Molecular Microbiology (1992) 6(1), 83-94

The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products: the antisense RNA II and the repressor protein, RepA G. del Solar* and M. Espinosa Centro de Investigaciones Bioiogicas, CSIC, Velazquez 144. 28006 Madrid. Spain. Summary The promiscuous plasmid pLS1 encodes two transacting elements that regulate its copy number: protein RepA and antisense RNA II. In vitro transcription showed that RNAs for both repressors are synthesized from two promoters, PAB and P,,. From PAB, genes encoding RepA (transcriptional repressor) and RepB (initiator of replication) are cotranscribed, the target of RepA being located within PAB- Mutants in repA or in PAB are still sensitive to RepA. However, cloning of the repA gene in a compatible repMcon did not result in incompatibility towards pLSI, From Pn, the 50-nucleotide RNA II is synthesized. The main incompatibility determinant towards pLS1 corresponds to the coding sequence for RNA II. The RNA II target could be reduced to 21 nucleotides, including the RepB initiation of translation signals. We propose that plasmids of the pLS1 family (pE194, pADB201, and pLB4) share functional and structural characteristics for the regulation of their copy numbers.

(According to Novick (1987), these antisense RNA species will be referred to as countertranscribed (ct) RNAs.) The only well-characferized regulatory circuit based on ctRNAs is that of the staphylococcal plasmid pTI 81 (Khan and Novick, 1983), in which replication is indirectly regulated by two ctRNAs starting at the same nucleotide (nt) and ending at different sites (Novick etai. 1984; Kumar and Novick, 1985: Novick etai. 1989). In the promiscuous streptococcal plasmid pLSI (Lacks etai, 1986), replication is initiated by protein RepB, which introduces a site- and strand-specific nick between coordinates 448 and 449 of hairpin I within the plasmid ori{+) (de la Campa et ai, 1990). Synthesis of the repB gene product is regulated by the plasmid-encoded 5.1 kDa transcriptional repressor, RepA (del Solar et ai, 1989). RepA is involved In pLSI copy-number control through its binding to a 13bp symmetric operator within which most of the - 3 5 region of the promoter PAB is included (del Solar etal.. 1990). Here we show that regulation of pLSI copy number is exerted at two levels; by the binding of protein RepA to PAB. and by a 50-nt ctRNA, RNA II. This RNA II would interact with a region of the repAB mRNA which includes the repB initiation of translation signals. We propose that both RepA and RNA II are regulatory elements belonging to the same circuit, and that ail plasmids of the pLSI family have similar mechanisms for maintaining their copy numbers.

Introduction Plasmid replication must be a highly regulated process because the number of copies of any replicon is kept constant under physiological conditions and in a given host (reviewed by Novick, 1987; Nordstrom and Austin, 1989). Plasmids regulate their copy number either by sequestering the initiator of replication (Rep) protein upon its binding to directly repeated sequences (iterons), or by binding a frans-acting repressor to a plasmid-speclfic target (Thomas, 1988). The latter includes several small multicopy plasmids from Gram-positive bacteria, which seem to regulate their copy number by one or two small RNAs synthesized In the opposite direction relative to the rep mRNAs (Kumar and Novick, 1985; Novick, 1989). Received 5 July, 1991; revised 23 September. 1991. *For correspondence. Tel. (1) 5854209; Fax (I) 2627518.

Resutts Transcriptional analysis of promoters PAB and P^^ Two mRNAs, repAB and tet, are synthesized in plasmid pLSI from promoters PAB (del Solar et ai, 1990) and Py (Ballester ef a/., 1990), respectively (Fig. 1A). The structure of promoter PAB (Fig. 1B), from which genes repA and repB are cotranscribed (del Solar et al., 1990), is such that the RepA operator overlaps the - 3 5 box, transcription starting at pLSI co-ordinate 633. From a third promoter, Pn (Fig. 1A and C), the putative RNA II should be transcribed in the opposite direction relative to the repAB mRNA (Fig. 1A). Part of the - 3 5 box of P,, is located in the loop of a possible hairpin structure, resembling the promoter region for the synthesis of the RNA primer of ColEI -type plasmid pMBI (Castagnoliefa/., 1985). At the end of the sequence


G. del Solar and M. Espinosa


Si on(-)

repAB mRNA










+i _,_,,_ ,^ repAB mRNA

- | T A C T A T | A G T T T T A T A A A A T T T T G A Q A 6 6 - 3'




Fig. 1. Features of pLSI. A. pLSI functional map showing the location of the plasmid origins of r^lication (empty lines), the direction of DNA and RNA synthesis (arrowheads), the RNAs (wavy lines), the identified promoters (•, tS^), and the location of the antisense RNA II relative to the fepA and repSgene products (magnified below). Only pertinent restriction sites are indicated. B. Nucleotide sequence of the pLSI coding strand from co-ordinates 590 to 648, showing the - 3 5 and - 1 0 regions ot promoter PAB- the RepA operator (dotted box) and the location of the transcription initiation (+1) of the rep^SmRNA. C. The nucleotfde sequence of the pLSI non-codmg strand between co-ordinates 790 and 900, indicating the - 3 5 and - 1 0 regions of promoter Pn. is shown, aiong with the possible secondary structure centred at co-ordinate 874, the location of the initiation of RNA II transcription ( + 1), and the structure of the proposed r,, transcription terminator.

from which RNA II would be synthesized, another secondary structure is located having the features of a rhoindependent transcription terminator. Applying the algorithm developed by d'Aubenton-Carafa et al. (1990), the calcuiated d value for the putative terminator of RNA II {Tn) was +59.6. with a predicted termination efficiency of over 90%. Both figures are among the highest values obtained for the 148 transcription terminators computed by the authors. No other plausible transcription terminator is observable past In. A putative fourth plasmid promoter {P\) is located between co-ordinates 162 and 191 (see Fig. 3 later), and could direct the synthesis of a small RNA (RNA I) in the opposite direction relative to the plasmid mRNAs. Deletions that remove RNA I resulted in plasmid mutants with twice the number of copies in the parental pLSI (Table 1), probably because of the removal of the pLS1endonuclease SI-sensitive major secondary structure

