Fig. 6: Activity of H1- and H3-T6SSs with chimeric TssA proteins.
To assess complementation of the tssA deletions, the pBBR1MCS4 vector was introduced either as EV, or encoding tssA1 (ptssA1), tssA3 (ptssA3) or chimeric tssAs. a Western blot of a H1-T6SS secretion assay in the PAO1ΔrsmAΔrsmNΔtssA1 background, to assess the levels of 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 PAO1ΔrsmAΔrsmNΔtssA1 background. (n = 3 independent experiments). c Quantification of the percentage of cells with TssB1-mScarlet sheath structures by fluorescence microscopy in the PAO1ΔretSΔtssA1 tssB1-mScarlet-I background. The percentage of cells with sheath structures was 0.37% (n = 10017 cells examined) with tssA1 deletion, upon introduction of tssA1 or tssA1Nt1tssA3CTD on a plasmid this rose to 4.95% (n = 8117 cells examined) and 5.26% (n = 1055 cells examined 3) respectively. Data was taken from two fields of view for three experiments. d Quantification of the percentage of cells with TssB3-mScarlet sheath structures by fluorescence microscopy in the PAO1ΔrsmAΔtssA3 tssB3-mScarlet-I background. Sheath structures were formed in 0.04% (n = 16822 cells examined) of cells in the absence of tssA3, this rose to 27.23% (n = 16253 cells examined) with expression of tssA3 on a plasmid and 28.07% (n = 10456 cells examined) with tssA3Nt1tssA1CTD on a plasmid. With expression of tssA3Nt1tssA2CTD or tssA3Nt1tssA2Nt2+CTD from a plasmid, 0.08% (n = 8751 cells examined) and 0.41% (n = 9472 cells examined) of cells formed sheath structures respectively. Data was taken from two fields of view for three experiments. e Western blot of a H1-T6SS secretion assay with cross-species chimeras, with domains switched between P. aeruginosa TssA1 (TssA1PA) and P. putida TssA1 (TssA1PP), in the PAO1ΔrsmAΔrsmNΔtssA1 background, to assess the levels of secretion marker Hcp1 in the supernatant sample. RpoB was used as a bacterial lysis control. Representative of 3 repeats. f H1-T6SS competition assay to assess the T6SS-dependent killing of a GFP-encoding E. coli prey by P. aeruginosa attacker strains expressing P. putida wildtype and cross-species chimeric TssA proteins in the PAO1ΔrsmAΔrsmNΔtssA1 background. (n = 3 independent experiments). Statistical testing was conducted by one-way ANOVA with Dunnett’s multiple comparisons test, each strain was compared to the parental tssA1 EV strain. Values are presented as means, error bars represent standard deviation. Source data are provided as a source data file.
