PLASMID

23, 1- 15 ( 1990)

Mutational

and Physiological Analyses of Plasmid p-T181 Functions Expressing Incompatibility

SARAHK.HIGHLANDER'

ANDRICHARDP.NOVICK

Department oJPlasmid Biology, The Public Health Research Inslitute of rhe City 455 First Avenue, New York, New York 10016

vf New

York, Inc.,

Received January 20, 1989; revised December 18, 1989 Plasmid pT18 1 is a small multicopy plasmid from Staphylococcus aureus that belongs to incompatibility group 3 and expresses two distinct types of incompatibility, Inc3A and‘Inc3B. Inc3A incompatibility is expressed by the primary replication control determinant, copA, which specifies two small transcripts, RNA I and RNA II, that jointly inhibit the synthesis of the ratelimiting initiator protein, RepC. Inc3B incompatibility is expressed by the leading strand replication origin and is due to competition for RepC. The copA region from each of 11 different pT18 1 copy number mutants was cloned onto the pT18 l-compatible vector, pE194, and tested for its ability to inhibit the replication of pT 18 1 and its copy number mutants. The pT 18 1 replication origin was also cloned and tested for its ability to inhibit the replication of the same plasmids. In general copA mutations that alter the production or sequence of RNA I and RNA II greatly reduced or completely eliminated Inc3A activity. Unlike the wild-type, all of the copy mutants were resistant to Inc3B inhibition. The separately cloned wild-type copA and ori regions each reduced the copy number of pT18 1 in proportion to their gene dosage, but neither blocked replication completely. It is proposed that the cloned Inc determinants cause incompatibility by interfering with the plasmid’s copy correction mechanism; this interference destabilizes the plasmid even under conditions where its average copy number is not greatly reduced. o 1990 Academic Press, Inc.

predicted to form a single-stranded loop in the countertranscript and to form the complementary loop transiently during transcription of the repC leader. In a previous study, we have shown that the cloned wild-type ctRNA gene (cop& and the cloned pT 18 1 replication origin (ori) are incompatibility determinants and that together they completely block replication of the wildtype plasmid (Novick et al., 1984). Plasmids with copy number mutations in the target region are resistant to this inhibition (incompatible-resistant) whereas most of those with copy mutations outside the target region are sensitive (incompatible-sensitive) (Carleton et al., 1984). In this sequel study, we examine the inhibitory activities of the two incompatibility determinants separately and analyze their tram effects on the replication of pT 18 1 and certain of its mutants. In the test system employed, the cloned incompatibility regions are maintained at a copy number of about 55, equivalent to 2.5X that

Plasmid pT 18 1 replication is controlled by a counter-transcript (ct)2 regulatory circuit which determines the rate of initiator protein RepC synthesis (Novick Ed al., 1984, 1985). Two small counter-transcript RMAs, RNA I and RNA II (inhibitors), are complementary to and can bind to the 5’ end of the repC mRNA leader (target). This interaction causes termination of repC mRNA transcription just prior to the translation start (R. P. Novick et al., submitted). As with the ColEl and the IncFII systems (Brady et al., 1983; Lacatena and Cesareni, 198 l), a cluster of copy mutations define a probable site for the primary inhibitor-target interaction (Carleton et al., 1984). This site is an RNA segment that is ’ Present address: Department of Biochemistry and Molecular Biology, University of Texas Medical School, 643 1 Fannin Street, Houston, TX 77030. ’ Abbreviations used: bp, base pair; ct, countertranscript; nt, nucleotide; Tsr, temperature sensitive for replication; Inc, incompatibility; Tc, tetracycline; Em, erythromycin.

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HIGHLANDER

of wild-type pT 181. At this gene dosagemost of the cloned mutant copA alleles exerted little or no inhibition of replication toward any of the copy mutant plasmids. The cloned origin and wild-type copA determinant each reduced the replication rate of the wild-type plasmid in proportion to their gene dosage.

