PLASMID lo,25 l-259 (1983)

Complete Nucleotide Sequence of pT181, a Tetracycline-Resistance Plasmid from Staphy/ococcus aureus

SALEEM A.KHAN*" ANDRICHARD P. NOVICK?

*Department of Microbiology, University of Pittsburgh, School of Medicine. Pittsburgh, Pennsylvania 15261, and tDepartment of Plasmid Biology The Public Health Research Institute of the

City of Nenl York, inc., New York, New York 10016

Received July 21, 1983

pT 18 I is a naturally occurring Staphylococcus aureus plasmid, encoding inducible resistance to tetracycline. The plasmid has a copy number of about 20 per cell, and belongs to the incom- patibility group inc3. The complete nucleotide sequence of pT I8 1 has been determined and consists of 4437 bp. The nucleotide sequence contains 69.8% A-T and 30.2% G-C pairs. pT 18 1 was found to contain four open reading frames capable of coding for polypeptides containing more than 50 ammo acids. All the putative polypeptides are coded by one strand. The molecular weights of the four putative polypeptides are (in daltons): A, 37,500; B, 35,000; C, 23,000, and D, 18,000. Polypeptide A corresponds to the repC protein, earlier shown to be specifically required for pTI 8 1 replication. Polypeptide B (and possibly polypeptide D) are involved in tetracycline resistance. No role has yet been established for polypeptide C; deletion of the coding sequence for the C polypeptide has no detectable effect on any property of the pTl8 1 plasmid. A region consisting of about 1200 bp contains information for the replication and copy number control of this plasmid. The sequencing results are discussed in relation to the replication properties and tetracycline resistance associated with the pT I8 1 plasmid.

pT18 1 is a small, naturally occurring tet- racycline resistance plasmid from Staphylo- coccus aureus. It is very similar to many other Tc’ plasmids like pT I27 and pSN1 isolated from S. aureus and Staphylococcus epider- midis (Iordanescu et al., 1978; Shafferman et al., 1978; Michel et al., 1980). Previous studies have identified and mapped the replication origin (Khan et al., 1982), repC, the deter- minant of a protein uniquely required for rep- lication (Khan et al., 198 1; Novick et al., 1982), inc3A, a determinant of incompatibility and copy control (Novick et al., unpublished results), and tet, a gene required for tetracy- cline resistance (Novick et al., 1982). To un- derstand the functional organization of this plasmid, we have determined its complete nu- cleotide sequence and here report these results

’ Author to whom all correspondence should be ad- dressed.

together with the composite functional map of the plasmid as it now stands.

MATERIALS AND METHODS

(a) Bacterial Strains and Plasmids

The S. aureus strains used were RN3259 (8325-4 (pT181)) and RN3268 (8325-4 (pT181 cop608)) (Khan et al., 1981). Media and growth conditions were as described (Novick and Brodsky, 1972).

(b) Enzymes and Chemicals

Restriction enzymes used were purchased from either Bethesda Research Laboratories (BRL) or New England Biolabs (NEB). Bac- terial alkaline phosphatase was from BRL. T4 polynucleotide kinase was a gift from Dr. Z. Humayun. [y-‘*P]ATP (specific activity 3000 Ci/mmol) was purchased from New England Nuclear. All other chemicals were from stan- dard commercial sources.

251 0147-619X/83 $3.00 Copyrigbl Q 1983 by Academic Press. Inc.

All ngbts of reproduction in any form reserved.

252 KHAN AND NOVICK

(c) Preparation of Plasmid DNA, Restriction Enzyme Mapping, and End- Labeling of DNA Fragments

Plasmid DNA was isolated by standard procedures involving CsCl-ethidium bromide density gradient centrifugation of cleared ly- sates (Clewell and Helinski, 1969). Plasmid DNA was digested with restriction enzymes in the buffers specified by NEB or BRL. Re- striction fragments were isolated by prepar- ative 4 or 5% slab polyacrylamide gel electro- phoresis using Tris-borate-EDTA buffer, pH 8.3 (Greene et al., 1974). DNA fragments were recovered from the gels as described (Maxam and Gilbert, 1980). Restriction fragments were dephosphorylated by alkaline phosphatase and then rephosphorylated with [y-32P]ATP and polynucleotide kinase. After subsequent digestion with appropriate restriction enzymes or strand separation, the S-end-labeled frag- ments were purified by polyacrylamide gel electrophoresis, autoradiography, and elution (Maxam and Gilbert, 1980).

