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Cleavage and Rejoining of DNA in the Shufflon-Specific Recombinase

Research Project

Project/Area Number 10216207
Research Category

Grant-in-Aid for Scientific Research on Priority Areas

Allocation TypeSingle-year Grants
Review Section Biological Sciences
Research InstitutionTokyo Metropolitan University

Principal Investigator

KOMANO Teruya  Tokyo Metropolitan University, Department of Biology, Professor, 理学研究科, 教授 (00087131)

Co-Investigator(Kenkyū-buntansha) FURUYA Nobuhisa  Tokyo Metropolitan University, Department of Biology, Assistant, 理学研究科, 助手 (50244413)
Project Period (FY) 1998 – 2002
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥37,600,000 (Direct Cost: ¥37,600,000)
Fiscal Year 2002: ¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 2001: ¥7,100,000 (Direct Cost: ¥7,100,000)
Fiscal Year 2000: ¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 1999: ¥7,800,000 (Direct Cost: ¥7,800,000)
Fiscal Year 1998: ¥8,500,000 (Direct Cost: ¥8,500,000)
Keywordsshufflon / site-specific recombination / plasmid R64 / conjugal transfer / cleavage and rejoining of DNA
Research Abstract

The pilV gene, the last gene of the pil operon of plasmid R64 is under the control of shufflon multiple inversion. R64 shufflon consists of four DNA segments, separated and flanked by seven sfx sequences. Site-specific recombinations mediated by the rci product occur between any inverted, sfx sequences, resulting. in inversions of the four segments independently or in groups. Thus the C-terminal segments of PilV adhesins interconvert among seven types, which determine the recipient specificity in R64 liquid matings.
Rci protein was overproduced and purified. Specific cleavages were introduced into the sfx sequence when a sfx DNA fragment was incubated with Rci. Cleavages occurred in the form of a 5' protruding 7-bp staggered cut, suggesting that DNA cleavage and rejoining in the shufflon recombination take place at these positions. The sfx sequences were shown to consist of central 7-bp spacer sequence and left and right 12-bp arms, where the sfx left arm sequence are not conserved. Rci tein was shown to bind to entire sfx sequences, suggesting that one Rci molecule binds to the conserved sfx right arm in a sequence-specific manner and the second Rci molecule binds to the sfx left arm in a non-sequence-specific manner. Different sfx left arm sequences exhibited different inversion frequencies. When a symmetric sfx sequence, where the left and right arm sequences are the same, was constructed, recombination between symmetric sfx sequences in direct orientation was observed. Asymmetry of the sfx sequence may be the reason why Rci protein acts on only the inverted sfx sequences. R64 liquid matings using various waa mutants of E. coli K-12 and S. enterica serovar Typhimurium LT2 as recipient strains indicated that the specific receptors for the PilV adhesins are LPS molecules. Binding of PilV adhesins to respective LPS molecules was demonstrated.

Report

(6 results)
  • 2003 Final Research Report Summary
  • 2002 Annual Research Report
  • 2001 Annual Research Report
  • 2000 Annual Research Report
  • 1999 Annual Research Report
  • 1998 Annual Research Report
  • Research Products

    (48 results)

All Other

All Publications (48 results)

