Vol. 147, No. 2JOURNAL OF BACTERIOLOGY, Aug. 1981, p. 297-3040021-9193/81 /080297-08$02.00/0

Identification of the Tetracycline Resistance Promoter andRepressor in Transposon TnlO

LEWIS V. WRAY, JR., RICHARD A. JORGENSEN,t AND WILLIAM S. REZNIKOFF*Department of Biochemistry, College ofAgriculture and Life Sciences, University of Wisconsin-Madison,

Madison, Wisconsin 53706

Received 22 January 1981/Accepted 7 May 1981

The structural and regulatory functions encoding tetracycline resistance intransposon TnlO lie within a 2,700-base pair region. Using recombinant plasmidswith different deoxyribonucleic acid sequences adjacent to a HincII site in thisregion, we located the promoter controlling the expression of tetracycline resist-ance. These various sequences conferred altered levels of tetracycline resistance.Plasmids containing deletions of a 695-base pair HinclI fragment were constitutiveand showed the loss of a 23,000-dalton tetracycline-inducible polypeptide, thusidentifying the repressor and the location of its gene.

TnlO is a 9,300-base pair transposon that en-codes resistance to tetracycline (16). The mech-anism of resistance involves an active efflux sys-tem which transports tetracycline out of the cell(20). In addition, resistant cells contain an intra-cellular component that antagonizes the abilityof tetracycline to inhibit translation (19, 44).The expression of tetracycline resistance is

inducible by subinhibitory levels of the anti-biotic (13, 14). Three different tetracycline-in-ducible polypeptides with molecular weights of36,000, 25,000, and 15,000 have been reported(14, 44, 46). The largest has been called the TETprotein and is regulated by a repressor which isinactive in the presence of tetracycline (44).Analysis of the tetracycline resistance pheno-types and TET protein-coding properties of var-ious TnlO deletions allowed the localization ofthe TET protein gene to the 1,275-base pairHincII fragment pictured in Fig. 1 (14). In ad-dition, these studies hypothesized that the pro-moter for the TET protein was located at ornear the HincII site on the left side of thisfragment (see Fig. 1) and that the repressor genewas located on the adjacent 695-base pair HincIIfragment. The experiments described in thiscommunication verify these hypotheses, and inaddition, they identify the TET repressor pro-tein and show that it is autoregulated.

MATERIALS AND METHODSBacterial strains and plasmids. Escherichia coli

strain JD600 is thr leu hsdR hsdM tonA supE lacYthi. DS410 is a minicell-producing strain of E. coli thatis minA minB ara lacY rpsL malA xyl azi tonA mtlthi. Plasmids pRT29 and pRZ112 have been described

t Present address: Department of Plant Biology, CarnegieInstitute of Washington, Stanford, CA 94305.

previously (14, 15). Plasmid pRT86 was obtained fromN. Panayotatos.DNA preparation and restriction enzyme

digestion. Plasmid DNA was prepared by the methodof Humphreys et al. (12). Enzymatic digestion of DNAwith the restriction endonucleases AluI, DdeI, HhaI,Hinfl, RsaI, Sau3AI, Sau96I, HincII, EcoRI, and XbaIwas performed in 60 mM NaCl-7 mM MgSO4-10 mMTris-hydrochloride (pH 7.9). Endonucleolytic diges-tion of DNA with the enzymes FnuDII, HaeII, HaeIII,and TaqI was performed in 6 mM MgCl2-0.5 mMdithiothreitol-10 mM Tris-hydrochloride (pH 7.9). Li-gations were performed with T4 DNA ligase in 10 mMMgCl2-1 mM dithiothreitol-0.05 mM ATP-20 mMTris-hydrochloride (pH 7.5).Enzymes. Restriction enzymes AluI, FnuDII,

