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. 1993 May;175(10):3161–3173. doi: 10.1128/jb.175.10.3161-3173.1993

Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene.

R Wu 1, X Wang 1, D D Womble 1, R H Rownd 1
PMCID: PMC204639  PMID: 8491730

Abstract

Replication-proficient (Rep+) revertants were isolated from mutants of IncFII plasmid NR1 that were replication defective (Rep-). The parental Rep- plasmids contained a mutation that inactivated promoter PE for transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1. The PE mutation also introduced a nonsense codon into a leader peptide gene that precedes and slightly overlaps the repA1 translation initiation site in the mRNA. This reduced the rate of synthesis of RepA1 by uncoupling its translation from that of the leader peptide. The reduced rate of RepA1 synthesis was responsible for the Rep- phenotype. All Rep+ revertants retained the PE mutation and contained second-site mutations responsible for suppression of the Rep- phenotype. One Rep+ revertant contained a second mutation adjacent to the Shine-Dalgarno sequence of repA1. Another Rep+ revertant contained a mutation in the repA2 gene, which encodes the trans-acting repressor of transcription of repA1. By using translational lacZ gene fusions, it was found that both kinds of suppressor mutation increased the expression of repA1 to a level sufficient to support replication. In both cases, the synthesis of RepA1 remained uncoupled from that of the leader peptide. The Shine-Dalgarno mutation increased the rate of leader peptide-independent translation of repA1 mRNA and also reduced the sensitivity of repA1 mRNA to inhibition by RNA-E. The repA2 mutation inactivated the RepA2 repressor and increased the rate of transcription of repA1 mRNA. The translational lacZ gene fusions were used to assess the range of regulation of expression of repA1 provided by each of the RNA-E and RepA2 regulatory circuits. By constructing miniplasmids that contained various combinations of the mutations, the contributions of the RNA-E and RepA2 regulatory circuits were assessed with respect to control of plasmid copy number and stable inheritance. Plasmids that lacked either circuit were less stable than wild-type plasmids.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Asano K., Kato A., Moriwaki H., Hama C., Shiba K., Mizobuchi K. Positive and negative regulations of plasmid CoLIb-P9 repZ gene expression at the translational level. J Biol Chem. 1991 Feb 25;266(6):3774–3781. [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blomberg P., Nordström K., Wagner E. G. Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation. EMBO J. 1992 Jul;11(7):2675–2683. doi: 10.1002/j.1460-2075.1992.tb05333.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blomberg P., Wagner E. G., Nordström K. Control of replication of plasmid R1: the duplex between the antisense RNA, CopA, and its target, CopT, is processed specifically in vivo and in vitro by RNase III. EMBO J. 1990 Jul;9(7):2331–2340. doi: 10.1002/j.1460-2075.1990.tb07405.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  6. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  7. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dong X. N., Womble D. D., Rownd R. H. In-vivo studies on the cis-acting replication initiator protein of IncFII plasmid NR1. J Mol Biol. 1988 Aug 5;202(3):495–509. doi: 10.1016/0022-2836(88)90281-1. [DOI] [PubMed] [Google Scholar]
  9. Dong X. N., Womble D. D., Rownd R. H. Transcriptional pausing in a region important for plasmid NR1 replication control. J Bacteriol. 1987 Dec;169(12):5353–5363. doi: 10.1128/jb.169.12.5353-5363.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dong X., Womble D. D., Luckow V. A., Rownd R. H. Regulation of transcription of the repA1 gene in the replication control region of IncFII plasmid NR1 by gene dosage of the repA2 transcription repressor protein. J Bacteriol. 1985 Feb;161(2):544–551. doi: 10.1128/jb.161.2.544-551.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  12. Easton A. M., Rownd R. H. The incompatibility product of IncFII R plasmid NR1 controls gene expression in the plasmid replication region. J Bacteriol. 1982 Nov;152(2):829–839. doi: 10.1128/jb.152.2.829-839.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Friedman S. A., Austin S. J. The P1 plasmid-partition system synthesizes two essential proteins from an autoregulated operon. Plasmid. 1988 Mar;19(2):103–112. doi: 10.1016/0147-619x(88)90049-2. [DOI] [PubMed] [Google Scholar]
  14. Hama C., Takizawa T., Moriwaki H., Mizobuchi K. Role of leader peptide synthesis in repZ gene expression of the ColIb-P9 plasmid. J Biol Chem. 1990 Jun 25;265(18):10666–10673. [PubMed] [Google Scholar]
  15. 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]
  16. Koop A. H., Hartley M. E., Bourgeois S. A low-copy-number vector utilizing beta-galactosidase for the analysis of gene control elements. Gene. 1987;52(2-3):245–256. doi: 10.1016/0378-1119(87)90051-5. [DOI] [PubMed] [Google Scholar]
  17. Light J., Molin S. Post-transcriptional control of expression of the repA gene of plasmid R1 mediated by a small RNA molecule. EMBO J. 1983;2(1):93–98. doi: 10.1002/j.1460-2075.1983.tb01387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lupski J. R., Ruiz A. A., Godson G. N. Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12. Mol Gen Genet. 1984;195(3):391–401. doi: 10.1007/BF00341439. [DOI] [PubMed] [Google Scholar]
