Vol. 174, No. 16
JOURNAL OF BACTERIOLOGY, Aug. 1992, p. 5475-5478
0021-9193/92/165475-04$02.00/0 Copyright © 1992, American Society for Microbiology
The Amount of RepR Protein Determines the Copy Number of Plasmid pIP501 in Bacillus subtilis SABINE BRANTL* AND DETLEV BEHNKE Institute for Molecular Biology, Beutenbergstrasse 11, PSF 73, 0-6900 Jena, Germany Received 30 March 1992/Accepted 3 June 1992
To prove the hypothesis that the amount of RepR protein is the rate-limiting factor for replication of plasmid pIP5Ol in Bacillus subtilis, the repR gene was placed under control of the inducible promoter Papac' The plasmid copy number of the pIP501 derivative pRS9 could be deliberately adjusted between approximately 1 and 50 to 100 molecules per cell by varying the concentration of the inducer isopropyl-13-D-thiogalactopyranoside. Construction of a repR-lacZ fusion proved that the increase in copy number was due to a proportional increase in the amount of RepR protein.
Plasmids pIP501 (2, 20), pSM19035 (3), and pAMP1 (12) form a group of streptococcal plasmids which replicate in a broad range of gram-positive bacteria, including Bacillus spp. (10, 13, 16, 30, 31). Unlike the small staphylococcal plasmids pC194, pE194, and pUB110, which are widely used as vectors in Bacillus spp. (1, 8, 18), these plasmids do not accumulate single-stranded intermediates during replication. Rather, they follow a theta-type mechanism of replication (9) and show high segregational and structural stability (21; unpublished results). It is therefore likely that this group of streptococcal plasmids has evolved properties and regulatory mechanisms that enable them to successfully interact with the pecularities of specific replication functions of different hosts. We have recently identified the pIP501 origin of replication (5) and established a working model for how the copy number of pIP501 is regulated (4). The only two plasmid functions that are essential for replication are the origin of replication, oriR, and a 57.4-kDa protein, RepR, which likely functions during the initiation of replication. On pIP501, the expression of repR is regulated by several mechanisms involving a small protein, CopR, an antisense RNA of 136 nucleotides (RNAIII), and an inverted repeat which is located upstream of and overlapping promoter pII. This promoter directs the transcription of the essential repR gene (7). The target of all regulatory mechanisms appears to be the expression of repR. It was therefore postulated that the amount of RepR is in fact the rate-limiting factor which determines the frequency of replication initiation and, therefore, the copy number of pIP501 (4). In order to test this hypothesis directly, a plasmid was constructed which allowed us to mimic regulated expression of repR by placing this gene under the control of the inducible Pspac promoter (33). For this purpose, a number of intermediate plasmids were generated. Initially, a 311-bp EcoRI-SalGI fragment encompassing the Pspac promoter and lac operator was subcloned from plasmid pSPAC (33) into pUC18 to yield plasmid pPS1. In a second subcloning step, the promoter-operator was recloned as a 140-bp EcoRVHindIII fragment into pUC19 that had been cleaved with SalGI (filled-in) and HindIII. The resulting plasmid, pPS3, was subsequently used as a template for a single polymerase chain reaction (PCR) together with the universal primer and *
the specific oligonucleotide 5'-CTGTTCTCClTlTCTAGAT TAATTGTTATC-3'. The amplified fragment was cleaved with XbaI and subcloned into the XbaI site of pUC19, giving rise to plasmid pPS4, which now carried the Pspac promoter and lac operator flanked by XbaI sites. The downstream XbaI site immediately followed the lac operator sequence. A 1,670-bp repR fragment (nucleotides 600 to 2269 of the pIP501 replication region [6]) was also initially amplified by PCR in order to place an XbaI site directly in front of the repR Shine-Dalgarno sequence. For this purpose, plasmid pUC118-F (6) served as the template, and amplification was accomplished by using the reverse sequencing primer and the specific oligonucleotide 5'-GGATAACAATTAATCTA GAAAGGAGAACAG-3'. The amplified fragment was then cleaved with XbaI and EcoRI and inserted into pUC19 to yield plasmid pPS2. The N-terminal region of the repR gene