MuhP1) that confers the P1 host range. A lysate prepared from this strainwas used to infect a P1-sensitive (i.e., galE), restriction-deficient, modification-proficient strain of S. typhimurium, and a double lysogen carrying Mu dl andMuhPl was isolated. Induction of the latter strain produced lysates capable ofinfecting and generating gene fusions in P1-sensitive strains of S. typhimurium.In this paper we describe the construction ofpyr::lac fusions by this technique.

The recently developed techniques for fusingin vivo the structural genes of the lactose (lac)operon to the promoter region of any gene oroperon of interest have provided a powerful toolfor studying the regulation of gene expression(5, 7-9). The products of such fused genes areeasily measured, and mutations affecting theirexpression can be readily selected. The simplesttechnique for fusing genes is the one-step pro-cedure developed by Casadaban and Cohen (9).This procedure uses a specialized transducingMu phage, Mu dl (Apr lac cts62), which carriesnear one end of the Mu genome the structuralgenes of the lac operon without the lac pro-moter. Upon infection of Mu-sensitive bacteria,the Mu dl genome can integrate into the hostchromosome at apparently random sites. If itbecomes integrated into a transcribed region inthe proper orientation, transcription directed bythe adjacent promoter-regulatory region pro-ceeds into the Mu dl genome to express the lacstructural genes.The one-step procedure has been useful in

constructing gene fusions in Escherichia coli K-12, but its use so far has been restricted to thisorganism because of the limited host range ofMu; for example, Mu does not infect Salmonellatyphimurium (23), E. coli B (23), or Klebsiellapneumoniae (4, 21). In this paper we describe atechnique for introducing Mu dl into S. typhi-murium. This technique takes advantage of thefact that the essential Mu genes that determinethe phage host range (24) are apparently deleted

t Present address: Department of Microbiology, Universityof Alabama in Birmingham, Birmingham, AL 35294.

from Mu dl (M. Casadaban, personal commu-nication). The host range of Mu dl is deter-mined by the helper phage which provides thedeleted functions. By using as the helper phagea Mu-P1 hybrid in which the Mu host rangegenes have been replaced by the analogous genesof phage P1, we have been able to prepare Mudl lysates capable of infecting P1-sensitive (i.e.,galE [19]) strains of S. typhimurium. In thispaper we describe the construction and charac-terization ofpyr::lac fusions that were obtainedby this technique.

MATERIALS AND METHODSChemicals and other materials. Dilithium car-

bamylphosphate (purity, 90 to 95%), penicillin G (so-dium salt), ampicillin trihydrate, o-nitrophenyl-/i-D-galactopyranoside, and X-gal (5-bromo-4-chloro-3-in-dolyl-p-D-galactoside) were purchased from SigmaChemical Co. L-[U-"4C]aspartic acid (>200 mCi/mmol) and ['4C]sodium carbonate (2 to 10 mCi/mmol)were from New England Nuclear Corp. The [14C]as-partic acid was purified before use (20). Analytical-grade cation-exchange resin AG 50W-X12 (200 to 400mesh, hydrogen form) was obtained from Bio-RadLaboratories and was washed before use (20). All otherchemicals were reagent grade and commerically avail-able.

Bacteria and bacteriophages. The bacterialstrains used in this study are listed in Table 1. Mucts62 hPl#1, called MuhPl, was isolated as an S'recombinant arising after infection by Mu cts62Saml004 (13) of an Su- E. coli K-12 strain lysogenicfor phage Plcl.100CMh (14, 24). MuhPl has Mu im-munity and forms plaques on a number of Pl-sensitive,Mu-resistant E. coli K-12 strains. Mu cts62 h7629 is aspontaneous mutant of Mu cts62 that can infect galE

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TABLE 1. Bacterial strainsStrain Genotype Source and comments

E. coli K-12MAL103 F- Mu dl (Apr lac cts62) Mu cts62 A(proAB lacIPOZYA) M. Casadaban (9)

XIII rpsLJA194 F- hsdR AtrpE5 thr-1 leuB6 thi-I lacYI supE44 J. JaynesJC3272/R751 F- his lys-301 trp rpsL maLA, t8x-317 lacX74gal-300/R751 R. A. J. Warren (15)MH2923 F' Mu cts62 hPl#1 araD M. HoweKC87 JC3272 [Mu dl (Apr lac cts62)]/R751 See ResultsKC89 MH2923/R751::Mu dl (Apr lac cts62) See Results

