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. Author manuscript; available in PMC: 2015 Apr 30.
Published in final edited form as: Nat Commun. 2014 Oct 31;5:5322. doi: 10.1038/ncomms6322

Figure 4.

Figure 4

Interaction of Osa with VirB/D4 T4SS machine components in vivo and in vitro. For in vivo study: DDAO-solubilized membrane protein material immunoprecipitated with (IP with) antibodies to VirB4 ATPase (a) or VirB11 ATPase (b) proteins and FLAG-tag (a and b) listed on the left were analyzed for (detected for) proteins listed on the right. Total DDAO-solubilized membrane preparation (MP, right) from the WT (A348) or WT expressing FLAG-Osa show position of VirB4/VirB11 and FLAG-Osa respectively. Absence or presence of FLAG-Osa expressing plasmid (pKA197) in all strains is indicated by − and + respectively. Molecular weight markers (M; lane 1) and their sizes in kiloDaltons (kDa) are shown in left. (a) Osa interacts with VirB4 independently of T4S machine and VirD4: Co-immunoprecipitation of VirB4 ATPase and FLAG-Osa in the absence of VirD4 coupling protein and T4S machine. Strains: WT with (lane 3) and without (lane 2) expressing FLAG-Osa (pKA197); ΔB4 (PC1004 (virB4 knockout (KO)) with (lane 5) and without (lane 4) expressing FLAG-Osa (pKA197); ΔD4 (Mx355 (virD4 KO) expressing (lane 6) FLAG-Osa (pKA197); A136 (A, G + B4) (KA2002 (WT deleted of pTiA6 plasmid and supplemented with VirA sensory kinase and VirG response regulator) expressing VirB4 (pKA93) with (lane 7) and without (lane 8) producing FLAG-Osa (pKA197). (b) Osa interacts with VirB11 independently of T4S machine and VirD4: Co-immunoprecipitation of VirB11 ATPase and FLAG-Osa in the absence of T4S machine. Strains: WT with (lane 3) and without (lane 2) expressing FLAG-Osa (pKA197); A136 (A, G + B11) (KA2002 (WT deleted of pTiA6 plasmid and supplemented with VirA sensory kinase and VirG response regulator) expressing VirB11 (pSR40) with (lane 5) and without (lane 4) producing FLAG-Osa (pKA197). For in vitro study: (c) Far-Western of purified MBP fused VirB/D4 T4SS and relaxosome components with purified Osa, Ice1056Fin and PifC, probed using HRP conjugated anti-MBP antibody. Osa and two other FIN factors were used as baits to trap the interacting partner from the T4SS machine and relaxosome.