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. 2020 Feb 28;11:277. doi: 10.3389/fmicb.2020.00277

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Copyright © 2020 Takada, Roghanian, Murina, Dzhygyr, Murayama, Akanuma, Atkinson, Garcia-Pino and Hauryliuk.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Deletion of the regulatory RRM domain leads to B. subtilis Rel toxicity due to ribosome-dependent (p)ppGpp overproduction. (A) Full-length (VHB155 and VHB183) as well as C-terminally truncated Rel variants [synthesis-competent ΔRRM (VHB159 and VHB184), ΔRRMΔZFD (VHB160 and VHB185) and Rel^NTD (VHB161 and VHB186), and the corresponding synthesis-inactive D264G mutants VHB162-164; VHB187-190] were expressed in either ppGpp⁰ (upper row; test for toxicity mediated by (p)ppGpp accumulation) or Δrel (lower row; test for HD functionality) B. subtilis growing on solid LB medium. Up-pointing arrows (↑) indicate ectopic expression. (B,C) Expression of Rel^ΔRRM causes a growth defect in liquid culture. Either wild-type rel (VHB183) (B) or rel^ΔRRM mutant (VHB184) (C) were expressed in ppGpp⁰ background grown in liquid LB medium at 37°C. Protein expression was induced by IPTG added to final concentration of 1 mM to exponentially growing bacterial cultures at OD₆₀₀ 0.2. Protein expression was monitored by Western blotting using anti-Rel antibodies (see also Supplementary Figure S2H). (D) The toxicity of mutant versions of Rel^ΔRRM tested in ppGpp⁰ B. subtilis growing on solid LB medium: wild type Rel^ΔRRM (VHB184), H420E (VHB231) defective in recognition of the tRNA 3’ CCA end, and ZFD mutants C602A C603A (VHB233) and R619E (VHB281) defective in 70S binding. LB plates were scored after 18 h incubation at 37°C. (E) C-terminally truncated Rel variants (either synthesis-competent (VHB155, VHB159-161) or synthesis-inactive D264G mutant versions (VHB156, VHB162-164) were expressed in ppGpp⁰ B. subtilis growing on either solid minimal medium or solid minimal medium supplemented with 0.025% casamino acids. Plates were scored after 36 h incubation at 37°C. Importantly, prior to experiment all strains were pre-cultured on solid minimal medium supplemented with 0.025% casamino acids. This was done in order to avoid the effects caused by the decreased fitness of the inoculum. (F) Synthesis activity of Rel mutants probed by amino acid auxotrophy assays. B. subtilis strains were constructed using either relP⁺ relQ⁺ wild-type 168 (upper row) or ΔrelP ΔrelP (lower row) background. The strains either expressed the indicated rel mutants [H420E (VHB68) and C602A C603A (VHB148), upper row, and H420E (VHB60), C602A C603A (VHB62), lower row] or contained an additional ΔrplK gene disruption (VHB47, relP⁺ relQ⁺ and VHB49, ΔrelP ΔrelP). The ppGpp⁰ mutant strain (ΔrelP ΔrelP Δrel, VHB63) was used as a control (highlighted with gray box). The strains were grown on either solid Spizizen minimal medium (right panel) or solid minimal medium supplemented with 0.025% casamino acids (left panel).