HhaI, Sau3AI, Sau96I, TaqI, and XbaI were obtainedfrom New England Bio-Labs. DdeI, HaeII, HaeIII,Hinfl, HincII, and T4 DNA ligase were obtained fromBethesda Research Laboratories. EcoRI and RsaIwere gifts from J. Gardner.Agarose and polyacrylamide gel electropho-

retic analysis of DNA. Horizontal agarose gel elec-trophoresis was carried out essentially as described byShinnick et al. (39). Polyacrylamide slab gel electro-phoresis was performed essentially as described byManiatas et al. (23), except that the gels were made20% in glycerol. DNA was visualized by photographyunder UV light after staining with ethidium bromide.Construction of mutants of pRT86. Plasmid

pRT201 was constructed by incubating 2 yg of pRT86DNA with 5 ,ug of RNA polymerase for 30 min in 60mM NaCl-7 mM MgSO4-10 mM Tris (pH 7.9). TheDNA was then digested with HincIl and XbaI. Thismixture was then phenol extracted and ether ex-tracted. The XbaI "sticky end" was filled in withMicrococcus luteus DNA polymerase. The DNA wasligated and used to transform CaCl2-treated JD600cells according to the method of Mandel and Higa(21). The cells were inoculated into 4 ml of L-broth(27) and shaken at 370C for 90 min. Transformantswere selected by treating 200 Al with colicin El for 30

297

298 WRAY, JORGENSEN, AND REZNIKOFF

Tn 10

H H IH 4 t- HS

H I m m Hjm =EC0 -0

c- o ao .x i o

I~~~~~~~~~1______,_11___I/1695 T 1275

z II ,2= z :\ z zI I

2025 - \

- 695 X 1275660 X 560-

._i,c o:,cpRT29 ± xI I

pRT86

pRT201

pRT202 _ ,_X

pRT203 4- i

pRT204 _

pRT205

pRT2O0_p

pRT2 11FIG. 1. Physical maps of recombinant plasmids containing portions of TnOO. The top line shows a partial

schematic map of TnlO (14). The thick lines represent the inverted repeat sequences. II refers to the HincIIcleavage sites. (I) refers to those HincII cleavage sites that are also recognized by HpaI. The lines below thatshou the portion of TnlO DNA in each plasmid. The hatched bars represent the lacP 789-base pair fragment,and the arrow above each refers to the direction of transcription of the lac promoter. P represents a promoteradjacent to the 695-base pair fragment in pRT210 and pRT21 1.

min. The cells were then spread on nutrient agarplates, which contained 0.5%k deoxycholate to preventthe growth of colicin-tolerant mutants. Colonies werescreened for a reduction in plasmid size, using a mod-ification of the procedure of Barnes (1).

Mutant pRT202 was constructed by digesting 1.5tig of pRT86 DNA with HincIl and then heating at70°C for 15 min to inactivate the restriction enzyme.The DNA was ligated and transformed, and the colo-nies were screened as described above. PlasmidspRT203, pRT204, and pRT205 were constructed byligating 0.24 ytg of HincII-digested pRT86 with 0.19tig of the lacP 789-base pair HincIl fragment (kindlyprovided by S. Hardies). The DNA was used for trans-formation, which was followed by selection as de-

scribed above, except that the plates contained 40 ygof 5-bromo-4-chloro-3-indolyl-,8-D-galactoside per ml.Colonies harboring lac operator-containing plasmidsare lac constitutive and thus will turn blue (11).

Both pRT210 and pRT211 were constructed byligating 0.4 jig of HpaI-cleaved pRZ112 DNA with 0.8Mg of HincII-cut pRT86 DNA. Transformants wereselected by plating on nutrient agar plates containing30 Mg of kanamycin per ml. Colonies were screened foran increase in plasmid size, and the presence of HinclIfragment 695 was confirmed by electrophoretic anal-ysis of HincII digests of the resulting plasmids.Measurement of tetracycline resistance levels.

The level of tetracycline resistance conferred by thevarious plasmids on JD600 was measured by a modi-

J. BACTF.RIOL.

TETRACYCLINE RESISTANCE PROMOTER AND REPRESSOR 299

fication of the procedure of Tait et al. (41). Overnightcultures were inoculated into fresh L-broth with andwithout 1 jig of tetracycline per ml and incubated at37°C with shaking. Log-phase cultures were diluted,and approximately 100 to 300 cells were plated on

nutrient agar plates containing various amounts oftetracycline. For plasmids pRT86, pRT201, pRT203,and pRT204, concentrations of 0, 25, 50, 75, 100, 125,and 150 fig of tetracycline per ml were used. Concen-trations of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 jig oftetracycline per ml were used for plasmids pRT202and pRT205. After 48 h of incubation at 37°C, thenumber of colonies was counted, and the concentrationof tetracycline necessary to reduce colony numbers by50% was determined graphically. This is the 50% effi-ciency of plating of a strain.

