JOURNAL OF BACTERIOLOGY, Dec. 1991, p. 7695-7697 0021-9193/91/237695-03$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 173, No. 23
The Escherichia coli terB Sequence Affects Maintenance of a Plasmid with the M13 Phage Replication Origin MARILYNE UZEST, S. DUSKO EHRLICH, AND BENEDICTE MICHEL* Laboratoire de Ge'ne'tique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France Received 1 July 1991/Accepted 26 September 1991 Replication initiated at the bacteriophage M13 origin can be affected by interaction of a properly oriented termination signal terB and the Tus protein. The effect can be alleviated by overproduction of the M13 replication gene protein II.
The replication terminus of the Escherichia coli chromosome is a large region (350 kb) flanked on both sides by terminator sites that arrest replication in a polar fashion (4, 10). These sequences impede replication initiated at the E. coli chromosomal origin or at the replication origins of phages or plasmids integrated into the chromosome (4, 5, 10). Homologous terminator sites were also identified on E. coli plasmid R6K (1, 15). The introduction of a ter site on ColEl-derived plasmids blocks their replication (9, 12, 22). This block is dependent on the orientation of the site relative to the direction of replication and requires the presence of a host-encoded protein named Tus (11). In vitro replication systems were used to characterize the mechanism of replication arrest: Tus protein binds to the ter sequence (13, 18), and the Tus-ter complex inhibits the progression of DNA helicases (17, 20). Strand displacement by DnaB helicase is inhibited as a result of the binding of Tus to either the R6K terR or the E. coli terB sequence. In addition, the Tus-terB complex also inhibits the action of two other E. coli helicases, UvrD (helicase II) and Rep. Most replicons used so far to study Tus-terB-dependent termination replicate by a theta mechanism and require the DnaB helicase. In contrast, the filamentous bacteriophage M13 (fl or fd) replicates by a rolling circle mechanism and requires the Rep helicase (24). In this study, we show that in vivo, Tus-terB interaction interferes with M13 replication. However, an excess of gene protein II (gpII) alleviates this
is less stable in the absence of selective pressure in tus cells than in tus+ cells. This plasmid does not carry a ter-like signal in the region deriving from pSC101 or in gpII, but the sequence of the spectinomycin resistance gene is not known. The reasons for the effect of the tus mutation on pSCgpII are not understood. It was reported that the stability of pSC101 derivatives lacking the par site is affected by plasmid supercoiling (21), which could possibly differ in tus+ and tus cells. The three plasmids pHV960, pHV960T+, and pHV96OTwere used to transform strain JJC40 (tus+ rep' [2]) harboring plasmid pSCgpII (Table 1). In the presence of 0.02 to 0.03 mM isopropyl-p-D-thiogalactoside (IPTG), pHV960T+ transformed much less efficiently than did pHV960 and pHV96OT-. In contrast, the three plasmids transformed a Atus::Kanr isogenic strain with the same efficiency (tus cells were poorly transformed by these plasmids, possibly because of the low copy number of pSCgpII; Fig. 2). These experiments show that the interaction of Tus and ter oriented to arrest replication initiated at the M13 origin interferes with plasmid transformation. This effect is not seen wheh the gpII protein is produced at a high level. JJC40 cells harboring pSCgpII and either pHV960, pHV960T+, or pHV96OT- were grown overnight in the presence of 0.03 mM IPTG without selective pressure (presence of spectinomycin and absence of ampicillin). More than 95% of cells harbor plasmids, as deduced from viable counts and replica plating on selective (ampicillin) and nonselective media. Plasmid DNA was extracted and analyzed by gel electrophoresis (Fig. 2). We estimate that the copy number of pHV960T+ is at least 10-fold lower than that of pHV960 or pHV96OT- (lanes 3 to 5). At this IPTG concentration, the results were irreproducible in the Atus strain, possibly because of the low copy number of pSCgpII. However, at 0.1 mM IPTG, a threefold difference was still observed with tus+ cells (lanes 6 to 8), whereas no difference