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. 2022 Nov 5;13:6684. doi: 10.1038/s41467-022-34607-9

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© The Author(s) 2022

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. 1 — a AI-2 regulates biofilm formation in S. Typhimurium independent of LsrB. Biofilms were stained with crystal violet and quantified using optical density measurement. Data were mean ± s.e.m. of five independent experiments. b AI-2 regulates the swimming motility of S. Typhimurium independent of LsrB. Data were mean ± s.e.m. of three independent experiments. c YeaJ-LBD is capable of retaining AI-2. Bioluminescence in V. harveyi MM32 (luxN⁻, luxS⁻) was induced by the addition of ligands released from purified proteins expressed in a luxS⁺ or luxS^- E. coli strain. LsrB from S. Typhimurium was used as a positive control. AI-2 activity is reported as fold induction relative to the light production induced by a buffer control. Data were mean ± s.e.m. of three independent experiments. d ITC assays for the specific interaction between YeaJ-LBD and AI-2. The data shown are representative of three independent experiments with similar results. The K_d and complex stoichiometry (n) values were presented as mean ± s.d. of three independent experiments. e AI-2 stimulates the DGC activity of YeaJ in vitro. Data were mean ± s.e.m. of three independent experiments. f Liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements of cellular levels of c-di-GMP in S. Typhimurium strains. The bacterial cultures grown in LB broth at 37 °C with shaking to an OD₆₀₀ of 1.3 were subjected to nucleotide extractions. Data represent mean ± s.d. from three biological replicates. g AI-2 increases cellular c-di-GMP concentration via YeaJ. The mutants ΔluxS and ΔyeaJΔluxS grown in LB medium to an OD₆₀₀ of 1.3 were induced by 10 μM DPD/AI-2 or a buffer control for 30 min, and then bacterial cells were collected to extract nucleotides for LC-MS/MS analysis. Data shown are mean ± s.d. of three biological replicates. a, b, e–g Statistical significance was evaluated using a two-tailed unpaired Student’s t-test and p < 0.05 was considered statistically significant. WT wildtype. Source data are provided as a Source Data file.