INTRODUCTION

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Plasmid replication by the rolling-circle (RC) mode has two initiation stages for the synthesis of the leading and the lagging strands. Leading-strand replication initiates through a nucleophilic attack exerted by the plasmid-encoded Rep protein on a specific dinucleotide, after recognition of a strand-specific sequence, the double-strand origin (dso). The 3'-OH end generated by Rep cleavage is thought to be elongated by conserved host-encoded proteins, such as DNA polymerase III and a helicase (reviewed in references 7, 11, 13, and 21). Termination of leading-strand synthesis leads to the generation of single-stranded plasmid DNA (ssDNA) intermediates which are the hallmark of this type of replicon; such plasmids are generically termed RCR (rolling-circle-replicating) plasmids (11, 20, 26). Lagging-strand synthesis usually starts from the single-strand origin, sso, which is a noncoding DNA region physically separate from the dso. The plasmid ssos operate in an orientation-dependent manner and have a high potential for intrastrand pairing (8, 10). The ssDNA intermediates are later converted to the double-stranded DNA (dsDNA) form solely by the use of the host machinery (1, 11, 13, 21).

Based on their sequence homologies, four groups of sso have been described so far: (i) ssoA, present in several plasmids from Staphylococcus aureus, such as pE194, pT181, and pSN2 (21); (ii) ssoU, described for pUB110 (2); (iii) ssoT, found in Bacillus plasmids such as pBAA1 and pT4010 (29); and (iv) ssoW, present in the lactococcal plasmid pWV01 (17, 28). The different types of sso can be distinguished not only by their nucleotide sequence and structure but also by the host range in which they are functional and by the effect of their deletion on plasmid copy number and segregational stability. In vivo and in vitro studies have shown either partial or total dependence on the host RNA polymerase (RNAP) in the first step of plasmid lagging-strand synthesis (2, 9, 14, 28, 29). In vitro, RNAP directs the synthesis of a 20-nucleotide (nt)-long primer RNA (pRNA) from within the ssoA of pMV158, which is later elongated by the host-encoded DNA polymerase I (15).

The streptococcal plasmid pMV158 is a promiscuous mobilizable multicopy RCR plasmid, originally isolated from Streptococcus agalactiae (3). It has the unique feature of having two ssos, namely ssoA and ssoU. This latter sso was removed when the pMV158-derivative plasmid pLS1 was constructed (8, 16, 23, 30). The pLS1-ssoA is included within a region of 199 bp, in which palindromic sequences able to generate a long stem-loop structure within the ssDNA are found (6, 14). Two sequences within this hairpin are conserved among ssoA-containing plasmids: (i) the recombination site B (RS_B), a 14-nt-long sequence located at the 5' end of the stem (22, 25), and (ii) a 6-nt conserved sequence, termed CS-6, located in the central loop (8, 10). The function of each of these conserved sequences has been assessed previously by construction of a set of mutants in the pLS1-ssoA. Changes in the RS_B region affect the binding of RNAP to the ssoA, while mutations in CS-6 interfered with pRNA termination without altering RNAP binding to the ssDNA (15). In the present work we have analyzed the in vivo effect of both types of mutations, carried by pLS1-derivative plasmids, in Streptococcus pneumoniae. We have also developed a cell-free system from S. pneumoniae and used it to study the effects of these mutations on the efficiency and specificity of lagging-strand initiation in vitro. The results obtained in vitro are consistent with those seen in vivo and demonstrate that RS_B plays a major role in lagging-strand replication while CS-6 has a minor influence on the ssoA activity in S. pneumoniae.

