Transfer protein TraY of plasmid R1 stimulates TraI-catalyzed oriT cleavage in vivo | JoVE Visualize

Transfer protein TraY of plasmid R1 stimulates TraI-catalyzed oriT cleavage in vivo

¹Institut für Molekularbiologie, Biochemie und Mikrobiologie, Karl-Franzens-Universität Graz, A-8010 Graz, Austria.

Journal of Bacteriology|February 24, 2001

View abstract on PubMed

Summary

The TraY protein enhances DNA cleavage at the R1 origin (oriT), but TraM protein is essential for plasmid DNA transfer during bacterial conjugation, suggesting a dual role.

Area of Science:

Molecular Biology
Bacteriology
Genetics

Background:

Plasmid conjugation is a key mechanism for horizontal gene transfer in bacteria.
The relaxosome complex, including proteins like TraI, TraY, and TraM, initiates DNA transfer at the origin of transfer (oriT).
The precise roles of TraY and TraM in relaxosome function and DNA processing remain incompletely understood.

Purpose of the Study:

To investigate the in vivo effects of TraY protein on TraI-catalyzed strand scission at the R1 oriT.
To elucidate the independent and overlapping functions of TraY and TraM in relaxosome assembly and DNA mobilization during bacterial conjugation.
To determine the essentiality of integration host factor (IHF) in TraI-mediated cleavage.

Main Methods:

In vivo cleavage assays in Escherichia coli using TraI, TraY, TraM, and IHF.
Mobilization experiments utilizing recombinant R1 oriT constructions and a heterologous conjugative helper plasmid.
Analysis of DNA strand scission and plasmid mobilization in wild-type and mutant strains.

Main Results:

TraY enhances TraI-catalyzed strand scission at oriT, with its presence inversely correlating with TraM dependence.
Both TraY and TraM can independently promote strand scission in vivo, but integration host factor (IHF) is essential for this activity.
While TraY is dispensable for mobilization when TraM is present, TraM remains essential for plasmid mobilization even when TraY is present, indicating a second, crucial function.

Conclusions:

TraY and TraM exhibit overlapping functions in promoting DNA strand scission at the R1 oriT.
TraM possesses an additional essential function beyond strand scission required for successful conjugative plasmid DNA transmission.
Integration host factor (IHF) plays a critical role in the in vivo TraI-mediated cleavage reaction at oriT.