(d) DNA Sequence Determination

DNA sequencing reactions were done as described by Maxam and Gilbert ( 1980). Di- methylsulfate was used for G reactions, formic acid for A + G, hydrazine for C + T, and hydrazine in the presence of 5 M NaCl for C reactions. The cleavage products were elec- trophoresed on 0.04 X 40-cm polyacrylamide gels (20% and 8%) containing 8.3 M urea and were autoradiographed on Kodak X-Omat XR-5 film. In most cases both strands (and overlapping fragments) were sequenced.

RESULTS AND DISCUSSION

The restriction map of the pT 18 1 plasmid was determined by digestion with restriction enzymes either separately or in combinations (Novick et al., 1982). A simple restriction map of pT 18 1 showing the regions involved in rep- lication, tetracycline resistance, and four open reading frames, as revealed by the sequence data, is shown in Fig. 1. Initial sequence de- terminations were made by using these re-

~Tl81

4437 bp

FIG. 1. Restriction endonuclease map of pTl81 DNA (4437 bp). The index point of the map is the single PvuI site and the distances are given in kilobases (kb). Location of the origin and direction of replication are shown. Also indicated is the copy control region and four open reading frames coding for polypeptides A, B, C, and D.

NUCLEOTIDE SEQUENCE OF pT18 1 PLASMID 253

PVUI Mb01 HpaII TaqI DdeI RSaI HpaI HhCII Hind111 KpnI XbaI AhI HinfI

1 500 1000 1500 2000 2500 3000 3500 4000 4437

I I I I I I I I I J

BASE PAIRS

FIG. 2. Sequencing strategy for pT 18 1. Arrows indicate the direction and extent of sequence determination from the S-end of restriction fragments. A, B, C, and D represent the open reading frames.

striction sites. New restriction sites revealed by the initial sequencing were subsequently used to determine the rest of the nucleotide sequence of the plasmid. The sequencing strategy and the extent of sequence determi- nation from each of the 5’-labeled ends are shown in Fig. 2. The complete nucleotide se- quence of pT18 1 is shown in Fig. 3. Parts of this sequence have been published earlier (Khan et al., 1982; Novick et al., 1982). The DNA sequence was analyzed by the NIH- Stanford Molgen Program for analysis of DNA sequences. This revealed the presence of four open reading frames (larger than 50 amino acids) capable of encoding polypeptides A, B, C, and D, having molecular weights of 37.5, 35,23, and 18 kDa, respectively. These coding sequences are also indicated in Fig. 3.

The Replication Region

A number of studies have been directed toward the understanding of the replication properties and copy control mechanism of the pT 18 1 plasmid. Cloning experiments, includ- ing insertional inactivation and in vitro dele- tions of the pT 18 1 replication region have lo- cated a region of about 1200 bp* (between

’ Abbreviations used: kb, 10’ base pairs; bp, base pairs; Tc, tetracycline; TC: tetracycline resistance.

nucleotides 3600 and 400 on the pT 18 1 map) that is involved in replication and its control (Khan et al., 1981, 1982; Novick et al., 1982; unpublished observations). The replication region has been shown to contain the coding sequence for the repC protein (polypeptide A) that is specifically required for pT 18 1 repli- cation (Novick et al., 1982). On the basis of transcription mapping (C. Kumar and R. P. Novick, unpublished observations) and the known DNA sequence, the putative - 10 and -35 sequences (Rosenberg and Court, 1979) for the repC mRNA are located at positions 322-328 and 345-35 1, respectively. The pu- tative ribosomal binding site (Shine and Dal- garno, 1974) for this protein is located at nu- cleotides 162- 168 and there is an open reading frame between nucleotides 15 1 to 3647 (counterclockwise orientation) coding for a 3 14-amino acid polypeptide corresponding to a molecular weight of 37,500 Da (Novick et al., 1982). The replication of pT18 1 is uni- directional and counterclockwise on the cir- cular map of the plasmid, with the functional origin, as defined by deletion analysis, lying between positions 16 and 183 and the start site between nucleotides 31 and 158 (Khan et al., 1982). The replication origin is con- tained within the coding sequence for the repC protein.

