Comnplete Nucleotide Sequence Macrolide-Lincosamide … · inducible resistance to the...

Vol. 164, No. 2JOURNAL OF BACTERIOLOGY, Nov. 1985, p. 782-7960021-9193/85/110782-15$02.00/0Copyright © 1985, American Society for Microbiology

Comnplete Nucleotide Sequence ofMacrolide-Lincosamide-Streptogramin fl-Resistance Transposon

Tn917 in Streptococcus faecalisJAY H. SHAWt AND DON B. CLEWELL*

Departments of MicrobiologylImmunology, a7nd Oral Biology, Schools ofMedicine and Dentistry, and the DentalResearch Institute, the University of Michigan, Ann Arbor, Michigan 48109-2007

Received 15 May 1985/Accepted 15 August 1985

Streptococcus faecalis transposon Tn917 was cloned in Escherichia coli on plasmid vector pBR325. Theerythromycin resistance determinant of Th917 was not expressed in the E. coil background. The nucleotidesequence of Tp917 was determine,d and found to be 5,257 base pairs in length. Six open reading frames (ORFs)were identified and designated 1 through 6, (5' to 3'); all were on the same DNA strand. A region exhibitingstrong homology with knowr promoters was identified upstream from ORF1. ORFs 1 to 3 were virtuallyidentical to the previously sequenced erythromycin resistance determinant on Streptococcus sanguis plasmidpAM77. .At.he 3 point, where the homology between Tn917 and pAM77 ends, was a 20-base-pair region about80% homologous with a component of the res site of Tn3. The amino acid sequence of ORF4 showed homologywith other site-specific recombinatioht ehzymes, including approximately 30% homology with the resolvase ofTn3. Contained within Tn917 was a directly oriented 73-base-pair duplication of the left terminus The Tn917sequence revealed that antibiotic-enhanced transposition might be due to eitension of transcription from theresistance-related genes (in ORFs 1 to 3) into transposition genes (in ORFs 4 to 6). Transcription analysesresulted in data consistent with this interpretation.

The streptococcal transposon Tn917 confers on its hostinducible resistance to the macrolide-lincosamide-streptogramin B (MLS) antibiotics. It was originally identi-fied on the 22-kilobase (kb), nonconjugative, multiple-resistance plasmid pAD2 in Streptococcus faecalis DS16(51-53). The size of Tn917 was estimated at 5 kb, andheteroduplex analyses revealed the presence of invertedrepeats at its termini (52). An especially interesting propertyof Tn917 is its ability to undergo enhanced transposition onexposure to erythromycin (5, 51, 52). Although a similarphenomenon is known to occur with regard to the gram-negative mercury-resistance transposon TnS01 (45), Tn917remains the only element reported to exhibit antibiotic-enhanced transposition. Both resistance and transpositionwere inducible at erythromycin concentrations as low as0.001 ,ug/ml, but not 0.0001 jig/ml, suggesting a common orsimilar regulatory mechahism.The termini of Tn917 have recently been sequenced by

Perkins and Youngman (39), who noted a significant degreeof homology with the ends of the Staphylococcus aureustransposon TnS51 (27) and the gram-negative transposonTn3 (20). Tn917 was also shown to generate a five-base-pairduplication on insertioti (39). In addition, it was shown byrestriction mapping arid heteroduplex analysis that Tn917and TnS51 exhibited extensive homology throughout theirentire lengths (39). (It is noteworthy that, in contrast to thecase of Tn917, the MLS resistance of TnS51 is expressedconstitutively [40].) Banai and LeBlanc (1) have identified onthe S. faecalis plasmid pJH1 a transposon (Tn3871) havingextensive homology with Tn9J7, and recent studies byLeBlanc and coworkers. (41) have shown that Tn917-likeelements are probably widespread in S. faecalis. Tn917 has

* Corresponding author.t Present address: Department of Medical Microbiology,

Stanford University, Stanford, CA 94305.

proven valuable as a mutagenic element in both S. faecalis(5, 25) and Bacillus subtilis (57), and derivatives recentlyconstructed by Youngman and coworkers (58, 59) shouldprove especially valuable for genetic and cloning studies ongram-positive bacteria.The determinant for MLS resistance, designated erm, has

been shown to encode a methylase which gives rise to an N6,N6 dimethylation of a specific adenine residue in 23S ribo-somal RNA (29-31). As first revealed from sequencingstudies of ermC of the staphylococcal plasmid pE194 (18,23), induction is the result of a posttran5criptional eventwhich corresponds to a translational attenuation. A leaderregion of the transcript exhibited a secondary structure ableto sequester the ribosome-binding site and initiation codonfor ermC (18, 23). A short open reading frame (ORF) whichcould encode a 19-amino-acid peptide is located within theleader region of the transcript. Induction is visualized asfollows. The antibiotic binds to and stalls ribosomes engagedin translation of this small peptide. This is followed by achange in mRNA secondary structure which, in turn, ex-poses the ribosome-binding and initiation sites of ermC.AInalysis of mutants which expressed MLS resistance con-stitutively provided strong support for this interpretation(18, 19, 23, 24). In additioh, studies with B. subtilis minicellshave shown that induction of the synthesis of the nmethylasecan occur after the minicells have been exposed to rifatnpin(49). Similar attenuational control mechanisms have beensuggested from the sequence of the erm locus of the Strep-tococcus sanguis plasmid pAM77 (22, 56), the chromosomalerm determinant of Bacillus licheniformis (17), and theTnSS4-bortne erm locus in Staphylococcus aureus (38). Ai-though there is significant homology between all of the errrdeterminants, homology between corresponding leader re-gions has been observed only between those of ermC and theermA of TnSS4 (38).The induction of Tn9O7 transposition in a manner which

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S. FAECALIS MLS-RESISTANCE Tn9J7 NUCLEOTIDE SEQUENCE

TABLE 1. Bacterial strains used in this study

Strain Host genotype Plasmid content Reference or source

S. faecalisOGlS(pAM307) str pAM307 5DS16 tet (Tn916) pAD1, pAD2 5DS16C2 tet (Tn916) pAD1 5FA3001 tet (Tn916) pAD1, pAD2 5PT411 str spc pAMal::Tn9J7 P. Tomich

S. sanguisChallis None F. Macrina (14)JS1 Same as Challis pAM238 This studyVA685 Same as Challis pVA380-1 F. Macrina (33)

E. coliDH1 F- recAl endAI gyrA96 thi-I hsdRJ7 supE44 None D. FriedmanDB11 met thi gal hsdR nal rif None F. Macrina (34), J. DaviesJS2 Same as DBll pAM225 This studyJS3 Same as DH1 pAM225 This studyDH1(pBR325) Same as DH1 pBR325 D. FriedmanJM103 Av(lac pro) thi rpsL supE endA sbcB hsdR F' D. Behnke

traD36 proAB lacI ZAMJ5

B. subtilisPT3 met trp lys pAM77 P. Tomich; transformed pAM77

from S. sanguis Al (56) intoBR151

seems to parallel the induction of MLS resistance (52) hasraised the question of whether transposition functions mayalso be controlled by translational attenuation in a coupledor even independent fashion. This question, as well as adesire to gain further insight into the general nature ofTn917, prompted us to conwduct a sequence analysis of theentire element. The results of such a study are presentedhere. The erm determinant, as well as an apparent leaderregion 5' to erm and a short ORF 3' to erm, were found to bealmost identical to sequences on pAM77 reported byHorinouchi et al. (22). Three additional reading frames werealso revealed, with one encoding a protein bearing resem-blance to previously reported resolvases of gram-negativeelements. Other interesting features relating to potentialrecombination or resolution sites 'or both were also ob-served. Finally, a likely explanation for coipling inductionof transposition to induction of resistance as a result oftranscriptional readthrough of a termination site was evidentfrom the sequence and ahalyses of transcription.

MATERIALS AND METHODS

Bacteria, media, and reagents. The bacterial strains andplasmids used in this study are listed in Table 1. Difcoantibiotic medium no. 3 was used as the growth medium forS. faecalis. Escherichia coli strains were grown in LBmedium (9). S. sanguis was grown in brain heart infusion(Difco Laboratories), and 1% horse serum (Colorado SerumCo.) was added when cells were grown for transformation.Cell growth was monitored with a Klett-Summersoncolorimeter with a no. 54 filter. When present in selectiveplates, tefracycline (Sigma Chemical Co,) was present at aconcentration pf 10 ,ug/ml, and erythromycin (Sigmna) w.aspresent at 50 ,ug/ml. M13 replicative form DNA, lambdaDNA, restriction endonucleases, and .)NA ligase werepurchased from Bethesda Research Laboratories, Inc.(BRL). Transformlation of E. coli and Sk isanguis were

performed as described elsewhere (14). Clindamycin,lincomycin, tylosin, and streptogramin B were a gift from B.Weisblum.