(hairpin III; Puyet etai, 1988). One of the plasmid mutants defective in RNA I (pLS5. Table 1) has a deletion spanning co-ordinates 34 to 365, which results in an increase in its copy number without affecting its replicative features (del Solar ef al., 1987b). To obtain plasmid mutations at the wild-type promoter PAB. we introduced a 4bp insertion (5'-TGCA-3') at the single ApaU site (pLSI co-ordinate 607; Fig. 1) of plasmid pLS5. Although the symmetry of the RepA operator is unchanged after this insertion, the spacing between the - 3 5 and - 1 0 boxes increases from 17bp

(in PAB) to 21 bp (in the mutant PABCORH)- The

resulting plasmid. pLS5copH, exhibited (in Streptococcus pneumoniae) 1.2 times more copies than its parental pLS5 (Table 1). Two sets of in W(ro transcription assays were performed (Fig. 2). The first was intended to show whether the mutant PABCOPII could direct the synthesis of the repAB mRNA,

Copy number control of pLS1 Table 1. Relevant features of plasmid pLSI and the derivatives used in this work. Ptasmid

Size (bp)

Oetetion (bp)


pLSI pLSIcop7 pLS5 pLS5cop11

4408 4408 4076 4080

PLS1AA4 pLS1dA12 pLS1dA21 pLS1AA34

4240 4256 4257 4245

332 332 4bp insertion 168 152 151 163

743 34-365 34-365 607 649-816 653-804 655-805 654-816


DNA sequence at the deletion junctions

Copy number"


22 110 52 62

±2 ±15 ±6 ±4

31 68 16 123

±4 ±4 ±2 ±12


a. Co-ordinates of pLSI (Lacks et al., 1986) included in the delation. Note that pLSScopI? has an insertion at co-ordinate 607. b. In S. pneumoniae.

and whether this synthesis was sensitive to RepA. We used the 842 bp Ban\-Rst\ fragment from pLS1 and the 723 bp Psfl B fragment from pLS5cop11 as templates (Fig. 2D). The results (Fig. 2A) showed that a main transcript of about 430nt (from pLSI, lane 4, and from pLS5cop11, lane 3) was synthesized, the amount of RNA being much lower from pLS5cop11 DNA than from pLSI DNA. This was not due to a reduced efficiency of the pLS5cop11 DNA template, as two small RNA species that were also synthesized (from Pn, see below) were found in similar amounts from both templates (lanes 3 and 4). The 430-nt RNAs have a size that coincides with run-off transcripts synthesized from PAB and from PABCOPH- These results indicate that PABCOPI I was still able to direct the synthesis of repAB mRNA, but with lower efficiency relative to the wild-type promoter. If the 430-nt transcripts derive specifically from PAB and PABCOPH. RepA should be able to repress their synthesis because the RepA-operator remains unaltered In the cop11 mutant. This was the observed result when the same in vitro transcription experiments were performed in the presence of increasing amounts of purified RepA protein (Fig. 2C, lanes 2-3 and 5-6). No indication of another repS mRNA, initiating from a promoter other than PABCOPH. was found (Figs 2A, lane 3, and 2C, ianes 1-3). In addition, by endonuclease SI mapping we have observed initiation of transcription in vivo from PAB, but not from PABCOPI I (not shown). Thus, we still do not know why the small amount of repAB mRNA synthesized from PABCOPH leads to viable plasmids with a slightly higher number of copies than the parental pLS5 (Table 1). In the second set of transcription experiments, we wanted to unequivocally assign the transcript corresponding to RNA II. DNA templates isolated from plasmids pLSI, pLS5cop11, and pCGA3 (see Fig. 3) were employed. When the pLSI Banl-Pst\ DNA fragment (842bp; Fig. 2D) was used, three main transcripts were synthesized: the 430-nt RNA and two nearly the same size, about 50nt long each (Fig. 2A, lane 4). The 430-nt transcript corresponded to RNA synthesized from PAB. as

it was repressed by RepA (Fig. 2C, lanes 4-6). The two small transcripts were insensitive to the addition of RepA. By long exposure of the autoradiograms (not shown), it was possible to visualize all of the nt ladder resulting from intermediates in the synthesis of the two small RNAs; both transcripts differed by only one nt, the longer one being more abundant. We estimate that the sizes of these two RNAs are 50 and 49 nt. The same efficiency of synthesis of these two small transcripts was observed when the pLS5cop11 Rst\B fragment (723 bp, with the A5 deletion; Figs 2D and 3) was employed as template (Fig. 2A, lane 3). Since the templates used above shared one of the Psfl ends, both small RNAs could correspond either to run-off transcripts starting 50 nt from this end or to full transcripts initiated and terminated within the region common to both templates. To distinguish between these two possibilities, we employed as template the 519bp Bgl\-Ssp\ fragment from pLSI because this DNA fragment lacks PAB and the 3' end of the coding region for RNA II (Fig. 2D). In this case there was no synthesis of the 50-nt RNAs, although a main transcript of about 30 nt and several less abundant transcripts up to 37 nt long were detected (Fig. 2A. lane 5 and Fig. 2B, lane 3). The 30-nt RNA could correspond to a run-off transcript synthesized from P,, (Fig. 2D), and the less abundant species could be artifacts resulting from the 3'-extension of the Bgl\ end (Hillen ef a/.. 1984). Since the pattern of RNA synthesis is different when using the Bgl\-Ssp\ template than when using the previous ones (compare lanes 1 and 2 with lane 3 in Fig. 2B), the 50- and 49-nt RNAs seem to arise from the termination at two contiguous sites rather than from two independent initiations. Thus it seems that both RNA species start about 30bp from the Bgl\ site (at around co-ordinate 836), ending 20bp past this site. On the basis of (i) the sites of initiation and termination of transcription of these small RNAs (around co-ordinates 836 and 787, respectively), (ii) the Insensitivity to repression by RepA, and (iii) the synthesis being in the opposite direction relative to the repAB mRNA, we may conclude that both RNAs are


G. del Solar and M. Espinosa

I 23 45



B 12

3 4

synthesized from Pn. and that they correspond to RNA II species which would end at two different sites. This oonctuston was confirmed by the use of the 702 bp Ssp\-Pvui\ fragment from plasmid pCGA3 (see Fig. 3), which contains PAB but lacks Pn (Fig. 2D). In this case, only a 480-nt run-off transcript, corresponding to RNA synthesized from PAB. was detected (Fig. 2A, lane 2). Consequently, an RNA with features coinciding with the data derived from the DNA sequence for RNA II is synthesized from promoter P||.

