3a). The TraJ–DNA complex could not be immunoprecipitated with anti-TraK antiserum used as a negative control (Fig. 3a). ChIP assays were also performed using wild-type (pILJ11) and mutant constructs pJ-M1T-G166R and pJ-M1T-G166A, as well as a

C-terminal deletion, pJ-M1T-C221*, in the presence of Flac traJ90 (Fig. 3b). The ability of pILJ11 and its mutant derivatives to complement Flac traJ90 was similar to that of pBADTraJ and its corresponding mutants. None of the pILJ11 mutant derivatives affected the production of TraJ as monitored by immunoblot (data not shown). Whereas the C-terminal deletion did not affect in vivo DNA binding, the G166A or G166R mutations reduced or eliminated TraJ binding, respectively. Thus, TraJ was Selumetinib mouse considered to be a DNA-binding protein with the GSK3 inhibitor C-terminal region (aa 154–180) containing an HTH DNA-binding motif. Because HTH DNA proteins usually bind to inverted repeats as dimers (Aravind et al., 2005) and because the predicted binding site for F TraJ is an inverted repeat, based on studies on R100 TraJ (Taki et al., 1998), we undertook cross-linking experiments to determine whether F TraJ was a dimer. Duplicate samples of MC4100/Flac traJ90/pBADTraJ, grown in LB with 0.1% arabinose to the late exponential phase (OD600 nm∼1.0),

were treated with or without the cross-linker DSS as described in Materials and methods. Immunoblot analysis showed a band corresponding to TraJ (26.7 kDa), which typically migrates at approximately 25 kDa (Fig. 4). Cross-linked

samples revealed a second band at 50 kDa that was consistent with a TraJ dimer. As the concentration of cross-linker was increased, bands at positions above 50 kDa were observed, suggesting that Nitroxoline TraJ could cross-link to form higher order complexes of unknown composition (data not shown). Dimerization was also observed in the analogous cross-linking experiment performed with purified TraJ protein (data not shown). In order to determine whether the C-terminal region is important for oligomerization, MC4100/Flac traJ90, carrying either pB24JΔ6 or pB24JΔ30, were cross-linked with DSS in the same manner (Fig. 4). The resulting patterns of bands were similar to those for wild-type TraJ monomer and dimmer, except that the bands migrated slightly faster, as expected. Because desilencing of H-NS-repressed promoters can occur via heterodimer formation (Fang & Rimsky, 2008), we assayed whether TraJ was able to dimerize with H-NS (15.6 kDa) to yield a heterodimer of ∼42 kDa. MC4100 or PD32 (hns) containing Flac or Flac traJ90 with pBAD33 (Cmr), pIJL14 (pBAD33TraJ) or pIJL14Δ6 were cross-linked with DSS and the resultant bands were identified by immunoblot with anti-H-NS and anti-TraJ antisera.