Phylogenic tree of the translocase domain of TraB and FtsK proteins. Multiple sequence alignments of the DNA-translocase domain of selected FtsK and TraB proteins for phylogenetic inference were built using PROMALS 3D (Pei et al, 2008). Actinomycetes are displayed in blue, Gram-positive bacteria in magenta and Gram-negative bacteria in green. The tree suggests that the paralogous function of TraB proteins derives from a gene duplication event of ftsK or a related gene only in actinomycetes.

The C-terminal winged-helix-turn-helix domain (wHTH) of selected DNA-translocator proteins. (A) Alignment of the wHTH motifs of selected TraB and FtsK proteins. Helices α1 and α2 are given in green letters, helix α3 in red letters. Blue letters indicate strand β1 and strand β2, which form the wings. A consensus of the predicted secondary structure is shown above the sequences (h, helix; s, strand). Letters in lower case denote residues of unsure equivalence. Sequences are ordered by similarity according to which wHTH domains of TraB and FtsK constitute the major groups. The order generally matches the pattern observed in the phylogenetic tree (Figure 1). The alignment shows pronounced differences in helix α3, which has a crucial role in specific DNA binding. This may reflect the affinity of a particular wHTH domain to the corresponding 8 bp repeats found in its cognate plasmid (Supplementary Figure S1). (B) Model of the TraB_pSVH1 wHTH domain generated by Modeller (Sali and Blundell, 1993) using the γ-domain of P. aeruginosa FtsK as a template. Helices α1 and α2 are given in green, while helix α3, proposed to determine sequence-specific DNA recognition is drawn in red colour.

Gel retardation assays demonstrating specific interaction of TraB with TraB-recognition sequences (TRS). (A) Gel shift with 58 nt oligonucleotides comprising the minimal TraB-binding site. clt⁺ oligonucleotide, containing two perfect and one imperfect TRS repeats or the complementary strand (clt⁻, lacking any TRS repeat) were Cy5 labelled and analysed for TraB (−: 0 μg, +: 1.5 μg, ++: 3 μg) binding. Visualization of the bands by fluorescence laser scanning revealed that TraB shifted only the clt⁺ strand (arrow) containing the TRS repeats. (B) Gel shift assay with synthetic double-stranded oligonucleotides. In all, 24 bp oligonucleotides TRS₀ and TRS₂ (containing two copies of the GACCCGGA sequence) were incubated with TraB_s (left) and TraB (right). TraB and TraB_s bind (arrow) only to TRS₂, while TRS₀ was not bound. The multiple retarded bands observed upon binding of full-length TraB probably reflect different oligomerization states of TraB. −: no protein added; +: 8 μg TraB_s or 0.5 μg TraB.

TraB_s also binds to clt-like chromosomal (clc) sequences. (A) Distribution of the clc_pSVH1 sequences on the linear chromosomal map of S. coelicolor. The clc sequences that were confirmed for TraB binding by gel retardation assays are highlighted in blue. (B) Gel retardation assay with subcloned clc sequences. S. coelicolor clc sequences present within SCO4481 and in the intergenic region between SCO4185 and SCO4186 were amplified, subcloned into a cloning vector and digested to yield three DNA fragments. The intermediate sized fragment contained the cloned clc sequence. Gel retardation assays with different amounts of TraB_s (0, 4, 8 μg) demonstrated that only the clc containing DNA fragment is retarded (arrows), while the other fragments that serve as negative controls are not shifted.

Helix α3 of the TraB wHTH motif determines sequence-specific DNA recognition. (A) Gel retardation experiment with chimeric TraB-IV_pIJ101 protein. clt sequences of plasmids pSVH1 and pIJ101 were subcloned into a cloning vector, digested to yield three DNA fragments and used in gel retardation assays with chimeric TraB proteins. The clt containing DNA fragment is indicated with an arrow. TraB-IV_pIJ101, containing the N-terminal 702 aa of TraB_pSVH1 and the 70 aa C-terminal wHTH domain of TraB_pIJ101 has lost its ability to interact with clt_pSVH1 (open arrow) but binds clt_pIJ101 (black arrow). (B) Exchange of helix α3 of TraB_pSVH1 against α3 of TraB_pIJ101 is sufficient to switch clt recognition. TraB_H3pIJ101 binds to the clt of plasmid pIJ101, while clt_pSVH1 is not recognized. −no protein; +: 0.1–0.3 μg protein.

Electron microscopy of TraB_pSVH1 shows hexameric ring-shaped structures with a central pore. (A) Elution profile of TraB_s on a Superose 6 column demonstrating oligomerization of TraB_s. Black lines indicate molecular weight standards. Under the assayed conditions (10 mM MgCl₂, 2 μM ATPγS), TraB_s mainly eluted as a high molecular weight oligomer (black arrow). Free ATPγS eluted at 21.3 ml (dashed arrow). (B, C) Negative stain electron micrographs of TraB. Manually selected TraB_s-hexamers (B) and full-length TraB-hexamers (C) greatly resemble each other in size and six-fold symmetry. (D, E) 2D averaging of electron microscopic images. Representative classes of averaged TraB_s-hexamers (D (n=33), E (n=90)) show a diameter of ∼12 nm and a pore size of about 3.1 nm (D) −3.5 nm (E). (F) Homology modelling of the TraB DNA-translocase domain. The TraB_pSVH1-translocase domain was modelled based on the P. aeruginosa FtsK DNA-translocase domain. The modelled TraB structure shows a six-fold symmetry with a diameter of ∼11.8 and an ∼3.1 nm pore, sufficient for translocation of a double-stranded DNA molecule.

TraB_pSVH1 forms pores in lipid bilayers in vitro. (A) Single channel recordings of TraB_pSVH1 pores. Representative traces (conductance level c1) of TraB single channel recordings at different voltages are given. The distribution of c1 conductance is shown in Supplementary Figure S10. (B) Current–voltage relationship of TraB channels. Symmetric but mostly non-linear behaviour of the measured current is observed in 200 mM NaCl (sigmoidal fit in green).