254 KHAN AND NOVICK

Pvul,Mbol Mb0 I IiyaJI HJgl CGATCGTCATTCCATCAAAACTTAATTTTGCATCAGATATGCACACAGTATGTGCGTCCAACCGGCTATTAGAGTAGCCGGTTTTAGAAAAATTGTCTAA

Taql/Hi"fI Hinfl ATCGTGATTTTCCAAATGATTTGAATGATTGCATGCATGATTGTTTTTATACATAAAAAATCGACTCCTTAATCTCAATTTCGTTTAAGGAATCGCTCACCCA

TAGCACTAAAAGCTTTACTPAACTTACTAAACGTACTAA~AAAAATATGTATTTTTTAG~TGAGGAATTAGAGTTAAAG~AAATTCCTTAGCGAGTGGG?

AspHisAsnGluLeuHisAsnSerHisAsnAlaHisAsnAsnL~sTyrMet START A m

MeI AATATATATCTTGATGTATATTTAAATATCGTTTAATATCTAAATATACAAGATTATAAAAACAACTCAGTGTTTTTTTCTTTGAATGATGTCGTTCACA TTATATATAGAACTACATATAAATTTATAGCAAATTATAGATTTATATGTTCTAATATTTTTGTTGAGTCACAAAAAAAGAAACTTACTACAGCAAGTG~

Hinfl HbOl AACTTTGGTCAGGGCGTGAGCGACTCCTTTTTATTTTG~ATTAATATAACACTATCAAAAGATTTGGTCTAATCAGATCAAGTCTTTTTTTATTTAAGC TTGAAACCAGTCCCGCACTFGCTGAGGAAAAATAAAACA~TAATTATATTGTGATAGTT?TCTAAACCAGATTAGTCTA~TTCAGAAAAAAATAAATTC~

Rsal ATTTGTATTATCTGGTAAACAGTTAAAACTACTAAAACACCAAGTACATACTTACTTGTTATAAAATTCTCAAAGGCTTCTCTGAATTTCATTTCTTCAA

TAAACATAATAGACCATTTGTCAATTTTGATGATTTTGT~GTTCATGTATGAATGAACAATATTTTAAGAGTTTCCGAA~AGACTTAAAGTAAAGAAGTT

Ria CTCCTTTTATTGCTTTATTCCAATTTCCTTATTGGTCCGTTCAAG GAGGAAAATAACGAAATAAGGTTAAAGGAATAACCAGCCTTGGATGTCCCGAAATACACAACAACTCAACCATGAAAGAACCCTAATTAGGGTTAAGTTC

AlUl TCCAACCAACTCGCTAACAAGTTAGCTAACACATAGCCCATTCCAACCAATAAGTTTTCTCGGCATAAATGCATGTTCTATAAGTGCATACCTCTATCTC AGGTTGGTTGAGCGATTGTTCAATCGATTGTGTATCGGG~AAGGTTGGTTATTCAAAAG~GCCGTATTTACGTACAAGA~ATTCACGTATGGAGATAGA~

C ENDLeuHisMetGlyArgAspAr

HinfI Hinfl Me I Al"1 GTTTTTCATTCTTTTGTTGTTCTTGTTGCTGTTCCTGTTCTGAATATAATTCTGGATTCATTTTTTTATCTAAGTTTCCTGCTAAACCATTT~TTGG

CAAAAACTAAGAAAACAAC4AGAACAACGACAAGGACAAGACTTATATTAAGACCTAAG?AAAAAAATAGATTCAAAGG~CGATTTGGTAAATCGAAAC~

gLysGl"As"LysGl"Gl"GluGl"Gl"Gl"G~~Gl"Gl~S~rT~rL~~G~~ProAs"~~tLvsLvsAspLeuAs"GlyAl~LeuGlvAs"LeuL~~Pr~