was detected with tus cells (lanes 9 to 11). Therefore, the Tus protein reduces specifically the copy number of pHV960T+. No difference was detected between the three plasmids when the DNA was extracted from cultures grown in 1 mM IPTG (not shown), which confirms that an excess of gpII alleviates the effect of Tus-ter interaction. On ColEl-derived plasmids, the arrest of replication at a ter site leads to the accumulation of molecules carrying a replication bubble, which can be detected by gel electrophoresis (16). Arrest of pHV960T+ replication at terB should lead to the formation of sigma-shaped molecules with the length of the linear tail corresponding to the distance between the M13 origin and terB. These should migrate slower
effect. Plasmid pHV960 carries the ampicillin resistance gene of plasmid pBR322 and the M13 plus and minus replication origins (Fig. 1). It replicates in E. coli strains possessing the Rep helicase and the M13 replication protein (gpII) but not in those lacking either gpII or Rep helicase (25). Two derivatives of pHV960 were constructed by cloning terB 2.25 kb downstream of the M13 plus replication origin. In pHV960T+, terB is oriented to arrest replication initiated at this origin, whereas in pHV96OT-, it is oriented in the opposite direction and should not arrest this replication (Fig. 1). Plasmid pSCgpII is a pCS101 derivative carrying a spectinomycin resistance gene (cloned from plasmid pHP45omega [23]) and gene II of fl under the control of the lacUV5 promoter (cloned from plasmid pLII [6]). pSCgpII and pHV960 are compatible and can thus be maintained in the same E. coli cell. In the course of this work, we observed that plasmid pSCgpII is present at a lower copy number and *
Corresponding author. 7695
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NOTES
J. BACTERIOL.
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p
NI
FIG. 1. Schematic representation of plasmid pHV960 (4,403 bp). Thin line, pBR322 sequences; hatched box, M13 sequences; open box, pC194-delta NsiI. The arrows indicate the direction of plus and minus M13 replication; the closed arrows represent the terB sequences (T+ and T-). Plasmid pHV960 was constructed in several steps. First, an M13 fragment carrying the replication origin (nucleotides 5614 to 5995 of M13 [27]) was inserted in the EcoRV site of pHV5O, a plasmid which can replicate in both E. coli and Bacillus subtilis (7). The resulting construct is maintained poorly in E. coli. A spontaneous deletant was isolated which is maintained well and in which nucleotide 2771 of pBR322 is linked to nucleotide 5823 of M13. Finally, an NsiI fragment of pC194 (nucleotides 113 to 576) which inactivates the replication of pC194 by deleting 154 amino acids from its replication protein (8, 14) was removed in vitro. Plasmids pHV960T+ and pHV96OT- were obtained by cloning in the Stul site of pHV960 the following oligonucleotide:
5'-GATCATAAAATAAGTATGTTGTAACTAAAGT-3' 3'-CTAGTATTTTATTCATACAACATTGATTTCA-5' The Sau3A site (5'-GATC) is proximal to the Apr gene in pHV960T+ and distal in pHV96OT-.
than monomeric plasmids on agarose gels. Total DNA was extracted by two different techniques from tus+ cells harboring pSCgpII plus pHV960, pHV960T+, or pHV96OT-, grown to the exponential phase or overnight, in the presence of 0.03 to 1 mM IPTG. The first technique was carried out according to the "ter-assay" (16), involving cell lysis by lysosyme-sodium dodecyl sulfate (SDS)-NaCl treatment and allowing the detection of D-loop replication intermediates. By this method, we detected pTH101 replication intermediates (12) in a control experiment (not shown). The second method was developed to detect single-stranded DNA molecules (26) and involves cell lysis by an SDS-Ficoll procedure. It allowed us to visualize the expected amounts of
FIG. 2. Evidence that the interaction of Tus and terB affects the copy number of plasmids with the M13 replication origin. DNA was extracted according to the "ter-assay" procedure (16) from 1.5 ml of
cells containing pSCgpII and pHV960 (0), pHV960T+ (+), or pHV96OT- (-) and linearized by SpeI. Lanes: 1 and 2, pSCgpII and pHV960 standard DNA, respectively; 3 to 8, tus+ cells; 9 to 11, tus cells; 3 to 5, 0.03 mM IPTG; 6 to 11, 0.1 mM IPTG.
