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. 2021 Jul 13;49(14):8384–8395. doi: 10.1093/nar/gkab608

Figure 1.

Figure 1.

The ART-HYD1 toxin domain of Rhs is an antibacterial toxin delivered by the type VI secretion system. (A) Schematic representation of the Photorhabdus laumondii TT01 T6SS cluster. Genes encoding core and accessory T6SS components are depicted as black arrows. Genes encoding the VgrG spike protein, the putative EagR chaperone, the Rhs effector protein and its putative immunity protein are shown in yellow, orange, red, and green, respectively. The repertoire of T6SS gene clusters and T6SS islands encoded on the P. laumondii TT01 genome is shown in Supplementary Figure S1. (B) Intra-species competition assay between P. laumondii with mutants deleted for effector/immunity (Δeff-imm) or promoter region (ΔP-T6SS), or inserted in an unrelated control locus (contr.) (recipients) and the wild-type parental strain (donor). The deleted regions are depicted under the scheme in (A). Recipient and donor competitors were mixed in a 1:1 ratio and the competitive index (surviving recipient mutant cells relative to total cell number (recipient + donor)) is calculated after 48 hours of co-incubation on LB agar plates. *** indicates statistically significant differences between Control and Δeff-imm (Tukey's range test, P = 0.0012) or ΔPT6SS (P = 0.00182). Difference between Δeff-imm and ΔPT6SS is not statistically significant (P = 0.597). (C) Toxicity assay in the heterologous host E. coli. E. coli cells producing the Tre23 toxin (ART-HYD1 domain of Rhs encoded by plu0353 as shown in (A)), or its variants in the H8 and Y33 putative catalytic residues, from pBAD33 vectors were serially diluted and spotted on LB-agar plates supplemented with glucose. (D) Tri23 protects cells against the action of Tre23. E. coli cells producing the Tre23 toxin from the low-copy number vector pNDM220 and Tri23 from the pBAD33 vector were serially diluted and spotted on LB agar supplemented with 0.05 mM IPTG and 0.5% arabinose to induce expression from pNDM220 and pBAD33, respectively. (E) Pull-down assay. Total cell extracts from E. coli BL21(DE3) cells producing His6-tagged VgrG (HVgrG), strep-tagged EagR (STEagR) and FLAG-tagged Rhs (RhsFL) (T) were subjected to purification on streptactin-agarose beads. Strep-tagged and co-precipitated proteins were eluted with desthiobiotin (IP). The two fractions were analyzed by SDS-PAGE and the co-purified proteins were stained by Coomassie blue (left panel) or immunodetected using anti-His, anti-Strep and anti-FLAG antibodies (right panels).