Preparation and labeling of minicells. Minicellgrowth and purification have been described previ-ously (14). The minicells were suspended in 2 ml of 1:4 methionine assay medium (Difco Laboratories) inM9 salts with 0.5% glucose and 40 jig of adenine perml. The minicells were divided into two separate ali-quots, one of which was induced with 1 jig of tetracy-cline per ml for 15 min. Both samples were labeledwith 50 ACi of [35S]methionine (600 to 800 Ci/mmol;Amersham Corp.) for 30 min, pelleted, and frozen. Forelectrophoresis, the pellets were suspended in 200 jilof water. Aliquots of this were dissolved in samplebuffer (18), and 40 jil was loaded on 10 to 15% gradientpolyacrylamide-sodium dodecyl sulfate slab gels (18).Electrophoresis was at 40 mA for 2 to 3 h, using a

constant-current power supply. The gel was stainedwith Coomassie blue, destained, and then treated withthe fluor EN;3HANCE (New England Nuclear Corp.).Gels were dried by heating under vacuum at 55°C, andfluorographs were prepared at -70°C, using Kodak X-Omat R film. Molecular-weight standards (DaltonMark VI, Sigma Chemical Co.) were electrophoresedin a lane adjacent to the minicell samples, and themobility of the stained standards in the gel was com-

pared with that of the labeled bands on the fluoro-graph.

RESULTS

Construction and characterization ofpRT86 mutants. In an earlier communication(14) the regulatory elements controlling tetra-cycline resistance were mapped in TnlO HpaIfragment 2025. HincIl fragment 695 was sug-

gested to be the location of the tet repressor

gene. Plasmid pRT86 is a derivative of pRT29that contains a deletion at the HpaI restrictionsite (see Fig. 1). Plasmid pRT86 was selected forstudy because it contains only two HincIl sites;this allowed for easy in vitro construction ofdeletion and substitution mutants of the controlregion.The structures of the TnlO-related portion of

these plasmids are shown in Fig. 1. In plasmidpRT201, the sequence between the HinclI andXbaI sites has been deleted, whereas pRT202contains a deletion of the entire HinclI fragment695. The three plasmids pRT203, pRT204, and

pRT205 contain substitutions in which theHincIl fragment 695 has been replaced with a789-base pair fragment containing the lac pro-moter region. The HincIL sites were regeneratedat both ends in pRT203 and pRT204, but onlyat one end in pRT205 (Fig. 2). The sequence ofthe lacP fragment (7, 25; W. Gilbert, personalcommunication) and the HpaI restriction mapof pRT29 (28) are known. Thus, the orientationof these substitutions could be determined bytheir HpaII restriction patterns (data notshown). Plasmids pRT210 and pRT211 containinsertions of HincIl fragment 695 into the HpaIsite of pRZ112. The orientation of the insertionwas determined by comparing the restriction

FIG. 2. EcoRI-HincII double-digestion cleavagepatterns of plasmids. 29 refers to pRT29, etc. Frag-ment size is given in base pairs. The bands labeled3,700 and 140 are vector fragments. The deletion ofpRT29, which generated pRT86, fuses the 3,700- and560-base pair fragments together, resulting in theappearance of the 2,500-base pair fragment.

TABLE 1. Tetracycline resistance phenotypesEOP-,,, ([Lg/MW)

Plasmid"Uninduced Induced

pRT29' 23 37pRT86 27 66pRT201 123 126pRT202 4 4pRT203 128 128pRT204 90 88pRT205 15 14None <2 <2

a All plasmids were in host strain JD600.hEOP50, Concentration of tetracycline necessary to

reduce colony numbers by 50%.' The levels of resistance reported for pRT29 are

from a previous communication (14).

VOL. 147, 1981

FIG. 3. Fluorograms ofpolyacrylamide-sodium dodecyl sulfate gels displaying plasmid-encoded polypep-tides labeled with [5S]methionine in minicells. Minicells were (+) or were not (-) induced with tetracyclineat the time of labeling.