Ddel Alul TCTCTCGTATTTCCTTATCTTTATCATCTAAGGCTCTACCAGACTTTATATACTCGTAATCTTCCGAAATATCTTGAGCAGCTTTTATCTGTTTCTGAAA

AGAGAGCATAAAGGAATAGAAATAGTAGATTCCGAGATGGTCTGAAATATATGAGCATTAGAAGGCTTTATACAACTCGTCGAAAATAGACAAAGACTTT

rGluArglleGluLysAspiysAspAspLeuAlaArgGliSerLyslleTyrGluTyrA;pGluSerIleAspGl"Ala~laLyslleGl"LysGl"Ph~

Alul TTCATTGAAATCTTTTTGGCTTATTAC~CATTTCCAGCTTCTTGTATCTCTTTGCTAAATAAACCACCTACTTTTTCAGTTTCTTGCTCATACGGAACA

AAGTAACTTTAGAAAAACCGAATAATGTTCTAAAGGTCGAAGAACATAGAGAAACGATT~ATTTGGTGGATGAAAAAGTCAAAGAACGAGTATGCCTTG~

GluAs"PheAspLysGl"S~'rlleValValAs"GlyAladluGl"lleGluLysSerPheLeuGlyGlyValLysGluTh'rGluGl"GluTyrProValA

Rsal TTTATAGGCTTTTTAAGCGTATTTAACGATTTTTGGTACTCTTGCATTAATTTATCGTTCTTTTGCTTTATATGGTCTGTTTTTTGGCTCTCACGTTCAT AAATATCCCAAAAATTCGCATAAATTGCTAAAAACCATGAGAACGTAATTAAATAGC~GA~ACGAAATATACCAGAC~~AACCGAGAGTGCAAGTA

s"~leProLysL~sL~~Th~AsnLeuSerLysGlnTyrCi~Gl"~~t START C S

ATTCTTGCTTATGATATTCTGTTTTTTGTTTATACTGACTTATTTGCTCATGTTTAGCATTTGTTACTTGTCTTGATTGCCCACGTTCT~ACCATATCC TAAGAACGAATACTATAAGACAAAAAACAAATATGACTGAATAAACGAGTACAAATCGTAAACAATGAACAGAACTAACGGGTGCAAGATTTGGTATAGG

Hi"d,lIlAlul TCGTTGTTTAACATGCTCATTAAATCTATCTTGAAACGCTGTTAAAGCTTTTTATTACCTACAACTTCTTTAGCACTTAAACGACCATCATCAAGTATTG AGCAACAAATTGTACGAGTAArTrAGATAGATAGAACTTTGCGAC~TTTCGA~AATAATGGATGTTGAAGAAATCGTGAATTTGCTGGTAGTAGTTCATAA~

100

200

300

400

500

600

700

800

900

IO00

1100

1200

1300

I400

1500

FIG. 3. Complete nucleotide sequence of pT 18 1 DNA, numbered from the single Paul site. Also shown are the presumed ribosomal binding sites and the potential amino acid sequences specified by open reading frames A, B, C, and D.

