Fig. 8: Exchange of TssA1 and TssA3 hairpin and loop regions disrupts TssA function.
a Alignment of TssA1 and TssA3 Nt1 domain models. TssA Nt1 and TssA3 Nt1 homology molecular models were structurally aligned to minimise the root mean square deviation (RMSD) between their corresponding Cα atoms. Superimposed structures were coloured according to the RMSD of Cα atoms. Blue indicates regions where the Cα atoms are within 1 Å of each other in the two models. Red highlights regions where the Cα atoms are 5 Å or more apart in the corresponding proteins. Yellow highlights regions where the two sequences differ, showing no correspondence between the residues due to the presence of an inserted or deleted region. b BTH interactions of TssA1 with TssA3 hairpin or loop regions with TssA1 and H1-T6SS sheath components. TssA1-TssA3hairpin and loop was not stable and therefore discarded. c BTH interactions of TssA3 with TssA1 hairpin and/or loop regions with TssA3 and H3-T6SS sheath components. d Quantification of the percentage of cells with TssB3-mScarlet sheath structures by fluorescence microscopy in the PAO1ΔrsmAΔtssA3 tssB3-mScarlet-I background. To assess complementation of a tssA deletion, the pBBR1MCS4 vector was introduced either as EV, encoding tssA1 (ptssA1), tssA3 (ptssA3) or tssAs with exchanged hairpin and/or loop sequences. 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. Upon introduction of tssA3-tssA1hairpin, tssA3- tssA1loop and tssA3-tssA1hairpin and loop sheath structures were detectable in 2.31% (n = 10760 cells examined), 1.60% (n = 13968 cells examined) and 0.08% (n = 11530 cells examined) of cells respectively. Data was taken from three fields of view for two experiments. e 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. f Quantification of the percentage of cells with TssB1-mScarlet sheath structures by fluorescence microscopy in the PAO1ΔretSΔtssA1 tssB1-mScarlet-I background. Sheath structures were formed in 0.37% (n = 10017 cells examined) of cells with tssA1 deletion, upon introduction of tssA1 on a plasmid this rose to 4.95% (n = 8117). Upon introduction of tssA1-tssA3hairpin 5.02% (n = 10683 cells examined) of cells formed sheath structures, this was 1.87% (n = 9471 cells examined) for introduction of tssA1-tssA3loop. Data was taken from three fields of view for two experiments. Values are presented as means, error bars represent standard deviation. Source data are provided as a source data file.
