, 2011a). For the membrane passage, one has to postulate a pore structure for TraB. This is in contrast to Escherichia coli FtsK that probably
translocates the chromosome before closure of the septum and therefore does not rely on a pore-forming ability (Dubarry & Barre, 2010). The ability of TraB to form pore structures was analysed by single channel recordings using planar lipid bilayers. These studies demonstrated that TraB spontaneously inserted into the membrane at various voltages GDC-0973 concentration and formed pores with an opening time of about 47–81 ms (positive voltage applied) and 105–200 ms, respectively, when a negative voltage was applied (Vogelmann et al., 2011a). Because only TraB and the non-coding clt region are required for plasmid transfer, it was studied whether clt represents the binding site of TraB. This hypothesis turned out to be correct, because gel retardation assays showed a specific interaction of TraB with a plasmid region at the 3′ end of traB, which represents the clt region of pSVH1 (Reuther et al., 2006a). The pSVH1 clt region contained nine imperfectly conserved copies of the GACCCGGA motif. Subcloning experiments revealed a minimal fragment containing only four copies, which still supported TraB binding. A more careful analysis detected even binding of TraB to a synthetic find more 20-bp fragment containing only two copies (Vogelmann et al., 2011a). This study confirmed
the GACCCGGA motif as the TraB Recognition Sequence (TRS). Although two copies of TRS were sufficient for TraB binding in vitro, binding of TraB to a larger clt fragment containing additional TRS copies was more efficient and required lower protein concentrations for retardation (Reuther et al., 2006a) indicating that in vivo only the complete
clt might be effective. Analysing other Streptomyces plasmids for the presence of 8-bp repeats also detected specific 8-bp repeats in the (predicted) clt regions (Franco et al., 2003; Vogelmann et al., HSP90 2011a). With the notable exceptions of pIJ101 (Kieser et al., 1982) and the highly related plasmid p1424 (G. Muth, unpublished), the clt localizes in all Streptomyces plasmids to the 3′ end of traB, forming a transfer module of only 2.5 kb in size consisting of the DNA-translocase-encoding traB gene and its binding site clt next to it. To characterize the TraB–clt interaction in more detail, TraB was incubated with covalently closed circular (ccc) DNA of the pSVH1 derivative pEB211 in the presence of ATP and divalent cations. An aliquot was directly loaded to the gel, while others were heat treated or phenol extracted to denature TraB previous to gel loading. These analyses revealed ccc-DNA that had not changed its conformation demonstrating that TraB binds noncovalently to plasmid DNA and that the plasmid molecule was not processed by TraB binding (Reuther et al., 2006a).