S. typhimurium LT2LT2-Z Wild type B. N. AmesLB82 zab-403::TnIO J. IngrhamJL3103 argil843::TniO J. IngrahamJL3404 galE1122 J. Ingraham

FOr and P1lJL3473 SL4213[Mu dl (Apr lac cts62) Mu cts62 hPl#1] See ResiltsJL3484 galEii22pyrAl5i7::Mu dl (Apr lac cts62) See ResultsJL3486 galE1122pyrB1518::Mu dl (Ap' lac cts62) See ResultsJL3487 pyrBi518::Mu dl (Apr lac cts62) P22(LT2-Z) x JL3486JL3500 galEII22pyrBi519::Mu dl (Apr lac cts62) See ResultsJL3501 galEII22pyrBi520::Mu dl (Apr lac cts62) See ResultspyrAA81 ApyrA81 B. N. AmespyrBA655 ApyrB655 J. RothpurFA145 ApurF145 B. N. AmesSL4213 galE496 metA22 metE55 rpsLi20 xyl-404 (Fels2)- Hl-b B. A. D. Stocker (10)

nml- H2-enx, (ilv?) hsdL6 hsdSA29TL154 JL3473 transduced to srl-2::TniO recAI P22(TT521) x JL3473TL15m SL4213 transduced to galE + P22(LT2-Z) x SL4213TL156 SL4213 (Mu cts62 h7629) See ResultsTT521 srl-2::TniO recAI J. Roth

strains of S. typhimurium. Phage P22 HT105/1 int201(3, 22) was provided by John Roth.Media and culture methods. Nutrient broth

(NB), VBCG (0.4% glucose) minimal medium, andN-C- minimal medium were prepared as previouslydescribed (25). LB medium, R medium, R top agarcontaining 6.5 g of agar per liter, and H top agar wereprepared according to Miller (17). SB medium con-tained 32 g of tryptone (Difco Laboratories), 20 g ofyeast extract (Difco), 5 g of NaCl, 2.5 g of MgSO4.7H20, and 0.2 g of NaOH per liter. Solid mediumcontained 15 g of agar (Difco) per liter. Ampicillin wasadded to liquid and solid media at 25 pg/ml. X-galindicator plates were N-C- medium supplementedwith 10 mM NH4Cl, 2% glucose, 0.5 mM arginine, and40 pg of X-gal per ml.

All liquid cultures and plates were incubated at30°C unless indicated otherwise. liquid cultures weregrown with shaking.

Preparation of Mu cta lysates by heat induc-tion. Mu cts lysogens were grown at 28 to 30°C in SBto a density of 108 cells/ml, shifted to 430C for 30 mi,and then incubated at 37°C until lysis (approximately1 h). Chloroform ('Ioo volume) was added, and thelysate was shaken vigorously. Cell debris was removedby centrifugation. Because Mu lysates lose infectivityrapidly (13), they were used immediately. Occasion-ally, complete clearing was not observed with heat-induced Mu cts lysogens of S. typhimurium, but titers

of approximately 10' plaque-forming units/ml wereobtained.

Titration of Mu lysates and Mu dl-mediatedtranaductions. Mu lysates were titrated by mixing0.1-ml samples of dilutions in LB with 0.1 ml of a freshculture of a recipient strain that had been grown tosaturation in LB plus 10 mM CaCl2 at 37°C. Afterincubation for 20 min at 370C, 2.5 ml of molten (450C)R top agar was added and the mixture was poured onR plates. Plates were incubated overnight at 37°C, andplaques were counted. Mu dl-mediated transductionswere performed by mixing 0.2-ml samples of appropri-ate dilutions of lysate with an equal volume of a freshculture of a recipient strain that had been grown tosaturation in LB plus 10 mM CaCl2 at 37°C. Themixtures were incubated at 30°C for 20 min, and 0.2ml was spread on an LB plus ampicillin plate.

Preparation of P22 lysates and P22-mediatedtransductions. P22 HT105/1 int201 lysates were pre-pared as described (12). Mu cts lysogenic donor strainswere grown at 28°C; all other donor strains were grownat 37°C. Transductions were performed by spreading108 recipient bacteria and dilutions of phage lysatesdirectly on selective medium plates. When the recipi-ent or donor stain was galE, it was grown in NBsupplemented with 0.2% glucose and 0.1% galactose,which restores P22 sensitivity (11).

