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. 1996 May;178(10):2902–2910. doi: 10.1128/jb.178.10.2902-2910.1996

Characterization of the primary immunity region of the Escherichia coli linear plasmid prophage N15.

M B Lobocka 1, A N Svarchevsky 1, V N Rybchin 1, M B Yarmolinsky 1
PMCID: PMC178027  PMID: 8631680

Abstract

N15 is the only bacteriophage of Escherichia coli known to lysogenize as a linear plasmid. Clear-plaque mutations lie in at least two regions of the 46-kb genome. We have cloned, sequenced, and characterized the primary immunity region, immB. This region contains a gene, cB, whose product shows homology to lambdoid phage repressors. The cB3 mutation confers thermoinducibility on N15 lysogens, consistent with CB being the primary repressor of N15. Downstream of cB lies the locus of N15 plasmid replication. Upstream of cB lies an operon predicted to encode two products: one homologous to the late repressor of P22 (Cro), the other homologous to the late antiterminator of phi 82 (Q). The Q-like protein is essential for phage development. We show that CB protein regulates the expression of genes that flank the cB gene by binding to DNA at symmetric 16-bp sites. Three sites are clustered upstream of cB and overlap a predicted promoter of the cro and Q-like genes as well as two predicted promoters of cB itself. Two sites downstream of cB overlap a predicted promoter of a plasmid replication gene, repA, consistent with the higher copy number of the mutant, N15cB3. The leader region of repA contains terminators in both orientations and a putative promoter. The organization of these regulatory elements suggests that N15 plasmid replication is controlled not only by CB but also by an antisense RNA and by a balance between termination and antitermination.