300

TETRACYCLINE RESISTANCE PROMOTER AND REPRESSOR 301

patterns from a double digestion with endonu-cleases EcoRI and XbaI (data not shown).

Effect of mutations on tetracycline re-sistance. Table 1 presents a comparison of thetetracycline resistance levels conferred on E. colicells by the mutant plasmids. The resistancelevels are given as the concentration of tetracy-cline at which the plating efficiency for a givenstrain is 50%. These were measured for culturesgrown in either the absence or the presence ofsubinhibitory levels of tetracycline.Both plasmids pRT29 and pRT86 encode an

inducible resistance phenotype, although theyconfer resistance to different levels of tetracy-cline. This is consistent with the previous report(14) that derivatives of pRT29 which containinsertions at the HpaI site also have alteredlevels of tetracycline resistance.Plasmid pRT201 confers a constitutive tetra-

cycline resistance phenotype. This level of re-sistance is higher than that observed for inducedcells carrying pRT86. This phenomenon hasbeen observed with other constitutive tetracy-cline resistance mutants (31). The constitutivephenotype of this mutant suggests that the dele-tion in pRT201 has removed either the sequencecoding for the repressor or the control site reg-ulating the expression of resistance or both.

Plasmids pRT202, pRT203, pRT204, andpRT205 also display a constitutive resistancephenotype, but the level of resistance is differentfor each plasmid. These mutants all differ withrespect to the DNA sequences adjacent to theHincII site of the left side of the 1,275-base pairfragment. An earlier study (14) has shown thatthis HincIl site is incompletely digested in thepresence of RNA polymerase and is thought tolie within the tet promoter. Thus, it appears thatthese mutants contain an altered tet promoterand that the different tetracycline resistancelevels are the consequence of promoters withaltered levels of expression.

Effect of mutants on plasmid-encodedprotein synthesis. The synthesis of polypep-tides encoded by plasmids can be studiedthrough the use of minicell-producing E. colistrains. Plasmids pRT29 and pRT86 code fortetracycline-inducible polypeptides with molec-ular weights of 37,000 and 34,000, respectively(37K and 34K proteins; Fig. 3A). The deletionwhich generated pRT86 removes a DNA se-quence from the region identified as coding forthe carboxy terminus of the TET protein (14).The 37K and 34K proteins are fusion polypep-tides of the 36K TET protein that are the resultof these different deletions in the carboxyl endof the TET protein-coding region. Both plasmidsdirect the synthesis of a 23,000-dalton polypep-tide (23K polypeptide). Qualitatively, it appears

that this polypeptide is tetracycline inducible.Plasmids pRT201, pRT203, and pRT204 all

code for a constitutive 34K polypeptide (Fig. 3Aand B). In addition, the 23K polypeptide is notpresent. From this observation it appears thatthe 23K polypeptide may function as the tetrepressor and be encoded in the 695-base pairHincII fragment. Plasmids missing this fragmentdo not direct the synthesis of the 23K polypep-tide and result in the constitutive expression ofthe resistance function.

Plasmids pRT202 and pRT205 do not directthe synthesis of the 23K polypeptide (Fig. 3Aand B). Moreover, they produce no detectableamount of the 34K TET fusion protein. This isconsistent with the idea that the low level oftetracycline resistance conferred by these plas-mids results from promoters with lowered levelsof expression.Recombinant plasmids pRT210 and pRT211

contain HincII fragment 695 inserted in oppositeorientations. The inserts are directly adjacent tothe Tn5 left inverted repeat promoter (34).pRT211 codes for a constitutive 23K polypep-tide, whereas this protein is not synthesized bypRT210. From this observation, we can concludethat the 695-base pair HincII fragment does notcontain the promoter controlling the expressionof the 23K polypeptide. In addition, the directionof transcription is from right to left, as the mapis drawn in Fig. 1.Restriction enzyme mapping of DNA

fragments containing the tet control re-gion. The restriction map of the control regionwas determined by making use of the XbaIrestriction site. This enzyme cleaves pRT86 atonly one site, and this site is directly adjacent tothe tet promoter (see Fig. 1). For a given restric-tion enzyme, the DNA fragment containing thecontrol region was identified by comparing thegel patterns of digestions of pRT86 with andwithout XbaI. Since there is a HinclI cleavagesite located about 30 base pairs from the XbaI

Alu IDde IFnu D IIHoe IIHoe IIIHha IHinf IRsa ISau 3AISOu 96ITaq I

190345

c.-

x a:

393

397

1581 269

359 > 1000522 691> 1000 L

9=5505 1

95 77

FIG. 4. Restriction enzyme cleavage map of DNAfragments containing the tet control region. Numbersrefer to distances, in base pairs, from the HincIIcleavage site.