Circumstantial evidence suggests that the protein binds to a 50-bp region within the repC protein is rate-limiting for pT 18 1 plasmid origin of replication (W. Rosenblum and S. replication (Novick et al., 1982). The repC Khan, unpublished observations). Synthesis

NUCLEOTIDE SEQUENCE OF pTl8 I PLASMID 255

Thai

TATTCAAAAAACTGCTTTGTATCTTCTGGCGTTTGATTATCAAAGAAATCATTGTCTGATGTAATTAAACCATCAATGTGTTTAAT~TCTGTTCTAA ,,C,O ATAAGTTTTTTGACGAAAC~TAGAAGACCGCAAACTAAT~GTTTCTTTAGT~CAGACT~CATTAATTTCGTAGGTACA~~ATTACtCCAGACAACA~

x TTTTTCTTTTGCCTGTATAATTCTGTTCGATTTTTTCATCAATCAAGTTATTAAAATTCTGTTTATTACCATTTACCAAATCATAATTTAAGTAACTTTT AAAAACAAAAcGCACATATTAAGACAAGCTAAAAAAGTAGTTAGTTC~TAATTTTAA6AC~AT~TCGT~ATGGTT?AGTATTA~TTCATTCAA~

I800

ACTATCGTCTATATCTTCATTTTCATAATTATTATTTTCTCTTTGAACATLTTTTTGTATGCCCGTTGTATTTGTTCCACATTTAACTTTTGAAACTCTA TCATACCAGATATACAAGT~AAGTATTAATAATAAAAGAGAAACTTGTACAAAAACAT~CGGGCAACATAAACAAGGTCTAAATTGAAAACTTTGAGAT

I900

Ddel ACAATGGAATAAtACATATAAAAATCACTC~CACAAAGGTTTTATGTGTATTTAATAC~TAAGTCTAACACACTAGACTTATTTTTAATAAGTCG 2ooo TCTTACCTTATTCTGTATATTTTTACTGAGCATTCGTGTTTCCAAAATACACATAAATTATCAAATTCAGATTGTtTGATCTGAATAAAAATTATTtAG~

TTAATACGTGTGCTCTGCCAGCCTTAAACCTGTTTTTGCTAACGCAARAAATTGCTAGCCACTCATAGTTCTAAACCAAAATATAATAT 2,00 AATTATGCACACGACACGCTCCGAATTTGGACAAAAACGATTGCGTTTTTTACTCACCG~TTTACGATCGGTGAGTATC~GA~TGGTTTTATATTAT~

AACTATTCAAACTGCTTTTCAGAACCTTTTAAATACAATAATCGTCAAAACACAACTTAAATAATAAttTTAGCCATtGCTACAGAATATTACTATACAC 2200 TTGATAAGTTTGACGAAAACTCTTGCAAAATTTATGTTATTAtCAtTTTTCTGTTGAATTTATTATTCC~TCGCTACC~TGTCTTATAATGATATGTG

0 ENDLeulleAsnSerTyrVal

ACTTTAATAAGAAAACGTATAGTTdCTATTGTAATCATAGGAAGTATAAGTAGGTAAGACCAATGAATATAT~TGTGCTATT ACTAAATGGTCCATGCTAA~G~ATTATTCTTTTCCATATCAATGATAACATTAtTATCCTTCATATTCATCCATTCTGGTTdCTTATATTACACGATAA 3000

SerLysGlyProValHetValLyslleLe"PheProlleihrValIleThrllenetPr~Le"lleLe"Le"TyrSerT~pHisIleTyrHisAlallei

FIG. 3-Continued.

of the repC mRNA may be attenuated by a rho-dependent mRNA termination structures termination structure shown in Fig. 4. This found in Escherichia coli (Rosenberg and G-C rich stem-loop structure followed by a Court, 1979). The synthesis of the repC protein stretch of Us (positions 152 to 192) resembles may also be regulated at the level of translation

256 KHAN AND NOVICK

Alul "b0 I Al"1 ATTCCCCCTATTCAAGGACCTAACCCTTCACCTA~~ACAATTGATCCTATAAAACCAAAGGCTTTGCCTTGTTTTTTTCTTGTAATATTTCTAGCTA 3100

TAAGGGGGATAACTTCCTGGATTCGGGAAGTGGATTTCGATGTTAACTAGGATATTTTGG~TTCCGAAACGGAACAAAAAAAGAACATTATAAAGATCGAT

leGlyClylleSerProGI;LeuClyCluGl~L~~AlaVdlIleSerClyllePheGlyPheAlaLysClyGinLysLy;ArgThrileAsnArgklaV~