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Selected References

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  1. Brendel V., Trifonov E. N. A computer algorithm for testing potential prokaryotic terminators. Nucleic Acids Res. 1984 May 25;12(10):4411–4427. doi: 10.1093/nar/12.10.4411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Béjar S., Bouché F., Bouché J. P. Cell division inhibition gene dicB is regulated by a locus similar to lambdoid bacteriophage immunity loci. Mol Gen Genet. 1988 Apr;212(1):11–19. doi: 10.1007/BF00322439. [DOI] [PubMed] [Google Scholar]
  3. Béjar S., Cam K., Bouché J. P. Control of cell division in Escherichia coli. DNA sequence of dicA and of a second gene complementing mutation dicA1, dicC. Nucleic Acids Res. 1986 Sep 11;14(17):6821–6833. doi: 10.1093/nar/14.17.6821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Campbell A. Comparative molecular biology of lambdoid phages. Annu Rev Microbiol. 1994;48:193–222. doi: 10.1146/annurev.mi.48.100194.001205. [DOI] [PubMed] [Google Scholar]
  5. Faubladier M., Bouché J. P. Division inhibition gene dicF of Escherichia coli reveals a widespread group of prophage sequences in bacterial genomes. J Bacteriol. 1994 Feb;176(4):1150–1156. doi: 10.1128/jb.176.4.1150-1156.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ghisotti D., Briani F., Forti F., Piazza F., Polo S., Sabbattini P., Sturniolo T., Terzano S., Zangrossi S., Zappone M. Multiple regulatory mechanisms controlling phage-plasmid P4 propagation. FEMS Microbiol Rev. 1995 Aug;17(1-2):127–134. doi: 10.1111/j.1574-6976.1995.tb00194.x. [DOI] [PubMed] [Google Scholar]
  7. Goliger J. A., Roberts J. W. Bacteriophage 82 gene Q and Q protein. Sequence, overproduction, and activity as a transcription antiterminator in vitro. J Biol Chem. 1987 Aug 25;262(24):11721–11725. [PubMed] [Google Scholar]
  8. Heinrich J., Riedel H. D., Rückert B., Lurz R., Schuster H. The lytic replicon of bacteriophage P1 is controlled by an antisense RNA. Nucleic Acids Res. 1995 May 11;23(9):1468–1474. doi: 10.1093/nar/23.9.1468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hinnebusch J., Barbour A. G. Linear plasmids of Borrelia burgdorferi have a telomeric structure and sequence similar to those of a eukaryotic virus. J Bacteriol. 1991 Nov;173(22):7233–7239. doi: 10.1128/jb.173.22.7233-7239.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hinnebusch J., Tilly K. Linear plasmids and chromosomes in bacteria. Mol Microbiol. 1993 Dec;10(5):917–922. doi: 10.1111/j.1365-2958.1993.tb00963.x. [DOI] [PubMed] [Google Scholar]
  11. Hiraga S., Sugiyama T., Itoh T. Comparative analysis of the replicon regions of eleven ColE2-related plasmids. J Bacteriol. 1994 Dec;176(23):7233–7243. doi: 10.1128/jb.176.23.7233-7243.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hubbard M. J., Klee C. B. Functional domain structure of calcineurin A: mapping by limited proteolysis. Biochemistry. 1989 Feb 21;28(4):1868–1874. doi: 10.1021/bi00430a066. [DOI] [PubMed] [Google Scholar]
  13. Kozyrev D. P., Dzhus M. G., Rybchin V. N. Lizogennaia konversiia, vyzyvaemaia fagom phi 80. Soobshchenie II. Kartirovanie lokusa cor. Genetika. 1983 Jun;19(6):940–944. [PubMed] [Google Scholar]
  14. Kozyrev D. P., Rybchin V. N. Lizogennaia konversiia, vyzyvaemaia fagom phi 80. Soobshchenie III. Utochnenie lokalizatsii gena konversii i dopolnitel'naia kharakteristika iavleniia. Genetika. 1987 May;23(5):793–801. [PubMed] [Google Scholar]
  15. Kozyrev D. P., Svarchevskii A. N., Zaitsev E. N., Rybchin V. N. Lizogennaia konversiia, vyzyvaemaia fagom phi 80. Soobshchenie I. Opisanie iavleniia i klonirovanie gena konversii. Genetika. 1982 Apr;18(4):555–560. [PubMed] [Google Scholar]
  16. Malinin A. Iu, Vasil'ev A. L., Kholodii G. A., Vostrov A. A., Rybchin V. N., Svarchevskii A. N. Struktura oblasti v genome bakteriofaga N15, neobkhodimaia dlia obrazovaniia shpilek na kontsakh lineinogo plazmidnogo profaga. Mol Gen Mikrobiol Virusol. 1992 May-Jun;(5-6):22–25. [PubMed] [Google Scholar]
  17. Malinin A. Iu, Vostrov A. A., Rybchin V. N., Svarchevskii A. N. Struktura kontsov lineinoi plazmidy N15. Mol Gen Mikrobiol Virusol. 1992 May-Jun;(5-6):19–22. [PubMed] [Google Scholar]
