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

<p>(A) Schematic depiction of the T6SS gene cluster of <i>S</i>. <i>marcescens</i> 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 (Δ<i>tssE</i>, Δ<i>ppkA</i>, Δ<i>pppA</i>, Δ<i>fha</i>, Δ<i>tagF</i>, Δ<i>ppkA</i>Δ<i>tagF</i>, Δ<i>pppA</i>Δ<i>tagF</i> and Δ<i>fha</i>Δ<i>tagF</i>) strains of <i>S</i>. <i>marcescens</i> Db10. (C) T6SS-dependent anti-bacterial activity as determined by recovery of target organism <i>P</i>. <i>fluorescens</i> following co-culture with wild type or mutant strains of <i>S</i>. <i>marcescens</i> Db10. Individual data points are overlaid with mean +/- SEM (n = 4). (D) Representative images of wild type and mutant strains of <i>S</i>. <i>marcescens</i> 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 Δ<i>fha</i>Δ<i>tagF</i> 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.