together with its natural Shine-Dalgarno sequence was subsequently combined with Pspac by inserting an XbaI (filled-in)-HindIII fragment from pPS2 into pPS4 cleaved with SalGI (filled-in) and HindIII, giving rise to plasmid pPS5. The pspac-repRoriR construct was finally completed by jointly cloning a BamHI-HindIII fragment from pPS5 and an EcoRI-HindIII fragment from pUC118-F, comprising the C terminus of repR and oriR, into pUC19 that had been cleaved with BamHI and EcoRI. From this plasmid, pPS6, the whole construct was transferred as a BamHI-EcoRI fragment onto plasmid pBT48 (6) to yield pPS9, which now carried a selective marker (phleomycin resistance gene of pUB110) for B. subtilis. A functional and restriction map of plasmid pPS9 and the DNA sequence of the pspac-repR junction are presented in Fig. 1 and 2. The oriR sequence and the coding region of repR were the only components of the pIP501 replication machinery present on pPS9. Nevertheless, this plasmid was found to replicate autonomously in B. subtilis. No structural alterations were observed upon reisolation of pPS9 from B. subtilis. The copy number of pPS9 was determined to be approximately equal to that of the pIP501 derivative pPR1 (Fig. 3) (4), which was estimated to be about 50 to 100 molecules per cell. Although repR was expressed constitutively by pPS9, no runaway replication phenomenon was observed. Plasmid pRS9 was constructed to allow regulated expression of the repR gene. A 1.3-kb BamHI-PstI fragment from plasmid pSPAC encompassing the lacI repressor gene under
Corresponding author. 5475
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- 44 lacZ ladi Ppac Ppcn repR FIG. 1. Linear maps of plasmids pPS9, pRS9, and pRS10. Plasmids pPS9 and pRS9 mediate constitutive or inducible expression of repR in B. subtilis under control of the p,pc promoter. Plasmid pRS10 carries an in-frame repR-lacZ translational fusion and can replicate only in a B. subtilis strain which provides the intact RepR protein in trans from a chromosomally integrated copy of the intact repR gene. phleo, phleomycin resistance gene from pUTB110; Papa,c, hybrid promoter inhibited by the LacI repressor (33); repR, gene coding for the essential RepR protein of pIP501; lacZ, -galactosidase-encoding sequences; oriR, origin of replication of pIP501; bla, ampicillin resistance gene; pp,,, promoter of the Bacillus licheniformis penicillinase gene; lacI, gene coding for the repressor of the Escherichia coli lactose operon. Restriction enzyme cleavage sites: B, BamHl; E, EcoRI; H, HindIII; P, PstI; Pv, PvuII; S, SphI; X, XbaI.
4-
phsoo
control of the penicillinase promoter (33) and a 2.8-kb PstI-EcoRI fragment of pPS9 (Fig. 1) were cloned together into pBR322 that had been cleaved with BamHI and EcoRI. The structure of the resulting plasmid, pRS9, is presented in Fig. 1. Like pPS9, plasmid pRS9 could be introduced into B. subtilis, but successful transformation was only observed in the presence of the inducer IPTG (isopropyl-3-D-thiogalactopyranoside). Plasmid pRS9 thus allowed us to artificially regulate the expression of repR by modulating the amount of the inducer IPTG added to the culture medium. Copy number estimations of pRS9 were therefore carried out in the presence of increasing amounts of IPTG. All copy number determinations were done in triplicate under standardized conditions as described previously (4; see also the legend to Fig. 3). Plasmids pCOP4 and pPR1 (4) were included as controls. Full induction of repR expression was expected to give rise to a copy number identical to that of pPS9 resulting from constitutive repR expression under the 5 -GATATCCTAA CAGCACAAGA GCGGAAAGAT GTTTTGTTCT ACATCCAGAA -35
CAACCTCTGC TAAAATTCCT GAAAAATTTT GCAAAAAGTT
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TAGGTCGA TAQAAAGQQ AACAGC TGA ATG AAT ATC. . repR XbaISalI
*
*
*
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610
620
-
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FIG. 2. DNA sequence at the junction between the pac promoter and the repR gene. Nucleotide numbers refer to the originally published sequence of the repR gene (5, 6); -35 and -10, conserved regions of the pac promoter; OP, lac operator; SD, Shine-Dalgarno sequence of repR. The N-terminal amino acids of RepR are shown above the DNA sequence.
