The F-Plasmid TraI Protein Contains Three Functional Domains Required for Conjugative DNA Strand Transfer

Abstract

The F-plasmid-encoded TraI protein, also known as DNA helicase I, is a bifunctional protein required for conjugative DNA transfer. The enzyme catalyzes two distinct but functionally related reactions required for the DNA processing events associated with conjugation: the site-and strand-specific transesterification (relaxase) reaction that provides the nick required to initiate strand transfer and a processive 5-to-3 helicase reaction that provides the motive force for strand transfer. Previous studies have identified the relaxase domain, which encompasses the first ϳ310 amino acids of the protein. The helicase-associated motifs lie between amino acids 990 and 1450. The function of the region between amino acids 310 and 990 and the region from amino acid 1450 to the C-terminal end is unknown. A protein lacking the C-terminal 252 amino acids (TraI⌬252) was constructed and shown to have essentially wild-type levels of transesterase and helicase activity. In addition, the protein was capable of a functional interaction with other components of the minimal relaxosome. However, TraI⌬252 was not able to support conjugative DNA transfer in genetic complementation experiments. We conclude that TraI⌬252 lacks an essential C-terminal domain that is required for DNA transfer. We speculate this domain may be involved in essential protein-protein interactions with other components of the DNA transfer machinery.Conjugative DNA strand transfer is a highly conserved mechanism for unidirectional transfer of genetic information among bacteria of the same species, from one species to another and, in some instances, between kingdoms (for reviews, see references 15, 22, 25, 30, 37, and 44). Importantly, this is the mechanism used by most plasmids and conjugative transposons to facilitate their spread throughout a bacterial population, and the same underlying mechanism is used to transfer T-DNA from Agrobacterium tumefaciens to plant cells (23,45).The process of conjugative DNA transfer (CDT) is initiated with the formation of a stable mating pair between donor and recipient to establish the close cell-cell contact required for physical transfer of single-stranded DNA (ssDNA) from one cell to another. This is followed by the production of a site-and strand-specific nick at a locus called nic within the origin of transfer (oriT) or at the ends of T-DNA and the unwinding/ replication of the duplex DNA molecule to provide the ssDNA that enters the recipient. Within the recipient cell, host enzymes (primarily) convert the transferred ssDNA into doublestranded DNA that either circularizes to form a plasmid or is recombined into the recipient chromosome (for reviews, see references 15 and 44).Although reasonably well understood at this "macroscopic" level, the molecular details of the process by which DNA is transferred from donor to recipient are still being resolved. Key players are the proteins that initiate the physical transfer of ssDNA, the conjugative initiator proteins (7,22,30). These proteins introduce a site-and strand-specific ...

Disrupting antibiotic resistance propagation by inhibiting the conjugative DNA relaxase

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“…Gel-based cleavage assays were performed as previously described (50). Fluorescent cleavage reactions were…”

“…4C). Thus, for the most extreme case of ETIDRO, the median EC 50 for transfer inhibition is 330 nM with a range of 1.1 M to Ͻ10 pM (SI Fig. 10).…”

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

“…The nicked plasmid is then unidirectionally transferred to the recipient cell as a singled-stranded DNA intermediate. In plasmid F, the relaxase is encoded by a gene called traI (Matson and Ragonese, 2005;Traxler and Minkley, 1988). This mechanism of plasmid conjugation appears to be conserved in many replicons isolated from Gram positive bacteria.…”

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

“…The solid lines are computer fits of the data using the Epstein combinatorial approach (Equations 7 and 8). The parameters were m ϭ 9 and g ϭ 1 for dsDNA 9 , dsDNA 11 , and dsDNA 15 and m ϭ 9, g ϭ 2 for dsDNA19, dsDNA 23 , and dsDNA 27 .…”