circular single-stranded plasmid molecules (not shown). DNA was analyzed by Southern hybridization with a pHV960-specific probe, either intact or cleaved by HindIII, PstI, PvuII, or PstI and PvuII (positions of the sites are shown in Fig. 1). No slowly migrating molecules were detected. In an attempt to protect the tail of the putative sigma-shaped molecules, similar experiments were performed (i) in the presence of the M13 single-stranded DNA binding protein, gpV, constitutively expressed from a compatible plasmid; (ii) after cloning, downstream of the M13 plus origin, of the primosome assembly site rriB of pBR322 (28) (a 180-bp fragment of pBR322, nucleotides 2066 to 2246, was inserted in the AlwNI site of pHV960T+), which should allow the conversion of the single-stranded tail to a doublestranded form; and (iii) in a recB sbcB strain, which is devoid of the two major E. coli exonucleases, ExoV and ExoL. Slowly migrating molecules were not detected under any conditions, which indicates that, if present, they represent less than 1% of plasmid DNA. In conclusion, our evidence indicates that interaction of terB and Tus interferes with maintenance of a plasmid with the M13 bacteriophage replication origin. The stability of the plasmid in nonselective media and the alleviation of the phenomenon by an excess of the replication protein gpII suggest that the interference takes place at the level of replication rather than plasmid partition. We consider two possibilities, inhibition of initiation or arrest of elongation in
TABLE 1. Relative efficiency of transformation in tus+ and tus cellsa Relative efficiency of transformation at indicated IPTG concn6 Plasmid
pHV960T+ pHV96OT-
tus+
0.02-0.03 mMc 5 x 10-3 0.6
0.05-0.1 mMc 0.2 0.7
tus
1 mMd
0.7 1.3
0.03 mMd 1 1
0.05 mMd 1 0.73
mMd 0.65 0.73
1
a Competent E. coli cells were prepared by overnight incubation at 0'C in 0.1 M CaCl2 (3); 150 1LI of competent cells was mixed with 0.05 to 50 ng of DNA and incubated for 10 min at 0°C and 10 min at 37°C. The mixture was then divided in three aliquots, which were added to 500 p.1 of LBT expression medium containing three different concentrations of IPTG. After 1 h of growth at 37°C, 100 pi1 of each culture was spread on plates containing the same concentration of IPTG as the expression medium and 100 ,ug of ampicillin per ml. b Relative to the value for pHV960, which was set at 1. Absolute values for pHV960 ranged from 0.5 to 2 x 106 Apr transformants per ,ug of DNA except when tus cells were transformed in the presence of 0.03 mM IPTG, in which case 102 transformants per ,ug of DNA were obtained (see text). c Average from five experiments. d Average from two or three experiments.