Cloning of Tn917. Tn917 was cloned in E. coli by thefollowing scheme. The plasmid pAM307 [in OGlS(pAM307)]is a derivative of the 58-kb hemolysin plasmid pAD1 with aTn917 insertion into a small (1.2-kb) EcoRI fragment (frag-ment H) within the hemolysin determinant (5). The H'fragment (H::Tn9J7) was extracted from a low melt agarosegel and ligated with EcoRI-digested pBR325 (4) [isolatedfrom DH1(pBR325)]. The EcoRI site of pBR325 is in thechloramphenicol resistance determinant. The ligation mix-ture was transformed into E. coli DB11, a strain initiallyselected because of its hypersensitivity to erythromycin (34).(Whereas most E. coli strains are resistant to about 100 ,ug oferythromycin p,r ml, DB11 is sensitive to 4 ,ug/ml.) Initialselection for transformants was on LB plates containing 10jig of tetracycline per ml. A chimera derived from a chlor-amphenicol-sensitive transformant and designated pAM225is shown in Fig. 1. Erythromycin resistance was not' ex-pressed in the DB11 host (i.e., strain JS2) or in a secondstrain, E. coli DH1 (i.e., JS3), even when the orientation ofthe cloned fragment was reversed after cleaving with EcoRIand religating. The MICs were the same (5 g±g/ml for DB11,150 ,uglml for DH1) for plasmid-free and plasmid-containingcells. In addition to erythromycin, other members' of theMLS family of antibiotics (clindamycin, tylosin, lincomycin,and streptogramin B) were also inhibitory. Paper discs (7mip diameter) containing either 2 or 10 ,ug of each antibioticv<ere placed on plates that had been spread with 0.25 ml of a1:20 dilution of an overnight culture of DB11 containingpAM225. After overnight incubation, zones of inhibitionwere observed in all cases.To ensure that the Em resistance determinant was intact,

we subcloned the EcoRI fragment from pAM225 that con-tained Tn917 into the single EcoRI site of pVA380-1 (33), asmall, cryptic plasmid isolated from S. sanguis VA685 (a

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784 SHAW AND CLEWELL

r_ X X Hind m

Sal I

FIG. 1. Physical map of pAM225. The orientation of the clonedfragment containing Tn917 was mapped by analysis of a HindIIIdigestion. The locations of the restriction sites were set by thesubsequent sequence data.

derivative of strain Challis). S. sanguis Challis was trans-formed and plated on medium containing erythromycin (50,g/ml). A chimera (designated pAM238) containing theexpected subcloned EcoRI was recovered (strain JS1). Theoriginally cloned segment of DNA, therefore, maintained theability to express Em resistance in streptococci.

Generation of M13 clones for sequencing. We generated aseries of bacteriophage M13mp8 clones containing Tn9J7DNA fragments (37, 43) by making use of the sonicationprotocol of Deninger (10). pAM225 was sonicated with aBranson Sonic Power Co. sonicator with a W-140 microtip.Plasmid DNA (4 ,ug) dissolved in 250 ,ul of buffer (10) wasplaced in a 1.5-ml Eppendorf tube for shearing. Sonicationwas calibrated with lambda DNA. A power setting of 70%was used with seven 5-s blasts. We monitored the amount ofshearing by sizing the DNA on an agarose gel with knownmolecular weight markers (pBR322 digested with AluI).Sonication was done until virtually all DNA was less than 1kb long. DNA fragment ends were repaired to blunt endswith T4 DNA polymerase from BRL. (We incubated soni-cated fragments with polymerase for 15 min before additionof the dNTPs to allow 3' to 5' exonuclease activity.) Wefractionated repaired fragments on an agarose gel to separateout the snialler fragments (10). Ligation to phage DNA andtransfection into E. coli JM103 were as recommended byBRL, and white plaques characteristic of clones havinginsertions (because of inability to express beta-galactosi-dase) were generated. The SmaI site of M13mp8 was usedfor cloning. Clones specifically containing Tn917 sequenceswere identified by plaque hybridization (2) with nick-translated pAMal::Tn9J7 that had been isolated from S.faecalis PT411. Insofar as pAMal sequences exhibit no

homology with Ml3 DNA, hybridization reflects the pres-

ence of Tn917 sequences. (pAMoal is a nonconjugative, 9-kb,multicopy plasmid isolated from S. faecalis [6, 7].) Nicktranslation of plasmid DNA with [32P]dCTP (670 Ci/mmol;New England Nuclear Corp.) was carried out with a nicktranslation kit from New England Nuclear. Autoradiography

was done at -70'C with a DuPont Cronex intensifyingscreen.A problem encountered in the ligation of sonication frag-

ments in the SmaI site of M13mp8 was the self-ligation ofphage DNA molecules that gave rise to white plaques. Anumber of these phage were sequenced, and it was foundthat a single base pair was lost at the site where SmaIcleaves. The deletion represented a change in the sequencefrom CCCCGGG to CCCGGG. Even with the loss of thisbase pair, the SmaI site is maintained. To alleviate thisproblem, once the ligation reaction was completed we addedSmaI to the ligation reaction mixture. Since Tn917 does notcontain any SmaI sites, this resulted in the cleaving of allself-ligated M13mp8 molecules that did not contain an insert.Chimeric molecules which are devoid of SmaI sites aretherefore selected during transformation. This method es-sentially eliminated the occurrence of false-positive whiteplaques.

For the region of the transposon around the SalI site, itwas necessary to clone with the SalI site along with theHindIII site into M13mpl9 and a SalI-to-KpnI fragment intoml3mpl8. In the case in which pAM77 sequences werecloned, plasmid DNA was isolated from B. subtilis PT3, andClaI fragments were cloned into AccI-digested M13mp8.DNA sequence analysis. The DNA sequencing protocol

was as described by Sanger et al. (43, 44) with modificationsto allow use of [35S]dATP (3). Sequencing reactions werecarried out at room temperature. Dideoxynucleotides anddeoxynucleotides were obtained from P-L Biochemicals,Inc. M13 synthetic sequencing primer was obtained fromNew England BioLabs, Inc. Klenow fragment was obtainedfrom BRL. [35S]dATP was obtained from Amersham Corp.The computer program of Larson and Messing (32) (pur-chased from the University of Minnesota) was used to filesequence data and locate restriction sites.A total of 120 clones containing DNA homologous with

Tn917 were sequenced. Overlapping of sequences was donevisually by using the computer-located restriction sites asreference points. A total of over 24,000 base pairs were readto ensure the accuracy of the sequencing data. Every regionof the transposon was sequenced at least twice, with theexception of the region close to the Sail site. The reading ofthis region, however, occurred at the bottom (and mostclearly readable region) of the corresponding sequence lad-ders. Sequences from both strands were determined for over90% of the transposon.

Transcription analyses. RNA was extracted by a hot-phenol protocol (8, 26). Cells (20 ml) were poured onto anequal volume of frozen, ctushed killing medium (0.02 M Trishydrochloride [pH 7.3], 0.005 M MgCl2, 0.02 M sodiumazide, 400 ,ug of chloramphenicol per ml). Cells werepelleted by spinning in an SS34 rotor (Beckman Instruments,Inc.) at 6,000 rpm for 5 min. The pellet was suspended in 2ml of 25% sucrose-50 mM Tris hydrochloride (pH 8.0). Twomicroliters of 0.25 M EDTA and 50 ,ul of lysozyme (20 mg/mlin water) were added, mixed gently, and placed on ice for 5min. After centrifugation at 5,000 rpm for 5 min, the peilet ineach tube was suspended in 0.3 ml of lysis buffer (20 mMTris hydrochloride [pH 8.0], 3.0 mM EDTA, 0.2 M NaCl).Lysis buffer (0.3 ml at 95°C) containing 1.0% sodium dodecylsulfate was added, and the mix was incubated at 95°C for 1min with gentle mixing. An equal volume of phenol saturatedwith lysis buffer and maintained at 65°C was added. Afterincubation at 65°C for 3 min with occasional mixing, thesamples were chilled and the phases were separated bycentrifugation at 10,000 rpm in an SS34 rotor. The aqueous

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S. FAECALIS MLS-RESISTANCE Tn9O7 NUCLEOTIDE SEQUENCE

* AVAGAATTAGTGTTTAGAGCGAACGATTAAATATTTATATTAGGCAAATATTGTCTCAAAAGGTAATAACAACGATTTAATAGATAGTCTTTTTTGGGGTCC

20 40 60 80 100

A 1 CLA 1 RSA 1CGAGCGCCTACGAGGAATTTGTATCGATAAGAAATAGATTTAAAAATTTCGCTGTTATTTTGTACATTTAMCTTGACGGTGACATCTCTCTATTGTGAGT

120 140 160 180 200

RSA 1TATTAGTGGTACAGTTTTCAACCGTTTTAATTATAAAAAAGTGGTGCATTTTTAAATTGGCACAAACAGGTAACGGTTATTGCAGGTGTATTTCTTATCT

220 240 260 280 300

HPA 1ATGGGTTTAACATGGATTTTATCATTAAAATCATGAGTATTGTCCGAGAGTGATTGGTCTTGCGTATGGTTAACCCTAAAGTTATGGAAATAAGACTTAG

320 340 360 380 400

-35 -10 S.D.AAGCAAACTTAAGAGTGTGTTGATAGTGCATTATCTTAAAATTTTGTATAATAGGAATTGAAGTTAAATTAGATGCTAAAAATTTGTAATTAAGAAGGAG

420 440 460 480 500ORF1MetLeuValPheGlnMetArgAsnValAspLysThrSerThrValLeuLysGlnThrLysAsnSerAspTyrAlaAspLysTyrValArgL