ATACA~TA~AAATGAAAGAATA~~AGC~~AATAAAA~G~TATAAAAC~A~~TTTAAATTTTTTATATAAACTAAA~~TTTTAC~T~TTTA~TTGA 3500 TATGTGATTTTTTACTTTCTTATGTTTCGGTTTTATTTTGCGATATTTTGTTTGGA~TTTAAAAAATATATTTGATTTGTTAA~TGGAGAAATGAAC~

uValSerPhePheSerLeuileC.,sLeuTrpPheLeuValSerTyrPheLeuGlyLysPh'=L~~L~~T~~L~"S~~Phe" START B S.D.

GGTGACTAAAGTTTATAGGGGTAAATTATTATTCTTTCACCTCATAT~ATTCCCCAAATTTTTAAATAGACACTTCATAAAAAAATCCTCCTAAATTAGAAT 3600 CCACTGATTTCAAATATCCCCATTTAATAAGAAAGTGGAGTATATTTAAGGGGTTT~AAATTTATCTGTGAAGTATTTTTTTAGGAGGATTTAATCTTP

Rsa I Clal/Taql AGTTTAAATATATCATTTTTGTTCAGTAATATTAATAT~ACTATTTCCAAAATTTAAATTCATGTTGCCAAAAATCGATTTGTTTTTGCAATTGTTT 3700 TCAAATTTATATAGTAAAAACAAGTCATTATAATTATACATGTGATAAAGGTTTTA~TTTAAGTACAACGGTTTTTAGCTAAACA~AACGTT~CA~

A ENDLysTrpPheLysP~~CluHisGl"T~pPheAspl'l~Gl"L~~Gl~Le~Gl~L~~

TTATATTTTGTTTTAACCAAATCATTTCATCTCGTGTAAGTTTGTTTGGATTMATTCAATACGCATATTACGTCTATCCCAACTATCTGCTTTCACTTT 4300 AATATAAAACAAAATTGGTTTAGT~AGTAGAGCACATTCAAACAAACCTAATTTAAGTTATGCGTATAATGCAGATAGFGTTGATAGACGAAAGTGAAA

elleAsnGlnLysLeuTrpile~etGluAspArgThrLe;LysAsnProAsnPheClulieArg~etAsnArgArgAspTrpSerPispl\laLysValL~~

AlUl GTCATATTCAATATAAACTTTTTCTTGAAGTGCTTT~TTAAACTTTGTTTGAAGAATATCCCAAAGTCTTATTTGGGGCTCTACACTCATAAATTTA 4400 CAGTATAAGTTATATTTGAAAAAGAACTTCACGA~TCGAAATTTGAAACAAACTTCTTATAGGGTTTCAGAATAAACCFCGAGATGTGAGTATTTAAAT

AspTyrG,u,leTyrVaIL;sGl"GlnLeuAlalysAlaLysPh~L~~Th~GlnLeulI;AspTrpLeuArgIleClnProGluValSer~etPheLvsS

GAAAGGGCTTGAGCGTTGTCTCGGTTGAGATTTCCAA CTTTCCCGAACTCGCAACAGAGCCAACTCTAAAGGTT 4437

erLeuAlaGlnAlaAs"AspArgAsnLeuAsnGlyVa

FIG. 3-Continued.

since the ribosomal binding site (Shine and involved in the control of pT 18 1 replication Dalgarno, 1974) for this protein is buried in which is thought to be by the regulation of a base-paired region (Fig. 4). Sequences in the the rate of repC protein synthesis and this long untranslated leader of the repC gene are region codes for a small, 80-nucleotides-long

NUCLEOTIDE SEQUENCE OF pT181 PLASMID 257

UA

I

AU AU

I AU CU UA.190

160.~1~ GC

3'...CAAAAAUAUGzUUUUUUA GAGUGGGUUU...5'

150 200

FIG. 4. Structure of possible transcription termination sequence in repC leader region. SD is the putative ribo- somal binding site for translation of the repC protein, initiating with the AUG codon at position I5 I.