VOL. 173, 1991
DNA synthesis. The binding of Tus protein could conceivably affect the efficiency of replication initiation at the M13 origin, by modifying superhelicity of the molecule. However, such an effect is not expected to be dependent on the orientation of the ter site. We therefore prefer to propose that the inhibition of replication is due to arrest of the replication forks initiated at the M13 replication origin. This confirms and extends the in vitro observation that the Tus-terB complex inhibits DNA unwinding by the Rep helicase (20) and the in vivo observation that it arrests the Rep-dependent replication of phage P2 integrated into the host chromosome (10). However, this block of replication does not seem to be very efficient, since the effects of terB on M13 replication are detected only in the presence of a low amount of the phage replication protein gpII. This finding may be correlated with the in vitro observation that the Tus-terR complex, which is less stable than the Tus-terB complex (19), does not inhibit the Rep helicase (17). The failure to detect the sigma-shaped structures expected from replication arrest at terB could be due to a low efficiency of the arrest or to a loss of these structures either in vivo by exonucleolytic degradation or during DNA extraction. The dependence on the orientation of terB and on the presence of Tus argues in favor of a similar mechanism of inhibition of replication for the M13 and oriC or ColEl replication forks. We are grateful to L. Janniere for helpful discussions and suggestions of experiments, to C. Anagnostopoulos for improvements on the manuscript, and to F. Haimet for producing the artwork. This work was supported by a grant from Fondation pour la Recherche Mddicale. B.M. is on the CNRS staff. REFERENCES 1. Bastia, D., J. Germino, J. H. Crosa, and J. Ram. 1981. The nucleotide sequence surrounding the replication terminus of R6K. Proc. Natl. Acad. Sci. USA 72:2095-2099. 2. Bierne, H., S. D. Ehrlich, and B. Michel. 1991. The replication terminator signal terB of the Escherichia coli chromosome is a deletion hot-spot. EMBO J. 10:2699-2705. 3. Dagert, M., and S. D. Ehrlich. 1979. Prolonged incubation in calcium chloride improves the competence of E. coli cells. Gene 6:23-28. 4. De Massy, B., S. Bejar, J. Louarn, J. M. Louarn, and J. P. Bouche. 1987. Inhibition of replication forks exiting the terminus of the Escherichia coli chromosome occurs at two loci separated by 5 min. Proc. Natl. Acad. Sci. USA 84:1759-1763. 5. Francois, V., J. Louarn, and J. M. Louarn. 1989. The terminus of the Escherichia coli chromosome is flanked by several polar replication pause sites. Mol. Microbiol. 3:995-1002. 6. Fulford, W., and P. Model. 1988. Regulation of bacteriophage fl DNA replication. I. New functions for genes II and X. J. Mol. Biol. 203:49-62. 7. Goze, A., and S. D. Ehrlich. 1980. Replication of plasmids from Staphylococcus aureus in Escherichia coli. Proc. Natl. Acad. Sci. USA 77:7333-7337. 8. Gros, M. F., H. te Riele, and S. D. Ehrlich. 1987. Rolling circle replication of single-stranded DNA plasmid pC194. EMBO J. 6:3863-3869. 9. Hidaka, M., M. Akiyama, and T. Horiuchi. 1988. A consensus sequence of three DNA replication terminus sites on the E. coli chromosome is highly homologous to the terR sites of the R6K
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plasmid. Cell 55:467-475. 10. Hill, T. M., J. M. Henson, and P. L. Kuempel. 1987. The terminus region of the Escherichia coli chromosome contains two separate loci that exhibit polar inhibition of replication. Proc. Natl. Acad. Sci. USA 84:1754-1758. 11. Hill, T. M., B. J. Kopp, and P. J. Kuempel. 1988. Termination of DNA replication in Escherichia coli requires a trans-acting factor. J. Bacteriol. 170:662-668. 12. Hill, T. M., A. J. Pelietier, M. L. Tecklenburg, and P. L. Kuempel. 1988. Identification of the DNA sequence of the E. coli terminus region that halts replication forks. Cell 55:459-466. 13. Hill, T. M., M. L. Tecklenburg, A. J. Pelletier, and P. L. Kuempel. 1989. tus, the trans-acting gene required for termination of DNA replication in Escherichia coli, encodes a DNAbinding protein. Proc. Natl. Acad. Sci. USA 86:1593-1597. 14. Horinouchi, S., and B. Weisblum. 1982. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150:815-825. 15. Horiuchi, T., and M. Hidaka. 1988. Core sequence of two separable terminus sites of the R6K plasmid that exhibit polar inhibition of replication is a 20 bp inverted repeat. Cell 54:515523. 16. Horiuchi, T., M. Hidaka, M. Akiyama, H. Nishitani, and M. Sekiguchi. 