GGATTCGTCATGTTGGTATTCCAAATGCGTAATGTAGATAAAACATCTACTGTTTTGAAACAGACTAAAAACAGTGATTACGCAGATAAATACGTTAGAT520 540 560 580 600

euIleProThrSerAsp

TAATTCCTACCAGTGACTAATCTTATGACTTTTTAAACAGATAACTAAAATTACAAACAAATCGTTTAACTTCTGTATTTATTTATAGATGTATCACTTC620 640 660 680 700

ORF2MetAsnLysAsnIleLysTyrSerGlnAsnPheLeuThrAsnGluLysValLeuAsnGlnIlleleLysGlnLeuAsnLeuLysGluT

S.D. RSA 1AGGAGTGATTACATGAACAAAAATATAAAATATTCTCAAAACTTTTTAACGAATGAAAAGGTACTCAACCAAATAATAAAACAATTGAATTTAAAAGAAA

720 740 760 780 800

hrAspThrValTyrGluIleGlyThrGlyLysGlyHisLeuThrThrLysLeuAlaLysIleSerLysGlnValThrSerIleGluLeuAspSerHisLeCCGATACCGTTTACGAAATTGGAACAGGTAAAGGGCATTTAACGACGAAMACTGGCTAAAATAAGTAAACAGGTAACGTCTATTGAATTAGACAGTCATCT

820 840 860 880 900

uPheAsnLeuSerSerGluLysLeuLysLeuAsnIleArgValThrLeuIleHisGlnAspIleLeuGlnPheGlnPheProAsnLysGlnArgTyrLys

ATTCAACTTATCGTCAGAAAAATTAAAACTGAACATTCGTGTCACTTTAATTCACCAAGATATTCTACAGTTTCAATTCCCTAACAAACAGAGGTATAAA920 940 960 980 1000

IleValGlyAsnIleProTyrHisLeuSerThrGlnIlleleLysLysValValPheGluSerHisAlaSerAspIleTyrLeuIleValGluGluGlyP

ATTGTTGGGAATATTCCTTACCATTTAAGCACACAAATTATTAAAAAMAGTGGTTTTTGAAAGCCATGCGTCTGACATCTATCTGATTGTTGAAGAAGGAT1020 1040 1060 1080 1100

heTyrLysArgThrLeuAspIleHisArgThrLeuGlyLeuLeuLeuHisThrGlnValSerIleGInGlnLeuLeuLysLeuProAlaGluCysPheHiRSA 1 TAQ 1 ALU 1

TCTACAAGCGTACCTTGGATATTCACCGAACACTAGGGTTGCTCTTGCACACTCAAGTCTCGATTCAGCAATTGCTTAAGCTGCCAGCGGAATGCTTTCA1120 1140 1160 1180 1200

sProLysProLysValAsnSerValLeuIleLysLeuThrArgHisThrThrAspValProAspLysTyrTrpLysLeuTyrThrTyrPheValSerLysALU 1 RSA 1

TCCTAAACCAAAAGTAAACAGTGTCTTAATAAAACTTACCCGCCATACCACAGATGTTCCAGATAAATATTGGAAGCTATATACGTACTTTGTTTCAAAA1220 1240 1260 1280 1300

TrpValAsnArgGluTyrArgGlnLeuPheThrLysAsnGlnPheHisGlnAlaMetLysHisAlaLysValAsnAsnLeuSerThrValThrTyrGluGTAQ 1 RSA 1

TGGGTCAATCGAGAATATCGTCAACTGTTTACTAAAAATCAGTTTCATCAAGCAATGAAACACGCCAAAGTAAACAATTTAAGTACCGTTACTTATGAGC1320 1340 1360 1380 1400

ORF3lnValLeuSerIlePheAsnSerTyrLeuLeuPheAsnGlyArgLys MetSerArgPheCysLysPheGlyLysLeuHisValThrLysGlyA

S.D.AAGTATTGTCTATTTTTAATAGTTATCTATTATTTAACGGGAGGAAATAATTCTATGAGTCGCTTTTGTAAATTTGGAAAGTTACACGTTACTAAAGGGA

1420 1440 1460 1480 1500

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snValAspLysLeuLeuGlylleLeuLeuThrAlaSerLysGluLeuLysArgserLeuAlaProThrGlyAsnLeuTyrArgALU 1 ALU 1 CLA 1

ATGTAGATAAMATTATTAGGTATACTACTGACAGCTTCCAAGGAGCTAAAGAGGTCCCTAGCGCCTACGGGGAATTTGTATCGATAAGGAATAGATTTAAA1520 1540 1560 1580 1600

RSA 1 KPN 1 RSA 1AATTTCGCTGTTATTTTGTACAATAAGGATAAATTTGAATGGTACCATAAACGACCGTTTATGGTACTTTTCATTTTCCTGCTTTTTCTAAATGTTTTTT

1620 1640 1660 1680 1700

RSA 1 -35 RSA 1 -10 S.D.AAGTAAATCAAGTACCAAAATCCGTTCCTTTTTCATAGTTCCTATATAGTATACTTAATGAGTTATGGTACATTTAAATTATAAAATTAAGGAGGTTTTT

1720 1740 1760 1780 1800ORF4MetIlePheGlyTyrAlaArgValSerThrAspAspGlnAsnLeuSerLeuGlnlleAspAlaLeuThrHisTyrGlylleAspLysLeuPheGlnGl

AVA 1 RSA 1 BCL 1TTATGATTTTTGGCTATGCTCGAGTGAGTACGGATGATCAAAATCTTAGT7TACAAATTGATGCACTTACTCATTATGGAATTGATAAATTATTTCAAGA

1820 1840 1860 1880 1900

uLysValThrGlyAlaLysLysAspArgProGlnLeuGluGluMetIleAsnLeuLeuArgGluGlyAspSerValValIleTyrLysLeuAspArglleBCL 1 TAQ 1

AAAAGTAACTGGTGCGAAAAAAGACCGACCGCAATTAGAAGAAATGATCAACCTACTACGTGAAGGAGATTCTGTTGTCATTTACAAGTTAGATCGAATT1920 1940 1960 1980 2000

SerArgSerThyLysHisLeuIleGluLeuSerGluLeuPheGluGluLeuSerValAsnPheIleSerIleGlnAspAsnValAspThrSerThrSerM

TCACGATCAACTAAACATTTGATTGAACTTTCTGAATTATTTGAAGAACTTAGTGTCAATTTTATATCTATTCAAGATAACGTAGATACTTCAACGTCTA2020 2040 2060 2080 2100

etGlyArgPhePhePheArgValMetAlaSerLeuAlaGluLeuGluArgAsplleIleIleGluArgThrAsnSerGlyLeuLysAlaAlaArgValAr

TGGGAAGATTCTTTTTCCGAGTTATGGCTAGTTTAGCAGAACTGGAACGGGATATTATTATTGAACGAACTAACTCTGGTCTTAAGGCAGCCAGAGTCCG2120 2140 2160 2180 2200

gGlyLysLysGlyGlyArgProSerLysGlyLysLeuSerIleAspLeuAlaLeuLysMetTyrAspSerLysGluTyrSerIleArgGlnIleLeuAspALU 1 ALU 1

AGGAAAAAAMAGGGGGCCGTCCAAGTAAMAGGTAAGCTATCAATTGATTTAGCTTTAAAAATGTATGACAGCAAAGAGTATTCTATTCGTCAAATTCTTGAT2220 2240 2260 2280 2300

ORF5AlaSerLysLeuLysAsnAsnLeuLeuProLeuProGln MetAlaMetLysArgIleLeuThrThrSerGlnArgGluGln

GCCTCTAAATTAAAAAACAACCTTTTACCGTTACCTCAATAAMAAGGTATGCTTAAGATATGGCTATGAAMAAGAATTTTAACTACTTCACAGCGTGAACAA2320 2340 2360 2380 2400

LeuLeuSerValAspHisLeuSerGluGluAspPheLysAlaTyrPheSerPheSerAspTyrAspLeuGluValIleAsnGlnHisArgGlyLysValAHPA 11

CTTCTTTCTGTAGACCACTTATCAGAMAGAGGATTTTAAAGCGTATTTTAGTTTTTCTGATTATGATCTGGAGGTTATTAATCAMACACCGTGGAAAGGTCA2420 2440 2460 2480 2500

snLySLeUbiyVheAlalleGlnLeuCysLeuAlaArgTyrProGlyCysSerLeuSerAsnTrpProIleLysSerThrArgLeuThrSerTyrValSeS

ATAAMACTAGGATTTGCGATACAACTTTGTTTGGCCCGGTATCCTGGGTGTTCTTTAAGTAATTGGCCGATTAAATCAACCAGACTAACTTCTTATGTGAG2520 2540 2560 2580 2600

rArgGlnLeuHisLeuAspAlalleAspLeuAsnSerTyrAspHisArgAsnThrArgAlaAsnHisPheAsnGluIleLeuGluValPheAsnTyrHisAL 1 XHO 11 CLTCGACAGCTCCATCTTGATGCAATTGATTTAAATTCATATGATCATAGAAATACACGTGCAAATCACTTCAACGAGATCTTAGAAGTATTCAACTATCAT