Penicilin counterselection of Mu dl-inducedpyrimidine auxotrophs. A stationary-phase LB cul-

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GENE FUSIONS IN S. TYPHIMURIUM USING Mu di 301

ture of S. typhimurium JL3404 (galE) (0.1 ml) was

plated with 2.5 ml of H top agar containing 10 mMMgSO4 and 5 mM CaCl2 on an LB plus ampicillinplate. Small drops (50 per plate) of a fresh Mu lysateprepared from JL3473 were spotted on the top agaroverlay, and the plate was incubated overnight at280C. Lysogenic cells were scraped from the centers of50 spots and used to inoculate a 5-ml VBCG plusampicillin, 0.2 mM uracil, and 1 mM arginine culture(note: pyrA strains require arginine and uracil), whichwas incubated at 280C with shaking until fully grown.Cells in a 0.1-ml sample of this culture were collectedby filtration on a sterile 0.45-,m membrane filter (Mil-lipore Corp.) and were washed with 30 ml of VBCG.The filter was placed in 10 ml of VBCG plus 1 mMarginine (note: arginine was added here and duringpenicillin treatment to select against arginine auxo-trophs) and incubated at 28°C for 2 h. One milliliter ofthis culture was added to 1 ml of VBCG containing 1mM arginine and 10,000 U of penicillin. (Mu dl-lyso-genic strains are sensitive to the high concentration ofpenicillin used here.) After incubating at 280C for 3 h,the entire penicillin-treated culture was collected byfiltration and washed as above. The filter was placedin 10 ml of VBCG plus 1 mM arginine and 0.2 mMuracil and incubated at 280C until fully grown. Theculture was diluted 106-fold, and 0.1 ml was spread on

VBCG plus ampicillin, 0.5 mM arginine, and 0.2 mMuracil plates. The plates were incubated at 280C for 2days, and colonies were screened for a requirement foruracil or uracil and arginine.

Preparation ofcell extracts for enzyme assays.Extracts for carbamylphosphate synthetase (CPSase)assays were prepared as described (1) with slight mod-ification. Harvested celLs were washed and extractedin buffer (400) without phenylmethylsulfonyl fluoride.Extracts were dialyzed (400) for at least 8 h againsttwo changes of buffer. For aspartate transcarbamylase(ATCase) assays, samples (40 ml) of culture were

centrifuged at 20,000 x g for 10 min at 40C. The cellswere washed with cold extraction buffer (0.1 M Tris-acetate-0.1 mM EDTA, pH 8.0), centrifuged as above,and stored at-20°C for 1 day with no loss of enzymaticactivity. The pelleted cells were suspended in 5 ml ofcold extraction buffer and disrupted by sonic oscilla-tion at 00C. Extracts were centrifuged at 17,000 x g

for 30 min at 40C, and the supernatant was used forenzyme and protein assays.Enzyme assays. ,B-Galactosidase activity was as-

sayed as described by Miller (17). CPSase activity wasdetermined at 300C as described (2). ATCase activitywas measured by a modification of the assay of Porteret al. (20). The reaction mixture contained 5 mMcarbamylphosphate, 20 mM L-aspartic acid, 100,000cpm of uniformly labeled L-['4C]aspartic acid, 0.2 Mimidazole acetate (pH 7.0), and cell extract (up to 150!L) in a final volume of 0.5 ml. Reaction mixtures wereincubated at 300C for 20 min, and the reaction was

stopped by the addition of 0.15 ml of 1 N acetic acid.The entire mixture was then applied to a 5-cm Pasteurpipette column ofAG 5OW (H' form) cation-exchangeresin, and the effluent along with three column wash-ings each with 0.7 ml of deionized water were collected,mixed with 10 ml of Bray scintillation fluid (6), andcounted. A control in which carbamylphosphate was

omitted from the reaction mixture was run for eachdetermination.

Protein determination. Protein was measured bythe method of Lowry et aL (16), using crystallinebovine serum albumin as the standard. Samples inTris buffer were precipitated in 5% trichloroacetic acidbefore being assayed.

