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. 2019 Nov 19;9:17016. doi: 10.1038/s41598-019-53171-9

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© The Author(s) 2019

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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LC-MS analysis of BpLOG physiological product. The growth medium was centrifuged to eliminate bacteria and then filtered with a membrane 3 K, the volumes reduced at 4 ml using SpeedVac, acidified with acid formic and cytokinins purified with solid phase extraction with a MCX matrix. The purified cytokinins were dried in SpeedVac, dissolved in 100 µl of 5% methanol in water and centrifuged. The supernatant was then analyzed in LC-MS. (A) The peak seen in the Tohama I growth medium was completely absent in the medium of the knock-out Tohama I strain. This peak isolated and analyzed in mass spectrometry in a positive ion mode with electrospray resulted in a compound of 166 Da. The 6-O-methylguanine standard was run on HPLC using the same conditions used for supernatant samples and the retention time obtained was similar to the physiological product. Experiments were performed in triplicate with identical results. (B) The MS/MS comparison analysis between the physiological product and the 6-O-methylguanine confirmed the identity of the product synthesized by BpLOG. The collision energy used for the fragmentation was 26 a.u.