1987. Replication intermediate of a hybrid plasmid carrying the replication terminus (ter) site of R6K as revealed by agarose electrophoresis. Mol. Gen. Genet. 210:394-398. 17. Khatri, G. S., T. MacAllister, P. R. Sista, and D. Bastia. 1989. The replication terminator protein of E. coli is a DNA sequencespecific contra helicase. Cell 59:667-674. 18. Kobayashi, T., M. Hidaka, and T. Horiuchi. 1989. Evidence of a ter specific binding protein essential for the termination reaction of DNA replication in Escherichia coli. EMBO J. 8:2435-2441. 19. Kuempel, P. L., A. J. Pelletier, and T. M. Hill. 1989. Tus and the terminators: the arrest of replication in procaryotes. Cell 59: 581-583. 20. Lee, E. H., A. Kornberg, M. Hidaka, T. Kobayashi, and T. Horiuchi. 1989. E. coli replication termination protein impedes the action of helicases. Proc. Natl. Acad. Sci. USA 86:91049108. 21. Miller, C. A., S. L. Beaucage, and S. N. Cohen. 1990. Role of DNA superhelicity in partitioning of the pSC101 plasmid. Cell 62:127-133. 22. Pelletier, A. J., T. M. Hill, and P. L. Kuempel. 1989. Termination sites Ti and T2 from the Escherichia coli chromosome inhibit DNA replication in ColEl-derived plasmids. J. Bacteriol. 171:1739-1741. 23. Prentki, P., and H. M. Krisch. 1984. In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303-313. 24. Takahashi, S., C. Hours, M. Iwaya, H. E. Lane, and D. T. Denhardt. 1978. The Escherichia coli rep gene, p. 393-400. In The single-stranded DNA phages. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 25. te Riele, H. Personal communication. 26. te Riele, H., B. Michel, and S. D. Ehrlich. 1986. Are singlestranded circles intermediates in plasmid DNA replication? EMBO J. 5:631-637. 27. Van Wezenbeek, P. M., T. J. M. Hulsebos, and J. G. G. Shoenmakers. 1980. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene 11:129-148. 28. Zipursky, S. L., and K. J. Marians. 1980. Identification of two Escherichia coli factor Y effector sites near the origins of replication of the plasmids ColEl and pBR322. Proc. Natl. Acad. Sci. USA 77:6521-6525.
Vol. 173, No. 23
The Escherichia coli terB Sequence Affects Maintenance of a Plasmid with the M13 Phage Replication Origin MARILYNE UZEST, S. DUSKO EHRLICH, AND BENEDICTE MICHEL* Laboratoire de Ge'ne'tique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France Received 1 July 1991/Accepted 26 September 1991 Replication initiated at the bacteriophage M13 origin can be affected by interaction of a properly oriented termination signal terB and the Tus protein. The effect can be alleviated by overproduction of the M13 replication gene protein II.
The replication terminus of the Escherichia coli chromosome is a large region (350 kb) flanked on both sides by terminator sites that arrest replication in a polar fashion (4, 10). These sequences impede replication initiated at the E. coli chromosomal origin or at the replication origins of phages or plasmids integrated into the chromosome (4, 5, 10). Homologous terminator sites were also identified on E. coli plasmid R6K (1, 15). The introduction of a ter site on ColEl-derived plasmids blocks their replication (9, 12, 22). This block is dependent on the orientation of the site relative to the direction of replication and requires the presence of a host-encoded protein named Tus (11). In vitro replication systems were used to characterize the mechanism of replication arrest: Tus protein binds to the ter sequence (13, 18), and the Tus-ter complex inhibits the progression of DNA helicases (17, 20). Strand displacement by DnaB helicase is inhibited as a result of the binding of Tus to either the R6K terR or the E. coli terB sequence. In addition, the Tus-terB complex also inhibits the action of two other E. coli helicases, UvrD (helicase II) and Rep. Most replicons used so far to study Tus-terB-dependent termination replicate by a theta mechanism and require the DnaB helicase. In contrast, the filamentous bacteriophage M13 (fl or fd) replicates by a rolling circle mechanism and requires the Rep helicase (24). In this study, we show that in vivo, Tus-terB interaction interferes with M13 replication. However, an excess of gene protein II (gpII) alleviates this