2620 2640 2660 2680 2700

ArgPheGlySerAlaAsnThrGlnLysGlnLeuIleGluTyrLeuIleGluLeuAlaLeuGluAsnAspAspSerIleTyrLeuMetLysLysThrIleAA 1 ALU 1CGATTCGGTAGTGCTAATACACAAAAACAGTTAMATAGAATATTTAATTGAACTAGCTTTAGAAAATGATGACTCTATCTATCTAATGAAAAAAACAATTG

2720 2740 2760 2780 2800

spPheLeuThrArgLysArgIlellePheProSerIleAlaThrLeuGluAspIleIleSerArgCysArgAspLysAlaGluAsnAsnLeuPheSerIllTAQ 1 ALU 1 TAQ 1

ATTTCTTAACTCGAAA GAATTATTTTTCCATCTATAGCTACACTTGAAGACATTATAAGCCGCTGTCGAGATAAAGCAGAAAAGAACTTATTTTCAAT2820 2840 2860 2880 2900

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S. FAECALIS MLS-RESISTANCE Tn917 NUCLEOTIDE SEQUENCE

eLeuLeuCysSerLeuThrAspIleGInIleGluLysLeuGluSerLeuPheGlnIleTyrGluGluThrLysIleThrLysLeuAlaTrpLeuLysAsp

ATTACTCTGTTCATTAACAGATATACAAATTGAAAAACTAGAGAGTTTGTTTCAAATTTATGAAGAGACGAAAATAACTAAACTCGCTTGGCTAAAAGAC2920 2940 2960 2980 3000

IleProGlyLysAlaAsnProGluSerPheMetSerIleCysLysLysValGluValIleAlaSerMetGlyLeuGlyThrIleAsnValSerHisIleAHIND 111 AVA 1

ATTCCAGGTAAGGCAAATCCAGAAAGCTTTATGAGTATTTGTAAAAAAGTGGAAGTGATTGCTTCCATGGGACTCGGGACAATTAATGTCTCCCATATTA3020 3040 3060 3080 3100

snArgAsnArgPheLeuGlnLeuAlaArgLeuGlyGluAsnTyrAspAlaTyrAspPheSerArgPheGluLeuGluLysArgTyrSerLeuLeuIleAlALUlALUl ALU 1

ATCGGAACAGGTTTCTTCAGCTAGCTAGACTAGGGGAAAATTATGATGCATATGACTTCTCCCGTTTTGAGCTTGAAAAAAGATACTCTTTACTTATTGC3120 3140 3160 3180 3200

aPheLeuValAsnHisHisGlnTyrLeuIleAspGlnLeuIleGluIleAsnAspArgIleLeuAlaSerIleLysArgLysGlyThrArgAspSerGlnCLA 1

TTTTTTAGTCAATCATCATCAATATCTGATCGATCAACTGATTGAGATTAATGACCGCATTTTAGCAAGTATTAAACGCAAAGGGACACGTGATTCACAA3220 3240 3260 3280 3300

GluGlnLeuLysGluLysGlyLysLeuAlaThrLysLysLeuGluHisTyrAlaSerLeuIleAspAlaLeuHisPheAlaLysAspAsnAspSerAsnP

GAACAGTTAAAAGAAAAAGGAAAATTGGCTACTAAAAAATTGGAACATTATGCTTCTTTAATTGATGCTCTTCACTTTGCAAAAGATAATGATAGTAATC3320 3340 3360 3380 3400

roPheAspGluIleGluArgIleMetProTrpGluAspLeuValGlnAspGlyGluGluAlaLysLysAlaIleThrGlyAsnLysAsnHisGlyTyrLeRSA 1 ALU 1 ALU 1

CTTTTGACGAAATTGAACGAATCATGCCTTGGGAAGATTTAGTACAAGATGGAGAAGAAGCTAAMAAAAGCTATTACAGGTAATAAAAATCATGGCTATTT3420 3440 3460 3480 3500

uGluMetValArgAsnLysAlaAsnTyrLeuArgArgTyrThrProMetLeuLeuArgThrLeuSerPheLysAlaThrProAlaAlaAsnProValLeuALU 1 HPA 11

AGAAATGGTTCGAAATAAAGCTAATTACCTCCGAAGATACACGCCAATGTTATTGAGGACCCTTTCGTTCAAAGCAACTCCGGCAGCAAATCCAGTCCTC3520 3540 3560 3580 3600

MetAlaLeuThrGlnLeuThrAspLeuHisAsnSerGlyLysArgLysIleProAlaAspThrSerThrAspPheValSerLysLysTrpLysSerLeuVHPA 11

ATGGCCCTAACTCAACTAACTGATTTACACAATAGTGGTAAAAGAAAAATACCGGCAGATACTTCTACTGATTTTGTGAGTAAAAAATGGAAAAGCCTTG3620 3640 3660 3680 3700

alArgProGluGluGlyLysIleAspArgSerTyrTyrGluLeuValAlaPheThrGluLeuLysAsnAsnIleArgSerGlyAspIleSerValGluGlALU 1 ALU TAQ 1

TTCGGCCAGAAGAGGGGAAAATAGATCGGTCTTACTATGAGTTAGTAGCTTTCACCGAGCTAAAGAACAATATTCGATCAGGAGATATTTCAGTTGAAGG3720 3740 3760 3780 3800

ySerMetIleHisArgAsnIleAspAspTyrLeuValAspLeuSerAlaCysIleAspSerGluThrl1eProAspThrPheGluAspTyrLeuLysAspTAQ 1

AAGTATGATCCATCGAAATATTGATGATTACTTAGTTGATTTATCTGCTTGTATTGATTCAGAAACTATTCCAGACACGTTTGAGGACTATTTAAAGGAT3820 3840 3860 3880 3900

ArgGluIleIleLeuAspLeuGlnLeuGlnPheTyrSerThrValAspLysArgIleSerArgAlaAsnLeuLysLysLeuGluLysValThrProSerAALU 1 TAQ 1

CGGGAAATAATTTTAGATTTACAGCTTCAATTTTATTCGACAGTTGATAAGAGAATTTCAAGAGCAAACCTTAAAAAGTTGGAAAAAGTTACACCTAGCG3920 3940 3960 3980 4000

spArgLysTyrIleGluLysAsnPhel1eGln

ACAGGAAATATATAGAAAAAAACTTTATTCAATAATTCCTAAGATAAGGCTTAGTGATCTTTTAATTGAGGTGGACAGTTGGACCAACTTTTCACAAGAA4020 4040 4060 4080 4100

TTTTAGTCATGATTCTACAGGGAAACCGCCGAGTGAACAAGAAAGAAAAATTATTTTTGCTGCTTTGCTGGGTTTAGGGATGAATATTGGTCTTGAAAAA4120 4140 4160 4180 4200

-35 -10 ALU 1ATGGCCCAATCAACTCCTGGAATTTCTTATTCTCAGTTAGCCAATGCCAAACAATGGCGCTTTTATAAAGAAGCTCTGACTCGTGCTC TCTGTTTTGG

4220 4240 4260 4280 4300

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HIND 111 ALU 1TTAATTATCAGTTAAAGCTTCCTGTTGCAGACTTTTGGGGTGAAGGAAAAACCACTGCTTCAGACGGAATGCGCGTCCCAGTGGCGTCTCAGCTCTAAAA

4320 4340 4360 4380 4400ORF6

S.D. MetIleArgSerIleAsnAspArgHisThrThrHisHisIleGluValAlaSerTALU 1 TAQ 1

TCCGATGTTAATCCACATTACAAAAGTATGGAAAAMGGAGCTACAATGATTCGATCAATAAATGATAGGCATACGACTCATCATATCGAGGTTGCTTCAA4420 4440 4460 4480 4500

hrAsnThrArgGluAlaThrHisThrLeuAspGlyLeuLeuTyrHi'sGluThrAspLeuAspIleGluGluHisPheThrAspThrAsnGlyTyrSerAsALU 1 XBA 1 BCL

CTAATACAAGGGAAGCTACTCATACCCTTGATGGCCTACTTTATCATGAAACAGATCTAGATATTGAGGAACATTTTACTGATACAAATGGGTATTCTGA4520 4540 4560 4580 4600

pGlnValPheGlyMetThrAlaLeuLeuGlyPheAspPheGluProArgIleArgAsnIleLysLysSerGlnLeuPheSerIleLysSerProSerTyr1

TCAGGTGTTTGGAATGACCGCATTACTAGGCTTTGATTTTGAACCTCGCATCAGAAATATAAAAMAATCACAATTATTTTCTATCAAATCACCTTCCTAC4620 4640 4660 4680 4700

TyrProAsnLeuSerGluAspIleSerGlyLysIleAsnValLysIlelleGluGluAsnTyrAspGluIleLysArgIleAlaTyrSerIleGlnThrGTAQ 1

TACCCTAACTTATCAGAAGATATAAGCGGAAAAATCAATGTAAAMATTATTGAAGAAAACTATGATGAAATTAAMCGAATCGCCTATTCGATTCAAACAG4720 4740 4760 4780 4800

lyLysValSerSerSerLeuLeuLeuGlyLysLeuGlySerTyrAlaArgLysAsnArgValAlaLeuAlaLeuArgGluLeuGlyArgIeGluLysSeALU 1 ALU 1

