Investigating Rate of TraI and DNA Transfer during Bacterial Conjugation

Abstract

F plasmid TraI (192 kD) is essential for DNA transfer during bacterial conjugation. TraI is composed of a relaxase domain, ssDNA binding domain, helicase domain, and C-terminal domain. Some TraI mutants with a 31 AA insertion (i31) in different domains exhibit higher mating efficiencies than wild-type (WT) TraI. To investigate the higher mating efficiency, we employed a live cell SeqA-YFP fusion protein system that resides in dam- recipient cells and tracks DNA transfer from a dam+ donor. After methylated ssDNA is transferred to the recipient and second strand synthesis begins, a fluorescent focus forms on the hemi-methylated dsDNA, allowing for near-real time analysis of bacterial conjugation. Mean foci formation time for the mutant TraI i31-681 was much lower than WT TraI. To determine if the change in foci formation time was due to second strand synthesis, the assay was repeated, but mating was disrupted at set times. Foci formation was followed over time and the results were similar, indicating the differences between mutants and WT was not due to a difference in second strand synthesis rate. To further investigate where the transfer process is altered, we employed a DNA nicking assay and determined that the nicking activity of the mutants is not altered. Addition of TraDΔN130, a pore coupling protein that increases nicking activity, to the nicking assay does not significantly alter nicking activity of the mutants over WT TraI. Nicking activity being unaffected, the 309-858 region of the TraI mutants will be tested to determine the folding profile of the i31 insertion. Currently we are exploring a possible correlation between increased transfer rates and increased unfolding rates of the domains.