RESULTSConstruction of an E. coli double lysogen

for making Mu dl lysates capable of infect-ing S. typhimuriunL The first step in intro-ducing Mu dl into S. typhimurium was theconstruction of an E. coli strain carrying bothMu dl and a Mu helper phage with the hostrange of P1. To do this, the E. coli K-12 strainJC3272/R751, which carries the self-transmissi-ble, broad-host-range plasmid R751 (15), wastransduced to ampicillin resistance by Mu dlfrom MAL103. The resultant strain, KC87,carrying Mu dl at an unknown site, was grownto a density of 10i cells/ml in LB at 30°C andthen shifted to 37°C for 5 h to effect limitedinduction of the prophage, thus generatingtranspositions to new sites (including R751). A0.5-ml sample of this culture was mixed with 1ml of a culture of E. coli K-12 strain MH2923growing exponentially in LB at a density of 109cells/ml. This strain is lysogenic for Mu cts62hPl#1 (MuhPl), a Mu phage with P1 hostrange. Dilutions of the mating mixture werespread on VBCG plus ampicillin plates to selectprototrophic, ampicillin-resistant transconju-gants which presumably arose by transfer of anR751::Mu dl to MH2923. Such transconjugantsoccurred at a frequency of 4 per 105 recipientcells; one of these was called KC89.Introduction of Mu dl into S. typhimu-

riumn The Pl-sensitive (i.e., galE), restriction-deficient, modification-proficient S. typhimu-rium LT2 strain SL4213 was transduced to am-picillin resistance using a heat-induced lysatefrom KC89. Fifty transductants were grown andheat induced to test for the presence of thehelper phage. One transductant produced a ly-sate capable of infecting SL4213. This putativedouble lysogen was designated JL3473; its recAderivative, TL154, was used to prepare lysatesto test whether other S. typhimurium strainswere sensitive to phage infection (Table 2, col-umn 1). Heat-induced lysates of TL154 formedplaques on the galE S. typhimurium strainsSL4213 and JL3404 but not on galE+ strainsTL155 and LT2-Z. Similar results were obtainedby selecting for ampicillin resistance: the galEstrains were transduced at a 105-fold-greater ef-ficiency than the galE' strains.The requirement for the recipient to carry

galE was also apparent when the Mu dl/

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TABLE 2. Infection of S. typhinurium with phage MuMu dl/Mu cts62 hPl#l Mu dl/Mu cts62 hPl#l Mu dl/Mu cts62 from E.from S. typhimurium from E. coli KC89 coli MAL103TL154

Recipient strainApr trans_ Apr trans- ApT trans-

Plaques/ml ductants/ Plaques/ml ductants/ Plaques/ml ductants/ml ml ml

JA194 (E. coli K-12) hsdR 1.4 X 1010 1.3 X 10" 2.1 X 1010 1.2 x 10i 6.8 X 1010 2.2 x 10"SL4213 (S. typhimurium) 5.8 X 10P 3.8 x 107 3.8 x 10" 2.9 x 107 4.2 x 104 4.5 x 106galE496 hsdL6 hsdSA29

JL3404 (S. typhi nuriurn) 1.8 x 109 3.0 x 107 5.8 X 104 7.5 X 103 <10 30galE1122 hsdL+ hsdSA+

TL155 (S. typhinurium) galE+ <10 90 <10 40 <10 9.5 X 103hsdL6 hsdSA29

LT2-Z (S. typhinuriurn) galE+ <10 70 <10 <10 <10 <10WdL + hsdA +

MuhPl lysate was derived from E. coli K-12strain KC89 (Table 2, column 2). The phageformed plaques on S. typhimurium strains onlyif they were P1 sensitive (galE), and the trans-ducing efficiency of P1-sensitive strains was atleast 5 x 103-fold greater than that of the cor-responding Pl-resistant strains. However, the E.coli-grown phage is apparently sensitive to theS. typhimurium restriction systems: the plaqu-ing as well as the transducing efficiency of thelysate was at least 4 x 103-fold greater on strainSL4213, which is deficient in DNA restriction,than on strain JL3404, which possesses intactDNA restriction. (We assume that the relevantdifference between the nonisogenic strainsSL4213 and JL3404 is the state of the restrictionsystems.)That the helper phage must have a P1 host