control Of Pspac. As shown in Fig. 3, the copy number of pRS9 increased in parallel to the amount of inducer added. Full induction of the ps promoter was obviously achieved at concentrations of >f,0O plM IPTG. The copy number of pRS9 at this inducer concentration was identical to that of pPS9 and only slightly lower than that of pPR1, a pIP501 derivative lacking the copR gene. A decrease in the IPTG
pRS9
pPR1
pCOP4
0
1000
100
25
100
100
50
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5
0 5
P&M Wm approxImate copy number
85,5
pgalacouldam unib per O.D. FIG. 3. Copy number of pRS9 (lanes 2 to 5) after induction with different IPTG concentrations. The copy numbers indicated below the lanes were estimated separately by linearizing the plasmids with EcoRI and subsequently diluting until they reached approximately equal plasmid concentrations. The copy number of plasmid pCOP4 (lane 6) was taken as a reference. Since variations by a factor of 2 are normal with this approach, copy numbers are indicated as approximate values. f-Galactosidase activity per unit of optical density (O.D.) were estimated for the repR-lacZ fusion present on plasmid pRS10. The values indicated were thus a direct measure for the amount of RepR expressed at the indicated inducer concentrations. Both ,3-galactosidase and copy number estimations were done in three independent experiments. 39,7 4,7 -1
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VOL. 174, 1992
concentration to 100 ,uM reduced the copy number by a factor of 2, and a 10-fold drop in pRS9 copy number (Fig. 3) was noted at 25 ,uM IPTG. At 25 ,uM IPTG, the copy number of pRS9 was comparable to that of pCOP4, a pIP501 derivative that carries all plasmid functions known to be involved in copy control. Further reduction of the IPTG concentration to 5 ,uM resulted in a further decrease in the pRS9 copy number, which now approached 1 molecule per cell (Fig. 3). To substantiate that the increase in copy number was in fact due to a stepwise increase in intracellular levels of RepR protein after induction with IPTG, a translational fusion between repR and lacZ was constructed. To this end, the lacZ gene was amplified by PCR with the oligonucleotides 5'-TCTAGAAAGCTTGCTTACAACGTCGTGAC-3' and 5'-TCrAGAAAGCTTATmTTTGACACCAGAC-3'. The amplified lacZ gene was subsequently inserted into the unique HindIII site within the repR gene of plasmid pRS9, giving rise to plasmid pRS10 (Fig. 1), which now carried an in-frame fusion between the two genes. The correct insertion of the lacZ gene was confirmed by DNA sequencing. Although the fusion protein included most of the RepR amino acid sequence, it was likely to be inactive, as could be concluded from previous results (6). Since plasmid pRS10 was unable to replicate in a normal Bacillus host strain, it was introduced into B. subtilis DB104, which carried an intact repR gene under control of the Pspac promoter inserted into the chromosomal amy locus. Supply of RepR in trans by this integrated copy of the repR gene has been demonstrated to be sufficient to support replication of plasmids that carried only the oriR region of pIP501 (5). Since both the integrated copy of repR and the repR-lacZ fusion on pRS10 were under control of the Pspac promoter, this arrangement mimicked most closely the above situation with pRS9. Measurements of j-galactosidase activity units per optical density unit after induction with 1,000, 100, 25, and 5 ,uM IPTG were carried out as reported elsewhere (27). A representative set of 0-galactosidase values at these inducer concentrations are presented in Fig. 3. All measurements were done in three independent experiments, and both stationary-phase and log-phase cultures showed the same rise in ,B-galactosidase levels in response to increasing IPTG concentrations. These results clearly confirmed that the amount of RepR protein increased proportionally to the amount of inducer added. Thus, the gradual increase in copy number observed with pRS9 was in fact due to increased amounts of the initiator protein RepR. In summary, these data confirmed our hypothesis that the amount of RepR is the rate-limiting factor in pIP501 replication and thus the factor that eventually determines the plasmid copy number. A direct correlation between the amount of replication initiator protein available and the plasmid copy number has also been reported for other plasmids, among them Rl, RSF1010, R1162, and pT181 (15, 19, 22, 26). However, overproduction of initiator proteins does not necessarily lead to an increase in copy number. The initiator proteins of plasmids P1, F, R6K, and Rtsl have been shown to directly exert both positive and negative effects on replication initiation (11, 14, 23, 28, 32). Although the mechanism of the direct inhibitory action of excess initiator protein is not yet fully understood, recent findings support the hypothesis that two different forms of the
initiator protein may be responsible and that proteolytic processing may be involved (17, 24). Constitutive as well as fully induced expression of repR by either pPS9 or pRS9 led to a final copy number of approxi-
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mately 100 molecules per cell. This copy number was, however, still two- to fourfold lower than the maximum copy number obtained with the pIP501 derivatives pPR11 and pPR12 (4). In addition, it was a surprising result that constitutive or fully induced expression of repR did not lead to an autocatalytic process of uncontrolled runaway replication. These discrepancies may be explained by (i) an as yet undetected mechanism of autoregulation of RepR expression at the translational level or (ii) a rapid rate of RepR degradation or inactivation, which is not overrun by the expression level mediated by the Pspac promoter. Other explanations, such as titration of an essential host factor or an inhibitory action of RepR at higher concentrations, similar to the situation with P1, F, and R6K, can be ruled out, as higher plasmid copy numbers have been obtained with pPR11 and pPR12. In particular, a high turnover rate of RepR would be consistent with its function as the rate-limiting factor in pIP501 replication. We have previously noted that one level of pIP501 copy number control is likely to involve transcriptional autoregulation of repR expression (4). The regulatory circuits involved in the control of pIP501 replication are thus reminiscent of features of the so-called iteron-regulated plasmids such as P1, R6K, and pSC101 (25, 29). On the other hand, copy number control of pIP501 also includes the negative regulators CopR and the antisense RNAIII (4), which resemble mechanisms known from the inhibitor-target regulation of plasmids such as ColEl, Rl, and pT181 (29). It therefore appears likely that the high segregational stability of pIP501 and its related plasmids pAM,B1 and pSM19035 is due to the efficient overlapping of two different types of regulatory circuits. This work was supported by a grant from the Max-Planck-Society to D.B. Helpful discussions with R. Breitling and the excellent technical assistance of Ina Poitz (deceased) and Barbel Ukena are gratefully acknowledged.
