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. 2017 Jul 20;7:6020. doi: 10.1038/s41598-017-06135-w

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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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Determination of orientation and localisation of LytU in the plasma membrane. (a, b, c) Trypsin treatment of protoplast surface proteins showing that LytU behaves like a protein outside on the plasma membrane surface. The three vertical immunoblotting lanes in the assay correspond to: P, protoplasts; PS, protoplast supernatant; P + T, protoplasts treated with 1 mg ml^-1 trypsin. As the level of production from the used expression vector is controlled by the amount of xylose inducer, 0.02% xylose was used to get very low amounts of LytU, resembling the natural conditions. The controls are shown in Supplementary Fig. S1. (d) Confocal microscopy image of S. aureus cells showing fluorescence of the N-terminal GFP-fusion to LytU protein. Scale bar, 2 μm. Clear localization of the fusion protein in the plasma membrane is seen as well as some possible accumulation at cell division sites (see text and Methods for details).