is less stable in the absence of selective pressure in tus cells than in tus+ cells. This plasmid does not carry a ter-like signal in the region deriving from pSC101 or in gpII, but the sequence of the spectinomycin resistance gene is not known. The reasons for the effect of the tus mutation on pSCgpII are not understood. It was reported that the stability of pSC101 derivatives lacking the par site is affected by plasmid supercoiling (21), which could possibly differ in tus+ and tus cells. The three plasmids pHV960, pHV960T+, and pHV96OTwere used to transform strain JJC40 (tus+ rep' [2]) harboring plasmid pSCgpII (Table 1). In the presence of 0.02 to 0.03 mM isopropyl-p-D-thiogalactoside (IPTG), pHV960T+ transformed much less efficiently than did pHV960 and pHV96OT-. In contrast, the three plasmids transformed a Atus::Kanr isogenic strain with the same efficiency (tus cells were poorly transformed by these plasmids, possibly because of the low copy number of pSCgpII; Fig. 2). These experiments show that the interaction of Tus and ter oriented to arrest replication initiated at the M13 origin interferes with plasmid transformation. This effect is not seen wheh the gpII protein is produced at a high level. JJC40 cells harboring pSCgpII and either pHV960, pHV960T+, or pHV96OT- were grown overnight in the presence of 0.03 mM IPTG without selective pressure (presence of spectinomycin and absence of ampicillin). More than 95% of cells harbor plasmids, as deduced from viable counts and replica plating on selective (ampicillin) and nonselective media. Plasmid DNA was extracted and analyzed by gel electrophoresis (Fig. 2). We estimate that the copy number of pHV960T+ is at least 10-fold lower than that of pHV960 or pHV96OT- (lanes 3 to 5). At this IPTG concentration, the results were irreproducible in the Atus strain, possibly because of the low copy number of pSCgpII. However, at 0.1 mM IPTG, a threefold difference was still observed with tus+ cells (lanes 6 to 8), whereas no difference was detected with tus cells (lanes 9 to 11). Therefore, the Tus protein reduces specifically the copy number of pHV960T+. No difference was detected between the three plasmids when the DNA was extracted from cultures grown in 1 mM IPTG (not shown), which confirms that an excess of gpII alleviates the effect of Tus-ter interaction. On ColEl-derived plasmids, the arrest of replication at a ter site leads to the accumulation of molecules carrying a replication bubble, which can be detected by gel electrophoresis (16). Arrest of pHV960T+ replication at terB should lead to the formation of sigma-shaped molecules with the length of the linear tail corresponding to the distance between the M13 origin and terB. These should migrate slower
effect. Plasmid pHV960 carries the ampicillin resistance gene of plasmid pBR322 and the M13 plus and minus replication origins (Fig. 1). It replicates in E. coli strains possessing the Rep helicase and the M13 replication protein (gpII) but not in those lacking either gpII or Rep helicase (25). Two derivatives of pHV960 were constructed by cloning terB 2.25 kb downstream of the M13 plus replication origin. In pHV960T+, terB is oriented to arrest replication initiated at this origin, whereas in pHV96OT-, it is oriented in the opposite direction and should not arrest this replication (Fig. 1). Plasmid pSCgpII is a pCS101 derivative carrying a spectinomycin resistance gene (cloned from plasmid pHP45omega [23]) and gene II of fl under the control of the lacUV5 promoter (cloned from plasmid pLII [6]). pSCgpII and pHV960 are compatible and can thus be maintained in the same E. coli cell. In the course of this work, we observed that plasmid pSCgpII is present at a lower copy number and *
Corresponding author. 7695
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FIG. 1. Schematic representation of plasmid pHV960 (4,403 bp). Thin line, pBR322 sequences; hatched box, M13 sequences; open box, pC194-delta NsiI. The arrows indicate the direction of plus and minus M13 replication; the closed arrows represent the terB sequences (T+ and T-). Plasmid pHV960 was constructed in several steps. First, an M13 fragment carrying the replication origin (nucleotides 5614 to 5995 of M13 [27]) was inserted in the EcoRV site of pHV5O, a plasmid which can replicate in both E. coli and Bacillus subtilis (7). The resulting construct is maintained poorly in E. coli. A spontaneous deletant was isolated which is maintained well and in which nucleotide 2771 of pBR322 is linked to nucleotide 5823 of M13. Finally, an NsiI fragment of pC194 (nucleotides 113 to 576) which inactivates the replication of pC194 by deleting 154 amino acids from its replication protein (8, 14) was removed in vitro. Plasmids pHV960T+ and pHV96OT- were obtained by cloning in the Stul site of pHV960 the following oligonucleotide:
5'-GATCATAAAATAAGTATGTTGTAACTAAAGT-3' 3'-CTAGTATTTTATTCATACAACATTGATTTCA-5' The Sau3A site (5'-GATC) is proximal to the Apr gene in pHV960T+ and distal in pHV96OT-.