range in order to form plaques with high effi-ciency even on galE strains of S. typhimuriumwas demonstrated using a mixed lysate of Mudl and Mu cts62 (wild-type host range) derivedby heat inducing E. coli K-12 strain MAL103(Table 2, column 3). This lysate formed plaouesat very low efficiency on SL4213. Also, the Mudl/Mu cts62 lysate transduced this strain at adecreased efficiency.Mutational events rendering S. typhimu-

rium sensitive to Mu. As demonstrated, agalE mutation is sufficient to render S. typhi-murium sensitive to phage Mu with P1 hostrange. However, Mu dl/MuhPl lysates cantransduce galE+ strains at reduced efficiency.To examine the genetic basis for this sensitivity,21 ampicillin-resistant transductants (12 derivedfrom TL155 and 9 derived from LT2-Z; Table 2,column 1) were further characterized. All werelysed at 430C, indicating the presence ofMu dl.Seven were Gal-, inhibited by galactose, sensi-tive to phage P1CM, and resistant to phages FOand P22-properties that are typical ofgalE or

galU strains (19). Nine were Gal' and sensitiveto phages P1CM, FO, and P22; two were Gal',sensitive to P1CM, and resistant to FO and P22.These strains may carry a mutation affectinglipopolysaccharide synthesis (26). The remain-ing three transductants were Gal', resistant toP1CM, and sensitive to FO and P22. They maybe products of rare infections ofgalE' S. typhi-murium by Mu with P1 host range.The low-level plaquing efficiency seen with

Mu cts62 (wild-type host range) from E. colistrain MAL103 on S. typhimurium strain SL4213(Table 2, column 3) is probably a consequenceof mutant Mu phages in the population. Exam-ination of five of the plaques revealed that theycontained phages that could plaque with highefficiency on galE S. typhimurium and E. coliK-12 strains, indicating that they carried a mu-tation extending their host range. Strain TL156(see below) was obtained by lysogenization ofSL4213 by one such mutant phage. In severalattempts, we were unable to find a spontaneousmutant ofMu cts62 or MuhPl that could plaqueon galE+ S. typhimurium.

Also, phage from MAL103 could transduce S.typhimurium strains at a low efficiency. To in-vestigate how this was possible, 20 ampicillin-resistant transductants of TL155 (Table 2, col-umn 3) were examined. All 20 were Gal' andresistant to phage FO 11 were sensitive to P1CMand P22 (at least partially); 5 were sensitive toP1CM and resistant to P22; 3 were resistant toP1CM and fully or partially sensitive to P22;and 1 was resistant to both P1CM and P22.Thus, several types of mutations in S. typhimu-rium appear to confer sensitivity to phage Mueven if it has the wild-type host range.Growth of Mu in S. typhimuriwn. The

growth of phage Mu with the host range of P1in galE straiis of S. typhimurium was similar tothat found in E. coli K-12. MuhPl formed turbid

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GENE FUSIONS IN S. TYPHIMURIUM USING Mu dl 303

plaques at 300C, clear plaques at 430C, andplaques of intermediate turbidity at 370C. In oneexperiment in which JL3404 was infected witha Mu dl/MuhPl lysate from TL154, the ampi-cillin-resistant transductants showed variableability to ferment lactose, 0.6% were shown tobe auxotrophic, and all were killed at 430C.Thus, as it does in E. coli K-12, Mu dl can

integrate into the chromosome of S. typhimu-rium at many sites.Construction and characterization of

pyr::lac gene fusions in S. typhiumurium us-ing Mu dl. Mu dl-induced pyrimidine auxo-

trophs were isolated as described in Materialsand Methods and were divided into four classes(tentatively identified as pyrA, pyrB, pyrC or

-D, and pyrE or -F) by virtue of their nutrientrequirements and growth characteristics (datanot shown). Only the putative pyrA and pyrBauxotrophs were characterized further. To de-termine whether Mu dl integrated in the ori-entation yielding pyr::lac fusions, cells werestreaked on X-gal indicator plates supplementedwith either 1 mM uracil or 1 mM orotate. Oro-tate is a poor pyrimidine source and causesderepressed pyr gene expression, whereas uracilis a good pyrimidine source and causes repres-

sion. Strains which were dark blue on orotateplates and light blue on uracil plates were judgedto be pyr::lac fusions. With only independentlyderived mutant strains, 7 of the 14pyrA and 3 ofthe 6 pyrB auxotrophs were scored as pyr::lac