	MATERIALS AND METHODS

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Bacterial strains. S. pneumoniae 708 (end-1 exo-2 trt-1 hex-4 malM594) was employed as host of plasmids with the pMV158 replicon and as the source of cell extracts. Pneumococcal cells were grown and transformed with plasmid DNA as previously described (5, 16). Escherichia coli JM109 {[endA1 recA1 gyrA96 thi hsdR17 (r_K m_K⁺) relA1 supE44 (lac-proAB) F'(traD36 proA⁺B⁺ lacI^q lacZM15)] (31) was used to prepare ssDNA from the recombinant phagemids based on the pALTER-1 (Promega) vector. Selective pressure was exerted by using tetracycline at concentrations of 1 µg/ml (S. pneumoniae containing pMV158-based replicons) and 15 µg/ml (Escherichia coli containing phagemids).

Construction of pLS1 derivatives and recombinant phagemids. Plasmids, phagemids, and the M13 coliphage containing the ssoA region of pE194 are listed in Tables 1 and 2. To construct pLS1 derivatives with mutations in the ssoA, site-directed mutagenesis in the pLS1-ssoA was performed by the use of the Altered Sites kit (Promega) designed for in vitro mutagenesis. The nucleotide changes introduced in conserved sequences CS-6 and RS_B are described in Table 1. The 1,243-bp EcoRI-PstI fragment of pLS1, containing the ssoA (the enzymes cut at pMV158 coordinates 3170 and 5, respectively) (16, 23), was cloned into pALTER-1 to perform the mutagenesis (14, 15). The changes introduced new restriction sites, which were used to select the desired recombinants (15). The altered EcoRI-PstI fragments were inserted back into pLS1 by swapping the same restriction fragment. The resulting pLS1-derivative plasmids containing each of the mutations (Table 1) were rescued by transformation of pneumococcal cells. For each plasmid, the entire nucleotide sequence of the region encompassing the altered ssoA was determined by using the T7 sequencing kit (Pharmacia). To construct recombinant phagemids from which ssDNA was isolated for the in vitro replication assays, the 1,134-bp HindIII-PstI fragments of the pLS1 derivatives (coordinates of cleavage 4407 and 5, respectively) containing the mutated ssoA were cloned into the phagemid pALTER-1 digested with the same enzymes (Table 2). The recombinant phagemids were transferred to E. coli JM109 cells by transformation (27).

Detection of ssDNA accumulated in vivo and measurement of plasmid copy number. Pneumococcal cultures containing plasmids were grown to mid-exponential phase (about 2 × 10⁸ CFU per ml of culture), and total DNA was prepared (8). DNAs were analyzed by electrophoresis on 0.7% agarose gels, followed by transfer to nitrocellulose filters with or without prior denaturation (26). DNA on the filters was hybridized by using ³²P-labeled pLS1 DNA as the probe. The amount of ss- and dsDNA transferred to the filters was directly quantified with the aid of a PhosphorImager and ImageQuant software (Molecular Dynamics). Plasmid copy number was determined by agarose gel electrophoresis of sheared whole-cell lysates, followed by ethidium bromide staining and fluorescence densitometry as previously described (24).

Isolation of ssDNA from phagemids. E. coli JM109 cells containing the recombinant phagemids were grown at 37°C to an optical density at 600 nm of 0.7 and infected with the RK408 helper bacteriophage at a multiplicity of infection of 10. Cultures were grown for 13 to 16 h, and the encapsidated ssDNA was purified as previously described (27). The DNA products were analyzed on 1% agarose gels, and the amount of ssDNA was directly quantified with the Gel Doc system and software (Bio-Rad Laboratories). When the recombinant pALTER-1-based phagemid contained the wild type (wt) or mutated pLS1-ssoA in the functional orientation, the ssDNAs obtained had the initiation signals properly positioned. ssDNA molecules from pA-pLS1ssoA contained the complementary sequence of the replication origin, i.e., the ssoA in the nonfunctional orientation.

