Fig. 7: Activity of H2-T6SS with chimeric TssA proteins.
To assess complementation of the tssA2 deletion, the pBBR1MCS4 vector was introduced either as EV, encoding tssA2 (ptssA2) or chimeric tssAs. a Western blot of a H2-T6SS secretion assay in the PAO1ΔrsmAΔtssA2 background to assess the levels of secretion marker Hcp2 in the supernatant sample. RpoB was used as a bacterial lysis control. Representative of 3 repeats. b H2-T6SS competition assay to assess the T6SS-dependent killing of a GFP-encoding E. coli prey by P. aeruginosa attacker strains in the PAO1ΔrsmAΔtssA2 background. (n = 4 independent experiments). c Quantification of the percentage of cells with TssB2-mScarlet sheath structures seen by fluorescence microscopy in the PAO1ΔrsmAΔtssA2 tssB2-mScarlet-I background. Sheath structures were formed in 0.64% (n = 9920 cells examined) of cells in the absence of tssA2, upon introduction of tssA2 on a plasmid this rose to 6.30% (n = 5412 cells examined). Upon introduction of chimeric tssA2Nt1tssA1CTD or tssA2Nt1tssA3CTD, 4.42% (n = 4687 cells examined) and 7.67% (n = 5387 cells examined) of cells formed sheath structures respectively. Data was taken from three fields of view for two experiments. Statistical testing was conducted by one-way ANOVA with Dunnett’s multiple comparisons test, each strain was compared to the parental EV strain. Values are presented as means, error bars represent standard deviation. Source data are provided as a source data file.
