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. 2020 Oct 23;10:18171. doi: 10.1038/s41598-020-72063-x

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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The H. pylori tfs4 ICE encodes a functional Type II toxin–antitoxin system. (a) Conserved position of the putative TA module within the tfs4 ICE gene cluster, comprising overlapping genes encoding toxin (denoted ‘TfiT’) and antitoxin (denoted ‘TfiA’). (b) Culture samples were taken at intervals post-induction for determination of viable counts. Expression of TfiT and TfiA individually was induced from pBAD18 constructs, whereas the TA module, comprising His-tagged TfiA and S-tagged TfiT was expressed from pACYCDuet-1 [TfiA, TfiT]. (-ara); no induction. (c) Relative expression of His-tagged TfiA or S-tagged TfiT (arrowheads) from the pACYCDuet-1 TA module construct in either uninduced (lanes 1) or induced (lanes 2) cultures. Western immunoblots were probed with either anti-His or anti-S-tag antibodies as appropriate. An unrelated ~ 40 kDa protein is detected in lysates by the His6 antibody. (d) Parent pUC18 or constructs comprising the entire TA module without (F0) or with either 213 bp (F1) or 160 bp (F2) of upstream sequence were passaged sequentially in the absence of antibiotic. Dilutions were made from daily cultures prior to passage for comparison of viable counts (CFU ml⁻¹) on both selective and non-selective plates from which the percentage of plasmid maintained was calculated. (e) In parallel column-pulldowns, clarified lysate from induced (lanes 5, 7) or uninduced (lane 6) cultures of pBAD-TfiT were passed over TALON resin prior to addition of lysate from a culture of either induced (lanes 6, 7) or uninduced (lane 5) S-tagged-TfiA. Elution fractions were assessed for co-purified protein in Western immunblots probed with either anti-His-tag (left panel) or anti-S-tag antibody (right panel). Samples from uninduced/induced cultures for both TfiT (lanes 1, 2 respectively) and TfiA (lanes 3, 4 respectively) were included to monitor expression. (f) Far Western. Western blots containing resolved lysate from cultures of SoluBL21 (lane 1), SoluBL21/pACYCDuet (lane 2), and either uninduced (lane 3) or induced (lane 4) SoluBL21/pACYCDuet-TfiA (S-tagged TfiA) were incubated with purified His-tagged TfiT then either anti-His-tag (left panel) or anti-S-tag antibody (right panel). Arrowheads indicate TA proteins. Error bars in (b,d) display standard deviations from three replicates. ara arabinose, Indⁿ induction.