REFERENCES 1. Alonso, J. C. 1989. DNA replication of plasmids from Grampositive bacteria in Bacillus subtilis. Plasmid pUBllO as a model system. Microbiol. Semin. 5:5-12. 2. Behnke, D., M. S. Gilmore, and J. J. Ferretti. 1981. Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamicin/kanamycin resistance determinant. Mol. Gen. Genet. 182:414-421. 3. Behnke, D., H. Malke, M. Hartmann, and F. Walter. 1979. Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid. Plasmid 2:605-616. 4. Brantd, S., and D. Behnke. 1992. Copy number control of the streptococcal plasmid pIP501 occurs at three levels. Nucleic Acids Res. 20:395-400. 5. Brantl, S., and D. Behnke. Submitted for publication. 6. Brantl, S., D. Behnke, and J. C. Alonso. 1990. Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM,B1 and pSM19035. Nucleic Acids Res. 18:4783-4790. 7. Brantl, S., B. Nuez, and D. Behnke. In vitro and in vivo analysis of transcription within the replication region of plasmid pIP501. Mol. Gen. Genet., in press. 8. Bron, S. 1990. Plasmids, p. 75-137. In C. R. Harwood and S. M. Cutting (ed.), Molecular biology methods for Bacillus. John Wiley & Sons Ltd., Chichester, England. 9. Bruand, C., S. D. Ehrlich, and L. Janniere. 1991. Unidirectional theta replication of the structurally stable Enterococcusfaecalis plasmid pAM,Bl. EMBO J. 10:2171-2177. 10. Buu-Hoi, A., G. Bieth, and T. Horaud. 1984. Broad host range of streptococcal macrolide resistance plasmids. Antimicrob.
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Agents Chemother. 25:289-291. 11. Chattoraj, D. K., K. M. Snyder, and A. L. Abeles. 1985. P1 plasmid replication: multiple functions of RepA protein at the origin. Proc. Natl. Acad. Sci. USA 82:2588-2592. 12. Clewell, D. B., Y. Yagi, G. M. Dunny, and S. K. Schultz. 1974. Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: identification of a plasmid determining erythromycin resistance. J. Bacteriol. 117: 283-289. 13. Engel, H. W. B., N. Soedirman, J. A. Rost, W. J. van Leeuwen, and J. D. A. van Embden. 1980. Transferability of macrolide, lincomycin, and streptogramin resistances between group A, B, and D streptococci, Streptococcus pneumoniae, and Staphylococcus aureus. J. Bacteriol. 142:407-413. 14. Filutowicz, M., M. McEachern, A. Greener, P. Mukhopadhyay, E. Uhlenhopp, R Durland, and D. R. Heinski. 1985. Role of the ir initiation protein and direct nucleotide sequence repeats in the regulation of plasmid R6K replication, p. 125-140. In D. R. Helinski, S. N. Cohen, D. B. Clewell, D. A. Jackson, and A. Hollaender (ed.), Plasmids in bacteria. Plenum Publishing Corp., New York. 15. Giskov, M., P. Stougaard, J. Light, and S. Molin. 1987. Identification and characterization of mutations responsible for a runaway replication phenotype of plasmid Rl. Gene 57:203-211. 16. Gonzalez, C. F., and B. S. Kunka. 1983. Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501. Appl. Environ. Microbiol. 46:81-89. 17. Greener, A., M. S. Fllutowicz, M. J. McEachern, and D. R. Helinski. 1990. N-terminal truncated forms of the bifunctional ir initiation protein express negative activity on plasmid R6K replication. Mol. Gen. Genet. 224:24-32. 18. Gruss, A., and S. D. Ehrlich. 1989. The family of highly interrelated single-stranded deoxyribonucleic plasmids. Microbiol. Rev. 53:231-241. 19. Haring, V., P. Scholz, E. Scherzinger, J. Frey, K. Derbyshire, G. Hatfull, N. S. Willetts, and M. Bagdarsarian. 1985. Protein RepC is involved in copy number control of the broad host range plasmid RSF1010. Proc. Natl. Acad. Sci. USA 82:6090-6094. 20. Horodniceanu, T., D. H. Bouanchaud, G. Bieth, and Y. A. Chabbert. 1976. R-plasmids in Streptococcus agalactiae (group B). Antimicrob. Agents Chemother. 10:795-801.
