Abstract

The experiments outlined in this proposal are directed toward an understanding of the DNA processing reactions that occur during conjugative transfer of the broad host-range plasmid R1162. A model is proposed in which the origin of transfer (oriT) consists of two domains, one required for initial nicking of the DNA prior to transfer, and the other for subsequent recircularization of the transferred but overlapping regions of a large open reading frame (ORFI). Mutations will be isolated in oriT to identify and map the different functional domains. Proteins fragments of ORFT that are involved in different processing events of oriT will be purified, and their biochemical activities tested in vitro. Protein-DNA relaxation complex will be characterized to identify the processing intermediate related to this structure. DNA synthesis occurs from within oriT in cell extracts and is dependent on the plasmid genes for transfer. The origin and direction of this synthesis will be determined. ORFI overlaps rep2, a gene required for plasmid DNA replication. The genetic relationship between rep2 and ORFI will be clarified, and the involvement of the rep2 region in DNA synthesis from oriT will be assessed. Mutations in oriT will also be tested to see whether nicking is required for synthesis. Another mobilization protein, MobI, complements the rep2 region of ORFI in vivo, and inhibits the processing of single-stranded oriT DNA. MobI will be tested for support of DNA synthesis from oriT, and the target of this protein will be identified. Finally, the R1162 DNA strands that are transferred during conjugation will be identified by capturing this DNA in infectious particles.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037462-07
Application #
3292738
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-12-01
Project End
1994-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
7
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
University of Texas Austin
Department
Type
Schools of Arts and Sciences
DUNS #
City
Austin
State
TX
Country
United States
Zip Code
78712

  Publications

Becker, Eric C; Meyer, Richard (2012) Origin and fate of the 3' ends of single-stranded DNA generated by conjugal transfer of plasmid R1162. J Bacteriol 194:5368-76
de la Cruz, Fernando; Frost, Laura S; Meyer, Richard J et al. (2010) Conjugative DNA metabolism in Gram-negative bacteria. FEMS Microbiol Rev 34:18-40
Meyer, Richard (2009) Replication and conjugative mobilization of broad host-range IncQ plasmids. Plasmid 62:57-70
Monzingo, Arthur F; Ozburn, Angela; Xia, Shuangluo et al. (2007) The structure of the minimal relaxase domain of MobA at 2.1 A resolution. J Mol Biol 366:165-78
Jandle, Sarah; Meyer, Richard (2006) Stringent and relaxed recognition of oriT by related systems for plasmid mobilization: implications for horizontal gene transfer. J Bacteriol 188:499-506
Parker, Christopher; Meyer, Richard (2005) Mechanisms of strand replacement synthesis for plasmid DNA transferred by conjugation. J Bacteriol 187:3400-6
Parker, Christopher; Becker, Eric; Zhang, Xiaolin et al. (2005) Elements in the co-evolution of relaxases and their origins of transfer. Plasmid 53:113-8
Zhang, Xiaolin; Zhang, Shuyu; Meyer, Richard J (2003) Molecular handcuffing of the relaxosome at the origin of conjugative transfer of the plasmid R1162. Nucleic Acids Res 31:4762-8
Becker, Eric C; Meyer, Richard J (2003) Relaxed specificity of the R1162 nickase: a model for evolution of a system for conjugative mobilization of plasmids. J Bacteriol 185:3538-46
Parker, Christopher; Zhang, Xiao-lin; Henderson, Dorian et al. (2002) Conjugative DNA synthesis: R1162 and the question of rolling-circle replication. Plasmid 48:186-92

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