Figure 4: Interaction of Osa with VirB/D4 T4SS machine components in vivo and in vitro. | Nature Communications

Figure 4: Interaction of Osa with VirB/D4 T4SS machine components in vivo and in vitro.

From: Multiple enzymatic activities of ParB/Srx superfamily mediate sexual conflict among conjugative plasmids

Figure 4

For in vivo study: N,N-Dimethyldodecylamine N-oxide (DDAO)-solubilized membrane protein material immunoprecipitated with (IP with) antibodies to VirB4 ATPase (a) or VirB11 ATPase (b) proteins and FLAG-tag (a,b) listed on the left were analysed 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 the 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 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 blot of purified MBP fused VirB/D4 T4SS and relaxosome components with purified Osa and ICE1056Fin, probed using horseradish peroxidase-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.

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