Functional dissection of the conjugative coupling protein TrwB

Abstract

The conjugative coupling protein TrwB is responsible for connecting the relaxosome to the type IV secretion system during conjugative DNA transfer of plasmid R388. It is directly involved in transport of the relaxase TrwC, and it displays an ATPase activity probably involved in DNA pumping. We designed a conjugation assay in which the frequency of DNA transfer is directly proportional to the amount of TrwB. A collection of point mutants was constructed in the TrwB cytoplasmic domain on the basis of the crystal structure of TrwB Delta N70, targeting the nucleotide triphosphate (NTP)-binding region, the cytoplasmic surface, or the internal channel in the hexamer. An additional set of transfer-deficient mutants was obtained by random mutagenesis. Most mutants were impaired in both DNA and protein transport. We found that the integrity of the nucleotide binding domain is absolutely required for TrwB function, which is also involved in monomer-monomer interactions. Polar residues surrounding the entrance and inside the internal channel were important for TrwB function and may be involved in interactions with the relaxosomal components. Finally, the N-terminal transmembrane domain of TrwB was subjected to random mutagenesis followed by a two-hybrid screen for mutants showing enhanced protein-protein interactions with the related TrwE protein of Bartonella tribocorum. Several point mutants were obtained with mutations in the transmembranal helices: specifically, one proline from each protein may be the key residue involved in the interaction of the coupling protein with the type IV secretion apparatus.

FIG. 1.

Western blots showing amounts of TrwB (A, B, and D) or TrwA (C and E) under different expression conditions. Total extracts from E. coli cells containing the trwB-expressing plasmid indicated at the top were processed as described in Materials and Methods, samples were run on SDS-PAGE, and proteins were detected with anti-TrwB (αB) or anti-TrwA (αA) serum. (A to C) SDS-PAGE with samples from cells harboring the trwB-expressing plasmids alone. (D and E) SDS-PAGE with samples from D1210 cells containing additional plasmids pKM101Δmob and pHP138, as in conjugation assay types DEF (Fig. 2). The position of TrwA and TrwB is indicated to the left. The white arrowhead points to unspecific bands visible in the strain with no plasmids. Molecular mass markers are indicated to the right (in kDa). BΔN70, purified TrwBΔN70 protein, shown in panel A for TrwB quantification and in panels B and D as a size marker. The induction conditions (IPTG for 0, 1, or 3 h) are indicated as −, +, or ++, respectively.

FIG. 2.

Scheme of the different conjugation assays used in this work and the transfer frequencies obtained. The elements of the transfer machinery present in each assay type (A to I) are represented as indicated in the lower part of the figure. The transposon insertion inactivating trwB in the R388 derivative is represented by a triangle. The different plasmids (separated by “+” symbols) present in the donor strains are the sum of those in the lane and column matching for each assay type; lanes also indicate the transferred product (either TrwC alone or with covalently attached DNA). Standard (A to F) or triparental (G to I) matings were performed as described in Materials and Methods. IPTG was added to the mating plate only (+) or was preceded by an additional 2-h incubation in liquid (++), as explained in Materials and Methods. Frequencies (as number of transconjugants per donor) are the mean of 3 to 5 independent experiments. Under these assay conditions, background transfer in the absence of a mobilizable plasmid is <10⁻⁷ transconjugants/donor and R388 transfer frequency is 10⁻¹ transconjugants/donor.

FIG. 3.

Representations of the 3D structure of TrwBΔN70 bound to ADP (PDB accession no. 1GKI), where the mutated residues are highlighted in the space-filled format. (A) Side view of two opposing monomers, showing in green the mutated residues which protrude into the ICH of the hexamer. The extent of the 12 and 17 C-terminal residues of the protein is indicated with arrows (12C and 17C, respectively). Mutated AAD residues are also indicated (space filled in gray). (B) TrwBΔN70 hexamer seen from the cytoplasmic side, with the selected residues from the AAD domain shown in the space-filled format. Residue R318, located in the interface between monomers, is also highlighted (in red). The region included in the quadrangle is amplified in panel C for better resolution of the mutated residues in the proximity of the nucleotide molecule.

FIG. 4.

Mutations affecting the TrwB-TrwE interaction. (Left) Scheme of the transmembranal regions of TrwE_Bt and TrwB. Predicted transmembranal segments are represented by helices embedded in the inner membrane (IM). C, cytoplasm; P, periplasm. N, N terminus of the proteins. The locations of the residues shown to be involved in the interaction are indicated with arrows. (Right) Protein-protein interactions tested by the two-hybrid assay. For each spot, the pairs of proteins produced as T25 and T18 fusions are indicated. The top panel shows the TrwE_Bt mutants tested against TrwB, the middle panel shows TrwB mutants tested against TrwE_Bt, and the bottom panel shows TrwB mutants tested against TrwB or themselves.

FIG. 5.

(Left) Representation of a TrwB protomer with the mutated residues shown in the space-filled format and their proposed function indicated in a color code. Within each proposed functional domain, the space-filled residues show brighter colors when their mutation affects the transfer efficiency. The 3D structure of the N-terminal TMD has been modeled as in reference . Each mutated residue is marked with a number. (Right) The table summarizes the results obtained from the in vivo assays for each mutant. Mating assay abbreviations: DNA transfer efficiency (DNA), TrwC transport efficiency (TrwC), and negative dominance (ND). Two-hybrid assay abbreviations: TrwB-TrwB interactions (B:B), TrwB-TrwE interactions (B:E), and TrwB-TrwE_Bt interactions (B:Eb).