Skip to main content
. Author manuscript; available in PMC: 2019 Apr 8.
Published in final edited form as: Environ Microbiol. 2018 Jan 19;20(2):903–919. doi: 10.1111/1462-2920.14039

Fig. 5. DotU2 phosphorylation at Thr4 and Thr28 enable its binding to Fha2 and T6SS2 activity.

Fig. 5

(a) Detection of the phosphorylation of DotU2-Flag or the related phosphorylation site mutants on a Phos-tag gel. Immunoblots were used to analyze the same lysate volumes with a Flag specific antibody. SDS-PAGE without Phos-tag is shown as a control.

(b) Western blot analyses of Hcp2 expression and secretion in EPGS, ΔdotU2, Δfha2 and complement strains. The ΔdotU2 mutant expressing DotU2 or the DotU2 T4A, T28A, or T4A T28A substitution variants, and the Δfha2 mutant expressing Fha2, as well as a strain containing a chromosomal fha2 R36A S52A substitution mutant are also included. RNAP was used as the loading control for the cell pellets.

(c) Pull-down analyses of DotU2- Fha2 interaction using purified Fha2-His6 protein and p-DotU2-Flag from cell lysates. The Fha2-His6 protein was first mixed with the His-beads and then cell lysates containing p-DotU2-Flag (or the mutant DotU2-Flag) proteins were added. After elution, the presence of DotU2-Flag (or the mutant DotU2-Flag proteins) and Fha2-His6 were detected with specific antibodies.

(d) CFU counts of E. coli before (t = 0) and after 4 h (t = 4) co-culture with the indicated V. alginolyticus predator strains.