  • [Publications] Gyohda, A., N.Furuya, A.Ishiwa, S.Zhu, T.Komano: "Structure and function of the shufflon in plasmid R64"Adv.Biophys.. 38(in press). (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ishiwa, A., T.Komano: "Thin pilus PilV adhesins of the plasmid R64 recognize specific structures of the lipopolysaccharide molecules of recipient cells"J.Bacteriol.. 185. 5192-5199 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Tojo, N., T.Komano: "The IntP C-terminal segment is not required for the excision of bacteriophage Mx8 from the Myxococcus xanlhw chromosome"J.Bacteriol.. 185. 2187-2193 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Gyohda, A., N.Furuya, N.Kogure, T.Komano: "Sequence-specific and non-specific binding of the Rci protein to the asymmetric recombination sites of the R64 shufflon"J.Mol.Biol.. 318. 975-983 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ishiwa, A., T.Komano: "The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement, in liquid matings with donors bearing the R64 plasmid"Mol.Gen.Genet.. 263. 159-164 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Komano, T.: "Shufflons : multiple inversion systems arid integrons"Annu.Rev.Genet.. 33. 171-191 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yoshida, T., N.Furuya, M.Ishikura, T.Isobe, K.Haino-Fukushima, T.Ogawa, T.Komano: "Purification and characterization of thin pili of Inc11 plasmids (ColIb-P9 and R64 : formation of PilV-specific cell aggregates by type IV pili."J. Bacteriol.. 180. 2842-2848 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Horiuchi T., T.Komano: "Mutational analysis of plasmid R64 thin pilus prepilin : the entire prepilin sequence is required for processing by type IV prepilin peptidase."J. Bacteriol.. 180. 4613-4620 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yoshida, T., S.-R.Kim, T.Komano: "Twelve pil genes are required for biogenesis of the R64 thin pilus."J. Bacteriol.. 181. 2038-2043 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Komano, T.: "Shufflons : multiple inversion systems and integrons."Annu. Rev. Genet.. 33. 171-191 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sakai, D., T.Komano: "The pilL and PilN genes of IncI1 plasmids R64 and ColIb-P9 encode outer membrane lipoproteins responsible for thin pilus biogenesis."Plasmid. 43. 149-152 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ishiwa, A., T.Komano: "The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement, in liquid matings with donors bearing the R64 plasmid."Mol. Gen. Genet.. 263. 159-164 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Komano, T., T.Yoshida, K.Narahara, N.Furuya: "The transfer region of IncI1 plasmid R64 : similarities between R64 tra and Legionella icm/dot genes."Mol. Microbiol.. 35. 1348-1359 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Gyohda, A., T.Komano: "Purification and characterization of the R64 shufflon-specific recombinase."J. Bacteriol.. 182. 2787-2792 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Furuya, N., T.Komano: "Initiation and termination of DNA transfer during conjugation of IncI1 plasmid R64 : the role of two sets of inverted repeat sequences within oriT in the termination of R64 transfer."J. Bacteriol.. 182. 3191-3196 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sakai, D., T.Horiuchi, T.Komano: "ATPase activity and multimer formation of PilQ protein are required for thin pilus biogenesis in plasmid R64."J. Biol. Chem.. 276. 17968-17975 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Sakai, D., T.Komano: "Genes required for plasmid R64 thin pilus biogenesis : identification and localization of products of the pilK, pilM, pilO, pilP, pilR and pilT genes."J. Bacteriol.. 184. 444-451 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Gyohda, A., N.Furuya, N.Kogure, T.Komano: "Sequence-specific and non-specific binding of the Rci protein to the asymmetric recombination sites of the R64 shufflon."J. Mol. Biol.. 318. 975-983 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Tojo, N., T.Komano: "The IntP C-terminal segment is not required for the excision of bacteriophage Mx8 from the Myxococcus xanthus chromosome."J. Bacteriol.. 185. 2187-2193 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Furuya, N., T.Komano: "NikAB-or NikB-dependent intracellular recombination between tandemly repeated oriT sequences of plasmid R64 in plasmid or single-stranded phage vectors."J. Bacteriol.. 185. 3871-3877 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ishiwa, A., T.Komano: "Thin pilus PilV adhesins of the plasmid R64 recognize specific structures of the lipopolysaccharide molecules of recipient cells."J. Bacteriol.. 185. 5192-5199 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Gyohda, A., N.Furuya, A.Ishiwa, S.Zhu, T.Komano: "Structure and function of the shufflon in plasmid R64."Adv. Biophys.. 38. (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Tojo, Komano: "The IntP C-terminal segment is not required for the excision of bacteriophage Mx8 from the Myxococcus"Journal of Bacteriology. 185(in press). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Horiuchi et al.: "Regulation of fruA expression during vegetative growth and development of Myxococcus xanthus"J. Mol. Microbiol. Biotech.. (in press). