Conjugation is an important means of genetic transfer between bacterial species and has been implicated in the spread of genes encoding antibiotic resistance. The goal of this research program is to understand the mechanism and regulation of plasmid nicking and of initiation of DNA transfer in bacterial conjugation. Plasmid nicking, a prerequisite of transfer of a single plasmid DNA strand, is performed by relaxases, nucleases that bind and cleave single-stranded DNA. TraI, the relaxase of conjugative plasmid F Factor, is essential to F conjugative transfer. TraI possesses both a relaxase activity and a helicase activity, the latter unwinding and separating the plasmid DNA strands as the cut strand is transferred to the recipient. How Tral is able to act against double-stranded DNA in vivo, and how TraI converts between its relaxase and helicase activities are unknown. Experiments are proposed to answer four key questions about Tral and conjugation initiation: How is TraI nicking and transfer initiation influenced by oriT DNA sequences and the proteins that bind them? What protein-protein interactions influence TraI nicking and the initiation of DNA transfer, and what is the effect of disrupting them? How does a protein recognize single-stranded DNA with exquisite sequence specificity? How does TraI structure and domain organization influence its activity? These questions will be answered through in vivo studies that will identify Tral proteins, protein interactions and plasmid DNA sequences that influence transfer, and will determine the effects of these proteins and DNA sequences on plasmid nicking, transfer initiation, and transfer termination. In vitro studies will focus on determining the DNA sequences and distortions that TraI can recognize, the energetics of the TraI-DNA interaction, the conformational or functional events that occur subsequent to TraI binding to DNA, and the role of structure and domain organization in determining TraI function.
| Gruber, Christian J; Lang, Silvia; Rajendra, Vinod K H et al. (2016) Conjugative DNA Transfer Is Enhanced by Plasmid R1 Partitioning Proteins. Front Mol Biosci 3:32 |
| Guja, Kip E; Schildbach, Joel F (2015) Completing the specificity swap: Single-stranded DNA recognition by F and R100 TraI relaxase domains. Plasmid 80:1-7 |
| Hemmis, Casey W; Wright, Nathan T; Majumdar, Ananya et al. (2014) Chemical shift assignments of a reduced N-terminal truncation mutant of the disulfide bond isomerase TrbB from plasmid F, TrbB?29. Biomol NMR Assign 8:435-8 |
| Clark, Nicholas J; Raththagala, Madushi; Wright, Nathan T et al. (2014) Structures of TraI in solution. J Mol Model 20:2308 |
| Hemmis, Casey W; Schildbach, Joel F (2013) Thioredoxin-like proteins in F and other plasmid systems. Plasmid 70:168-89 |
| Buller, Andrew R; Labonte, Jason W; Freeman, Michael F et al. (2012) Autoproteolytic activation of ThnT results in structural reorganization necessary for substrate binding and catalysis. J Mol Biol 422:508-18 |
| Wright, Nathan T; Raththagala, Madushi; Hemmis, Casey W et al. (2012) Solution structure and small angle scattering analysis of TraI (381-569). Proteins 80:2250-61 |
| Zechner, Ellen L; Lang, Silvia; Schildbach, Joel F (2012) Assembly and mechanisms of bacterial type IV secretion machines. Philos Trans R Soc Lond B Biol Sci 367:1073-87 |
| Buller, Andrew R; Freeman, Michael F; Wright, Nathan T et al. (2012) Insights into cis-autoproteolysis reveal a reactive state formed through conformational rearrangement. Proc Natl Acad Sci U S A 109:2308-13 |
| Wright, Nathan T; Majumdar, Ananya; Schildbach, Joel F (2011) Chemical shift assignments for F-plasmid TraI (381-569). Biomol NMR Assign 5:67-70 |
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