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21. Janniere, L., C. Bruand, and S. D. Ehrlich. 1990. Structurally stable Bacillus subtilis cloning vectors. Gene 87:53-61. 22. Kim, K., and R. J. Meyer. 1985. Copy number of the broadhost-range plasmid R1162 is determined by the amounts of essential plasmid-encoded proteins. J. Mol. Biol. 185:755-767. 23. Kline, B. C. 1985. A review of mini-F plasmid maintainance. Plasmid 14:1-16. 24. Kline, B. C., G. S. Sandhu, B. W. Eckloff, and R. A. Aleff. 1992. Site-specific proteolysis of mini-F plasmid replication protein RepE destroys initiator function and generates an incompatibility substance. J. Bacteriol. 174:3004-3010. 25. Kues, U., and U. Stahl. 1989. Replication of plasmids in gramnegative bacteria. Microbiol. Rev. 53:491-516. 26. Manch-Citron, J., M. L. Gennaro, S. Majumder, and R. P. Novick. 1986. RepC is rate-limiting for pT181 plasmid replication. Plasmid 16:108-115. 27. Msadek, T., F. Kunst, D. Henner, A. Klier, G. Rapoport, and R. Dedonder. 1990. Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU. J. Bacteriol. 172:824-834. 28. Muraisio, K., G. Mukhopadbyay, and D. K. Chattoraj. 1990. Location of a P1 plasmid replication inhibitor determinant within the initiator gene. J. Bacteriol. 172:4441 4447. 29. Novick, R. P. 1987. Plasmid incompatibility. Microbiol. Rev. 51:381-395. 30. Oultram, J. D., and M. Young. 1985. Conjugal transfer of plasmid pAM,B1 from Streptococcus lactis and Bacillus subtilis to Clostfidium acetobutylicum. FEMS Microbiol. Lett. 27:129134. 31. Perez-Diaz, J. C., M. F. Vicente, and F. Baquero. 1982. Plasmids in Listeria. Plasmid 8:112-118. 32. Terawald, Y., H. Nozue, H. Zeng, T. Hayashi, Y. Kamio, and Y. Itoh. 1990. Effects of mutations in the repA gene of plasmid Rtsl on plasmid replication and autorepressor function. J. Bacteriol. 172:786-792. 33. Yansura, D. G., and D. J. Henner. 1984. Development of an inducible promoter for controlled gene expression in Bacillus subtilis, p. 249-263. In A. T. Ganesan and J. A. Hoch (ed.), Genetics and biotechnology of bacilli. Academic Press, Inc., New York.
JOURNAL OF BACTERIOLOGY, Aug. 1992, p. 5475-5478
0021-9193/92/165475-04$02.00/0 Copyright © 1992, American Society for Microbiology
The Amount of RepR Protein Determines the Copy Number of Plasmid pIP501 in Bacillus subtilis SABINE BRANTL* AND DETLEV BEHNKE Institute for Molecular Biology, Beutenbergstrasse 11, PSF 73, 0-6900 Jena, Germany Received 30 March 1992/Accepted 3 June 1992
To prove the hypothesis that the amount of RepR protein is the rate-limiting factor for replication of plasmid pIP5Ol in Bacillus subtilis, the repR gene was placed under control of the inducible promoter Papac' The plasmid copy number of the pIP501 derivative pRS9 could be deliberately adjusted between approximately 1 and 50 to 100 molecules per cell by varying the concentration of the inducer isopropyl-13-D-thiogalactopyranoside. Construction of a repR-lacZ fusion proved that the increase in copy number was due to a proportional increase in the amount of RepR protein.
Plasmids pIP501 (2, 20), pSM19035 (3), and pAMP1 (12) form a group of streptococcal plasmids which replicate in a broad range of gram-positive bacteria, including Bacillus spp. (10, 13, 16, 30, 31). Unlike the small staphylococcal plasmids pC194, pE194, and pUB110, which are widely used as vectors in Bacillus spp. (1, 8, 18), these plasmids do not accumulate single-stranded intermediates during replication. Rather, they follow a theta-type mechanism of replication (9) and show high segregational and structural stability (21; unpublished results). It is therefore likely that this group of streptococcal plasmids has evolved properties and regulatory mechanisms that enable them to successfully interact with the pecularities of specific replication functions of different hosts. We have recently identified the pIP501 origin of replication (5) and established a working model for how the copy number of pIP501 is regulated (4). The only two plasmid functions that are essential for replication are the origin of replication, oriR, and a 57.4-kDa protein, RepR, which likely functions during the initiation of replication. On pIP501, the expression of repR is regulated by several mechanisms involving a small protein, CopR, an antisense RNA of 136 nucleotides (RNAIII), and an inverted repeat which is located upstream of and overlapping promoter pII. This promoter directs the transcription of the essential repR gene (7). The target of all regulatory mechanisms appears to be the expression of repR. It was therefore postulated that the amount of RepR is in fact the rate-limiting factor which determines the frequency of replication initiation and, therefore, the copy number of pIP501 (4). In order to test this hypothesis directly, a plasmid was constructed which allowed us to mimic regulated expression of repR by placing this gene under the control of the inducible Pspac promoter (33). For this purpose, a number of intermediate plasmids were generated. Initially, a 311-bp EcoRI-SalGI fragment encompassing the Pspac promoter and lac operator was subcloned from plasmid pSPAC (33) into pUC18 to yield plasmid pPS1. In a second subcloning step, the promoter-operator was recloned as a 140-bp EcoRVHindIII fragment into pUC19 that had been cleaved with SalGI (filled-in) and HindIII. The resulting plasmid, pPS3, was subsequently used as a template for a single polymerase chain reaction (PCR) together with the universal primer and *