In vivo influence of RepA and RNA II on pLS 1 copy number


842 repAB mRNA




Fig. 2. In vitro transcription from promoters PAB and Pn. and the effect of purified RepA protein on transcription. RNA was synthesized from DNA fragments (outlined in Panel D), containing one (PAB or P,,) or two (PAB and Pii or PABCOPM and P,,) promoters. A. Transcripts synthesized from the following D^4A fragments are shown: Ssp\~Pvu\\ (702bp) from pCGA3 (lane 2), Psti B (723bp) from pLS5copii (lane 3). Banl-Pstt (842bp) from pLSI (lane4), and Bgl-Ssp\ (842bp)1rompLS1 (lane 5). Lane 1: DNA standards (labelled Odel restriction fragments from pLS1AA12) with their sizes indicated on the left. B. Synthesis of RNA II as observed in a long-exposure autoradiogram. The templates used were the same DNA fragments as above. Lanes 1-3 con^espond to lanes 3-5 in Panel A, and lane 4 corresponds to the DNA standards. C. tn vitro transcription from promoter Pn in the presence of RepA protein, used at concentrations that inhibit synthesis of repAB mRNA. The DNA templates were fragments: Pst\ B {723 bp) was from pLS5copU (lanes 1-3). and Ban\-Pst\ (842bp) was from pLSl (lanes 4-6). Transcription assays were performed in the absence (lanes 1 and 4) or in the presence of 50ng (lanes 2 and 5) and of 100ng (lanes 3 and 6) of purified R ^ A protein. Lane 7: DNA standards (as in Panel A), with relevant sizes indicated on the right. D. Maps of the DNA fragments that were employed as templates, showing the relative positions of the promoters (filled boxes). In the pCGA3 fragment, DNA sequences con-esponding to plasmid pCI 94 are shadowed. Expected run-off transcripts (wavy lines) and full transcripts (indicated by an'owheads) are shown. The position of the deletion in pLS5copI r (A5}, and the siz:es (in bp) of the fragments are also indicated.

On the basis of previous results (del Solar etai, 1990) and the above data, it seemed that two pLSI-encoded products would regulate the piasmid copy number (the RepA repressor protein and the RNA II). If this were the case, both should be frans-acting elements that can influence the number of copies of pLSI -based replicons when their genes are cloned into compatible piasmids. To perform these in vivo experiments, we chose S. pneumoniae because of the higher copy number of plasmids with the pLS1 repiioon in this host. The DNA fragments to be cloned were from pLS1 or from its derivative, pLSl cop7. The cop7 mutation (A—»C, co-ordinate 743) ieads to a single amino acid substitution (Aia-30-^-Glu) in the proposed recognition helix of RepA (del Solar et ai, 1990). Plasmids bearing this mutation exhibit a high copynumber phenotype (Tabie 1), probabiy because of the synthesis of a defective RepA repressor (RepA7; del Solar etai, 1990). As a vector for cloning pLSI regions we used pC194cop (bearing a copy-number mutation; Ballester et al.. 1990) to supply a high gene dosage of repA. repA7, and/or of mall. Several DNA fragments were cioned into the single H/ndlll site of pC194cop (Fig. 3). The recombinant plasmids (Fig. 3) were isolated, characterized by detailed restriction mapping, and used to transform competent S. pneumoniae cultures harbouring pLSI or pLS1 cop7. Transformants were seieoted for chtorampheniool resistance (Cm^, the marker of the incoming plasmid, this being a Type i incompatibiiity test {Nordstrom et al.. 1984). We have caiculated that 25 generations are needed for one pneumococcal transformant to give rise to a colony, and that 12 more generations are sufficient to obtain a culture grown to 4 x 10^ o.f.u. m!"^ from the portion of the colony picked. Consequently, the incompatibiiity tests were performed after about 37 generations in the presence of Cm, and several clones were analysed from each transformation. To analyse the frans-effect of RepA and RepA7 on pLS1 and pLSI cop7, two recombinant plasmids were used: pCGA3 and pCGA3cop7'. These plasmids contain the Alu\-Bgl\ fragment (co-ordinates 505-804) from pLSI

Copy number control ofpLSI

I ,

I , I

I t



i I

pCGA3cop7 . I




Fig. 3. Physical and functional maps of the recombinant plasmids that were constructed to characterize the trans activity of RepA and RNA II onpLSI.Acircular map of pC194 with indications of the car gene orientation and the position of the relevant restriction sites is shown on top. At the single Hindlll site of pC194. different subfragments of the Ps(l B fragment (drawn to a different scale) of pLSI were cloned to construct the derivatives depicted below. The relative position of the cop7 mutation is indicated by a vertical an-ow. The indicated restriction sites (co-ordinate of cleavage in parenthesis) within the pLSI region (and those in pC194) were used to perform a detailed restriction map of the recombinant plasmids. Below the Psfl B pLSI fragment, a functional map of the region is shown, indicating the relative positions of hairpins I to III, the three 11 bp iterons (11-13). promoters P,. PAB. and P». and genes repA and repB. RNA il and the putative RNA I are indicated as wavy lines.

and pLSI cop7, respectively (Fig. 3). The cloned fragment includes the repA gene (wild type or cop7) with its own transcription/translation signals and the three iterons, but it lacks P|| and most of the coding sequence for RNA II (Fig. 3). The results (Fig. 4) showed that neither RepA nor RepA7 significantly changed pLSI copy number. However, RepA (but not RepA7) reduced the pLSI cop7 copy number below the levels of wild-type pLSI. No further significant reduction in the number of copies of the resident plasmid (pLSI or pLSI cop7) was observed after 30 more generations (not shown). Total compatibility between pC194copand the pLS1 replicons was observed (Figs 4 and 5). Therefore, we may draw the following