  18. Malinin A. Iu, Vostrov A. A., Vasil'ev A. L., Rybchin V. N. Kharakteristika gena lizogennoi konversii bakteriofaga N15 i identifikatsiia ego produkta. Genetika. 1993 Feb;29(2):257–265. [PubMed] [Google Scholar]
  19. Miozzari G. F., Yanofsky C. The regulatory region of the trp operon of Serratia marcescens. Nature. 1978 Dec 14;276(5689):684–689. doi: 10.1038/276684a0. [DOI] [PubMed] [Google Scholar]
  20. Mulligan M. E., Hawley D. K., Entriken R., McClure W. R. Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):789–800. doi: 10.1093/nar/12.1part2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nichols B. P., Yanofsky C. Plasmids containing the trp promoters of Escherichia coli and Serratia marcescens and their use in expressing cloned genes. Methods Enzymol. 1983;101:155–164. doi: 10.1016/0076-6879(83)01011-3. [DOI] [PubMed] [Google Scholar]
  22. Poteete A. R., Hehir K., Sauer R. T. Bacteriophage P22 Cro protein: sequence, purification, and properties. Biochemistry. 1986 Jan 14;25(1):251–256. doi: 10.1021/bi00349a035. [DOI] [PubMed] [Google Scholar]
  23. Ravin V. K., Shulga M. G. Evidence for extrachromosomal location of prophage N15. Virology. 1970 Apr;40(4):800–807. doi: 10.1016/0042-6822(70)90125-x. [DOI] [PubMed] [Google Scholar]
  24. San'kova T. P., Svarchevskii A. N., Rybchin V. N. Poluchenie, kharakteristika i kartirovanie insertsionnykh mutatsii fazmidy N15. Genetika. 1992 Jul;28(7):66–76. [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sauer R. T., Anderegg R. Primary structure of the lambda repressor. Biochemistry. 1978 Mar 21;17(6):1092–1100. doi: 10.1021/bi00599a024. [DOI] [PubMed] [Google Scholar]
  27. Sauer R. T., Pan J., Hopper P., Hehir K., Brown J., Poteete A. R. Primary structure of the phage P22 repressor and its gene c2. Biochemistry. 1981 Jun 9;20(12):3591–3598. doi: 10.1021/bi00515a044. [DOI] [PubMed] [Google Scholar]
  28. Schuler G. D., Altschul S. F., Lipman D. J. A workbench for multiple alignment construction and analysis. Proteins. 1991;9(3):180–190. doi: 10.1002/prot.340090304. [DOI] [PubMed] [Google Scholar]
  29. Siemering K. R., Praszkier J., Pittard A. J. Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication. J Bacteriol. 1993 May;175(10):2895–2906. doi: 10.1128/jb.175.10.2895-2906.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Takechi S., Yasueda H., Itoh T. Control of ColE2 plasmid replication: regulation of Rep expression by a plasmid-coded antisense RNA. Mol Gen Genet. 1994 Jul 8;244(1):49–56. doi: 10.1007/BF00280186. [DOI] [PubMed] [Google Scholar]
  31. Versalovic J., Koeuth T., Britton R., Geszvain K., Lupski J. R. Conservation and evolution of the rpsU-dnaG-rpoD macromolecular synthesis operon in bacteria. Mol Microbiol. 1993 Apr;8(2):343–355. doi: 10.1111/j.1365-2958.1993.tb01578.x. [DOI] [PubMed] [Google Scholar]
  32. Vostrov A. A., Malinin A. Iu, Rybchin V. N., Sravchevskii A. N. Konstruirovanie lineinykh plazmidnykh vektorov dlia klonirovaniia v kletkakh Escherichia coli. Genetika. 1992 Jul;28(7):186–188. [PubMed] [Google Scholar]
  33. Vostrov A. A., Vostrukhina O. A., Svarchevsky A. N., Rybchin V. N. Proteins responsible for lysogenic conversion caused by coliphages N15 and phi80 are highly homologous. J Bacteriol. 1996 Mar;178(5):1484–1486. doi: 10.1128/jb.178.5.1484-1486.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wold M. S., McMacken R. Regulation of expression of the Escherichia coli dnaG gene and amplification of the dnaG primase. Proc Natl Acad Sci U S A. 1982 Aug;79(16):4907–4911. doi: 10.1073/pnas.79.16.4907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yarmolinsky M. B., Hansen E. B., Jafri S., Chattoraj D. K. Participation of the lytic replicon in bacteriophage P1 plasmid maintenance. J Bacteriol. 1989 Sep;171(9):4785–4791. doi: 10.1128/jb.171.9.4785-4791.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ziegelin G., Lanka E. Bacteriophage P4 DNA replication. FEMS Microbiol Rev. 1995 Aug;17(1-2):99–107. doi: 10.1111/j.1574-6976.1995.tb00191.x. [DOI] [PubMed] [Google Scholar]
  37. del Solar G., Moscoso M., Espinosa M. Rolling circle-replicating plasmids from gram-positive and gram-negative bacteria: a wall falls. Mol Microbiol. 1993 May;8(5):789–796. doi: 10.1111/j.1365-2958.1993.tb01625.x. [DOI] [PubMed] [Google Scholar]

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