Killing with proficiency: Integrated post-translational regulation of an offensive Type VI secretion system
Fig 1
Phosphorylation of Fha promotes formation of Fha and TssB foci in S. marcescens Db10.
(A) T6SS-dependent secretion of Hcp by wild type (WT) S. marcescens Db10 or mutants lacking the kinase PpkA (ΔppkA) or the phosphatase PppA (ΔpppA), and by derivatives expressing fusions of GFP to the C-terminus of TssB (TssB-GFP) and mCherry to the C-terminus of Fha (Fha-mCherry), in an otherwise wild type, ΔppkA or ΔpppA mutant background. The T6SS inactive mutant ΔtssE is a negative control and cellular and secreted fractions were subjected to immunoblotting using anti-Hcp antisera. (B) T6SS-dependent anti-bacterial activity of fluorescent reporter strains against a P. fluorescens target. Number of recovered target cells following 4 h co-culture with wild type or mutant strains of S. marcescens Db10 as indicated. Individual data points are overlaid with mean +/- SEM (n = 4). (C) Representative images of cells expressing TssB-GFP and Fha-mCherry fusions, in a wild type, ΔppkA or ΔpppA mutant background. From left to right, panels show individual fluorescence channels (TssB-GFP, Fha-mCherry), an overlay of the fluorescent channels (Merge; GFP signal false-coloured green and mCherry red), and the corresponding DIC image. Inset panels show a zoomed-in view of the region represented by the white box; for the ΔppkA mutant, the contrast has been increased in the inset panels to reveal non-focal TssB-GFP fluorescence. Scale bars, 5 μm. (D) Fluorescent signal intensity measurement of the segmented TssB-GFP signal in wild type, ΔppkA, ΔpppA, ΔtagF and ΔppkAΔtagF mutants (au, arbitrary fluorescence units). The boundaries of the boxes represent the 25 and 75 percentiles and the horizontal lines are the median values of the analysed population. The whisker bars represent standard error (10 and 90 percentiles). Regions of interest from at least 32 fields of view were analysed in each case. Relative frequency of TssB-GFP foci (expressed per cell at a single timepoint) were estimated by combining automated focus detection with manual cell counting in a representative 15 of these images.