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. 2023 Jul 21;13:11771. doi: 10.1038/s41598-023-38924-x

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© The Author(s) 2023

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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recA induction and ROS and R-loop accumulation in PNPT1_Ec. (A) ROS accumulation. Normalized fluorescence (N. F.) of C-1a (pnp⁺, Ec); C-5691 (Δpnp, Δ); C-6001 (PNPT1_Ec, Hs) cultures (N = 5) in presence of DCFH-DA. Significance was evaluated with ANOVA and Tukey post hoc analysis. ****, P < 0.0001. (B) RT-qPCR analysis of recA expression. RNA was extracted from replicate cultures (N = 3) grown in aerobiosis (AER) or anaerobiosis (ANA). ΔCt between the gene of interest and the 16S gene was arbitrarily set at 1 for one of the samples extracted from C-1a AER (reference condition). The bars represent relative amount (R.A.) with respect to the reference condition and show the average with standard deviations of the values obtained on three biological replicates, each performed in duplicate. Bars sharing the same letter represent averages not significantly different from each other according to ANOVA and Tukey post hoc comparisons. ***, P ≤ 0.001; *, P < 0.05. (C, D) Dot blot quantification of R-loops. Strains are indicated as in A. DNA extracted from cultures grown at high (C) or low (D) oxygen were analysed. C, left panel. Dot blot signals obtained by immunostaining of genomic DNA with the S9.6 antibody were quantified with ImageQuant. The median (N = 6) is reported inside the boxes (line). The whiskers represent the minimum and maximum values observed. Boxes sharing the same letter are not significantly different from each other according to ANOVA and Tukey posthoc comparisons. (C) Right panel, and (D) left panel. Genomic DNA samples were dot blotted after incubation with RNase H or III, as described in Methods. –, mock incubation without RNase addition. Immunostaining (IS) with S9.6 antibody. MB, staining of the filter with methylene blue as loading control. (D) Right panel. Dot blot signals obtained by immunostaining of genomic DNA with the S9.6 antibody were quantified with ImageQuant. Bars represent average (N = 3) with standard deviation. Significance of the difference was estimated with two-tail t-test. **, P < 0.01. (E) Serial dilution of C-1a (Ec), C-5691 (Δ) and C-6001 (Hs) grown on LD-agar at 37 °C in aerobiosis (AER) or anaerobiosis (ANA). Duplicate cultures are shown in the ANA panel.