  19. Masai H., Arai K. RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4781–4785. doi: 10.1073/pnas.84.14.4781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  21. Miki T., Easton A. M., Rownd R. H. Cloning of replication, incompatibility, and stability functions of R plasmid NR1. J Bacteriol. 1980 Jan;141(1):87–99. doi: 10.1128/jb.141.1.87-99.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miller J., Manis J., Kline B., Bishop A. Nonintegrated plasmid-folded chromosome complexes: genetic studies on formation and possible relationship to plasmid replication. Plasmid. 1978 Jun;1(3):273–283. doi: 10.1016/0147-619x(78)90045-8. [DOI] [PubMed] [Google Scholar]
  23. Miyazaki C., Kawai Y., Ohtsubo H., Ohtsubo E. Unidirectional replication of plasmid R100. J Mol Biol. 1988 Nov 20;204(2):331–343. doi: 10.1016/0022-2836(88)90580-3. [DOI] [PubMed] [Google Scholar]
  24. Nordström K., Molin S., Light J. Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system. Plasmid. 1984 Sep;12(2):71–90. doi: 10.1016/0147-619x(84)90054-4. [DOI] [PubMed] [Google Scholar]
  25. Novick R. P. Plasmid incompatibility. Microbiol Rev. 1987 Dec;51(4):381–395. doi: 10.1128/mr.51.4.381-395.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ohman M., Wagner E. G. Secondary structure analysis of the RepA mRNA leader transcript involved in control of replication of plasmid R1. Nucleic Acids Res. 1989 Apr 11;17(7):2557–2579. doi: 10.1093/nar/17.7.2557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ortega S., Lanka E., Diaz R. The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA. Nucleic Acids Res. 1986 Jun 25;14(12):4865–4879. doi: 10.1093/nar/14.12.4865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Praszkier J., Wilson I. W., Pittard A. J. Mutations affecting translational coupling between the rep genes of an IncB miniplasmid. J Bacteriol. 1992 Apr;174(7):2376–2383. doi: 10.1128/jb.174.7.2376-2383.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rosen J., Ryder T., Inokuchi H., Ohtsubo H., Ohtsubo E. Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis. Mol Gen Genet. 1980;179(3):527–537. doi: 10.1007/BF00271742. [DOI] [PubMed] [Google Scholar]
  30. Salser W. Globin mRNA sequences: analysis of base pairing and evolutionary implications. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):985–1002. doi: 10.1101/sqb.1978.042.01.099. [DOI] [PubMed] [Google Scholar]
  31. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  32. Tabuchi A., Min Y. N., Kim C. K., Fan Y. L., Womble D. D., Rownd R. H. Genetic organization and nucleotide sequence of the stability locus of IncFII plasmid NR1. J Mol Biol. 1988 Aug 5;202(3):511–525. doi: 10.1016/0022-2836(88)90282-3. [DOI] [PubMed] [Google Scholar]
  33. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  34. Wagner E. G., Blomberg P., Nordström K. Replication control in plasmid R1: duplex formation between the antisense RNA, CopA, and its target, CopT, is not required for inhibition of RepA synthesis. EMBO J. 1992 Mar;11(3):1195–1203. doi: 10.1002/j.1460-2075.1992.tb05160.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Womble D. D., Dong X., Luckow V. A., Wu R. P., Rownd R. H. Analysis of the individual regulatory components of the IncFII plasmid replication control system. J Bacteriol. 1985 Feb;161(2):534–543. doi: 10.1128/jb.161.2.534-543.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Womble D. D., Dong X., Wu R. P., Luckow V. A., Martinez A. F., Rownd R. H. IncFII plasmid incompatibility product and its target are both RNA transcripts. J Bacteriol. 1984 Oct;160(1):28–35. doi: 10.1128/jb.160.1.28-35.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Womble D. D., Rownd R. H. Genetic and physical map of plasmid NR1: comparison with other IncFII antibiotic resistance plasmids. Microbiol Rev. 1988 Dec;52(4):433–451. doi: 10.1128/mr.52.4.433-451.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Womble D. D., Rownd R. H. Regulation of IncFII plasmid DNA replication. A quantitative model for control of plasmid NR1 replication in the bacterial cell division cycle. J Mol Biol. 1986 Dec 5;192(3):529–547. doi: 10.1016/0022-2836(86)90274-3. [DOI] [PubMed] [Google Scholar]
  39. Womble D. D., Sampathkumar P., Easton A. M., Luckow V. A., Rownd R. H. Transcription of the replication control region of the IncFII R-plasmid NR1 in vitro and in vivo. J Mol Biol. 1985 Feb 5;181(3):395–410. doi: 10.1016/0022-2836(85)90228-1. [DOI] [PubMed] [Google Scholar]
  40. Wu R. P., Womble D. D., Rownd R. H. Incompatibility mutants of IncFII plasmid NR1 and their effect on replication control. J Bacteriol. 1985 Sep;163(3):973–982. doi: 10.1128/jb.163.3.973-982.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wu R., Wang X., Womble D. D., Rownd R. H. Expression of the repA1 gene of IncFII plasmid NR1 is translationally coupled to expression of an overlapping leader peptide. J Bacteriol. 1992 Dec;174(23):7620–7628. doi: 10.1128/jb.174.23.7620-7628.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

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