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. 2021 Feb 22;12:1210. doi: 10.1038/s41467-021-21306-0

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© The Author(s) 2021

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a The domain organization of Rel and the truncated Rel variants used in this study. HYD, hydrolase domain; SYN, synthetase domain; TGS, TGS domain (for: ThrRS, GTPase, and SpoT); ZFD, a zinc finger domain; RRM domain (for RNA Recognition Motif). b Bacterial two-hybrid (BACTH) assay to test for the interaction between DarB and the full-length and truncated Rel-variants. N- and C-terminal fusions of DarB and the Rel variants to the T18 or T25 domain of the adenylate cyclase (CyaA) were created and the proteins were tested for interaction in E. coli BTH101. Dark colonies indicate an interaction that results in adenylate cyclase activity and subsequent expression of the reporter β-galactosidase. The experiment was conducted three times and a representative plate is shown. c In vitro pulldown experiment with the NTD of Rel. Strep-Rel^NTD was immobilized onto a StrepTactin column and incubated with DarB, DarB preincubated with c-di-AMP, or the control protein CcpC. The eluate and wash fractions were analyzed by SDS-PAGE and the presence of DarB in the elution fractions was further verified by MS analysis (excised gel bands are numbered with 3 and 4). The experiment was conducted three times and a representative gel is shown. d The DarB–Rel^NTD complex was analyzed by size exclusion chromatography and multi-angle light scattering (SEC-MALS). Rel^NTD and DarB were used in equimolar concentrations. Dark blue line, DarB; black line, Rel^NTD; blue line, mixture of DarB and Rel. The calculated molar masses determined by MALS are listed below the chromatogram. e The molar ratio of the DarB–Rel^NTD-complex was assessed by Isothermal titration calorimetry (ITC). The cell and the syringe contained 10 µM Rel^NTD and 100 µM DarB (blue) or 100 µM c-di-AMP-bound DarB (DarB^cdA) (pink), respectively. cdA, c-di-AMP. Source data are provided as a Source data file.