RNA which is likely to be involved in copy control (Novick et al., 1982; C. Kumar and R. Novick, unpublished observations).

Tetracycline Resistance Region

The tetracycline resistance encoded by pT181 is inducible (Iordanescu, 1979). Ex- amination of the DNA sequence in this region revealed two open reading frames capable of coding for 295 amino acids (polypeptide B, molecular weight 35 kDa) and 152 amino ac- ids (polypeptide D, molecular weight 18 kDa), respectively. It should be noted that the pos- sible initiating codon for polypeptide B is UUG which has earlier been identified in Gram-positive bacteria, including S. aureus (McLaughlin et al., 1981; Lofdahl et al., 1983). This is supported by the presence of a strong ribosome binding site (Shine and Dalgamo, 1974) (positions 3494-3487) close to the UUG codon and the absence of strong ribosome binding site next to internal AUG codons (Fig. 3). Studies on the expression of tetracycline resistance in E. coli have demonstrated the involvement of at least two proteins: an in- ducible 36K membrane protein (TET protein) which is required for resistance, and a 23K repressor protein that acts as a negative reg- ulator of its own synthesis and that of the TET protein (Yang et al., 1976; Curiale and Levy, 1982; Altenbuchner et al., 1983). Examination of the amino acid sequence of polypeptide B

reveals an abundance of hydrophobic amino acids (Fig. 3). It is likely, therefore, that this protein is also a membrane protein (similar to the TET protein). The KpnI site lies within the structural gene for polypeptide B (position 29 1 l-29 16, Fig. 3). Disruption of this coding sequence at the KpnI site results in the loss of Tc’ (Novick et al., 1982) showing that this protein is involved in Tc resistance. Polypep- tide D (18 kDa) is also hydrophobic and is encoded by a region close to the KpnI site. Based on the DNA sequence, there may be a short overlap in the genes coding for poly- peptides B and D. Whether polypeptide D is also involved in Tc resistance and/or corre- sponds to the repressor protein in E. coli is not yet known.

A translational attenuation mechanism has been proposed for the ermC (ribosomal meth- ylase) system involved in erythromycin resis- tance coded by the pE 194 plasmid (originally isolated from S. aureus) in Bacillus subtilis (Gryczan et al., 1980; Horinouchi and Weis- blum, 1980; Shivakumar et al., 1980). The main features of this model include the pres- ence of a leader with extensive potential sec- ondary structure, and the possible translation of a short polypeptide within the leader se- quence. This model of translational atten- uation suggests that the rate of methylase syn- thesis depends upon mRNA folding and erythromycin induction causes stalling of the ribosomes during translation of a 19-amino acid peptide, resulting in refolding of the mRNA into an active configuration for meth- ylase synthesis.

A close analysis of the leader sequence for polypeptide B (Figs. 3 and 5) shows the striking presence of potential, alternating mRNA sec- ondary structures similar to the ones found in the ermC gene of pE 194. Two possible hair- pin structures (A and B) for the 5’-end leader sequence for the TET protein containing 13 1 nucleotides (positions 3590 to 3460) are shown in Fig. 5. S.D. 1 and S.D.2 are Shine-Dalgamo sequences (Shine and Dalgamo, 1974) rep- resenting possible ribosomal binding sites and contain 6 and 8 base complementarities with the 3’-terminal sequences of E. coli and Bu-

258 KHAN AND NOVICK

“”

AGuCAC

C

“C

ABA”? A:

UA A A

UA CC

C A S.D.2

CG CC AU

II UA UA

“AA AU

III

A I I AU UC AU AU

GU A AUG AU

I A”A GU UA

AGCffiGAUUUUUUUAUGAAGUGUCUAUUUAAAAAUUUCGCGAAUUUAUAUGAGG UAAAAAAUU -

S.D.1

AGGUGAA

GU GA AuAA

AU UA UA I

B u u

I AU II

AU A GC CC CC III CC

AGGAGGAUUUUUUUAUGAAGUGUCUAUUUAAAAAUUU UAUAAACUUUAGUCACCUCAAGU~GAGGU~UUGUUUAGUUUAUAUAA~UU - - - S.D.1 S.D.2

FIG. 5. Hypothetical hairpin structures for the S-end of the TET mRNA. S.D. 1 and S.D.2 are putative ribosomal binding sites. The probable start and stop codons are underlined. Structures A and B correspond to the inactive and active forms, respectively.