than monomeric plasmids on agarose gels. Total DNA was extracted by two different techniques from tus+ cells harboring pSCgpII plus pHV960, pHV960T+, or pHV96OT-, grown to the exponential phase or overnight, in the presence of 0.03 to 1 mM IPTG. The first technique was carried out according to the "ter-assay" (16), involving cell lysis by lysosyme-sodium dodecyl sulfate (SDS)-NaCl treatment and allowing the detection of D-loop replication intermediates. By this method, we detected pTH101 replication intermediates (12) in a control experiment (not shown). The second method was developed to detect single-stranded DNA molecules (26) and involves cell lysis by an SDS-Ficoll procedure. It allowed us to visualize the expected amounts of
FIG. 2. Evidence that the interaction of Tus and terB affects the copy number of plasmids with the M13 replication origin. DNA was extracted according to the "ter-assay" procedure (16) from 1.5 ml of
cells containing pSCgpII and pHV960 (0), pHV960T+ (+), or pHV96OT- (-) and linearized by SpeI. Lanes: 1 and 2, pSCgpII and pHV960 standard DNA, respectively; 3 to 8, tus+ cells; 9 to 11, tus cells; 3 to 5, 0.03 mM IPTG; 6 to 11, 0.1 mM IPTG.
circular single-stranded plasmid molecules (not shown). DNA was analyzed by Southern hybridization with a pHV960-specific probe, either intact or cleaved by HindIII, PstI, PvuII, or PstI and PvuII (positions of the sites are shown in Fig. 1). No slowly migrating molecules were detected. In an attempt to protect the tail of the putative sigma-shaped molecules, similar experiments were performed (i) in the presence of the M13 single-stranded DNA binding protein, gpV, constitutively expressed from a compatible plasmid; (ii) after cloning, downstream of the M13 plus origin, of the primosome assembly site rriB of pBR322 (28) (a 180-bp fragment of pBR322, nucleotides 2066 to 2246, was inserted in the AlwNI site of pHV960T+), which should allow the conversion of the single-stranded tail to a doublestranded form; and (iii) in a recB sbcB strain, which is devoid of the two major E. coli exonucleases, ExoV and ExoL. Slowly migrating molecules were not detected under any conditions, which indicates that, if present, they represent less than 1% of plasmid DNA. In conclusion, our evidence indicates that interaction of terB and Tus interferes with maintenance of a plasmid with the M13 bacteriophage replication origin. The stability of the plasmid in nonselective media and the alleviation of the phenomenon by an excess of the replication protein gpII suggest that the interference takes place at the level of replication rather than plasmid partition. We consider two possibilities, inhibition of initiation or arrest of elongation in
TABLE 1. Relative efficiency of transformation in tus+ and tus cellsa Relative efficiency of transformation at indicated IPTG concn6 Plasmid
pHV960T+ pHV96OT-
tus+
0.02-0.03 mMc 5 x 10-3 0.6
0.05-0.1 mMc 0.2 0.7
tus
1 mMd
0.7 1.3
0.03 mMd 1 1
0.05 mMd 1 0.73
mMd 0.65 0.73
1
a Competent E. coli cells were prepared by overnight incubation at 0'C in 0.1 M CaCl2 (3); 150 1LI of competent cells was mixed with 0.05 to 50 ng of DNA and incubated for 10 min at 0°C and 10 min at 37°C. The mixture was then divided in three aliquots, which were added to 500 p.1 of LBT expression medium containing three different concentrations of IPTG. After 1 h of growth at 37°C, 100 pi1 of each culture was spread on plates containing the same concentration of IPTG as the expression medium and 100 ,ug of ampicillin per ml. b Relative to the value for pHV960, which was set at 1. Absolute values for pHV960 ranged from 0.5 to 2 x 106 Apr transformants per ,ug of DNA except when tus cells were transformed in the presence of 0.03 mM IPTG, in which case 102 transformants per ,ug of DNA were obtained (see text). c Average from five experiments. d Average from two or three experiments.
