Abstract

Conjugative mobilization of broad host-range plasmid R1162 requires both oriT, a cis-active, 38 base-pair site, and the products of at least four plasmid mob genes. Mutagenized oriT DNA will be tested to identify the base-pairs important for mobilization, conjugation-dependent recombination, and nicking by relaxation complex. In addition, the sites of recombination, and relaxation complex-induced nicking within oriT will be mapped. The mob gene products will be identified by examining the polypeptides made in minicells containing cloned fragments R1162 DNA; the corresponding open reading frames will then be located by DNA base sequencing. The direction and strandedness of plasmid DNA transfer during mobilization will be determined by hybridizing newly-transferred DNA, isolated from minicells, with specific DNA probes. Mutations in mob genes whose products interact with oriT will be obtained as pseudorevertants of nonmobilizable, oriT-plasmids. The altered mob genes will be mapped by complementation and recombination. Finally, the polypeptide covalently joined at the relaxation complex nick site will be isolated, and the amino acid sequence partially determined. This sequence will then be compared to the base sequence of the mob genes, in order to identify the gene encoding the polypeptide.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM037462-01
Application #
3292732
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-12-01
Project End
1989-11-30
Budget Start
1986-12-01
Budget End
1987-11-30
Support Year
1
Fiscal Year
1987
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
78713

  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

Showing the most recent 10 out of 31 publications