conclusions: (i) in vitro, RepA represses synthesis of the repAB mRNA not only from wild-type PAB but also from the weak PABCOPH; (ii)'" vivo, the region encoding RepA. although involved in the control of pLS1 copy number, does not express incompatibility towards pLSI; (iii) RepA7 is almost (or totally) inactive, the mutation being recessive relative to the wild-type allele; and (iv) the binding site of the initiator RepB protein (the three iterons) is not enough, perse, to show any incompatibility towards pLSI, which contrast with the results found for other iteron-containing plasmids (Novick, 1987; Thomas, 1988). The three Iterons alone, cloned in a compatible replicon, also failed to exhibit incompatibility towards pLSI, indicating that only an intact ori{+) may be required for incompatibility (G. del Solar and M. Espinosa, in preparation). Next, we cloned a pLS1cop7 Alu\ fragment (co-ordinates 505-921) to construct pCGAI cop7 (Fig. 3), in which P,, and the entire sequence coding for RNA II are cloned (in addition to the regions present in pCGA3cop7). The resutts of incompatibility tests (Fig. 4) showed that pCGA1cop7 dramatically eliminated the resident pLSI from the heteroplasmid cultures. Determining the number of tetracycline-resistant fjc^ cells in these cultures showed that, after 37 generations in the presence of Cm, only 1-8% of the cells still harboured pLS1. The above results indicate that, in addition to RepA, RNA II is involved in the control of the pLSI number of copies, being the main incompatibility (inc) determinant of the plasmid. To further dissect the inc region of pLSI, another derivative (pCGA9; Fig. 3) was constructed by DNA 'swapping' of the 163 bp Sty\-Alu\ fragment (co-ordinates 758-921 OfpLSI) from pCGA1cop7intopC194cop. Thus, the only pLS1-coding sequences cloned into pCGA9 are those corresponding to RNA II (Fig. 3). Type I incompatibility tests were performed with cells harbouring pLSI or pLS1 cop7. Analysis of the DNA content of several clones (After 37 generations with selection for Cm*^, showed that pLSI was eliminated from the heteroplasmid population (Fig. 5). A different result was obtained for pLS1cop7: out of 12 clones analysed, 5 showed no resident plasmid but it could be observed in 7 clones, either at a very low copy number (3 clones) or with a number of copies similar to that of pLSI (4 clones). Clones representing the three categories are depicted in Fig. 5 (left panel). The presence of the resident pLS1cop7 could be expected because of its high copy number, since plasmid maintenance should depend on the amount of replication-productive target (i.e. not subjected to repression).

Deletion mapping of the target of RNA II Computer searches showed no significant homology between the pLSI inc region (co-ordinates 787-871) and


G. del Solar and M. Espinosa Fig. 4. Trans-effect of the increase in the gene dosage of rapA, repA7, and of repA7-rnall on copy number and stability of pLSI replicons. Cells harbouring pLSi or pLSI cop7 were transformed with the indicated donor DNAs and plated into selective media containing chloramphenicol (Cm) (marker of the incoming plasmid). Several clones were grown in Cm-containing medium, and total DNA was prepared and electrophoresed in 1.2% agarose gels. The homoplasmid strains are indicated by a (-) for the donor plasmid. The positions of the supercoiled monomeric forms of the different replicons are marked. Other plasmid DNA torms (open circles, dimers) and chromosomal DNA (upper part of the gels) are not indicated.

— PLSI pLSI,pL9cop7 —

— pCGAIcop7 — pCI94cop

3 S


Fig. 5. The effect in trans ol the region coding RNA II on copy number and stability of plasmids bearing the pLSl replicon. Cells harbouring the indicated plasmids were transformed with plasmid pCGA9 (see Fig. 3) DNA and selected for Cm". Several clones were grown, and total DNA was prepared and electrophoresed in 1.2% agarose gels. Initially, heteroplasmid clones obtained from the combination pCGA9/pLS1AA4 (three clones) or from the combination pCGA9/pLS1iA34 (two clones) were subcultured from g = 0 to g = 50 in the absence of tetracycline (Tc) (the marker of the pLSI-based plasmids). and their DNA content was also analysed. Note that generation g = 0 corresponds to 37 generations in the absence of Tc (from one transformant to a fullgrown culture).

Copy number control of pLS 1 89 the rest of the plasmid. For this reason, and by analogy with other welt-known replicons (Novick. 1987), the simplest hypothesis was to assume that RNA II should interact with the complementary region in the repAB mRNA. To define the minimal target of RNA II, we constructed a series of deletion derivatives at the plasmid inc region by BAL31 digestion of Sg/1-linearized pLSI DNA {co-ordinate 804; Fig. 1). Several derivatives were isolated, characterized, and the nt sequence of the deletion junctions of four of them was determined (Table 1). These four pLSI-derivatives have an intact repB and lack most repA (and consequently PAB is closer to repB). Since they also lack the putative terminator T^ of RNA II (Fig. 1), synthesis from P|| of transcripts longer than RNA II cannot be dismissed. If such new ctRNAs exist, their length would depend on the nt sequence, and their synthesis could interfere with transcription from PAB- This would account for the differences in the number of copies of the deleted plasmids (Table 1); two have a high copy number (pLS1AA12 and pLS1AA34), and two have a number of copies close to that of pLSI (pLS1AA4 and pLS1i\A21). The four repA~ plasmids exhibited total stability (as the parental pLSI) in homoplasmid strains of S. pneumoniae, which argues against strong interierence of CtRNA synthesis with replication. If RNA II starts at cc-crdinate 836, the length of the repB mRNA complementary to the wild-type RNA II is reduced from 50nt (pLSI) to 32nt (pLS1dA12), 31 nt (pLS1AA21) and 21 nt (pLS1AA4 and pLSI AA34). To test whether the complementarity of these regions is still enough for 'sensing' an increase in the dosage of wild-type RNA II, Type I incompatibility tests (using pCGA9 as incoming plasmid) were performed. Analysis of the DNA content of various clones, representatives of each transformation, showed that the presence (or absence) of the resident plasmid depended upon the repA~ derivative copy number in the homoplasmid strains (Fig. 5). For pLSI AA21, no resident plasmid was detectable, and less than 1 % of the cells were Tc'^. For pLS1AA4, clones apparently without resident plasmid (1 % of Tc'^ cetls) or with a reduced amount of it (20-60% of Tc^ cells) were observed. In the latter case, three clones (I to III; Fig. 5. lower panei) were selected and grown in the absence of Tc for several more generations (g = 0 to p = 50). As can be observed (Fig. 5), subsequent generations in the absence of Tc led to the resident plasmid being eliminated from the population. The differences found for the two resident plasmids (pLS1AA21 and pLS1AA4) can be ascribed to their different copy number (Table 1). In the case of the high copy-number repA~ derivatives (pLS1AA12 and pLS1AA34), the resident piasmid was always observable at 9 = 0 (50-100% of Tc'^ cells). Densitometric scanning of the gels (not shown) indicated that in the clones with about 100% of Tc'^ celts, the number of copies of the resident