AND

NOVICK

gels, mixed with 1 ml water, and autoclaved 5 min to melt the agarose.The melted agarose was combined with 15 ml Hydrofluor (National Diagnostics) aqueous scintillant and counted in a Beckman LS200 scintillation counter. Copy numbers were calculated on the basis of the ratio of plasmid to total cellular DNA and are given as copies per cell. For strains containing plasmid cointegrates, single MATERIALS AND METHODS colony isolates were grown, with selection for Bacterial strains and plasmids. The staph- the cointegrate, for four to five generations in ylococcal strains used are derivatives of strain aerated CY broth at 37°C and then used to NCTC 8325 (Novick, 1967). RN27 is lyso- prepare whole cell lysates for copy number genie for bacteriophage 8Oa and was used as determinations. All values are the average of a recipient for transductions performed either measurementsfrom at leasttwo independently with this phage or with phage 4 11. Plasmids isolated transductants and are accurate to are listed in Table 1. within 20% of the mean. Media and growth conditions. CY broth Restriction mapping and cloning. Restric(Novick and Brodsky, 1972) was used for liq- tion endonucleases were purchased from uid cultures, shaken at 32°C and monitored Boehringer-Mannheim Biochemicals and were turbidimetrically with a KIett-Summerson used as described by the manufacturer. Rephotoelectric calorimeter read at 540 nm. striction mapping and fragment isolations When constant density cultures were required, were performed with ethidium bromide-CsClcells were grown in a manually operated tur- purified plasmid DNA samples (Clewell and bidostat at 42 or 32°C. GL agar (Novick and Helinski, 1969; Novick et al., 1979). For moBrodsky, 1972) was supplemented with anti- lecular cloning, specific fragments were eluted biotics as indicated. Tetracycline (Tc) and from polyacrylamide gels, phenol extracted, erythromycin (Em) were each used at 5 and ethanol precipitated (Maxam and Gilbert, 1980). For ligation, samples were combined b&ml. Protoplast transformation was performed in approximately equimolar ratios and incuaccording to the method of Chang and Cohen bated 16-24 h at 14’C at a DNA concentra(1979) for Staphylococcus aureus (Novick et tion of at least 10 pg/ml and a ligase concentration of 40 units/ml. Ligated samples were al., 1984). Analysis of plasmid DNA. Plasmid DNA used to transform S. aureus protoplasts with was prepared and analyzed as previously de- selection for the appropriate antibiotic resisscribed (Novick et al., 1979; Projan et al., tance marker. Transformants were screened 1983). Plasmid DNA replication was moni- for plasmid content and those carrying plastored by [methyl-3H]thymidine labeling. One- mids of the expected size were used to prepare milliliter samples of exponentially growing plasmid DNA for confirmatory restriction cells were labeled for 5 min, with shaking at analysis. Construction of copA and cointegrate clones. 32°C using 10 &i/ml [methyl-3H]thymidine. Labeling was stopped by freezing the cells on The pT 181 copA gene maps between nucleodry ice/ethanol and the cells were used to pre- tides 158 and 570 on the plasmid and is compare whole cell lysates as described previously pletely contained within the 1568-bp TaqI-A (Projan et al., 1983). Aliquots of the lysates fragment (Novick et al., 1984). Mutant copA were electrophoresed on agaroseand plasmid alleles were cloned by inserting the correcopy numbers were determined by fluoro- sponding TaqI-A fragments of each of 10 difmetric densitometry of the ethidium bromide- ferent copy mutants into the TaqI-cohesive stained gels (Projan et al., 1983). Plasmid and ClaI site of a Tsr pE 194 copy mutant plasmid, chromosomal bands were excised from the pRN5 110 (S. J. Projan, unpublished results),

PLASMID

pT18 1 INCOMPATIBILITY TABLE

Plasmid pT181 pE194 pRN5 101 pRN5110 pRN60 19 pRN6116 pRN6264 pRN6287 pRN630 1 pRN6302 pRN6303 pRN6304 pRN6305 pRN6306 pRN6307 pRN6308 pRN6323 pRN635 1 pRN6326 pRN8008 pRN6350 pRN80 12 pRN6352 pRN8020 pRN6355 pRN8023 pRN6358 pRN8024 pRN6359 pRN8025 pRN6360 pRN8026 pRN636 1 pRN8027 pRN6362 pRN806 1 pRN6363 pRN8115 pRN6364

(FUNCTIONS)

1

Source or reference

Description 4.4 kb naturally occurring Tc’, Inc3 3.7 kb naturally occurring Em’, Incl 1 pE 194 Tsr pE 194 Tsr cop pTlSl::pE194” pTlSl::pE194,” Aon’pT181 pT181 cop608::pE194” pTlSl::pRN5101” pT181 cop-612::pE194’ pT181 cop615::pE194” pT181 cop-618::pE194” pT181 cop619::pE194” pT181 cop-620::pE194” pT 18 1 cop-621 : : pE 194” pT181 cop-622::pE194” pT18 1 cop-623::pE194” pE194::pTlSl TaqI-A pRN5110::pTlSl TaqI-A pE194::pTlSl TuqI-C pT 18 1 cop-608 pRN5llO::pRNSOOS TaqI-A pT181 cop-612 pRN51 lO::pRN8012 TuqI-A pT181 cop-615 pRN5 1 lO::pRN8020 TuqI-A pT181 cop-618 pRN51 lO::pRN8023 TaqI-A pT181 cop-619 pRN5 1 lO::pRN8024 TuqI-A pT 18 1 cop-620 pRN5 1 lO::pRN8025 TuqI-A pT 18 1 cop-621 pRN5 110: :pRN8026 TuqI-A pT 18 1 cop-622 pRN5 110: :pRN8027 TuqI-A pT 18 1 cop-623 pRN5 1 lO::pRN8061 TuqI-A pT 18 1 cop-639 pRN51 lO::pRN8115 TuqI-A