fusions, consistent with two possible Mu dl ori-entations.One pyrA::lac fusion strain (JL3484) and all

three pyrB::lac fusion strains (JL3486, JL3500,and JL3501) were tested for the presence ofhelper phage. Cultures of each strain were heatinduced, but none produced a lysate capable offorming plaques on JL3404, indicating that theylacked the helper phage. To test for the presenceof additional copies of Mu dl, each strain wastransduced to Pyr+ with P22 grown on LT2-Z(pyrA+ pyrB+). All transductants tested (16from each fusion strain) were ampicillin sensitiveand white on the X-gal indicator plates, indicat-ing that they had no additional copies ofMu dl.Two strains, JL3484 (pyrA::lac) and JL3486

(pyrB::lac), were used to characterize furtherthe pyr::lac fusions. The tentative pyr gene as-

signments for the fusions in these strains wereconfirmed by the mapping experiments sum-marized in Table 3 and by showing that P22grown on strains pyrAA81 and pyrBA655 couldnot transduce JL3484 and JL3486, respectively,to Pyr+. The synthesis of ,B-galactosidase,CPSase, and ATCase (encoded by lacZ, pyrA,and pyrB, respectively) was compared in thefusion strains grown under conditions that resultin either repression or derepression ofpyr geneexpression (Table 4). The repression/derepres-sion ratios for fi-galactosidase in JL3484 (pyrA::lac) and CPSase in JL3486 (pyrA+) were essen-tially identical, as were the ratios for fl-galacto-

TABLE 3. P22 transductional mapping ofpyr::lac fusionsSelected pheno- Distribution of unselected

type phenotype in recipientsLB82 (zab-403::TnlO) pyrAA81 Tetr 83/100 Pyr+

Pyr+ 77/100 TetrJL3484 (pyrA::lac) Tetr 89/100 Pyr+

Pyr+ 55/100 TetrJL3103 (arg11843::TnlO) pryBA655 Tetr 24/100 Pyr+

Pyr+ 28/100 TetrJL3486 (pyrB::lac) Tetr 58/100 Pyr+

Pyr+ 31/100 Tetr

TABLE 4. Synthesis of CPSase, ATCase, and /3-galactosidase in pyr::lac fusion strainsa

Strain Pyrimidine surce CPSase(rmol/min ATCase (mnol/min per f,-Galactosidase (U)per mg of protein) mg of protein)JL3484 (pyrA::lac) Uracil NDb 14.0 (1)e 128 (1)

Orotate ND 1,695 (121) 4,575 (36)JL3486 (pyrB::lac) Uracil 0.408 (1) ND 21.8 (1)

Orotate 16.82 (41) ND 2,542 (117)a Cultures were grown in MOPS (morpholinepropanesulfonic acid) minimal medium (18) supplemented with

0.5mM arginine, 0.4% glucose, and either 0.2 mM uracil or 0.5 mM orotate as indicated. Samples were harvestedat an optical density (650 nm) of 0.5.

b ND, Not detectable.' Numbers in parentheses represent relative specific activities for that enzyme in the indicated strain.

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304 CSONKA ET AL.

sidase in JL3486 (pyrB::lac) and ATCase inJL3484 (pyrB+).Phage P22-mediated transduction ofpyr:

.lac fusions. A P22 lysate grown on JL3487(pyrB::lac), a galE+ transductant of JL3486,was used to transduce TL156 (Mu cts62 h7629lysogen) to ampicillin resistance. Twelve of 13transductants characterized were Pyr- and wereshown to contain the intact pyrB::lac fusion.The sole Pyr+ transductant did not carry thepyrB::lac fusion. This transductant may havearisen after homologous recombination betweenMu dl and the endogenous Mu cts62 h7629 orby the translocation ofMu dl from a transduc-ing particle to a nonessential region on the chro-mosome. When the JL3487 lysate was used totransduce nonlysogenic strains to ampicillin re-sistance, none ofthe transductants characterizedcontained the pyrB::Iac fusion. Of 56 transduc-tants of strainpurFA145 and 16 transductants ofSL4213, all were pyrB+. Three transductants,two from purFAl45 and one from SL4213, hadacquired an additional Mu dl-induced auxo-trophic requirement. It appears likely that all 72transductants of purFA145 and SL4213 weregenerated by translocation ofMu dl to randomsites on the chromosome after zygotic inductionof Mu dl-carrying transducing particles. Thenumber of tranaductants acquiring new auxo-trophic requirements (3 of 72) is consistent withthis interpretation (23).