VOL. 173, 1991
DNA synthesis. The binding of Tus protein could conceivably affect the efficiency of replication initiation at the M13 origin, by modifying superhelicity of the molecule. However, such an effect is not expected to be dependent on the orientation of the ter site. We therefore prefer to propose that the inhibition of replication is due to arrest of the replication forks initiated at the M13 replication origin. This confirms and extends the in vitro observation that the Tus-terB complex inhibits DNA unwinding by the Rep helicase (20) and the in vivo observation that it arrests the Rep-dependent replication of phage P2 integrated into the host chromosome (10). However, this block of replication does not seem to be very efficient, since the effects of terB on M13 replication are detected only in the presence of a low amount of the phage replication protein gpII. This finding may be correlated with the in vitro observation that the Tus-terR complex, which is less stable than the Tus-terB complex (19), does not inhibit the Rep helicase (17). The failure to detect the sigma-shaped structures expected from replication arrest at terB could be due to a low efficiency of the arrest or to a loss of these structures either in vivo by exonucleolytic degradation or during DNA extraction. The dependence on the orientation of terB and on the presence of Tus argues in favor of a similar mechanism of inhibition of replication for the M13 and oriC or ColEl replication forks. We are grateful to L. Janniere for helpful discussions and suggestions of experiments, to C. Anagnostopoulos for improvements on the manuscript, and to F. Haimet for producing the artwork. This work was supported by a grant from Fondation pour la Recherche Mddicale. B.M. is on the CNRS staff. REFERENCES 1. Bastia, D., J. Germino, J. H. Crosa, and J. Ram. 1981. The nucleotide sequence surrounding the replication terminus of R6K. Proc. Natl. Acad. Sci. USA 72:2095-2099. 2. Bierne, H., S. D. Ehrlich, and B. Michel. 1991. The replication terminator signal terB of the Escherichia coli chromosome is a deletion hot-spot. EMBO J. 10:2699-2705. 3. Dagert, M., and S. D. Ehrlich. 1979. Prolonged incubation in calcium chloride improves the competence of E. coli cells. Gene 6:23-28. 4. De Massy, B., S. Bejar, J. Louarn, J. M. Louarn, and J. P. Bouche. 1987. Inhibition of replication forks exiting the terminus of the Escherichia coli chromosome occurs at two loci separated by 5 min. Proc. Natl. Acad. Sci. USA 84:1759-1763. 5. Francois, V., J. Louarn, and J. M. Louarn. 1989. The terminus of the Escherichia coli chromosome is flanked by several polar replication pause sites. Mol. Microbiol. 3:995-1002. 6. Fulford, W., and P. Model. 1988. Regulation of bacteriophage fl DNA replication. I. New functions for genes II and X. J. Mol. Biol. 203:49-62. 7. Goze, A., and S. D. Ehrlich. 1980. Replication of plasmids from Staphylococcus aureus in Escherichia coli. Proc. Natl. Acad. Sci. USA 77:7333-7337. 8. Gros, M. F., H. te Riele, and S. D. Ehrlich. 1987. Rolling circle replication of single-stranded DNA plasmid pC194. EMBO J. 6:3863-3869. 9. Hidaka, M., M. Akiyama, and T. Horiuchi. 1988. A consensus sequence of three DNA replication terminus sites on the E. coli chromosome is highly homologous to the terR sites of the R6K
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