Cell-free replication extracts from S. pneumoniae. Cell extracts were prepared essentially as described previously for Staphylococcus aureus (1). One liter of exponentially growing S. pneumoniae culture (about 2 × 10⁸ CFU per ml) was centrifuged (5,000 × g for 10 min at 4°C), and the cell pellet was washed in buffer (50 mM Na-phosphate [pH 6.9], 1 mM EDTA, 5 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride). Cells were resuspended in 4 ml of the same buffer to which KCl was added (final concentration, 150 mM). After two rounds of freezing (70°C) and thawing (10°C), autolysin was added to a final concentration of 10 µg/ml, and the mixtures were incubated at 37°C for 7 min. Cell debris was removed by centrifugation (100,000 × g for 10 min at 4°C). To remove nucleic acids, 400 µl of streptomycin sulfate (30% in distilled water) was slowly added to the supernatant (3.6 ml) in an ice-chilled tube. After 30 min on ice, the precipitate was separated by centrifugation (20,000 × g), and ammonium sulfate (0.472 g/ml) was added to the supernatant. The protein precipitate was collected by centrifugation (20,000 × g) and dissolved in the assay buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 100 mM KCl, 1 mM dithiothreitol, 10% ethylene glycol). After dialysis against the same buffer, proteins were stored at 70°C. Protein concentration (usually 55 to 60 mg/ml) was measured by the bicinchoninic acid protein assay (Pierce).

In vitro lagging-strand synthesis. The in vitro replication assays were performed essentially as previously described (1). Reaction mixtures (30 µl) contained 150 ng of ssDNA in buffer (40 mM Tris-HCl [pH 8.0], 100 mM KCl, 12 mM magnesium acetate, 1 mM dithiothreitol), 50 µM NAD, 50 µM cyclic AMP, 2 mM ATP, 0.5 mM (each) UTP, CTP, and GTP, 50 µM (each) dCTP, dGTP, and dTTP, 20 µM [-³²P]dCTP (3,000 Ci/mmol), and 240 µg of cell extract proteins (unless otherwise stated). Mixtures were incubated at 32°C for 20 min in the experiments designed to map the lagging-strand initiation sites or for 60 min when total DNA replication was tested. DNA was recovered by phenol-chloroform extraction and ethanol precipitation. The replication products were separated by electrophoresis on 1% agarose gels in the presence of ethidium bromide (0.5 µg/ml) or on 8% acrylamide-8 M urea sequencing gels. When total replication levels were to be determined, the DNA products were linearized with HindIII (which cuts approximately 1,000 nt downstream from the ssoA, in the direction of replication) to convert the various forms of DNA (double-stranded open circles and covalently closed supercoiled and replicative intermediates) to a single band before electrophoresis. Bands were visualized under UV irradiation and by autoradiography of the dried gels. As size markers, linearized dsDNA phagemids were run on the same gel. To map the initiation sites of lagging-strand synthesis, the procedure described by Dempsey et al. (9) was followed. Partially replicated ssDNA samples (20-min reaction) were treated with AflII (which cuts approximately 100 bp downstream from the expected initiation site of DNA synthesis) before electrophoresis. To determine the distance from the lagging-strand initiation start points to the restriction site, known nucleotide sequencing reactions were run as size markers.

	RESULTS AND DISCUSSION

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In vivo characterization of ssoA mutants. The boundaries of the pLS1-ssoA have been defined between the recognition sites for AflII and NcoI (pMV158 coordinates 5150 and 5349, respectively) (Fig. 1). Deletion of this fragment results in plasmids which accumulate large amounts of ssDNA in S. pneumoniae (14). The two conserved sequences of the ssoA, RS_B and CS-6, are located within this 199-bp region (Fig. 1). This region is predicted to fold into an extensive secondary structure (Fig. 2). The functionality of the ssoA of RCR plasmids is orientation dependent, indicating that sequences important in their function are located in the unpaired regions within the ssoA secondary structure (8, 10). The CS-6 and part of the RS_B region comply with these requirements. Mutations were introduced in these two regions to change the sequence or the local structure of the RS_B (mutations G5, G6, and G7), the CS-6 (mutations CM, G1, G3, and G4), or both (G3G7 double mutant), without a gross alteration of the global pLS1-ssoA structure (15) (see Table 1). When ssDNA preparations containing these mutations were tested in vitro for their interactions with the Bacillus subtilis RNAP, two types of results were obtained: mutations in the RS_B severely impaired the binding of the enzyme to the DNA, whereas alterations in the CS-6 led to a twofold reduction in the total amount of primer RNA (pRNA) synthesized and resulted in the synthesis of pRNAs longer than 20 nt (size of the pRNA from the wt substrate [15]).

