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. Author manuscript; available in PMC: 2021 Aug 31.
Published in final edited form as: Nat Microbiol. 2021 Aug 19;6(9):1199–1210. doi: 10.1038/s41564-021-00950-8

Figure 1. Tse8-Tsi8 is a H1-T6SS toxin-immunity pair.

Figure 1

a-b, Expression of Tse8 (either HA tagged or untagged) in PAKΔretSΔtsei8 is toxic when expressed in trans from pMMB67HE ((-) no induction; (+) with induction) (a) or when delivered by the H1-T6SS into a recipient strain lacking tsi8 (b). c-d, Tsi8 can rescue Tse8 toxicity in competition assays with donors PAKΔretS or PAKΔretSΔH1 and recipient PAKΔretSΔtsei8 expressing either pJN105 or pJN:tsi8 (c) and in growth assays with PAKΔretSΔtsei8 expressing pMMB:tse8 or pJN:tsi8 (d). e, Bacterial-Two-Hybrid (BTH) assays were used to quantify the level of interaction between Tse8 and Tsi8 with β-galactosidase activity assays performed on the cell lysates of each interaction pair. f, Tse8-HA-Strep interacts directly and specifically with Tsi8-His. Proteins were added to His-Tag Dynabeads as indicated. Lane 1: Tsi8-His (as bait) interacts with Tse8-HA-Strep. Lane 2: Tse8-HA-Strep alone does not interact with the Dynabeads. Lane 3: Tse8-HA-Strep does not interact with a different His-tagged bait protein, CcmE. Molecular weight markers positions are indicated on the left in kDa. Black vertical lines indicate where a lane was removed. Statistical analyses: (a) mean OD600 ± SEM is plotted over time from three independent replicates; (b) Mean CFUs/mL ± SEM of recipient cells in competition/alone are represented from three independent replicates performed in triplicate (n=3). Two-tailed student’s t-test, *** P<0.001; * P<0.05; ns between PAKΔretS and PAKΔretSΔH2ΔH3 (P=0.436); (c) Mean CFUs/mL ± SEM of recipient cells in competition/alone are represented from three independent replicates performed in triplicate (n=3). Two-tailed student’s t-test, * P<0.05 for each sample to PAKΔretS and ns between PAKΔretSΔH1 [pJN105] and PAKΔretS [pJN:tsi8] (P=0.598); (d) Mean OD600 ± SEM is plotted over time from three independent replicates; (e) Mean ± SEM of three biological replicates performed in triplicate (n=3). One-way Anova with Tukey’s multiple comparison post-test, * P<0.05 compared to the Miller units for T18c + T25 for Zip + Zip, or compared to Tsi8 + T25 and T18c + Tse8 for Tsi8 + Tse8; (f) Representative blot from one independent replicate (n=1).