cillus 16 s rRNA (Shine and Dargarno, 1975; Sprague et al., 1977). These sites are followed by AUG and UUG codons, respectively. The AUG codon can potentially inmate translation of a 16-amino acid polypeptide, while the UUG can initiate the synthesis of the TET protein. Similar to the ermC system, in the inactive structure (A) the SD.2 and initiating UUG codon for the TET protein are buried in a base-paired region, whereas they are ex- posed in the active structure (B). In both A and B structures, the S.D.l and the nearby AUG are exposed and therefore available for

translation. It will be interesting to see whether mRNA folding plays a role in the regulation of the synthesis of the TET protein.

A fourth open reading frame (nucleotides 1247 to 68 1) can code for polypeptide C con- sisting of 189 amino acids and having a mo- lecular weight of 23,000 Da. However, deletion of a region from nucleotides 770 to 1931 (in- cluding most of the C reading frame) had no effect on the replication properties, stability, copy control, and Tc resistance of this plasmid (data not shown). Therefore, no function has yet been assigned to polypeptide C.

NUCLEOTIDE SEQUENCE OF pT18 1 PLASMID 259

ACKNOWLEDGMENTS

We thank Thomas Aldrich for excellent technical as- sistance, Warren Rosenblum for the computer analysis of DNA sequences, David Dubnau for stimulating dis- cussions, and Betty Rooney for typing the manuscript. We thank the Stanford Molgen project and the National Institutes of Health Sumex-Aim facility for the use of the computer program for analysis of DNA sequences. This work was supported by Public Health Service Grant GM 3 1685 (to S.A.K.) from the National Institute of General Medical Sciences and Grant MV-IA from the American Cancer Society (to R.P.N.).

REFERENCES

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CuRtALE, M. S., AND LEW, S. B. (1982). Two comple- mentation groups mediate tetracycline resistance de- termmod by T&O. J Bacterial. 151, 209-2 15.

GREENE, P. J., BETLACH, M. C., GOODMAN, H. M., AND BOYER, H. W. (1974). The EcoRI restriction endo- nuclease. Methods Mol. Biol. 7, 87-l 11.

GRYCZAN, T. J., GRANDI, G., HAHN, J., GRANDI, R., AND DUBNAU, D. (1980). Conformational alteration of mRNA structure and the posttranscriptional regulation of erythromycin-induced drug resistance. Nucleic Acids Res. 8, 608 I-6097.

HORINOUCHI, S., AND WEISBLUM, B. (1980). Post- transcriptional modification of mRNA conformation: Mechanism that regulates erythromycin-induced resis- tance. Proc. Natl. Acad. Sci. USA 77, 7079-7083.

IORDANEXU, S. (1979). Incompatibility-deficient deriv- atives of a small staphylococcal plasmid. Plasmid 2, 207-215.

IORDANESCU, S., SURDEANU, M., DELLA LAITA, P., AND NOVICK, R. P. (1978). Incompatibility and molecular relationships between small staphylococcal plasmids carrying the same resistance marker. Plasmid 1, 468- 479.

KHAN, S. A., CARLETO N,S.M.,ANDNOVICK,R.P.(I~~~). Replication of plasmid pT 18 1 DNA in vitro; Require- ment for a plasmid-encoded product. Proc. Natl. Acad. Sci. USA 78.4902-4906.

KHAN, S. A., ADLER, G. K., AND NOVICK, R. P. (1982). Functional origin of replication of pT 18 1 plasmid DNA

is contained within a 168-basepair segment. Proc. Natl. Acad. Sci. USA 79, 4580-4584.

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