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 approximately 310 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 (TraIDelta252) 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, TraIDelta252 was not able to support conjugative DNA transfer in genetic complementation experiments. We conclude that TraIDelta252 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.

FIG. 1.

Diagram of TraI and ClustalW alignments. (A) Schematic diagram of TraI. The relaxase domain is wholly contained within the region spanned by residues 1 to 310 (6, 11). The helicase-associated motifs are located within the region spanned by residues 990 to 1450. The functional role of the central region (residues 310 to 990) and the C-terminal region (residues 1450 to 1756) is unknown. (B) ClustalW alignment of selected sequences obtained from a BLAST search using TraI (NP_862951.1) as the query sequence. The relaxase domain is shown in red, the central region with unknown function is shown in green, and the region containing the helicase-associated motifs is in blue. The sequences used in the alignment shown are TraI (F plasmid), TraI (plasmid R100) (NP_052981.1), TraI (Salmonella enterica serovar Typhimurium) (AAM90727.1), oriT nicking/unwinding protein fragment (Shigella flexneri) (NP_085415.1), putative conjugative transfer protein TraI (Vibrio vulnificus YJ016) (NP_932226.1), putative DNA helicase (TraI) (Photobacterium profundum SS9) (YP_015476.1), putative TraC protein (Pseudomonas putida) (AAP57243.1), TrwC (Xanthomonas citri) (NP_942625.1), TrwC protein (IncW plasmid R388) (S43878), and TraI [IncN plasmid R46] (NP_511201.1).

FIG. 2.

SDS-polyacrylamide gel analysis of the purification and expression of TraI and TraIΔ252. (A) One microgram each of purified TraI (lane 2) and purified TraIΔ252 (lane 3) was resolved on an 8% polyacrylamide gel run in the presence of SDS and stained with Coomassie brilliant blue. High-range molecular mass standards (Bio-Rad) with sizes in kilodaltons indicated to the left are shown in lane 1. (B) Whole-cell lysates of HMS174/pOX38T (lane 1), HMS174/pOX38TΔtraI (lane 2), HMS174/pET11d-traI (lane 3), and HMS174/pET3c-traIΔ252 (lane 4) were resolved on an 8% polyacrylamide gel run in the presence of SDS, transferred to nitrocellulose, and probed with anti-TraI antisera. Prestained molecular mass standards (Bio-Rad) with sizes in kilodaltons are shown on the right. The slight difference in migration between TraI in lane 1 and TraI in lane 3 is due to a gel artifact.

FIG. 3.

DNA helicase activity of TraI and TraIΔ252. DNA unwinding assays using a 93-bp partial duplex substrate (A) or an 851-bp partial duplex substrate (B) were performed as described in Materials and Methods, using the indicated amount of TraI (•) or TraIΔ252 (○). Reaction mixtures were incubated for 10 min at 37°C, and the products were resolved on a native polyacrylamide gel. The fraction of the substrate unwound was calculated by the formula [(U − B_u)/(S − B_s + U − B_u)] × 100, where U represents the DNA in the product band, S represents DNA in the substrate band and B_u and B_s represent background levels of DNA at the position of the product (B_u) or the substrate (B_s) determined from reaction mixtures containing no protein that were either incubated as described above or incubated at 95°C for 5 min to denature the substrate.

FIG. 4.

Relaxase activity of TraI and TraIΔ252. (A) The relaxase activity assays using pBSoriT DNA were performed as described in Materials and Methods. A representative experiment is shown. The upper half of the gel represents a titration of TraI, and the lower half represents a titration of TraIΔ252. Lane 1, no-enzyme control; lane 2, 3.9 nM TraI (TraIΔ252); lane 3, 7.8 nM TraI (TraIΔ252); lane 4, 15.6 nM TraI (TraIΔ252); lane 5, 31.3 nM TraI (TraIΔ252); lane 6, 62.5 nM TraI (TraIΔ252); and lane 7, 125 nM TraI (TraIΔ252). (B) The gel shown in panel A was quantified as described previously (29) for TraI (•) and TraIΔ252 (○), and the data were plotted as a fraction of total plasmid in the relaxed species.

FIG. 5.

Cleavage activity of TraI and TraIΔ252. (A) Schematic illustration of the oriT cleavage reaction catalyzed by TraI. The 22-nucleotide substrate was 5′-end labeled and contained the binding site recognized by TraI and the scissile phosphodiester bond (nic). The reaction requires the addition of MgCl₂ and results in the release of a 5′-end-labeled 14-mer and an 8-mer that is covalently bound to TraI (TraIΔ252). (B) Cleavage assays were performed as described in Materials and Methods and were incubated for 20 min at 37°C prior to denaturation and resolution of products on a 16% denaturing polyacrylamide gel. Lane 1, no-protein control; lane 2, 1.7 nM TraI; lane 3, 4.1 nM TraI; lane 4, 12.3 nM TraI; lane 5, 37 nM TraI; lane 6, 111 nM TraI; lane 7, 333.3 nM TraI; lane 8, 1,000 nM TraI; lane 9, 1.7 nM TraIΔ252; lane 10, 4.1 nM TraIΔ252; lane 11, 12.3 nM TraIΔ252; lane 12, 37 nM TraIΔ252; lane 13, 111 nM TraIΔ252; lane 14, 333.3 nM TraIΔ252; and lane 15, 1,000 nM TraIΔ252. This is a representative experiment. (C) Quantitation of multiple (three to five) oligonucleotide cleavage assays using TraI (•) and TraIΔ252 (○) by PhosphorImager analysis using ImageQuant software.

FIG. 6.

Cleavage/ligation activity of TraI and TraIΔ252. (A) Schematic representation of the strand exchange assay. See text for details. (B) Strand exchange assays were performed as described in Materials and Methods. The substrate (22-mer) is indicated on the left, and the cleavage product (14-mer) and expected recombinant product (24-mer) are indicated on the right. Lane 1, no-protein control; lane 2, 1.7 nM TraI; lane 3, 4.1 nM TraI; lane 4, 12.3 nM TraI; lane 5, 37 nM TraI; lane 6, 111 nM TraI; lane 7, 333.3 nM TraI; lane 8, 1,000 nM TraI; lane 9, 1.7 nM TraIΔ252; lane 10, 4.1 nM TraIΔ252; lane 11, 12.3 nM TraIΔ252; lane 12, 37 nM TraIΔ252; lane 13, 111 nM TraIΔ252; lane 14, 333.3 nM TraIΔ252; and lane 15, 1,000 nM TraIΔ252. This is a representative experiment.

FIG. 7.

TraIΔ252 is capable of assembling into a functional relaxosome. The relaxosome reconstitution experiments were performed as described in Materials and Methods with the indicated proteins at the following concentrations: TraI (TraIΔ252), 62.5 nM; IHF, 49.4 nM; and TraY, 170 nM. The positions of relaxed and supercoiled DNA are indicated on the left. Lane 7 contains a linear DNA marker that migrates just slightly faster than nicked DNA on an agarose gel.