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. 2018 Sep 14;8:13815. doi: 10.1038/s41598-018-32164-0

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© The Author(s) 2018

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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Construction of disruption mutants. (A) Schematic diagram of the loci targeted for disruption. The target genes are in gray. The black arrows indicate the primers for RT-PCR analysis (panel C and Table S3). (B) Normalized reads’ coverage as a function of the median coverage observed for all four deleted T. kodakarensis strains. Colors correspond to the coverage observed for ΔTK1509/cbf5 (blue), ΔTK0903/pus10 (red), ΔTK1101/nop10 (yellow) and ΔTK2286/gar1 (green) strains. Identity of outlier points is indicated. TK2276 (pyrF) is affected in all four strains analyzed. (C) Disruption of each targeted ORFs does not impair transcription of their respective downstream contiguous ORF. RT-PCR reactions (gel + RTase) were performed with oligonucleotide primers (Table S3) complementary to the coding sequence of the target gene’s adjacent ORFs (indicated below each lane). The RT-PCR products were fractionated by 0.8% agarose gel electrophoresis. The bands correspond to RNA amplification since no amplified products were obtained in a reaction mixture lacking reverse transcriptase (-RTase). The asterisk indicates non-specific amplification.