the specific oligonucleotide 5'-CTGTTCTCClTlTCTAGAT TAATTGTTATC-3'. The amplified fragment was cleaved with XbaI and subcloned into the XbaI site of pUC19, giving rise to plasmid pPS4, which now carried the Pspac promoter and lac operator flanked by XbaI sites. The downstream XbaI site immediately followed the lac operator sequence. A 1,670-bp repR fragment (nucleotides 600 to 2269 of the pIP501 replication region [6]) was also initially amplified by PCR in order to place an XbaI site directly in front of the repR Shine-Dalgarno sequence. For this purpose, plasmid pUC118-F (6) served as the template, and amplification was accomplished by using the reverse sequencing primer and the specific oligonucleotide 5'-GGATAACAATTAATCTA GAAAGGAGAACAG-3'. The amplified fragment was then cleaved with XbaI and EcoRI and inserted into pUC19 to yield plasmid pPS2. The N-terminal region of the repR gene together with its natural Shine-Dalgarno sequence was subsequently combined with Pspac by inserting an XbaI (filled-in)-HindIII fragment from pPS2 into pPS4 cleaved with SalGI (filled-in) and HindIII, giving rise to plasmid pPS5. The pspac-repRoriR construct was finally completed by jointly cloning a BamHI-HindIII fragment from pPS5 and an EcoRI-HindIII fragment from pUC118-F, comprising the C terminus of repR and oriR, into pUC19 that had been cleaved with BamHI and EcoRI. From this plasmid, pPS6, the whole construct was transferred as a BamHI-EcoRI fragment onto plasmid pBT48 (6) to yield pPS9, which now carried a selective marker (phleomycin resistance gene of pUB110) for B. subtilis. A functional and restriction map of plasmid pPS9 and the DNA sequence of the pspac-repR junction are presented in Fig. 1 and 2. The oriR sequence and the coding region of repR were the only components of the pIP501 replication machinery present on pPS9. Nevertheless, this plasmid was found to replicate autonomously in B. subtilis. No structural alterations were observed upon reisolation of pPS9 from B. subtilis. The copy number of pPS9 was determined to be approximately equal to that of the pIP501 derivative pPR1 (Fig. 3) (4), which was estimated to be about 50 to 100 molecules per cell. Although repR was expressed constitutively by pPS9, no runaway replication phenomenon was observed. Plasmid pRS9 was constructed to allow regulated expression of the repR gene. A 1.3-kb BamHI-PstI fragment from plasmid pSPAC encompassing the lacI repressor gene under
Corresponding author. 5475
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- 44 lacZ ladi Ppac Ppcn repR FIG. 1. Linear maps of plasmids pPS9, pRS9, and pRS10. Plasmids pPS9 and pRS9 mediate constitutive or inducible expression of repR in B. subtilis under control of the p,pc promoter. Plasmid pRS10 carries an in-frame repR-lacZ translational fusion and can replicate only in a B. subtilis strain which provides the intact RepR protein in trans from a chromosomally integrated copy of the intact repR gene. phleo, phleomycin resistance gene from pUTB110; Papa,c, hybrid promoter inhibited by the LacI repressor (33); repR, gene coding for the essential RepR protein of pIP501; lacZ, -galactosidase-encoding sequences; oriR, origin of replication of pIP501; bla, ampicillin resistance gene; pp,,, promoter of the Bacillus licheniformis penicillinase gene; lacI, gene coding for the repressor of the Escherichia coli lactose operon. Restriction enzyme cleavage sites: B, BamHl; E, EcoRI; H, HindIII; P, PstI; Pv, PvuII; S, SphI; X, XbaI.
4-
phsoo
control of the penicillinase promoter (33) and a 2.8-kb PstI-EcoRI fragment of pPS9 (Fig. 1) were cloned together into pBR322 that had been cleaved with BamHI and EcoRI. The structure of the resulting plasmid, pRS9, is presented in Fig. 1. Like pPS9, plasmid pRS9 could be introduced into B. subtilis, but successful transformation was only observed in the presence of the inducer IPTG (isopropyl-3-D-thiogalactopyranoside). Plasmid pRS9 thus allowed us to artificially regulate the expression of repR by modulating the amount of the inducer IPTG added to the culture medium. Copy number estimations of pRS9 were therefore carried out in the presence of increasing amounts of IPTG. All copy number determinations were done in triplicate under standardized conditions as described previously (4; see also the legend to Fig. 3). Plasmids pCOP4 and pPR1 (4) were included as controls. Full induction of repR expression was expected to give rise to a copy number identical to that of pPS9 resulting from constitutive repR expression under the 5 -GATATCCTAA CAGCACAAGA GCGGAAAGAT GTTTTGTTCT ACATCCAGAA -35
CAACCTCTGC TAAAATTCCT GAAAAATTTT GCAAAAAGTT
GSTA
>>
Asn Ie
TAGGTCGA TAQAAAGQQ AACAGC TGA ATG AAT ATC. . repR XbaISalI
*
*
*
603
610
620
-
3
FIG. 2. DNA sequence at the junction between the pac promoter and the repR gene. Nucleotide numbers refer to the originally published sequence of the repR gene (5, 6); -35 and -10, conserved regions of the pac promoter; OP, lac operator; SD, Shine-Dalgarno sequence of repR. The N-terminal amino acids of RepR are shown above the DNA sequence.