GAAAAGTATCTAGTTCTTTACTATTAGGAAAGCTAGGCTCATACGCACGTAAGAATAGAGTAGCTCTTGCACTGAGAGAACTAGGTCGCATTGAAAAGAG4820 4840 4860 4880 4900

rIlePheMetIleAspTyrIleThrAspSerGluLeuArgArgArgIleThrHisGlyLeuAsnLysThrGluAlaIleAsnAlaLeuArgArgGluLeuALU 1

CATTTTTATGATAGATTATATTACAGATAGTGAGCTACGGCGAAGGATCACTCATGGACTAAATAAGACAGAAGCGATTAATGCTTTACGTAGAGAACTA4920 4940 4960 4980 5000

PhePheGlyAspAlaTluAsnLeuTrpSerAlaIlePheAlaAspAsnPheLysValLeuValArgLeuMetCys

TTTTTTGGCGACGCGGAAAATTTATGGAGCGCGATATTCGCCGACAACTTCAAAGTGCTAGTGCGCTTAATGTGTTAATAAATGCAATAAGTATATGGAA5020 5040 5060 5080 5100

ALCGCCGTCTACTTACAAGCAGCTTATAATTATCTCGTCAAMTAGATCCCGAAGTAACTAAGTATATGAAGCATGTATCTCCTATTAATTGGGAGCATATC

5120 5140 5160 5180 5200

ACTTTTCTTGGAGAGTATAAATTTGACTTGTTATCTATTCCTAAACACTTAAGAGAATTGAATATAAAAAATAAAAGGCCTTGAAACATTGGTTTAGTGG5220 5240 5260 5280 5300

CLA 1 AVA 1 * HINC 11GAATTTGTACCCCTTATCGATACAAATTCCCACTAAGCGCTCGGGACCCCTTTTTTAGGATATATTTGTTTTTAATGGTTAACTATTCTATTTTACTGAC

5320 5340 5360 5380 5400

ALU 1AATAATAGCTCTTTTCTAATCTCTTTAATAGCTTTTTTAAGTATTATAAATTCGCATACAATAAMAAGATTTGTAGATAAMGAAATAATGGAACAAGGAA

5420 5440 5460 5480 5500

ACGTCCAGAGAATTATTACAGAAGCAATTGAAGGAATTGAAACCATTAAATCTGAATGCAGAAAAGAGTTTTTTGTTAAATTGGAAAAACATGTTTACGT5520 5540 5560 5580 5600

CTCAA

FIG. 2. Nucleotide sequence of Tn917. The sequence of Tn9O7 begins at base number 94 and ends at base number 5350. * indicates thefirst and last bases of Tn9O7. The terminal inverted repeats (94 to 131, 5313 to 5350) and the internal repeat (1550 to 1587) are underlined. Alsounderlined are potential Shine-Dalgarno sequences and the potential promoter sequences (indicated as -35 and -10). The predicted aminoacid sequence based on the nucleotide sequence is indicated above potential ORFs. Also pointed out are some of the key restriction enzymesites.

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phase was reextracted with an equal volume of phenol asdescribed above, followed by four extractions with an equalvolume of ether. We precipitated RNA by adding 10% of thevolume of 20%Wo sodium acetate and two volumes of ethanol.After overnight incubation at -20°C, the RNA was pelletedby spinning at 12,000 rpm for 10 min in an SS34 roto'r. Thepellet was suspended in water.The RNA was separated with a 1.0% agarose gel contain-

ing 2.2 M formaldehyde (35). The RNA was transferred tonitrocellulose (Micro Filtration Systems) with 3.0 M NaCl-0.3 M sodium citrate (pH 7.5) as the blotting buffer, and thetransfer was allowed to go overnight (35). The filters werebaked for 2 h in a vacuum oven at 80°C. Hybridization withprobe was at 65°C for 18 h. The probe was pAM225 DNAlabeled with [32PIdATP (800 Ci/mmol) with a nick translationkit purchased from BRL. After washing, the filters weredried and autoradiography was performed at -70°C with aDuPont Cronex intensifying screen. For molecular weightmarkers, pAM225 fragments generated by AvaI and EcoRIdigestion were deniatured and analyzed on agarose gels aswas the case for RNA.

RESULTS

Tn917 sequence. The entire sequence of Tn9J7, along withflanking DNA sequences, is shown in Fig. 2. The transposonis 5,257 base pairs long. Tn917 is 33% G+C, and the flankingDNA derived from pAD1 is 25% G+C. Since Perkins andYoungman (39) had previously sequenced the inverted ter-minal repeats of Tn917, identification of the transposontermini here was greatly simplified. In total agreement withtheir data, the inverted repeats of Tn917 are 38 base pairslong and are referred to here as LR38 (38-base-pair leftrepeat) and RR38 (38-base-pair right repeat). The repeats arenot identical and differ by 4 base pairs. Tn917 was shown byPerkins and Youngman (39) to generate a 5-base-pair dupli-cation on insertion. This particular insertion is flanked bytwo runs of six thymidines, five of which are presumed tocorrespond to duplicated target DNA (Fig. 2).A computer search (32) for ORFs yielded six likely re-

gions, all found on the same DNA strand. These are indi-cated in Fig. 2 and 3. Figure 2 shows the predicted aminoacid sequence of the six ORFs contained within Tn917, andTable 2 gives the relative amino acid compositions andcorresponding molecular weights for these potential pro-teins. Figure 3 diagramatically shows the location of theORFs within Tn917. ORFs 1 through 6 are each preceded bya sequence corresponding to Shine-Dalgarno (48) ribosome-bindihg sites. ORF5, which is the largest ORF, is not

ORF3

LR ORFI ORF2 ,IR ORF40o r | I1*f

o000

Af.AvoI HpaI

2000

preceded by such a sequence; it is noteworthy, however,that the stop codon for ORF4 is only 19 nucleotides from thestart codon of ORF5.Regions characteristic of a promoter (36, 42) are present at

locations preceding ORFs 1, 4, and 6 (Fig. 2). The potentialpromoter preceding ORFi is located 55 base pairs upstreamfrom the ORFi start codon and has characteristic -10(TATAAT) and -35 (TTGATA) sequences (42). ORFs 4 and6 both have the -10 sequence TATAAA; the -35 hexamerswere TTAATG and ATGCCA, respectively. In the case ofORF4, the "promoter" is only 5 base pairs upstream fromthe Shine-Dalgarno sequence; whereas the ORF6 promoterprecedes the ribosome-binding site by 167 nucleotides. It isnoteworthy that an octamer sequence (AGTTATGG) lo-cated 32 base pairs upstream from the ORF1 -35 hexamer isalso located within the ORF4 promoter (between the -10and -35 hexamers), and the first 5 base pairs of this octamercan also be found 4 base pairs upstream from the ORF6 -35hexamer. The significance of these sequences is not known,but their proximity to potential promoter sequences suggeststheir possible role in transcription initiation or control.As previously noted by Perkins and Youngman (39), the

right terminal repeat is capable of forming a stem-loopstructure that is reminiscent of a factor-independent tran-scription terminator. This potential transcription stop site isdownstream from all of the ORFs contained within Tn917.The internal sequence now reveals that 59 base pairs down-stream from the ORF3 translation stop codon is a perfect12-base-pair inverted repeat that is separated by 2 base pairs.This inverted repeat is followed by a string of fourthymidines, making the structure a potential factor-independent transcription terminator.

Identification of the methylase gene. Sequence data fromPerkins and Youngman (39) showed that a region of Tn917near the left terminus showed almost perfect homology (169of 170 base pairs) to the methylase (erm) regulatory region ofpAM77 that was sequenced by Horinouchi et al. (22). Acomparison of sequences here shows that the entire ORF2 isalmost identical to the pAM77 erm gene; it differs by only 4base pairs. Table 3 shows the nucleotide differences and thepredicted amino acid changes. Of the 4 base pair differences,one results in no change in the amino acid encoded. Theasparagine-to-serine and threonine-to-serine differences be-tween Tn917 and pAM77, respectively, mnaintain unchargedamino acid residues at the same site. The asparagine-to-lysine shift results in an uncharged amino acid being re-placed by a basic amino acid.Upstream from the erm gene ofpAM77 is a region capable

of encoding a short peptide believed to participate in trans-lational attenuation control of methylase synthesis (22). The

ORF5

3000 4000

KpjnI XhoI/AvaI Sa I Hindm AvaI

ORF6 RR' *

5000.a 1

r

Iiodm KbVI Ava IFIG. 3. Diagram of Tn917. The locations of the six ORFs are shown. Key restriction enzyme sites are indicated. All ORFs are on the same

DNA strand. The AvaI sites within the terminal inverted repeats are 7 nucleotides from the ends of the transposon. The location andorientation of the left, internal, and right repeats are indicated as LR, IR, and RR, respectively. Position numbers correspond to those of Fig.2. Although not apparent from the figure, the IR is actually almost completely within the 3' end of ORF 3.

