Fig. 2: Functional specificity of TssA proteins for their cognate T6SS.

To assess complementation of a tssA deletion, the pBBR1MCS4 vector was introduced either as empty vector (EV) or encoding tssA1, tssA2 or tssA3 (ptssA1/2/3). a Western blot of a H1-T6SS secretion assay in the PAO1ΔrsmAΔrsmN background to assess the levels of the secretion marker Hcp1 in the supernatant sample. RpoB was used as a bacterial lysis control. Representative of 3 repeats. b H1-T6SS competition assay to assess the T6SS-dependent killing of a GFP-encoding E. coli prey by P. aeruginosa attacker strains in the PAO1ΔrsmAΔrsmN background. (n = 3 independent experiments). c Western blot of a H2-T6SS secretion assay in the PAO1ΔrsmA background to assess the levels of the secretion marker Hcp2 in the supernatant sample. RpoB was used as a bacterial lysis control. Representative of 3 repeats. d H2-T6SS competition assay to assess the T6SS-dependent killing of a GFP-encoding E. coli prey by P. aeruginosa attacker strains in the PAO1ΔrsmA background. (n = 4 independent experiments). e Quantification of the percentage of cells with TssB3-mScarlet sheath structures by fluorescence microscopy in the PAO1ΔrsmA tssB3-mScarlet-I background. In the parental strain, sheath structures were formed in 38.9% of cells (n = 10582 cells examined), upon deletion of tssA3 with the EV present this fell to 0.036% (n = 16822 cells examined), upon introduction of tssA1 0.14% of cells formed sheath structures (n = 11205 cells examined) and with tssA2 this was 0.082% (n = 8500 cells examined). Upon introduction of tssA3 27.2% of cells formed sheath structures (n = 16253 cells examined). Data were taken from two fields of view for three experiments. Statistical testing was conducted by one-way ANOVA with Dunnett’s multiple comparisons test, each strain was compared to parental EV strains. Values are presented as means, error bars represent standard deviation. Source data are provided as a source data file.