plasmids was reduced (on average) to levels similar to those of homoplasmid cultures harbouring pLS1. Since pLSI is fully stable in S. pneumoniae (del Solar et ai, 1987a), these findings indicate that the frans-acting RNA II produces a reduction in the plasmid copy number per cell, accompanied by slight instabitity which cannot be attributabte to the reduction in their copy number. This Kind of instability coutd be due to a defective self-correction in the fluctuations of the plasmid copy number because of the presenceof a frans-acting inhibitor source (Novick, 1987). After growing these initiatly heteropiasmid cultures for 20 and 50 more generations, the resident plasmid was being stowty eliminated from the cultures, concomitantty with a reduction in the number of Tc^ cells. The incompatibility behaviourofthesetwo high copy-number repA"" plasmids is consistent with the resutts obtained for pLSI cop7. However, the proportion of clones with a low resident plasmid content was higher for pLSI cop7 than for pLSI AA34, in spite of the similar number of copies of both repltcons in homoplasmid strains (Table 1). This difference could be due to a stronger repression by RNA It on pLSIcop/' than on the repA~ derivatives, probably because of the longer interaction region between RNA II and its target in the former than in the latter plasmids. In short, we may conclude that pLSI derivatives lacking repA and the putative terminator Tu are still susceptible to an increase in RNA it dosage (when supplied in trans) and that a comptementary 21 -nt stretch is enough to act as a target for the inhibitor. Discussion Two trans-acting plasmid-encoded products (RepA protein and RNA II) regulate pLSI copy number. RepA regulates synthesis of the initiator protein RepB by hindering transcription from PAB, whereas RNA II probably interacts with the repAB mRNA. How could there be RNA II interaction with its target? Computer analysis of the most favourable folding of the repAB mRNA showed that different conformations can be generated as the mRNA grows in length (not shown). However, the secondary structure complementary to Tj, (structure u in Fig. 6A). once generated, was always invariable. Generation of two other hairpin structures ([3 and 7) was predicted when the repAB mRNA reached co-ordinate 905 (Fig. 6A). On the loop of structure p is located the atypical ribosome-binding site of repB (Lacks ef ai, 1986); around co-ordinate 871, within structure -y, there is a segment comptementary to the - 3 5 region of Pn. For RNA II, the only predicted structure was that of Tu. tn view of this, direct inhibition by pairing of RNA tl with the repB ribosome-binding site (Fig. 6A) seems to be the simplest exptanation. Other plausible interstrand pairing between RNA It and repAB mRNA, teading to the inhibition of RepB synthesis, was not found.


G. del Solar and f\A. Espinosa B






0 11-13



I »• I' RNA


874 Rg. 6. Regulatory ctrcutts in pLS1. A. The proposed structure of the repAB mRNA at the 5' end of repS, and the proposed (arget of RNA II on the rer>AB mRNA. The three possible stem-loop structures (o, p. and -y) and the 9bp direct repeat {broken line) are indicated. The region complementary to RNA II is shown by a solid line. The termination codon of RepA (UAA), the proposed atypical ribosame-binding site (AUUUCU-4nt~UAUA; Lacks et a/., 1986) and ttie initiation codon of RepB (AUG) are depicted in bold. Numbers correspond to pLSI co-ordinates. B. Regulatory elements in pLSI. RNAs are shown as wavy lines, promoters as filled circles, and proteins as shaded arrows; arrowheads point to 3' ends. The positions of the three plasmid iterons (II - 13), the origins of replication, the hairpins, and the deletions that remove hairpin III are also depicted. Hairpin IV corresponds to the plasmid or\-). Broken lines indicate protein-DNA Interactions.

Direct inhibition by hindrance of the transtation initiation signals of the Rep protein by a ctRNA has been reported for the Pseudomonas ptasmid R1162 (Kim and Meyer, 1986). Both pLSI and R1162 have the ribosome-binding site tocated in a possible toop of the rep mRNA, suggesting that competition between ctRNAs and ribosomes for the translation of the initiator protein may be the mechanism used to eontrot ptasmid copy number, tncompatibitity was stitt observed when structure a was removed without affecting the repB atypicat ribosome-binding site (Fig. 5), suggesting that the RNA It-target interaction woutd not necessarity require pairing by the loops of hairpins, as postulated for other plasmids (Wagner and Nordstrom, 1986; Novick, 1987;Thomas, 1988).AnotherpLSI feature is the existence of a 9bp directty repeated sequence: one repeat is located in the region overtapping with RNA tl, and the other within the first codons of repB (Figs 6A and 7). Atthough these repeats participate in possible intrastrand repAB mRNA pairing (Fig. 6A), we have not found any ptausible alternative structure involving pairing of any of these repeats with the complementary counterpart region in repAB mRNA. Thus, the role of these repeats (if any) remains to be clarified. The repA gene is not essential for

pLSI, since its deletion does not affect ptasmid reptication or maintenance. Furthermore, an increase in the repA dosage (when cloned under its own promoter) does not result in incompatibitity towards pLSI or in any significant reduction in its copy number (Fig. 4). Thus, RepA is not abte to efficiently correct fluctuations in plasmid copy number, probabty because of its autoregulatory rote. Some repA gene features resemble gene copB of ptasmid RI: (i) the products of both genes share homologies (del Solar etal., 1989); (ii) as repA, copB is not essential, and its deletion yietds ptasmids with increased copy number (Nordstrom and Nordstrom, 1985), and (iii) frans-acting CopB does not affect replication of wild-type RI (Nordstrom and Nordstrom, 1985). However, CopB does not regulate its own synthesis (as RepA does) and the CopB-repressed promoter is totatty sitent, being activated onty when the ptasmid copy number drops dramatically. In contrast, PAB from pLSI is never totalty btocked and it seems to be the only promoter invotved in repB expression. We may envisage the fotlowing general mechanism for the pLS1 reptication control invotving RepA and RNA It (Fig. 6B): the protein woutd keep the tevets of the repAB mRNA within certain timits, whereas RNA tl woutd be