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TABLE 2. Amino acid composition of ORFsa

No. of residues (%) present in ORF:Amino acid

1 2 3 4 5 6

Alanine 1(3) 5(2) 2(5) 9(5) 32(6) 12(6)Arginine 2(6) 8(3) 3(7) 13(7) 35(6) 17(8)Asparagine 2(6) 17(7) 2(5) 7(4) 32(6) 12(6)Aspartic acid 4(11) 7(3) 1(2) 13(7) 40(7) 13(6)Cysteine 0(0) 1(0,4) 2(5) 0(0) 6(1) 1(0.4)Glutamine 2(6) 16(6) 0(0) 7(4) 19(3) 3(1)Glutamic acid 0(0) 11(4) 1(2) 12(7) 40(7) 16(8)Glycine 0(0) 7(3) 4(9) 10(6) 17(3) 11(6)Histidine 0(0) 11(4) 1(2) 2(1) 14(2) 7(3)Isoleucine 1(3) 19(7) 1(2) 15(8) 48(9) 21(10)Leucine 3(8) 31(12) 8(18) 23(13) 66(12) 22(10)Lysine 4(11) 27(11) 6(14) 15(8) 45(8) 12(6)Methionine 2(6) 2(0.8) 1(2) 5(3) 10(2) 4(2)Phenylalanine 1(3) 12(5) 2(5) 7(4) 24(4) 10(5)Proline 1(3) 6(2) 1(2) 4(2) 14(2) 3(1)Serine 3(8) 15(6) 3(7) 17(9) 42(8) 17(8)Threonine 4(11) 19(7) 3(7) 7(4) 28(5) 14(7)Tryptophan 0(0) 2(0.8) 0(0) 0(0) 4(0.7) 1(0.4)Tyrosine 2(6) 12(5) 1(2) 5(3) 23(4) 8(4)Valine 4(11) 17(7) 2(5) 8(4) 19(3) 7(3)

a Total molecular weight was obtained with the use of a computer program(32). The molecular weights of ORFs 1 through 6 were 4,201, 28,785, 4,799,20,342, 64,780, and 24,119, respectively.

homology between Tn917 and pAM77 includes this leadersequence, with ORFi corresponding to the control peptide.The published sequence of pAM77 (22) extends 124 basepairs upstream from the start codon of the methylase controlpeptide. This entire region is homologous to Tn917, withdifferences at only 3 base pairs. We extended the upstreamsequence ofpAM77 another 125 base pairs (data not shown),and homology with Tn917 was found for another 72 nucleo-tides, at which point homology with the transposon ended.The region of pAM77 sequenced did not contain a copy ofthe terminal inverted repeat upstream from the methylasestructural gene.On the 3' side of erm in pAM77 is a region that potentially

encodes a protein made up of 43 amino acids (22). This exactsame region, corresponding to ORF3, is found in Tn917.Two base pairs after the stop codon of this potential peptide,the homology between these elements ends.Tn917 contains an internal repeat. Beginning in ORF3, a

sequence designated I1,38 (38-base-pair internal repeat) ispresent that is nearly identical to LR38 and is in the sameorientation. In addition to the 38 base pairs corresponding toLR38, the homology extends an additional 35 base pairs(Fig. 4); this sequence is designated IR73 (73-base-pairinternal repeat). The corresponding terminal sequence isdesignated LR73. There are four base pair differences be-tween LR73 and IR73 (Fig. 4). Only a portion of the internalrepeat (the equivalent of IR38) is present in pAM77. To theright of this region is where homology between Tn9O7 andpAM77 ends; beginning at this posint, however, pAM77exhibits homology to an extension of RR38. This similarityextends for 20 base pairs (R58 in Fig. 4). A 29-base-pairsequence (R29) on the 3' end of the region in pAM77 shownin Fig. 4 represents an inverted repeat of the first 5' 29nucleotides shown.ORF4 homology with Tn3 resolvase, Diver et al. (11) have

compared the resolvases of Tn3, Tn2J, TnSOJ, TnJ721, andthe Hin protein (Salmonella phase variation) and haveshown that these proteins share homology. The amino-terminal ends of these proteins show a much higher degree of

similarity than the rest of the molecule. ORF4 has a similarsize and exhibits significant homology to these recombina-tion enzymes; comparisons with the resolvase of Tn3,Tn501, and Hin are shown in Fig. 5. Of the first 15 residuesin ORF4, 12 are identical with residues in the Tn3 resolvase.Further homplQgy is scattered throughout the protein. Totalhomology between the ORF4 product and the Tn3 resolvaseis about 30%.Tn917 has a site homologous with the res site of Tn3. The

internal res site is the location where cointegrate intermedi-ates of an interplasmid tranposition event resolve by asite-specific recombination event catalyzed by the trans-poson-%Jetermined resolvase. The exact site of resolution hasbeen identified in Tn3 (16, 21). Interestingly, a 20-base-pairregion exhibiting about 80% homology with part of the ressite of Tn3 is present within the two directly repeatedsequences LR73 and IR73 (Fig. 4 and 6). There is a 1-base-pair difference between the res-like sequences in LR'73 andIR73. In the case of IR73, it is noteworthy that the pointwhere the res begins corresponds to the point where homol-ogy between Tn917 and pAM77 ends.

Potentially significant features of the sequence precedingORF4. The structure of Tn917 presented thus far suggestedthat drug-induced transposition may result from a transcrip-tional readthrough from the erm gene promoter into ORFs 4,5, and 6, which are presumed to encode transpositionfunctions. Consideration of this notion requires a closerexamination of the region preceding the start codon for ORF4. This region is focused on in Fig. 7 and 8. The latter reflectsa potential secondary structure that might be exhibited by acorresponding transcript of this region. Three adjacent stem-loop structures are shown and designated loops 1, 2, and 3.Loop 1 corresponds to the potentfal factor-independenttranscription termination site (see above). Within, and par-tially within, the 11-base-pair stem are two direct repeats(ATGGTAC). Loop 2 has a stem corresponding to 5 contig-uous base pairs with 14 nucleotides in the loop; calculationof the free energy values indicate that the stem would not, onits own, be thermodynamically stable (50). Loop 3 caninvolve up to 21 base pairs with some additional nonpairingbases occurring at several places within the stem; the loopcontains 39 nucleotides. The stem is followed by TTAA,which leads into the potential Shine-Dalgamo site for ORF4.It is noted that the potential independent promoter whichprecedes ORF4 occurs within the region corresponding tothe stem of loop 3.

In a region ppstream from the transcription terminationsequpnce (Fig. 7) are sequences resembling components ofthe nut site (13) that is found in bacteriophage lambda. Partof the lambda nut site includes a sequence called box A,believed to be important to the activity of the E. coli nusAprotein (15) which, in turn, is necessary for an antitermina-tion of transcription that also requires the N protein (a107-amino-acid, relatively basic protein). (There are other

TABLE 3. Differences between the erm determinants of Tn917and pAM77

Base no. in Base in Base in Amino acid Amino acidTn917a Tn917 pAM77 in Tn9O7 in pAM77

718 C A Asn Lys1011 A G Asn Ser1129 A T Arg Arg1130 A T Thr Ser

a Base number corresponds to that used in Fig. 2.

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A RR38 5'GGGGTCCCGAGCGCTTAGTGGGAATTTGTATCGATAAG* ***

LR73 5'GGGGTCCCGAGCGCCTACGAGGAATTTGTATCGATAAGAAATAGATTTAAAAATTTCGCTGTTATTTTGTACA* * * *

I R73 5'GAGGTCCCTAGCGCCTACGGGGAATTTGTATCGATAAGGAATAGATTTAAAAATTTCGCTGTTATTTTGTACA

B PAM77 5'GAGGTCCCTAGCGCCTACGGGGAATTTGTAATCGATAAGGGGTACAAATTCCCACTAAGCGCTCGGGACCCC* * * **

R58 5'GGGGTCCCGAGCGCTTAGTGGGAATTTGTAATCGATAAGGGGTACAAATTCCCACTAA

R29* * *

LR38 5 GGGGTCCCGAGCGCCTACGAGGAATTTGTAATCGATAAG

c Tn917LR

erm k

LR38LR73

IR

IR38IR73

5'ACAAATTCCCACTAAGCGCTCGGGACCCC

RR

RR38

pAM77 erm =

FIG. 4. The internal repeat of Tn917 and the corresponding region of pAM77. LR38 is within LR73, with both sharing the same 5' end.R58 is a 58-base-pair sequence whose first 38 base pairs correspond to RR38. R29 is a 29-base-pair sequence present in pAM77 that is invertedwith respect to the first 29 base pairs of the pAM77 sequence. A. The nucleotide sequences RR38, LR73, and IR73 are shown. * indicatesbase pair differences between adjacent sequences. B. The nucleotide sequences of homologous regions of pAM77 and Tn917 are shown. *

indicates base pair differences with pAM77. C. Diagrams show the homologous regions of Tn917 and pAM77.

examples of box A sequences found near the 3' end ofmRNAs [55].) There are actually three regions that make upthe nut site (13), designated boxes A, B, and C. Theconsensus box A sequence corresponds to 5'C/TGCTCTT(T)A3'. Box B corresponds to a region of dyad symmetry,and box C is a G+T-rich region. Sequences resembling allthree boxes can be found in Tn917 (Fig. 7). The box B regioncorresponds to inverted repeats which flank the Shine-

TN91Z M-*IFGYARVSTDDQNLSLQIDALTHTGIDKLFQELVTGALLDRPQLEEMINLLR EGDSWVIYKLDRTNI MRIFGYARVSTSQQSLDIGIRALKDAGV-K-ANRIFTDKASGSSTUREGLDLLRMKVEEGDVI LVKKLDRH IN MATIGYI RVSTIIDQNIDLQRiALTSAiRC----DRIFEDRI SGKIANRPGLKRALKYVNKGDTLVVWKLDRTNUQ1 MQGHRIGYVRYSSFDQNPERQLEQTQVS-------KVFTOKASGKDTQRPALEALLSFVREGDTVWVIHSMDR

