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. 2021 Jul 20;9:161. doi: 10.1186/s40168-021-01095-w

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© The Author(s) 2021

Open AccessThis 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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 8 — Role of ObgE in cFP-mediated change in the membrane permeability of B. vulgatus. A Transcript levels of obgE, recA, and cidA in cFP-treated B. vulgatus. The total RNAs were extracted from B. vulgatus cells treated with 4 mM cFP for 0, 2, and 4 h. Then, cDNAs were produced through the reverse transcriptase reaction and subjected to quantitative PCR using primer sets specific to obgE, recA, or cidA (Additional file: Table S3). The transcript contents of each gene were normalized with the gap transcript levels. The ratios of normalized contents in cFP-treated cells to those in the control cells were plotted. B Protein levels of ObgE and RecA in cFP-treated B. vulgatus. Fifteen micrograms of protein lysates of B. vulgatus, which had been exposed to 0 or 4 mM cFP for 4 h, were fractionated in SDS-PAGE and subjected to western blotting analysis using anti-ObgE and anti-RecA polyclonal antibodies. As a positive control, rRecA (38 kDa) and rObgE (45 kDa) were included in the blots. Densitometric readings of the bands corresponding to each protein in the control and cFP-treated cells were plotted. C Specific binding of cFP to ObgE. Isothermal titration calorimetry (ITC) experiments were performed by titrating the recombinant ObgE (40 μM) with 400 μM cFP at 25°C. As a control, rObgE was titrated with 400 μM cPT under the same condition for ITC. D Differential expression of ObgE in B. vulgatus cells. Freshly grown B. vulgatus carrying the vector plasmid (pBAD/Myc-His B) or pBAD-obgE_Bv was added with various concentrations of L-arabinose ranging from 0% to 1.0% to differentially induce obgE expression. Upon 1 h induction, bacterial lysates (2 μg) were subjected to western blotting along with the known concentrations (from 0.2 to 10 ng) of rObgE to estimate the cellular contents of ObgE. E Effect of the cellular levels of ObgE on membrane permeability. The same cells prepared in Fig. 8D were exposed to 4 or 0 mM cFP for 6 h then mixed with 0.4 μΜ DiSC3(5). As described in Fig. 7, the values of released DiSC3(5) from 0.002% (Δ_0.002%) and 0.1% Triton X-100-treated cells (Δ_0.1%) were estimated. The ratios ([Δ_0.002%/Δ_0.1%] × 100) were plotted against the cellular contents of ObgE. shown in Fig. 8D. P-values for the comparison of 4 mM cFP-treatment with no cFP-treatment are indicated (Student’s t-test; ns, not significant; *0.001 < P < 0.01; **P < 0.001; ^#no p value determined)