DISCUSSIONIn this paper we have presented a method for

introducing the coliphage Mu d 1 (Apr lac cts62)into S. typhimurium in order to construct genefusions. This method extends the host range ofMu dl by using a Mu helper phage (MuhPl)that confers the P1 host range and by using P1-sensitive strain of S. typhimurium as recipients.To overcome DNA restriction, a Mu dl/MuhPlmixed lysate from E. coli was used to infect aPl-sensitive, restriction-deficient, modification-proficient strain of S. typhimurium. A doublelysogen of this strain carrying Mu dl andMuhPl was then used to make lysates capableof infecting and generating fusions between S.typhimurium genes and the lac operon in otherPl-sensitive strains of S. typhimurium.This procedure for constructing gene fusions

is simple and appears to be generally useful. Theonly limitation in using this method is that therecipient bacteria be sensitive to phage P1. AllgalE strains of S. typhimurium are P1 sensitive,and these strains are common in many labora-tories. They can also be isolated conveniently byselecting strains that are resistant to phage FO(26). In addition, P1 sensitivity can be trans-

J. BACTERIOL.

duced into strains of S. typhimurium by exploit-ing the close proximity of galE to the bio-203::TnlO insertion; in P22-mediated transduction,the linkage of bio-203::TnlO to galE1122 is 15%(unpublished data).In addition to the method presented here, two

other procedures for introducing Mu dl into S.typhimurium have been developed recently. Leeand co-workers (J. H. Lee, L. Heffeman, and G.Wilcox, manuscript in preparation) constructedan F::Mu dl in E. coli which was then trans-ferred into S. typhimurium. They obtained genefusions by inducing Mu dl to translocate fromthe F to other sites on the chromosome. Asecond technique (S. Rosenfeld and J. Bren-chley, personal communication) used P1 totransduce Mu dl from E. coli into a galE,restriction-deficient strain of S. typhimurium.Then a P1 lysate grown on this strain was usedto transduce Mu dl to other galE S. typhimu-rium strains, and gene fusions were obtained asa consequence of zygotic induction.Using the procedure described in this paper,

we constructedpyr::lac fusions for several of thepyr genes in S. typhimurium. Two ofthese fusionstrains, JL3484 (pyrA::lac) and JL3486 (pyrB::lac), were characterized with respect to the syn-thesis of f-galactosidase (encoded by lacZ) un-der conditions of pyrimidine limitation and ex-cess. The regulation of,-galactosidase synthesisin JL3484 and JL3486 was identical to that ofCPSase (encoded by pyrA) and ATCase (en-coded by pyrB), respectively. These pyr::lacfusion strains should be useful for the furtherstudy of the regulation of the expression of pyrgenes and for the selection of regulatory muta,tions.

In such studies, it would be useful to movegene fusions constructed by our procedure intoother strains of S. typhimurium. We have shownthat this is possible by P22-mediated transduc-tion, if the recipient strain is lysogenic for Mu.If the recipient is nonlysogenic, it appears thatthe Mu dl phage on infecting transducing par-ticles translocates to other sites on the chromo-some, probably as a consequence of zygotic in-duction. Although this makes it difficult to trans-fer fusions into nonlysogenic strains, it providesyet another way of constructing gene fusions.

ACKNOWLEDGMENTSWe acknowledge the advice and encouragement of Ray-

mond C. Valentine. We would also like to thank Stan Artz,Sydney Kustu, Linda McCarter, and Bruce Stocker for theirhelpful comments on the manuscript.

This work was supported by Public Health Service grantAI 05526 from the National Institutes of Health (to J.L.I.),National Science Foundation grant PFR 77-07301 (to R.C.V.),the College of Agriculture and Life Sciences, University ofWisconsin, Madison (M.M.H.), and National Science Foun-

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GENE FUSIONS IN S. TYPHIMURIUM USING Mu dl 305

dation grant PCM 75-02465 (to M.M.H.). M.M.H. is the recip-ient of Public Health Service research career developmentaward Al 00274 from the National Institutes of Health.

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