VOL. 147, 1981

302 WRAY, JORGENSEN, AND REZNIKOFF

TET protein

PO

I I i I Irepressor

FIG. 5. Model for organization of the tetracycline resistance gene and its regulatory elements. P, Promoter;0, operator; I, HpaI cleavage sites in TnlO. Unlabeled uertical bars are HincII cleauage sites in TnlO.

site, the orientation of the two fragments pro-duced by Xbal cleavage was determined by com-paring the XbaI double digest with a HincIldouble digest. The results for 11 different restric-tion enzymes are summarized in Fig. 4.

DISCUSSIONThe promoter controlling the expression of

tetracycline resistance in TnlO has been identi-fied by placing different DNA sequences on theupstream side of the RNA polymerase-protectedHinclI site. These mutations conferred alteredlevels of tetracycline resistance and directed thesynthesis of altered levels of the resistance pro-tein. E. coli promoter sites are known to containareas of sequence homology known as the Prib-now or Schaller box (29, 30, 36) and the -35 orrecognition region (38, 42). Several promotersare known to contain HinclI cleavage siteswithin the recognition region, e.g., the trp operonpromoter (2), the bacteriophage X Pi, and pRpromoters (22, 24, 43), the bacteriophage pX174D promoter (35), and the rRNA promoters D2,E2, and X2 (6, 45). Point mutations which alterpromoter activity have been localized in therecognition region for a number of different pro-moters, e.g., the bacteriophage X pi,, PRM, andPIE promoters (17, 26, 27), the lacI gene pro-moter (4), and the lac operon promoter (7, 32).In addition, Rodriguez et al. (33) have reportedthat the insertion of DNA into the recognitionregion of the tetracycline resistance gene pro-moter in pBR313 alters the level of tetracyclineresistance. Thus, it seems plausible that theRNA polymerase-protected HinclI site in TnlOlies within the recognition region of the TETgene promoter and that the insertion of DNA atthis site could alter promoter activity.The tetracycline resistance repressor is a poly-

peptide with an apparent molecular weight of23,000 and is most likely identical to the 25Ktetracycline-inducible polypeptide identified byZupancic et al. (46). The observation that it istetracycline inducible suggests that the repres-sor regulates its own synthesis. Other repressormolecules have been demonstrated or suggestedto be autogenously regulated, e.g., the bacterio-

phage X repressor (26), the lexA protein (3), theTn3 transposase repressor (5), and the trp op-eron repressor (40). The repressor gene is locatedwithin HinclI fragment 695 and is transcribed ina direction that is opposite that of the tetracy-cline resistance gene. The possibility that thetranscription of these genes originates from acommon control region is suggested by the ob-servation that the 695-base pair HincIl fragmentdoes not contain the promoter controlling re-pressor synthesis. Such divergently transcribedcontrol regions have been described for bacterio-phage X (22, 26, 43), transposon Tn3 (10), andbiosynthetic gene clusters argECBH (8) andbioABFCD (9).

In Fig. 5 we present a simple model for thelocation and organization of the tet resistancegene and its repressor on Tnl 0. The presentationof two operators is arbitrary and is based on theobservation that uninduced minicells producerepressor but do not produce resistance protein(Fig. 3). This suggests that the two genes havedifferent regulatory sites.

In a subsequent communication (K. P. Ber-trand, L. V. Wray, Jr., and W. S. Reznikoff,manuscript in preparation), we shall describethe construction and use of X phages in whichthe TET protein promoter and the tet repressorpromoter have each been fused to the lacZ gene.The properties of these fusions have confirmedthe localization and regulatory properties of thetwo promoters and the repressor described inthis communication.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grantGM1970 from the National Institutes of Health and by grantPCM7910686 from the National Science Foundation. L.V.W.was supported by Public Health Service training grantGM07215 from the National Institutes of Health.

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