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. 2024 Oct 3;14:22998. doi: 10.1038/s41598-024-73879-7

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© The Author(s) 2024

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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Fig. 1 — Importance of single amino acids for TisB functionality. (a) Conservation analysis of TisB. Conservation levels of TisB were determined via BLAST. Amino acids with 95% conservation are in bold. E. coli K-12 (Ec), Salmonella enterica serovar Typhimurium (Se), Shigella flexneri (Sf), Citrobacter freundii (Cf), Citrobacter koseri (Ck), and Klebsiella pneumoniae (Kp). Amino acids color code: nonpolar (yellow), polar (purple), acidic (red), and basic (blue). (b) Helical wheel projection of TisB. Same color code as in (a). (c) Western blot analysis of TisB localization. Wild type MG1655, harboring p0SD-3xFLAG-tisB (3xFLAG-TisB) and variants with different amino acid substitutions, were treated with L-ara (0.2%) during exponential phase for one hour. p0SD-tisB (TisB) and an empty pBAD plasmid (CTR) were used as controls. Cytoplasmic (C) and membrane (M) fractions were isolated from total protein samples using ultracentrifugation, followed by Tricine-SDS-PAGE and western blot detection. An anti-3xFLAG antibody was used for detection of 3xFLAG-TisB. Anti-YidC (membrane) and anti-YchF (cytoplasm) antibodies were used as fractionation controls. For unedited western blot images, see Supplementary Fig. S2. (d) Growth inhibition by TisB. Wild type MG1655, harboring p0SD-tisB (TisB) and variants with different amino acid substitutions, were treated with L-ara (0.2%) during exponential phase (arrow). An empty pBAD plasmid (CTR) was used as control. Data points of OD₆₀₀ measurements represent the mean of at least three biological replicates (TisB: n = 9; CTR: n = 9; D5L: n = 3; K12L: n = 3; Q19L: n = 3; D22L: n = 6; K26L: n = 3; K29L: n = 3). (e) TisB-dependent ATP depletion. Wild type MG1655, harboring p0SD-tisB (TisB) and variants with different amino acid substitutions, were treated with L-ara (0.2%) during exponential phase for one hour. An empty pBAD plasmid (CTR) was used as control. Pre- and post-treatment samples were analyzed using a luciferase-based assay to measure cellular ATP levels (nM per OD₆₀₀). Bars represent the mean of at least three biological replicates (TisB: n = 7; CTR: n = 9; D5L: n = 4; K12L: n = 9; Q19L: n = 4; D22L: n = 6; K26L: n = 4; K29L: n = 3). Error bars indicate the standard deviation. ANOVA with post-hoc Tukey HSD test was performed (*** p < 0.001; ** p < 0.01).