TN917 ISRSTLHLIELSELFEELSVNFISIQDNVDTS--TS-MGRFFFPVMASLAELERDI tIEERTNSGLKAARVTNH LGRDTADMIIQLI I(EFDAQGVAVRFIDDGISTDGD ---MGQMWTI LSAVAQAERRRI LERTNEGRQEA'\LHI N LGRSVKNLVALI SELHERGAHFHSLTDSIDTS---SAMGRFFFHVMSALAEMERELIVERTLAGLAAARATN5Q LARNLDDLRR[VQKLTARGVRI EFLKEGLVFTGEDSPMANLNLSVMGAFAEFERALI RERQREGITLA('Q

TN91Z RGKKGGRPSKGKLSIDLALKMTDSKEYSI ROIL-----.DASKLNNNLLPLPQTNI KGI KFGRRRTVDRNVVLTLHQI'GTGATEIAHQL---SIARSTVYKI LEDERASHIN QGRLGGRPRAITKHEQEQISRLLEKGHPRQQLAI IFGIGVSTLYRYFPASSIKRMNTNSI RGAYRGRI\KALSDEQMTLRQRATAGEPKAQLAREFNI SRETLYQYLRTDD

FIG. 5. ORF 4 amino acid homology with other recombinases.The amino acid sequences of the resolvases of Tn3 and TnSOI andthe Hin protein are shown (beginning with the N terminus in eachcase) in comparison with the predicted amino acid sequence ofORF4. * indicates where ORF4 shares homology with at least two ofthree of the other proteins.

Dalgarno sequence of ORF3. The occurrence of sequencesresembling components of the lambda nut site suggests thatevents leading to antitermination might occur in this regionof Tn917.

Analysis of mRNA before and after exposure to erythromy-cin. RNA was isolated from S. faecalis DS16 cells whichcontain plasmids pAD1 and pAD2 (the original location ofTn917). The cells were grown under three different condi-tions: (i) no exposure to erythromycin and harvested at 80Klett units (mid-log phase); (ii) 0.5 ,ug of erythromycin perml added at 40 Klett units and cells harvested at 80 Klettunits; (iii) 0.5 pg of erythromycin per ml added at the time ofinoculation (<5 Klett units) and harvested at 80 Klett units.After electrophoresis on an agarose gel containing formalde-hyde, the RNA was transferred to a nitrocellulose filter andhybridized with a pAM225 probe. The results are shown inFig. 9. Lane 6, which shows no hybridization, is a DS16isolate (DS16C2) not containing pAD2. Three bands ofparticular interest are thos'e designated A, B, and C. Band C

TN917 "TNPR" - 5'CGAAAT-TTTTAAATCTATTC-ERM

TNO TNPA - 5'CGAAATATTATAAAT-TAT-C-TNPRFIG. 6. Potential res site of Tn917. A region upstream of ORF4

that has homology with the res 'site of Tn3 is shown. The orientationrelative to tnpR is the opposite for Tn917. * indicates differencesbetween the two sequences.

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S.D. STOP START(ORF3) (ORF2) (ORF3) 1478

GCAAGTATTGTCTATTTTTAATAGTTATCTATTATTTAACGGGAGGAAATAATTCTATGAGTCGCTTTTGTAAATTTGGA

----_-- ------- "boxA"A A'

"boxB" "boxB"

1558AAGTTACACGTTACTAAAGGGAATGTAGATAAATTATTAGGTATACTACTGACAGCTTCCAAGGAGCTAAAGAGGTCCCT

------"boxcC"- B1 1

STOP(ORF3) 1638

AGCGCCTACGGGGAATTTGTATCGATAAGGAATAGATTTAAAAATTTCGCTGTTATTTTGTACAATAAGGATAAATTTGAB' "res"

2 "boxA" 2

1718ATGGTACCATAAACGACCGTTTATGGTACTTTTCATTTTCCTGCTTTTTCTAAATGTTTTTTAAGTAAATCAAGTACCAA

C C' H G E4 4 4 ---- ____

_______------- 5 5

3 3_

S.D.(ORF4)1798

AATCCGTTCCTTTTTCATAGTTCCTATATAGTATACTTAATGAGTTATGGTACATTTAAATTATAAAATTAAGGAGGTTT---- ---- --------- G' H'6 6 ------ E' -----

F 5 F'

START(ORF4) 1838

TTTTATGATTTTTGGCTATGCTCCAGTGAGTACGGATGAT

FIG. 7. Possible control region of Tn917. Numbers identify direct repeats and letter sets indicate inverted repeats. Other importantsequences are also indicated.

was estimated to have a size of 1.2 kb. This approximates thesize calculated for an mRNA that would initiate at thepromoter upstream of ORFi and terminate at the potentialtermination site downstream of ORF3. The band shows adifferehce in intensity when cells unexposed to erythromycin(less intense, lane 1) are compared with cells exposed to thedrug (more intense, lanes 2 and 3). This band appears to bespecifically related to induction of resistance and may rep-resent an enhanced rate of transcription or a stabilization ofRNA owing to enhanced translation (or a combination ofboth). In the case in which cells were diug induced, a newband (band A), not detectable in the uninduced cells, wasapparent. Band A was estimated to have a size of approxi-mately 4.8 kb, corresponding to almost the entire length ofthe transposon and consistent with a transcript that initiatedat the promoter preceding ORFi and extended through thetertnination site after ORF3 proceeding on to the terminationsite at the end of the transposon. Band B is detectable inboth uninduced and induced cells and corresponds to a sizeof approximately 3.6 kb. It is possible that this band corre-sponds to transcripts initiated at the potential promoterpreceding ORF4 and extending to the end of the transposon.Its intensity does not differ significantly in induced and

unindUced cells. The nature of a 2.2-kb band located be-tween bands B and C is not clear. It may represent read-infrom an outside promoter or the use of an as yet unknowntermination site within the transposon. Lanes 4 and 5 repre-sent unexposed and drug-exposed cells, respectively, of S.faecalis FA3001. This variant of DS16 is characterized by anenhanced rate of Tn917 transposition (5); however, theobserved similarity to its parent implies that its alteredbehavior is not due to major differences in transcription.

DISCUSSIONThe sequence of Tn917 has revealed a number of signifi-

cant features. First, the transposon appears to have sixsignificant ORFs (designated ORFs 1 through 6), all of whichoccur on the same strand and are read from left to right (Fig.2 and 3). A consensus promoter site precedes a Shine-Dalgamo sequence that is close to the start site of ORF1.ORFs 4 and 6 are also preceded by potential promotersequences. The erm determinant and flanking regions areessentially identical to those of pAM77 previously se-quenced by Horinouchi et al. (22). This finding is in agree-ment with a receht report by Perkins and Youngman (39),who sequenced from the left end of Tn917 into the N

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A CLoop 1 G C T

C G C TA T G TA T T T Loop 2A T C TT A C CA T T TC G T AC G T AA T T AT A A T

AAATTTGAATGGCTTTTCGTTTT TTAAAAAA TATTT

G T AAT CA AGTACC AAAA

AA ATTTA CATGGTATTTAAGGA S.D. (ORF4)GGTTTTTTTAT Start (ORF4)GA

Loop 3

FIG. 8. Possible secondary structure. The stem-loop structure that can be formed by the region shown in Fig. 7 is shown. The AG, inthousands of calories (1 calorie = 4.184 J) per mole, of stem-loop 1 is -17.6; that of stem-loop 2 is +0.4; and that of stem-loop 3 is -3.9.

terminus of a reading frame resembling that of the pAM77leader sequence. The translational attenuation believed to beassociated with the control of pAM77 methylase induction isof a more complex nature than that of the staphylococcalpE194 (18, 23); within the 155-nucleotide leader regionpreceding the methylase structural gene is a control peptide36 amino acid residues in length (in contrast to 19 in thepE194 peptide) (18, 23). This region corresponds to ourORF1; as pointed out by the Weisblum group (22), thesequence appears capable of at least 14 possible classes ofoverlapping inverted complementary repeat sequences (incontrast to 3 in the pE194 control region), one of whichwould sequester the Shine-Dalgarno sequence which closelyprecedes the start codon of the methylase (ORF2 in the caseof Tn917). It was speculated that this greater degree ofcomplexity might relate to the ability of essentially all MLSantibiotics to induce, a phenomenon also observed for Tn9J7(unpublished data) but contrasting with pE194, which isinduced only by certain macrolides (54). (This behaviorprecludes the use of such drugs for the selection of mutantsthat constitutively express resistance.) The similarity be-tween Tn917 and pAM77 extends upstream from the ermgene; however, the latter does not contain a copy of theterminal repeat of the transposon in a corresponding loca-tion. The downstream similarity of the two elements extends2 base pairs beyond the stop codon of ORF3. The highdegree of homology between Tn917 and pAM77 suggeststhat the erm determinant (and flanking regions) on pAM77may have been the result of an abortive transposition or arecombination involving a Tn9J 7-like element. Alterna-tively, Tn917 may have evolved from a pAM77-like elementvia linking up with DNA having transposition functions.The occurrence of LR73 and IR73 flanking the ORFl-