Iordanescu, 1976 Novick and Bouanchaud, 197 1 Novick et al., 1984 Projan, unpublished Novick et al., 1984 Novick et al., 1984 Novick et al., 1984 Novick et al., 1984 This work This work This work This work This work This work This work This work Novick et al., 1984 This work Novick et al., 1984 Khan et al., 1981 This work Carleton et al., 1984 This work Carleton et al., 1984 This work Carleton et al., 1984 This work Carleton et al., 1984 This work Carleton et al., 1984 This work Carleton et al., 1984 This work Carleton et al., 1984 This work Carleton et al., 1984 This work Highlander and Novick, 1987 This work

0 XbaI cointegrate.

as shown in Fig. 1. pRN5 110 was chosen as a vector because it is compatible with pTI 8 1 and its temperature sensitivity allows manipulation of the countertranscript gene dosage. In addition, cointegrates containing the entire pT 181 plasmid, interrupted in the repC gene, were formed between XbaI-linearized wildtype pE 194 and pT 181 copy mutant plasmids, as shown. Tetracycline-sensitive cointegrate

derivatives arose from spontaneous, homologous recombination between homologous RSAsites flanking the pT 181 tet gene (Novick et al., 1981). The mutant copA alleles cloned are described in Table 2 and their map locations are shown in Fig. 2 (Carleton et al., 1984). Each of the copA clones expressedthe countertranscripts produced by the corresponding copy mutant plasmid (not shown,

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HIGHLANDER

AND NOVICK

Pvu Xba I

Taq I

Pvu

FIG. 1. Cloning scheme for the construction of pT 181 TuqI-A clones and pT 18I : : pE 194 cointegrates. A complete TuqI digest of pT181 or a copy number mutant was ligated to cohesive CluI linearized pRN5 110 (pE194), and clones carrying the TaqI-A fragments were isolated and mapped. Clones of the cop-612 and cop-615 alleles also contained the TuqI-E fragment (not shown). Cointegrate plasmids were formed between XbaI-linearized pT 18I copy mutant and pE 194 DNAs. Tc’ derivatives were formed by homologous recombination at the RSA sites (dashed lines).

S. K. Highlander, Ph.D. thesis, New York University, 1985).

and is maintained at about 55 copies per cell. In these studies, it was found that cointegrates constructed with either Inc-resistant or Incsensitive pT 181 copy mutant plasmids or with RESULTS the wild-type plasmid expressed incompatibility toward a wild-type pT 181 test plasmid. Initial studies of pT 181 incompatibility made use of pT 181: : pE 194 cointegrates con- Deletion analysis of a pT 181: :pE 194 cointestructed by XbaI digestion and ligation (No- grate containing the wild-type pT18 1 sevick et al., 1984). pE194 is compatible with quences showed that this incompatibility acpT18 1, has an unrelated replication system, tivity had two components, one, Inc3A, at-

PLASMID pT18 1 INCOMPATIBILITY TABLE 2 CHARACTERISTICS OF pT 18 1 COPY MUTATIONS *

Allele

Location of mutation

cop-608 cop-612 cop-615 cop-618 cop-619 cop-620 cop-621 cop-622 cop-623 cop-639

Deletes RNA I and RNA II Target Target Target Defect in RNA I/II promoter Target Defect in RNA II termination Defect in RNA I termination Target Target

Copy number 820 290 260 320 200 73 170 200 610

1100

0 Data are summarized from analyses of pT18 1 DNA sequences(Carleton et al., 1984;Novick et al., 1984),putative RNA structures (Carleton et al., 1984; Novick et al., 1984, Recsei et al., 1986), and Northern blot analysis (S. K. Highlander, Ph.D. thesis, New York University, 1985).

tributable to the copy control determinant, copA, and the other, Inc3B, associated with the replication origin (Carleton et al., 1984). In the following seriesof experiments, we have analyzed these incompatibility properties in detail, utilizing three sets of plasmid clones. One set contained the copA determinant only, the second contained ori, and the third contained both. The first and third setswere constructed with pT18 1 copy mutants as well as wild-type; the second with wild-type only. A similar set of experiments analyzing the Inc3B activity of the pT 181 origin cloned to pE 194 has recently been reported (Iordanescu, 1987) and is discussed below. Incompatibility

properties of copA and ori.