control Of Pspac. As shown in Fig. 3, the copy number of pRS9 increased in parallel to the amount of inducer added. Full induction of the ps promoter was obviously achieved at concentrations of >f,0O plM IPTG. The copy number of pRS9 at this inducer concentration was identical to that of pPS9 and only slightly lower than that of pPR1, a pIP501 derivative lacking the copR gene. A decrease in the IPTG
pRS9
pPR1
pCOP4
0
1000
100
25
100
100
50
5
5
0 5
P&M Wm approxImate copy number
85,5
pgalacouldam unib per O.D. FIG. 3. Copy number of pRS9 (lanes 2 to 5) after induction with different IPTG concentrations. The copy numbers indicated below the lanes were estimated separately by linearizing the plasmids with EcoRI and subsequently diluting until they reached approximately equal plasmid concentrations. The copy number of plasmid pCOP4 (lane 6) was taken as a reference. Since variations by a factor of 2 are normal with this approach, copy numbers are indicated as approximate values. f-Galactosidase activity per unit of optical density (O.D.) were estimated for the repR-lacZ fusion present on plasmid pRS10. The values indicated were thus a direct measure for the amount of RepR expressed at the indicated inducer concentrations. Both ,3-galactosidase and copy number estimations were done in three independent experiments. 39,7 4,7 -1
NOTES
VOL. 174, 1992
concentration to 100 ,uM reduced the copy number by a factor of 2, and a 10-fold drop in pRS9 copy number (Fig. 3) was noted at 25 ,uM IPTG. At 25 ,uM IPTG, the copy number of pRS9 was comparable to that of pCOP4, a pIP501 derivative that carries all plasmid functions known to be involved in copy control. Further reduction of the IPTG concentration to 5 ,uM resulted in a further decrease in the pRS9 copy number, which now approached 1 molecule per cell (Fig. 3). To substantiate that the increase in copy number was in fact due to a stepwise increase in intracellular levels of RepR protein after induction with IPTG, a translational fusion between repR and lacZ was constructed. To this end, the lacZ gene was amplified by PCR with the oligonucleotides 5'-TCTAGAAAGCTTGCTTACAACGTCGTGAC-3' and 5'-TCrAGAAAGCTTATmTTTGACACCAGAC-3'. The amplified lacZ gene was subsequently inserted into the unique HindIII site within the repR gene of plasmid pRS9, giving rise to plasmid pRS10 (Fig. 1), which now carried an in-frame fusion between the two genes. The correct insertion of the lacZ gene was confirmed by DNA sequencing. Although the fusion protein included most of the RepR amino acid sequence, it was likely to be inactive, as could be concluded from previous results (6). Since plasmid pRS10 was unable to replicate in a normal Bacillus host strain, it was introduced into B. subtilis DB104, which carried an intact repR gene under control of the Pspac promoter inserted into the chromosomal amy locus. Supply of RepR in trans by this integrated copy of the repR gene has been demonstrated to be sufficient to support replication of plasmids that carried only the oriR region of pIP501 (5). Since both the integrated copy of repR and the repR-lacZ fusion on pRS10 were under control of the Pspac promoter, this arrangement mimicked most closely the above situation with pRS9. Measurements of j-galactosidase activity units per optical density unit after induction with 1,000, 100, 25, and 5 ,uM IPTG were carried out as reported elsewhere (27). A representative set of 0-galactosidase values at these inducer concentrations are presented in Fig. 3. All measurements were done in three independent experiments, and both stationary-phase and log-phase cultures showed the same rise in ,B-galactosidase levels in response to increasing IPTG concentrations. These results clearly confirmed that the amount of RepR protein increased proportionally to the amount of inducer added. Thus, the gradual increase in copy number observed with pRS9 was in fact due to increased amounts of the initiator protein RepR. In summary, these data confirmed our hypothesis that the amount of RepR is the rate-limiting factor in pIP501 replication and thus the factor that eventually determines the plasmid copy number. A direct correlation between the amount of replication initiator protein available and the plasmid copy number has also been reported for other plasmids, among them Rl, RSF1010, R1162, and pT181 (15, 19, 22, 26). However, overproduction of initiator proteins does not necessarily lead to an increase in copy number. The initiator proteins of plasmids P1, F, R6K, and Rtsl have been shown to directly exert both positive and negative effects on replication initiation (11, 14, 23, 28, 32). Although the mechanism of the direct inhibitory action of excess initiator protein is not yet fully understood, recent findings support the hypothesis that two different forms of the
