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. 2017 May 15;7:1887. doi: 10.1038/s41598-017-01692-6

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© The Author(s) 2017

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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Assessment of a transcriptional terminator is present in the region 298–397. RT-PCR amplification of inter-cistronic region between orfX and ltxC in RhAa3, RhAa-ltxP530 and RhAa-ltxP298-397 with increasing concentrations of cDNA are shown as follows. Lane 1. 1 ng; Lane 2. 2 ng; Lane 3. 4 ng; Lane. 4 5 ng; Lane. 5 7.5 ng and Lane. 6 10 ng. Note that a clear signal was obtained from the 5 ng template in RhAa-ltxP298-397 (A). qRT-PCR shows significant increase in ltxC expression as compared to orfX expression in RhAa-ltxP298-397 further indicating a potential weak terminator, a negative regulatory element, within the region 298–397. Results are means ± SD for triplicate cultures normalized to 5S rRNA. The significant fold change was calculated by One-way ANOVA test. * indicates P < 0.001 ± SD with Tukey’s multiple comparison post-hoc test (B).