View larger version (35K): [in this window] [in a new window]   FIG. 1.   Plasmid pMV158 and its ssoA. (Top) Schematic map of pMV158 with some of the plasmid-encoded genes, relevant restriction sites (coordinates of cleavage sites in parentheses), and the relative positions of the dso and the two ssos (ssoU and ssoA). The position of the small EcoRI fragment (deleted in plasmid pLS1) is indicated. (Bottom) Nucleotide sequence of the plasmid ssoA between restriction sites AflII and NcoI (underlined). The two conserved sequences, RSB and CS-6, are boxed. The 3' ends of the deletions in plasmids pLS113 and pLS114 are indicated (arrows).

View larger version (8K): [in this window] [in a new window]   FIG. 2.   Predicted secondary structure of the pLS1-ssoA and initiation of lagging-strand synthesis in the wt pLS1 plasmid. The proposed structure of the pMV158-ssoA between the indicated coordinates, and the positions of the CS-6 and RSB sequences, and of the 3' end of the deletions in plasmids pLS113 and pLS114 are shown. The initiation points and direction of lagging-strand synthesis, mapped in Fig. 6, are indicated by filled arrowheads along with the sizes of the bands obtained after digestion with AflII. The same bands were observed in a time course analysis from the pLS1-ssoA (15).

View larger version (50K): [in this window] [in a new window]   FIG. 3.   Copy number and intracellular ssDNA accumulation of pLS1 and its derivatives in S. pneumoniae. Total DNA was isolated from the indicated plasmid-containing cultures, electrophoresed on 0.7% agarose gels in the presence of ethidium bromide (A), and transferred to nitrocellulose with (B) or without (C) prior denaturation. Note that in the latter case, only ssDNA is visible. The different plasmid forms were detected by using radiolabeled pLS1 as a probe. Both autoradiograms were overexposed to reveal the weak bands corresponding to ssDNA from plasmids with mutations in the CS-6 region. The positions of the supercoiled monomeric plasmid form (ccc), open circular dsDNA (oc) and ssDNA are indicated. chr, chromosomal DNA.

Development of an S. pneumoniae replication system for in vitro DNA synthesis. Plasmid pLS1 accumulated large amounts of ssDNA when transferred to S. aureus (not shown), as was the case with E. coli and B. subtilis (6, 8), indicating that the pLS1-ssoA was poorly functional in these hosts. Consequently, we decided to develop an in vitro replication system from S. pneumoniae, the host in which the pLS1-ssoA is fully functional. Based on previously described cell-free plasmid replication systems from E. coli (5) and S. aureus (1), we set up a new cell-free system from S. pneumoniae. Pneumococcal cells were subjected to lysis by the use of purified pneumococcal autolysin, since neither lysozyme nor lysostaphin (used for E. coli and S. aureus, respectively) acts on the pneumococcal cell wall. To optimize the lysis conditions, we tested different amounts of autolysin, reaction times, and temperatures of assay. Optimal conditions were as described in Materials and Methods.

View larger version (26K): [in this window] [in a new window]   FIG. 4.   In vitro replication from pLS1-ssoA and pE194-ssoA in the pneumococcal cell-free system (A) and inhibition of replication from the pLS1-ssoA by rifampin (B). (A) A total of 150 ng of ssDNA isolated from pA-pLS1ssoA and M13-pE194ssoA was used as the template. As negative controls, ssDNAs from both vectors pALTER-1 and M13mp19 (pA and M13, respectively) were employed. Each reaction was carried out in a volume of 30 µl, incubated for 60 min at 32°C with different concentrations of proteins from the cell extract (Ext). The reaction products were digested with HindIII and electrophoresed on a 1% agarose gel for 12 h at 2.5 V/cm. The autoradiogram of the dried gel is shown. (B) Similar assays were performed with DNA from pA-pLS1ssoA in the absence () or in the presence (+) of rifampin (Rif) (100 µg/ml).

