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. 2019 Jan 10;9:34. doi: 10.1038/s41598-018-37321-z

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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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Characterization of A. aurita QQ ORFs. (A) Arrangements of predicted ORFs on original metagenomic fosmid inserts (supplemental dataset) are depicted, carrying the genes aaqq1, aaqq2 and aaqq3 linked to QQ phenotypes (black arrows, Accession Nos. JX274296-JX274298). (B) QQ-ORFs were cloned into pMAL-c2X and purified as MBP-fusion proteins by affinity chromatography. 5 µg of elution fractions were analyzed on 12% SDS-PAGE. Purified MBP-QQ proteins (0.1 µg, 1 µg and 10 µg) were tested regarding their QQ activities using reporter strains AI1-QQ.1 and AI2-QQ.1 (AHL, left panel; AI-2, right panel) monitoring QQ activities by growth. (C) QQ proteins were analyzed using secondary structure prediction software PredictProtein (http://www.predictprotein.org/) with following visual output in Profsec: yellow, strand; red, helix. InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) and GlobPlot (http://globplot.embl.de/) were used for functional protein analysis. (D) Fragments of QQ-ORFs were PCR-amplified from genomic DNA as well as cDNA transcribed from RNA of antibiotic-treated polyps (P) and ephyrae (E). C, non-template control. The presented gel grouping is cropped from different gels.