ORF2-ORF3 cluster suggests a potential for recombinational

amplification or deletion events or both. The fact that thesetwo repeats each contain a region resembling the res of Tn3raises the interesting question of whether a transposon-encoded resolvase might participate in such events. The factthat deletions of the erm gene from Tn917 have not beenencountered suggests that this phenomenon, if it in factoccurs, does not occur at a high frequency. A recombi-national amplification system has been observed in S.faecalis in connection with tetracycline resistance plasmidpAMal, in which directly repeated 380-base-pair recombi-nation sequences flank the tet determinant (6, 7). In thatsystem, deletions of the tet determinant occur spontaneouslyat a frequency of about 1%; the possibility of a lowerfrequency of deletion of erm in Tn917 (perhaps due torecombination sequences [i.e., LR73 and IR73] that aremuch shorter?) has not yet been examined. Amplification-deletion systems are well known for a number of resistancedeterminants in gram-negative plasmids (12), and the tetdeterminant within transposon Tnl721 has been shown to beflanked by direct repeats in a similar fashion and to beamplifiable (46, 47). It should be kept in mind that, sinceinduction leads to a relatively high level of resistance (>2mg/ml for erythromycin), selection for elevated resistancearising from an increased gene dosage would not be ex-pected. However, in another host (species) in which induc-tion may not operate efficiently, the ability to amplify couldbe important.The existence of IR38 inverted with respect to the distal

terminal repeat (RR38) suggests that the intervening segmentmight be capable of independent transposition. An analo-gous situation is known to occur in the case of Tnl721, inwhich an internal minor transposon designated Tnl722 ispresent (46, 47).We have noted a similarity between the predicted protein

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1 2 3 4 5 6 7 8 9 Between ORFs 5 and 6(25 kilodaltons) is a span of over 300nucleotides with no apparent coding capacity. The relation-ship, if any, of this region to the control of transposition isunknown.The presence of a ribosome-binding site preceding the

start site ofORF6 implies that translation of the latter occursindependently of ORFs 4 and 5. The upstream occurrence ofa potential promoter raises the possibility that expression ofthis protein can be governed independently. The recentconstruction and examination of a mutation at the XbaI sitein the N-terminal region ofORF6 has shown that this gene isessential for transposition (manuscript in preparation).

AM" Z The sequence data presented here clearly confirm thatTn917 is a member of the Tn3 family (20, 21, 28) oftransposons. Tn917 shares many common features withthese other transposons, including generation of a 5-base-pair duplication on insertion, sequence homology within theterminal inverted repeats, amino acid homology of theresolvases, and homology at a potential res site. The pres-ence of an internal copy of the terminal inverted repeat,resulting in flanking of the resistance determinant by directrepeats and defining a potential minor transposon, resemblesa feature also present in Tnl 721 (a member of the Tn3family). In addition, erythromycin-enhanced transposition ofTn917 may resemble mercury-enhanced transposition ofTnSOJ (45) (also a member of the Tn3 family).

FIG. 9. Analysis of Tn917-specific transcripts in S. faecalis. Mechanism of erythromycin-enhanced transposition. TheRNA was separated electrophoretically on an agarose gel, blotted to sequence of Tn917 suggested that erythromycin-enhanceda nitrocellulose filter, and hybridized to nick-translated pAM225. transposition might occur as a result of a downstreamLanes 1 to 6 were loaded with 10 ,ug of RNA. Lane 1 contains RNA extension of the erm-related transcription unit, and an ex-from DS16 (contains pAD1 and pAD2) cells not exposed to eryth- amination of RNA synthesized in both the absence andromycin. Lane 2 contains RNA isolated from DS16 cells that had presence of erythromycin revealed data consistent with thisbeen exposed to erythromycin from the time of culture inoculation. interpretation. A Tn9J7-specific transcript approximatingLane 3 contains RNA from DS16 exposed to erythromycin at 40 the entire length of the transposon was detectable only afterKlett units. Lane 4 contains RNA from S. faecalis FA3001 not exposure tonthe tra n w a ting terexposed to erythromycin. Lane 5 contains RNA from FA3001 exposure to the antibiotic. An RNA approximating the sizeexposed to erythromycin at 40 Klett units. Lane 6 contains RNA of a transcription unit corresponding to ORF4-ORF5-from DS16C2 (devoid of pAD2). Lane 7 contains 36 jig of the same ORF6 was also detected, but at similar levels in the absenceRNA used in lane 1. Lane 8 contains 20 ,ug of the RNA used in lane and presence of erythromycin. Conceivably, the latter tran-3. Lane 9 contains 25 ,ug of the RNA used in lane 4. The arrows script is governed by the promoter preceding ORF4.point to transcripts estimated on the basis of molecular weight A transcription unit corresponding in size to ORFl-markers (not shown; see Materials and Methods) to have sizes of 4.8 ORF 2-ORF3 (erm expression) was detectable in the absence(A), 3.6 (B), and 1.2 (C) kb. of the drug but greatly enhanced in its presence. Since

induction of resistance is believed to be posttranscriptionalsequence of ORF4 (20 kilodaltons) and specific recombi- (22), the extent to which this enhancement is due to in-nases of other systems (e.g., Tn3 resolvase). The occurrence creased transcription or stabilization of RNA (owing toof a Tn3-like res between ORFs 3 and 4 adds more to the enhanced translation?) or both is not known.likelihood of this protein being a resolvase. In addition, One straightforward explanation for drug-induced tran-Perkins and Youngman reported (39) that insertions of a scriptional extension is that the transcription terminatorheterologous segment of DNA into the KpnI site just to the located downstream from the ORF3 stop site always allowsright of the putative res site significantly reduced the ability a certain low level of readthrough, and that increased levelsof intermediate cointegrate structures to resolve in B. subti- of the extended transcript result from an increase in tran-lis. It is also noteworthy that a sequence that precedes the scription from the erm-related promoter. It is not apparent,-10 promoter region of ORF4 bears some resemblance however, what the basis for enhanced transcription at the(ITTTAAATT) to a central portion of the potential res site erm promoter would be, if it does in fact occur. It is(TTTAAAAATTT). Perhaps the ORF4 product recognizes conceivable that the ORF3 product, whose synthesis isthis region and participates in autorepression similar to that probably affected by the translationally induced ORF2 (erm)believed to occur with the resolvase of Tn3 (16). product, acts as a positive regulator at this promoter.The ORF5 (65-kilodalton) segment appears somewhat Another hypothesis has extended transcriptional read-

dependent on the ORF4 sequence for translation, since there through being controlled by events blocking transcriptionis no Shine-Dalgarno sequence preceding the translation termination. The presence of upstream sequences (boxes A,start site. It is likely that translation of the two reading B, and C) resembling those known to be involved in tran-frames is coupled or closely coordinated. An insertion into scription antitermination in bacteriophage lambda (13) addthe SalI site (Fig. 3), which is in the N-terminal region of credence to such a possibility. A possible candidate forORF5, has been reported to prevent transposition (39), and antitermination activity would be the ORF3 product (analo-a deletion of DNA between the two HindIII sites, which gous to lambda N protein), whose increased synthesis aris-includes most of ORF5 (Fig. 3), also blocks activity (39). ing from its coupling to the translationally induced erm

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product, leads to enhanced antitermination. It is noteworthythat, like the N protein of lambda, the ORF3 product is arelatively basic protein (Table 2).Whatever the function of the ORF3 product, it should be

kept in mind that its determinant is also present adjacent tothe erm determinant in pAM77 (22), in which transposition-related genes presumably do not exist. This might arguemore for a role of the ORF3 product in erm expression andwould be consistent with the first model presented above(i.e., that in which the ORF3 product acts as a positiveregulator of transcription). However, it does not rule out thepossibility of transcription antitermination activity useful togenes (of unknown function) downstream on pAM77 or thatit is simply unutilized because the pAM77 segment mighthave recently evolved from a Tn917-like structure via dele-tion or recombination resulting in a loss of the adjacentsequence. Finally, it is possible that the ORF3 product playsno role at all in transcription. Since it has its own ribosome-binding site, it might act to protect the 3' end of the ermtranscription unit as a result of ribosome stalling in thepresence of inducing drug concentrations. Additional workwill be necessary to distinguish between these or otherpossible explanations for the role of the ORF3 product andthe control mechanism of drug-enhanced transposition ofTn917. The numerous direct and inverted repeats present inthe region preceding and including the transcription termi-nation sequence following ORF3 suggests that this region isrelatively active. It is also interesting that the 7-base-pairdirect repeat that occurs within and partially within the stemof the transcription termination loop could give rise torecombinational amplification or deletion, resulting in astronger terminator or loss of the terminator, respectively.

ACKNOWLEDGMENTSWe thank Wes Dunnick and Michael Mowatt for technical advice

and assistance. We thank D. Friedman, J. Perkins, and P. Youngmanfor valuable comments and observations. We also thank C.Gawron-Burke, E. Ehrenfeld, F. Macrina, and F. An for helpfuldiscussions.

This work was supported by U.S. Public Health Service grantsDE02731 and A110318 from the National Institutes of Health. J.H.S.was supported by a U.S. Public Health Service predoctoral traininggrant (genetics).

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