initiator protein may be responsible and that proteolytic processing may be involved (17, 24). Constitutive as well as fully induced expression of repR by either pPS9 or pRS9 led to a final copy number of approxi-
5477
mately 100 molecules per cell. This copy number was, however, still two- to fourfold lower than the maximum copy number obtained with the pIP501 derivatives pPR11 and pPR12 (4). In addition, it was a surprising result that constitutive or fully induced expression of repR did not lead to an autocatalytic process of uncontrolled runaway replication. These discrepancies may be explained by (i) an as yet undetected mechanism of autoregulation of RepR expression at the translational level or (ii) a rapid rate of RepR degradation or inactivation, which is not overrun by the expression level mediated by the Pspac promoter. Other explanations, such as titration of an essential host factor or an inhibitory action of RepR at higher concentrations, similar to the situation with P1, F, and R6K, can be ruled out, as higher plasmid copy numbers have been obtained with pPR11 and pPR12. In particular, a high turnover rate of RepR would be consistent with its function as the rate-limiting factor in pIP501 replication. We have previously noted that one level of pIP501 copy number control is likely to involve transcriptional autoregulation of repR expression (4). The regulatory circuits involved in the control of pIP501 replication are thus reminiscent of features of the so-called iteron-regulated plasmids such as P1, R6K, and pSC101 (25, 29). On the other hand, copy number control of pIP501 also includes the negative regulators CopR and the antisense RNAIII (4), which resemble mechanisms known from the inhibitor-target regulation of plasmids such as ColEl, Rl, and pT181 (29). It therefore appears likely that the high segregational stability of pIP501 and its related plasmids pAM,B1 and pSM19035 is due to the efficient overlapping of two different types of regulatory circuits. This work was supported by a grant from the Max-Planck-Society to D.B. Helpful discussions with R. Breitling and the excellent technical assistance of Ina Poitz (deceased) and Barbel Ukena are gratefully acknowledged.
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21. Janniere, L., C. Bruand, and S. D. Ehrlich. 1990. Structurally stable Bacillus subtilis cloning vectors. Gene 87:53-61. 22. Kim, K., and R. J. Meyer. 1985. Copy number of the broadhost-range plasmid R1162 is determined by the amounts of essential plasmid-encoded proteins. J. Mol. Biol. 185:755-767. 23. Kline, B. C. 1985. A review of mini-F plasmid maintainance. Plasmid 14:1-16. 24. Kline, B. C., G. S. Sandhu, B. W. Eckloff, and R. A. Aleff. 1992. Site-specific proteolysis of mini-F plasmid replication protein RepE destroys initiator function and generates an incompatibility substance. J. Bacteriol. 174:3004-3010. 25. Kues, U., and U. Stahl. 1989. Replication of plasmids in gramnegative bacteria. Microbiol. Rev. 53:491-516. 26. Manch-Citron, J., M. L. Gennaro, S. Majumder, and R. P. Novick. 1986. RepC is rate-limiting for pT181 plasmid replication. Plasmid 16:108-115. 27. Msadek, T., F. Kunst, D. Henner, A. Klier, G. Rapoport, and R. Dedonder. 1990. Signal transduction pathway controlling synthesis of a class of degradative enzymes in Bacillus subtilis: expression of the regulatory genes and analysis of mutations in degS and degU. J. Bacteriol. 172:824-834. 28. Muraisio, K., G. Mukhopadbyay, and D. K. Chattoraj. 1990. Location of a P1 plasmid replication inhibitor determinant within the initiator gene. J. Bacteriol. 172:4441 4447. 29. Novick, R. P. 1987. Plasmid incompatibility. Microbiol. Rev. 51:381-395. 30. Oultram, J. D., and M. Young. 1985. Conjugal transfer of plasmid pAM,B1 from Streptococcus lactis and Bacillus subtilis to Clostfidium acetobutylicum. FEMS Microbiol. Lett. 27:129134. 31. Perez-Diaz, J. C., M. F. Vicente, and F. Baquero. 1982. Plasmids in Listeria. Plasmid 8:112-118. 32. Terawald, Y., H. Nozue, H. Zeng, T. Hayashi, Y. Kamio, and Y. Itoh. 1990. Effects of mutations in the repA gene of plasmid Rtsl on plasmid replication and autorepressor function. J. Bacteriol. 172:786-792. 33. Yansura, D. G., and D. J. Henner. 1984. Development of an inducible promoter for controlled gene expression in Bacillus subtilis, p. 249-263. In A. T. Ganesan and J. A. Hoch (ed.), Genetics and biotechnology of bacilli. Academic Press, Inc., New York.