Influence of ssoA mutations on in vitro DNA synthesis. Cells containing pLS1 derivatives with mutations in the ssoA exhibited two categories of phenotypes related to the amount of intracellular ssDNA accumulated (Fig. 3): moderate (CS-6 mutants) or high (mutations in the RS_B). To test whether this situation could be reproduced in the in vitro replication system, we constructed phagemids with some of the ssoA mutations representing each category (Tables 1 and 2). Thus, the following phagemids were constructed: pA-G1, changes in the CS-6; pA-G6, changes in the local structure of RS_B; pA-G7, changes in the conserved sequence of the RS_B; and pA-G3G7, changes in both the conserved sequences. In addition, we cloned a DNA region encompassing the pLS1-ssoA in the nonfunctional orientation (pA-pLS1ssoA) or a DNA region from a plasmid lacking ssoA (pA-NA) in the vector pALTER-1. From the various phagemids, ssDNA was generated and used as the substrate for in vitro DNA synthesis in the pneumococcal extracts as described above. The results (Fig. 5) can be summarized as follows. The efficiency of in vitro ss-  dsDNA conversion obtained with the G1 mutant was very similar to that with the wt origin. However, alterations in the RS_B region led to 17- and 20-fold decreases in the level of lagging-strand synthesis for the G7 and G6 mutants, respectively. These results agree with those obtained in vivo, supporting a major role of the RS_B region in the activity of the ssoA. No DNA synthesis was observed when the substrates lacked ssoA (pA-NA) or contained it in a nonfunctional orientation (pA-pLS1ssoA), confirming the specificity and the orientation dependence of the pLS1-ssoA in the in vitro pneumococcal system.

View larger version (39K): [in this window] [in a new window]   FIG. 5.   Lagging-strand synthesis from the mutagenized ssoA in S. pneumoniae. Reactions were performed as described in the legend for Fig. 4 but 8 µg of protein per 30 µl of reaction mixture (240 µg) and the ssDNA generated from the indicated recombinant phagemids were used. The autoradiogram was overexposed to visualize the bands corresponding to the RSB mutants (pA-G6 and pA-G7) and to the double mutant (pA-G3G7).

Specificity of in vitro lagging-strand synthesis: influence of ssoA mutations. In vitro DNA synthesis from the pLS1-ssoA initiates specifically at four major positions in S. pneumoniae (15). These sites were mapped by employment of partially replicated molecules obtained in the in vitro extracts which were then treated or not treated with a restriction enzyme that cleaves downstream of the expected initiation sites of DNA replication from the ssoA (9, 15). In the case of the pLS1-ssoA, the enzyme chosen was AflII, which cleaves downstream of the replication start points (Fig. 1). Denaturation of the DNA releases replication products whose sizes correspond to the distance between the initiation sites of lagging-strand synthesis and the enzyme cleavage site. Lack of specificity in initiation of DNA synthesis is revealed either by lack of defined bands in the reaction products or by the presence of the same bands in both the digested and undigested samples. Bands that are present in the undigested samples indicate random initiation and/or end points of ss-  dsDNA synthesis and do not reveal the specific initiation sites (9).

View larger version (77K): [in this window] [in a new window]   FIG. 6.   Determination of the initiation points of lagging-strand synthesis from the mutagenized pLS1-ssoA in S. pneumoniae. The ssDNAs generated from the indicated recombinant phagemids were employed as replication templates. After a 20-min reaction, the DNA was purified and one half of each sample was digested with AflII (A) or left undigested (). DNA in the samples was denatured by boiling prior to separation of the reaction products on an 8% polyacrylamide-urea gel. ACGT, sequencing ladder generated by the Sanger method (27). Numbers indicate sizes of bands in nucleotides.