Copy number control of pLS 1 91










a.c c.c 1'-














c c A S G U 'tl.ll C.C


cC19t (ctRMA)


U.A B.C V e C.B U.A

«.u u.a




Rg. 7. Comparison of the pLSI replication control region with analogous regions of plasmids of the pLSI family. Structural and functional elements similar to those shown for pLSI can be observed as follows: (i) a promoter (bold letters), similar to PAB, for the synthesis of a small repressor protein (equivalent to RepA) and the plasmid Rep protein; (ii) a ribosome-binding site (underlined) and an initiation codon (bold) for the synthesis of RepA-like proteins; (iii) a promoter with a defined -10 {bold) and an unclear - 3 5 (not marked) region, having the same location as Pn, which could direct the synthesis of a small ctRNA (equivalent to RNA tl); (iv) an inverted repeat (broken lines) which could generate a rho-independent transchption terminator for the CtRNAs (equivalent to TK). and (v) a directed repeat (underlined) affecting two different regions in the rep mRNA of these plasmids. Below each DNA sequence is the proposed secondary structure for the ctRNAs of the four plasmids. Co-ordinates are taKen from the nucleotide sequences of the plasmids; pLSI (Lacks ef al., 1986); pLB4 {Bates and Gilbert. 1989); pADB20i (Bergemann ef aL. 1989). and pE194 {Horinouchi and Weisbtum, 1982. with the modifications reported by Villafane etaL, 1987).


G. del Solar and M. Espinosa

synthesized constitutively, its concentration being proportional to plasmid copy number. Consequently, variable levels of RNA II could act on a rather constant concentration cf the target mRNA, allowing very efficient correction of the copy number fluctuations. A defective repA gene (as the cop7mutation) would break this equilibrium, leading tc an increase in copy number. Several homologies have been observed between pLSI and other Gram-positive plasmids at the Rep proteins, the ori{+), and the postulated RepA-like proteins (Mintcn et al., 1988; Bates and Gilbert, 1989; Bergemann ef ai, 1989; del Solar ef a/., 1990; de la Campa etai, 1990; Sozhamannan ef ai, 1990). Thus, pLS1 can be considered as the prototype of a family of plasmids replicating by a rollingcircle mechanism. This family also includes pE194 (from Sfap/7y/ococcusaureus; Horinouchi and Weisblum, 1982), pADB201 {irom Mycoplasma mycoides] Bergemann e(a/., 1989) and pLB4 (from Lactobacillus plantarum\ Bates and Gilbert, 1989). On this basis, we searched for possible small CtRNAs in those plasmids, trying to find putative promoters and secondary structures which coutd resemble those of pLSI RNA II. The results ofthis search (Fig. 7) shewed that, indeed, such small ctRNAs could exist in all the members of the pLS1 family. No homology at the nucieotide level existed, but the striking following similarities at structural level can be observed: the small size and simple structure of the ctRNAs, their genetic location, and the existence of one putative transcription terminator. In addition, a 9-11 bp direct repeat, located in a position similar to that of pLSI, exists in those plasmids (Fig. 7). In the case of pE194, Villafane et ai (1987) proposed that a long CtRNA could start at around co-ordinate 1245 (Fig. 7), ending at a secondary structure that was later proposed tc be the or/(-i-)cf the plasmid (Dempsey and Dubnau, 1989; de la Campa etai, 1990). A more recent report (Byeon and Weisbium, 1990) demonstrated the existence of a RepAlike protein (Cop) in pE194, which was proposed to be the cnly plasmid element involved in copy-number control, since analyses aimed at the detection cf ctRNAs in pE194 were unsuccessful. If our hypothesis of the existence of small CtRNAs in the plasmids of the pLSI family is correct we may reconcile the above reports since both a small CtRNA (ending at around cc-ordinate 1180 rather than at co-ordinate 880) and a repressor protein similar to the pLSI RepA would exist in plasmid pE194, as well as in the other members of the pLSI family. The similarities at the nucleotide, structural, and functional levels between four plasmids isolated from different hosts suggest a recent evolutionary divergency from a common ancestor. Experimental procedures Bacterial strains and plasmids S. pneumoniae 708 (end-1, exo'2. trt-1. hex-4, malM594) was used for the preparation of plasmid DNAs and for the in vivo

assays. E co/fBL21(DE3) (F , rgmB, ga/, ompT./nf..-P,acuvs T7 gene / imm21 nin5: Studier and Moffal (1986), a gift of B. Studier) was used as host for the expression of the repA gene, present in plasmid pLS19(deiSolarefa/., 1989). Plasmids used were: pLSI and pLS5 (Lacks etai., 1986), pLS1cop7and pLS19 (del Solar ef ai, 1989). and pC194cop (Ballester ef al.. 1990); the other plasmids described in this work (Table 1 and Fig. 3) were constructed and analysed in the pneumococcal host. Selective pressure in S. pneumoniae was: Tc (1 M-gml ') for pLSI-based plasmids and Cm (3jxgml ') for pC194-based replicons. Selection in E. coli cells harbouring pLS19 was for ampicillin at 200M.gml ', to avoid the overgrowth of plasmid-free cells (Studier and Moffat, 1986).

RIasmid DNA preparations and DNA manipulations Purified plasmid DNA was prepared by two consecutive CsCIethidium bromide gradients (del Solar et al., 1987b). Total DNA was analysed from pneumococcal cells harbouring the plasmids indicated in the Results. The cultures were grown to 4 x io^ c.f.u. ml ' and extracts containing plasmid and chromosomal DNA (crude extracts) were prepared and electrophoresed as described (del Solar et ai, 1987a). Restriction endonucleases were purchased from New England Biolabs or Boehringer Mannheim, and were used as specified by the suppliers. Restriction fragments were purified from agarose gels with the GeneClean kit. (Bio-101 Inc.). Filling-in of DNA fragments with cohesive ends with Pol IK enzyme (Boehringer Mannheim) was performed as described (del Solar and Espinosa, 1991).

