Fig. 2

Interaction of TssA1^B with other T6SS subunits. a Two-hybrid analysis (maltose phenotypes). Hybrid proteins are represented by a green or yellow coloured motif representing the CyaA fragment (T25 and T18, respectively) linked to a white rectangle labelled according to the fused T6SS subunit, as shown in the key at the bottom of (b). T6SS subunits are indicated by a single letter corresponding to the suffix used in the Tss nomenclature (i.e. A1 corresponds to TssA1^B, B corresponds to TssB etc) except for H (Hcp), V (VgrG) and V_C (VgrG core region). M_N and M_C represent the N-terminal cytoplasmic and C-terminal periplasmic regions of TssM, respectively. The efficiency of complementation of representative combinations (phenotypes shown in red font) were determined by β-galactosidase assay. b Two-hybrid analysis (β-galactosidase activity). Data is representative of three independent experiments (n = 3) performed in duplicate and values correspond to the mean ± standard deviation. Nomenclature as in (a). Z represents the Zip control. Values are presented in Supplementary Data 1. c Co-immunoprecipitation analysis. FLAG-tagged Bc TssA1^B and potential interacting T6SS subunits (TssX) tagged with the VSV-g or HA epitope tags, or with MBP, were co-expressed in E. coli. FLAG.TssA1^B was immunoprecipitated from cell lysates and recovered prey proteins (IP) were detected with the appropriate epitope antibody by western blotting. MBP was included as a control and was detected with MBP pAb. Proteins present in the cell lysate (Tot) and the unbound material (UB) were also analysed. Blots were also probed with FLAG mAb (an example of such a blot following co-expression of FLAG.TssA1^B and VSVg.TssM_N is shown in the bottom panel). Uncropped images of the blots are shown in Supplementary Fig. 4. d Bc TssA1^B interaction network. Interactions between Bc TssA1^B and other T6SS subunits are indicated by red arrows. Previously reported interactions are indicated by grey connectors. Note that VgrG and TssK have been shown to interact with the TssF-TssG complex²² but for simplicity are linked to TssF in this figure