The separate activities of the two incompatibility loci were first compared by performing transductional displacement tests using the copA’, ori- cointegrate, pRN6116, and the A copA ori+ cointegrate, pRN6264 (Novick et al., 1984). In these tests (Table 3), pRN6116 displaced pT 181 and all of the recessivecopy mutants, but pRN6264 eliminated only the wild-type plasmid from heteroplasmid strains. None of the Inc-resistant copy mutants was displaced by either plasmid. The incompatibility activities of cointegrate plasmids were next compared with those of the separately

(FUNCTIONS)

5

cloned copA and ori regions. Unlike the copA+ ori- cointegrate, pRN6 116, the TaqI-A fragment containing the wild-type copA gene, excluded only pT 181 from heteroplasmid strains (Table 3) and did not affect any of the mutant plasmids. Of the cloned TaqI-A fragments containing mutant copA alleles, only those of cop-621, -623, and -639 completely excluded pT 181. Unexpectedly, the cop-623 copA clone had an inhibitory activity greater than that of the wild-type, as it excluded the recessivecopy mutants pRN8024 (cop-619), pRN8026 (cop621) and pRN8027 (cop-622). None of the other copy mutant test plasmids were displaced by any of the cloned copA alleles. The inhibitory activity of the cointegrate pRN6264 (A copA ori+) was compared to that of pRN6326, a pE 194 derivative carrying the pT18 1 TaqI-C fragment which includes the functional pT181 origin (Novick et al., 1984). Whereas pRN6264 excluded wild-type pT 181 from heteroplasmid strains (Table 3), pRN6326 did not completely exclude any of the test plasmids. However, only 20% of the pRN6326 transductants of a strain carrying wild-type pT 181 grew on tetracycline, indicating an intermediate level inhibition of pT 181 by the cloned ori. Copy numbers. In caseswhere heteroplasmids were established,the interaction between the incoming clone and the resident test plasmid was quantitated by a determination of the copy numbers of the two plasmids. The copy numbers of pT18 1 and its copy mutants measured in the presence of copA- and ori-containing clones are presented in Table 4. Note that the copy numbers of the pE 194 cop-6 derivatives carrying the cloned incompatibility determinants were not significantly different from that of wild-type pE 194: thus, cloning at the ClaI site of pE194 cop-6 suppressesthe copy mutant phenotype of the vector plasmid. The basis for suppression of the copy mutant phenotype is not understood, but it has been observed for pE194 cop-6 derivatives with DNA cloned at sites other than CZuI(S. J. Projan and A. Gruss, personal communication). With very few exceptions, none of the cloned pT 181 Inc determinants caused a significant (i.e., greater than twofold) change in

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HIGHLANDER

Taq I TTTTTATACA TAAAAAA TCG ACTCCTTAAT

STTTTTTAGC RepC Start

TBTTA S-D

AND NOVICK 210

TTTAAGGAAT CGCTCACCCA AATATATATC GAGTTAAAGC AAATTCCTTA GCGAGTGGGTTTATATATAG * * A 623 I----> 608 (A144)

CTCAATTTCG

RNA I/II Start 280 ,-----------__--------~~~~-----~~~~~~~ -35 -10 TTGATGTATA TTTAAATATC GTTTAATATC TAAATATACA AGATTATAAA AACAACTCAG TGTTTTTTTC AACTACATAT AAATTTATAG CAAATTATAG ATTTATATGT TCTAATATTT TTGTTGAGTC ACARAAAAAG G 619 End of RNA I

---____------_____----~~~~-----~~~~---~~~-~~~~~~~~~--------~---------~~~----

330

TTTGAATGAT GTCGTTCACA A A C T T T G G T C A G G G C G T G A G CGACTCCTTT AAACTTACTA CAGCAAGTGT T T G A A A C C A G T C C C G C A C T C GCTGAGGAAA

*

I-As-1 639

-----------------------------------------~--------~~--------~~~-,

*

*

*

A 615

A 618

C 620

A 622

End of RNA II

400

TTATTTTGTT ATTAATATAA CACTATCAAA AGATTTGGTC TAATCAGATC AAGTCTTTTT TTATTTAAGC AATAAAACAA TAATTATATT GTGATAGTTT TCTAAACCAG ATTAGTCTAG TTCAGAAAAA AATAAATTCG *

Mutational and physiological analyses of plasmid pT181 functions expressing incompatibility.

Plasmid pT181 is a small multicopy plasmid from Staphylococcus aureus that belongs to incompatibility group 3 and expresses two distinct types of inco...
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