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Gyohda et al.: "Sequence-specific and non-specific binding of the Rci protein to the"Journal of Molecular Biology. 318. 975-983 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Horiuchi et al.: "Role of fruA and csgA genes in gene expression during development of Myxococcus xanthus : analysis by two-dimentional gel electrophoresis"Journal of Biological Chemistry. 277. 26753-26760 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Horiuchi et al.: "Analysis of dofA, a fruA-dependent developmental gene and its homologue"Journal of Bacteriology. 184. 6803-6810 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Sakai, Komano: "Genes required for plasmid R64 thin pilus biogenesis : identification and localization of products of the pilK, pilM, pilO, pilP, pilR, pilT"Journal of Bacteriology. 184. 444-451 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Sakai, Komano: "Genes required for plasmid R64 thin pilus biogenesis:identification and localization of products of the pilK,pilM,pilO,pilP,pilR,pilT"Journal of Bacteriology. 184. 444-451 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Sakai et al.: "ATPase activity and multimer formation of PilQ protein are required for thin pilus biogenesis in plasmid R64"Journal of Biological Chemistry. 276. 17968-17975 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Komano et al.: "The transfer region of Incl1 plasmid R64 : similarities between R64 tra and Legionella icm/dot genes."Molecular Microbiology. 35. 1348-1359 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Furuya and Komano: "Initiation and termination of DNA transfer during conjugation of Incl1 Plasmid R64 : the role of two sets of inverted repeat sequences."Journal of Bacteriology. 182. 3191-3196 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Gyohda and Komano: "Purification and characterization of the R64 shufflon-specific recombinase."Journal of Bacteriology. 182. 2782-2792 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Ishiwa and Komano: "The lipopolysaccharide of recipient cells is a specific receptor for PilV proteins, selected by shufflon DNA rearrangement."Molecular and General Genetics. 263. 159-164 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Sakai and Komano: "The pilL and pilN genes of Incl1 plasmids R64 and Collb-P9 encode outer membrane lipoproteins responsible for thin pilus biogenesis."Plasmid. 43. 149-152 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Komano: "Shufflons : multiple inversion systems and integrons."Annual Review of Genetics. 33. 171-191 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] T. Komano: "Shufflons : multiple inversion systems and integrons"Annual Review Genetics. 33. 171-191 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] T. Yoshida et al.: "Twelve pil genes are required for biogenesis of the R64 thin pilus"Journal of Bacteriology. 181. 2038-2043 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Sakai and Komano: "The pilL and pilN genes of IncI1 plasmids R64 and ColIb-P9 encode outer membrane lipoproteins responsible for thin pilus biogenesis"Plasmid. 43. 149-152 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Ishiwa and Komano: "Lipopolysaccharide of recipient cells is a specific receptor for PilV proteins selected by shufflon DNA rearrangement in R64 liquid matings"Molecular and General Genetics. (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] T. Komano et al.: "The transfer region of IncI1 plasmid R64 : similarities between R64 tra and legionella icm/dot genes"Molecular Microbiology. 35. 1348-1359 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Gyohda and Komano: "Purification and characterization of the R64 shufflon-specific recombinase"Journal of Bacteriology. 182 (in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Gyohda et al.: "Analysis of DNA inversions in the shufflon of Plasmid R64." Journal of Bacteriology. 179. 1867-1871 (1997)

    • Related Report
      1998 Annual Research Report
  • [Publications] Kim and Komano: "The plasmid R64 thin pilus identified as a type IV pilus." Journal of Bacteriology. 179. 3594-3603 (1997)

    • Related Report
      1998 Annual Research Report
  • [Publications] Horiuchi and Komano: "Mutational analysis of plasmid R64 thin pilus prepilin : the entire prepilin sequence is required for processing by type IV prepilin peptidase." Journal of Bacteriology. 180. 4613-4620 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] H.Sekimoto et al.: "Molecular clonig and Characterization of aldehyde oxidase in Arabidopsis thaliana." Plant and Cell Physiology. 39. 433-442 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] T.Yoshida et al.: "Purification and characterization of thin pili of lncll plasmids Collb-P9 and R64:formation of PilV-specific cell aggregates by type IV pili." Journal of Bacteriology. 180. 2842-2848 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] T.Yoshida et al.: "Twelve pil genes are required for the biogenesis of R64 thin pilus." Journal of Bacteriology. 181 in press. (1999)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2018-03-28  

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