Construction of deletions from pLSI and DNA nucleotide sequence determination Controlled deletions were prepared as follows: plasmid pLSI DNA (4 H-g) was linearized with Bgl\ (co-ordinate 804) and treated with 1 unit of BAL31 nuclease (New England Biolabs) as specified by the supplier, to allow digestion of 50-100bp. After incubation (for 0.5,1, 1.5 and 2min), reaction was stopped and the extent of digestion was detected by agarose gel electrophoresis. The appropriate samples were pooled, and the DNA ends were filled in with Pol IK. Samples were subjected to ligation, and used to transform competent pneumococcal cultures. One hundred clones were examined for their plasmid DNA content, and several of them were selected for further analysis. Purified plasmid DNA was prepared from the selected clones, and the nucleotide DNA sequence between the restriction sites ApaU (co-ordinate 607) and Psfl (co-ordinate 1056) was determined by the chemical method of Maxam and Gilbert (1980).

Cloning pLSI DNA fragments intopC194 cop DNA (lOM-g) from either pLSI or pLS1cop7 was digested with Psfl, and fragments B (1051 bp) from both plasmids were purified and separately subjected to total digestion with Alu I and to partial digestion with Bgl\. To make the cohesive ends blunt, the mixture of fragments (from pLSI or from pLSI copT) was treated with nuclease SI and iigated to DNA (0.5 ^-g) from pC194cop which had been linearized with H/ndlll and SI-treated. The ligation mixtures were used to transform competent pneumococcal cultures, and transformants were selected for Cm'' and screened

Copy number control ofpLSI 93 for the presence of recombinant plasmids. The desired plasmids were characterized by detailed restriction mapping. Plasmid pCGA9 was constructed by ligation of the fragment Sty\ A (2324bp) from plasmid pCGAlcop7 with the small Sty\-Hin6i\\ (743bp) fragment of plasmid pCI 94cop. Ligation was performed in three steps; first, the Sfyl site included within the cat gene of pC194cop was regenerated, next the Sty\ and H/ndlll non-compatible ends were treated with Pol IK and, finally, a second ligation was performed in the conditions necessary to obtain circular DNA molecules. The ligation mixture was used to transform competent pneumococcal cultures, as above.

Soft laser densitometric scannings were performed in an LKB Ultroscan 2202 coupled to an Apple II computer. Searches for homologies and secondary structures were done with the DiwsTAR computer programs (DI^STAR Inc. UK). Some of the predictions on folding in the repAB mRNA were kindly made by Dr Sierd Bron at the Groningen University (The Netherlands). Independently, we performed the predictions for repAB mRNA

Incompatibility tests and copy number determinations

Notes added in proof

Type I quantitative incompatibility Tests (Nordstrom etai. 1980) were performed by using the above mentioned recombinant plasmids (carrying the cloned pLSI-based DNA fragments) as incoming replicons. Competent pneumococcal cultures (1 ml) harbouring pLS1 or its derivatives were transformed with 0.5 ^g of the donor piasmid DNA. After allowing for phenotypic expression (75min), cultures were induced with 0.1 (igml"' of Cm (15min) and transformants were selected in plates containing 3 ^g ml" •" of Cm. Several colonies of the same size and incubated for the same period of time were picked, and grown (37 generations, see Results). Total DNA content was analysed by preparation of crude extracts from exponentially growing cultures, and eiectrophoresis in agarose gels. Several amounts of each extract were run and various pictures were taken (with different exposition times) to avoid saturation of the negatives, which were later subjected to densitometric scanning. Plasmid copy number in the homoplasmid strains was determined as previously reported (Projan et ai, 1983; Lacks era/.. 1986).

The recently sequenced lactococcal plasmid, pWVOi (Leenhouts, K.J., Tolner, B., Bron, S.. Kok, J., Venema, G., and Seegers, J.F.M.L. (1991) Plasmid 26: 55-66), belonging to the pLSI family, could also encode a small ctRNA (co-ordinates 832-735) not reported by the authors. The genetic location ofthis region and the features of this putative ctRNA are analogous to those depicted in Fig. 7.

Computer work

and for RNA II with the aid of PCFOLD and MOLECULE programs

developed by M. Zucker and J. R. Thompson, respectively, and provided by the authors.

Acknowledgements Thanks are due to B. Peral for the construction of the copii mutation, to members of our laboratory for discussions, to M. T. Alda, P. Valiente, and R. Galan for technical assistance, and to A. Hurtado for the art work. Special thanks are due to Dr S. Bron who aided us with computer analyses. Research was financed by CiCYT (Grant BIO88-0449), and by the CSIC (Grant 88DK-391).

References Purification of RepA Hyperexpression of repA was done in Escherichia coii BL21(DE3) cultures harbouring plasmid pLS19. as described (del Solar etai, 1989). RepA protein was purified essentially as reported (del Solar et ai, 1989), except that the DEAE-Sephacel column was omitted, and a second heparin-agarose column was used as the final purification step. The molar concentration of the purified RepA protein was calculated by amino acid analysis of a known volume of the protein preparation. The final concentration of the RepA preparation used in this work was 20ng(j..l"\

In vitro transcription assays Full or run-off transcripts were synthesized from the purified DNA fragments (lOnM) indicated in the Results under published conditions (del Solar ef a/., 1990). RNA synthesis was initiated by the addition of 0.2 units of E. coli RNA polymerase (Promega) followed by incubation of the reaction mixtures for 10min at 37''C. Samples treated with purified RepA (50 and 100 ng) were preincubated with the protein for 10 min at 2GX prior to the addition of the RNA polymerase. Transcripts were detected by electrophoresis in 8% polyacrylamide, 8M urea sequencing gels (Maxam and Gilbert, 1980). As molecular weight standards, Ddel fragments from pLSI AA12 DNA. labelled at their 5' ends, were used.

