Fig 3. Phosphorylation of Fha and formation of Fha foci is no longer required for T6SS activity in the absence of the post-translational regulator TagF.

(A) Schematic depiction of the T6SS gene cluster of S. marcescens Db10 showing the genes encoding components of the T6SS post-translational regulatory system in this organism (in red). Core T6SS components are shown in blue, with letters indicating TssA-M, and effector/immunity pairs are shown in purple/pink. (B) T6SS-dependent Hcp secretion as measured by immunoblot detection of Hcp in cellular and secreted fractions of wild type (WT) or mutant (ΔtssE, ΔppkA, ΔpppA, Δfha, ΔtagF, ΔppkAΔtagF, ΔpppAΔtagF and ΔfhaΔtagF) strains of S. marcescens Db10. (C) T6SS-dependent anti-bacterial activity as determined by recovery of target organism P. fluorescens following co-culture with wild type or mutant strains of S. marcescens Db10. Individual data points are overlaid with mean +/- SEM (n = 4). (D) Representative images of wild type and mutant strains of S. marcescens Db10 expressing TssB-GFP and Fha-mCherry fluorescent fusions. Panels show individual fluorescence channels (TssB-GFP, Fha-mCherry) and an overlay of the fluorescence channels and the DIC channel (Merge; GFP signal false-coloured green and mCherry red). Additionally, TssB-GFP was monitored in the ΔfhaΔtagF background. The mCherry channel image in this case illustrates the low level but frequently observed background signal unrelated to mCherry expression. Scale bar, 5 μm.