Abstract
Second-site mutations that allow stable inheritance of partition-defective pSC101 plasmids mapped to seven distinct sites in the 5' half of the plasmid repA gene. While the mutations also elevated pSC101 copy number, there was no correlation between copy number increase and plasmid stability. Combinations of mutations enabled pSC101 DNA replication in the absence of integration host factor and also stabilized par-deleted plasmids in cells deficient in DNA gyrase or defective in DnaA binding. Our findings suggest that repA mutations compensate for par deletion by enabling the origin region RepA-DNA-DnaA complex to form under suboptimal conditions. They also provide evidence that this complex has a role in partitioning that is separate from its known ability to promote plasmid DNA replication.
Full Text
The Full Text of this article is available as a PDF (177.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Armstrong K. A., Acosta R., Ledner E., Machida Y., Pancotto M., McCormick M., Ohtsubo H., Ohtsubo E. A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1. J Mol Biol. 1984 May 25;175(3):331–348. doi: 10.1016/0022-2836(84)90352-8. [DOI] [PubMed] [Google Scholar]
- Austin S. J., Eichorn B. G. Random diffusion can account for topA-dependent suppression of partition defects in low-copy-number plasmids. J Bacteriol. 1992 Aug;174(16):5190–5195. doi: 10.1128/jb.174.16.5190-5195.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Beaucage S. L., Miller C. A., Cohen S. N. Gyrase-dependent stabilization of pSC101 plasmid inheritance by transcriptionally active promoters. EMBO J. 1991 Sep;10(9):2583–2588. doi: 10.1002/j.1460-2075.1991.tb07799.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Biek D. P., Cohen S. N. Identification and characterization of recD, a gene affecting plasmid maintenance and recombination in Escherichia coli. J Bacteriol. 1986 Aug;167(2):594–603. doi: 10.1128/jb.167.2.594-603.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Biek D. P., Cohen S. N. Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF. J Bacteriol. 1989 Apr;171(4):2056–2065. doi: 10.1128/jb.171.4.2056-2065.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Biek D. P., Cohen S. N. Propagation of pSC101 plasmids defective in binding of integration host factor. J Bacteriol. 1992 Feb;174(3):785–792. doi: 10.1128/jb.174.3.785-792.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gustafsson P., Wolf-Watz H., Lind L., Johansson K. E., Nordström K. Binding between the par region of plasmids R1 and pSC101 and the outer membrane fraction of the host bacteria. EMBO J. 1983;2(1):27–32. doi: 10.1002/j.1460-2075.1983.tb01375.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
- Humphreys G. O., Willshaw G. A., Smith H. R., Anderson E. S. Mutagenesis of plasmid DNA with hydroxylamine: isolation of mutants of multi-copy plasmids. Mol Gen Genet. 1976 Apr 23;145(1):101–108. doi: 10.1007/BF00331564. [DOI] [PubMed] [Google Scholar]
- Ingmer H., Cohen S. N. Excess intracellular concentration of the pSC101 RepA protein interferes with both plasmid DNA replication and partitioning. J Bacteriol. 1993 Dec;175(24):7834–7841. doi: 10.1128/jb.175.24.7834-7841.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ingmer H., Cohen S. N. The pSC101 par locus alters protein-DNA interactions in vivo at the plasmid replication origin. J Bacteriol. 1993 Sep;175(18):6046–6048. doi: 10.1128/jb.175.18.6046-6048.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Magee T. R., Asai T., Malka D., Kogoma T. DNA damage-inducible origins of DNA replication in Escherichia coli. EMBO J. 1992 Nov;11(11):4219–4225. doi: 10.1002/j.1460-2075.1992.tb05516.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Manen D., Goebel T., Caro L. The par region of pSC101 affects plasmid copy number as well as stability. Mol Microbiol. 1990 Nov;4(11):1839–1846. doi: 10.1111/j.1365-2958.1990.tb02032.x. [DOI] [PubMed] [Google Scholar]
- Meacock P. A., Cohen S. N. Partitioning of bacterial plasmids during cell division: a cis-acting locus that accomplishes stable plasmid inheritance. Cell. 1980 Jun;20(2):529–542. doi: 10.1016/0092-8674(80)90639-x. [DOI] [PubMed] [Google Scholar]
- Miller C. A., Beaucage S. L., Cohen S. N. Role of DNA superhelicity in partitioning of the pSC101 plasmid. Cell. 1990 Jul 13;62(1):127–133. doi: 10.1016/0092-8674(90)90246-b. [DOI] [PubMed] [Google Scholar]
- Miller C. A., Cohen S. N. The partition (par) locus of pSC101 is an enhancer of plasmid incompatibility. Mol Microbiol. 1993 Aug;9(4):695–702. doi: 10.1111/j.1365-2958.1993.tb01730.x. [DOI] [PubMed] [Google Scholar]
- Orr E., Fairweather N. F., Holland I. B., Pritchard R. H. Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12. Mol Gen Genet. 1979;177(1):103–112. doi: 10.1007/BF00267259. [DOI] [PubMed] [Google Scholar]
- Roberts R. C., Helinski D. R. Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2. J Bacteriol. 1992 Dec;174(24):8119–8132. doi: 10.1128/jb.174.24.8119-8132.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Samitt C. E., Hansen F. G., Miller J. F., Schaechter M. In vivo studies of DnaA binding to the origin of replication of Escherichia coli. EMBO J. 1989 Mar;8(3):989–993. doi: 10.1002/j.1460-2075.1989.tb03462.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stenzel T. T., MacAllister T., Bastia D. Cooperativity at a distance promoted by the combined action of two replication initiator proteins and a DNA bending protein at the replication origin of pSC101. Genes Dev. 1991 Aug;5(8):1453–1463. doi: 10.1101/gad.5.8.1453. [DOI] [PubMed] [Google Scholar]
- Tautz D., Renz M. An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem. 1983 Jul 1;132(1):14–19. doi: 10.1016/0003-2697(83)90419-0. [DOI] [PubMed] [Google Scholar]
- Tucker W. T., Miller C. A., Cohen S. N. Structural and functional analysis of the par region of the pSC 10 1 plasmid. Cell. 1984 Aug;38(1):191–201. doi: 10.1016/0092-8674(84)90540-3. [DOI] [PubMed] [Google Scholar]
- Vocke C., Bastia D. DNA-protein interaction at the origin of DNA replication of the plasmid pSC101. Cell. 1983 Dec;35(2 Pt 1):495–502. doi: 10.1016/0092-8674(83)90183-6. [DOI] [PubMed] [Google Scholar]
- Vocke C., Bastia D. The replication initiator protein of plasmid pSC101 is a transcriptional repressor of its own cistron. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2252–2256. doi: 10.1073/pnas.82.8.2252. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xia G. X., Manen D., Goebel T., Linder P., Churchward G., Caro L. A copy-number mutant of plasmid pSC101. Mol Microbiol. 1991 Mar;5(3):631–640. doi: 10.1111/j.1365-2958.1991.tb00734.x. [DOI] [PubMed] [Google Scholar]