Ballester, S., Alonso, J.C.. L6pez. P., and Espinosa. M. (1990) Comparative expression of the pC194 caf gene in Streptococcus pneumoniae, Bacillus subtiiis and Escherichia coli Gene 86; 71-79. Bates, E.E.M.. and Gilbert, H.J. (1989) Characterization of a cryptic plasmid from Lactobacillus plantarum. Gene 85: 253258. Bergemann, A.D., Whitley, J.C. and Finch, LR. (1989) Homology of mycoplasma plasmid pADB201 and staphylococcal plasmid pE194. JSacf6nD/171; 593-595. Byeon, W.-H., and Weisblum, B. (1990) Replication genes of plasmid pE194-cop and repF: transcripts and encoded proteins. J Bacteriol 172: 5892-5900. Castagnoli, L., Lacatena, R.M., and Cesareni, G. (1985) Analysis of dominant copy number mutants of the plasmid pMBI. NucI Acids Res 13: 5353-5367. d'Aubenton-Carafa, Y.. Brody, E., andThermes. C. (1990) Prediction of rho-independent Escherichia co/i transcription terminators. A statistical analysis of their RNA stem-loop structures. J /Wo/e/o/216: 835-868. de la Campa, A.G., del Solar, G., and Espinosa, M. (1990) Initiation of replication of plasmid pLSI. The initiator protein RepB acts on two distant DNA regions. J/Wo/B/o/213: 247-262. del Solar, G., and Espinosa, M. (1991) Labelling DNA ends with the Klenow fragment of the E. coli DNA polymerase I: a cautionary note. NucI Acids Res 19: 1956. del Solar, G., Puyet, A., and Espinosa, M. (1987a) Initiation signals for the conversion of single stranded to double stranded DNA


G. del Solar and M. Espinosa

forms in the streptococcal plasmid pLSI. NucI Acids Res 15: 5561-5580. del Solar. G., Diaz, R., and Espinosa, M. (1987b) Replication of the streptococcal plasmid pMV158 and derivatives in cell-free extracts of Escherichia coli. Mol Gen Genet 206: 42&-435. del Solar, G., de la Campa, A.G., Perez-Martin, J., Choli, T., and Espinosa, M. (1989) Purification and characterization of RepA, a protein involved in the copy number control of plasmid pLSl. NucI Acids Res 17: 2405-2420. del Solar, G.. Perez-Martin, J.. and Espinosa, M. (1990) Plasmid pLSI-encoded RepA protein regulates transcription from repAB promoter by binding to a DNA sequence containing a 13-base pair symmetric element. J Biol Chem 265: 1256912575. Dempsey, L.A., and Dubnau, D.A. (1989) Localization of the replication origin of plasmid pE194. J Bacterioi 171: 28662869. Hillen, W., Schollmeier, K., and Gatz, C. (1984) Control of expression of the Tn/O-encoded tetracycline resistance operon. II. Interaction of RNA polymerase and TET repressor with the (efoperon regulatory region. JMolBioiM2:185-201. Horinouchi, S.. and Weisbium, B. (1982) Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. J Bacteriol 150: 804-814. Khan, S.A.. and Novick, R.P. (1983) Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus. Piasmid 10: 251-269. Kim. K., and Meyer, R.J. (1986) Copy-number of broad-hostrange plasmid R1162 is regulated by a small RNA. Nud Acids Res 14: 8027-8046. Kumar, C. and Novick, R.P. (1985) Plasmid pT181 replication is regulated by two countertranscripts. Proc NatI Acad Sci USA 82: 638-642. Lacks, S.A., Lopez, P., Greenberg, B., and Espinosa, M. (1986) Identification and analysis of genes for tetracycline resistance and reptication functions in the broad-host-range plasmid pLSI. J Mol Biol 192: 753-765. Maxam, A.H., and Gilbert, W. (1980) Sequencing end-labelled DNA with base-specific chemical cleavages. Meth Enzymol65: 499-560. Minton, N.P.. Oultram, J.D., Brehm, J.K., and Atkinson, T. (1988) The replication proteins of plasmid pE194 and pLSI have N-terminal homology. NucI Acids Res 16: 3101. Nordstrom, K., and Austin, S.J. (1989) Mechanisms that contri-

bute to the stable segregation of plasmids. Annu Rev Genet 23: 37-69. Nordstrom, M., and Nordstrom, K. (1985) Control of replication of Fll plasmids: comparison of the basic replicons and of the copS systems of plasmids R100 and R I . Plasmid A3: 81-87. Nordstrom, K., Molin, S., and Aagaard-Hansen, H. (1980) Partitioning of plasmid R1 in Escherichia coii: II. Incompatibility properties of the partitioning system. Plasmid A: 332-349. Nordstrom, K., Motin, S., and Light, J. (1984) Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid RI system. Ptasmid 12: 71 -90. Novick, R.P. (1987) Plasmid incompatibility. Microbiol Rev 5i: 381-395. Novick, R.P. (1989) Staphylococcal plasmids and their replication. Annu Rev Microbiol 43: 537-565. Novick. R.P., Adier, G.K., Projan, S.J., Carleton, S., Highlander, S.K., Gruss, A.. Khan, S.A.. and lordanescu, S. (1984) Control of pT181 replication. 1. The pT181 copy control function acts by inhibiting the synthesis of a replication protein. EMBO J 3: 2399-2405. Novick, R.P., lordanescu, S., Projan, S.J., Komblum, J., and Edelman, I. (1989) pT181 plasmid replication is regulated bya countertranscript-driven transcriptional attenuator. Cell 59: 395-404. Projan, S.J., Carleton, S., and Novick, R.P. (1983) Determination of plasmid copy number by fluorescence densitometry. Plasmid 9: 182-190. Puyet, A., del Solar. G.. and Espinosa, M. (1988) Identification of the origin and direction of replication of the broad-host-range plasmid pLSI. NucI Acids Res 16: 115-133. Sozhamannan, S., Dabert, P., Moretto, V., Ehrlich, S.D., and Gruss, A. (1990) Plus-origin mapping of single-stranded DNA piasmid pEI 94 and nick site homologies with other plasmids. J Bacteriol 172: 4543-^548. Studier, F.W.. and Moffat. B.A. (1986) Selective expression of cloned genes directed by T7 RNA polymerase. J Mol Biol 189: 113-130. Thomas, C M . (1988) Recent studies on the control of piasmid replication. Bioehem Biophys Acta 949: 253-263. Villafane. R., Bechhofer, D.H., Narayan. C.S., and Dubnau, D.A. (1987) Replication control genes of plasmid pE194. J Bacterioi 169:4822-4829. Wagner. E.G.H., and Nordstrom, K. (1986) Structural analysis of a molecule involved in replication control of plasmid R I . NucI Acids Res 14: 2523-2538.

The copy number of plasmid pLS1 is regulated by two trans-acting plasmid products: the antisense RNA II and the repressor protein, RepA.

The promiscuous plasmid pLS1 encodes two transacting elements that regulate its copy number: protein RepA and antisense RNA II. In vitro transcription...
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