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. 2020 Aug 24;10:14089. doi: 10.1038/s41598-020-71059-x

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© The Author(s) 2020

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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New surface S-layer colocalizes with areas of new peptidoglycan synthesis. (a) Examples of timepoints from real-time widefield fluorescent HADA signal (left panels) and phase contrast (center panels) of C. difficile 630 cells chased for HADA stain. Frame time represented in minutes, scale bar indicates 6 µm. (b) Airyscan confocal image of a C. difficile 630 cell grown with HADA to label peptidoglycan (Blue) and chased to reveal darker areas of newly synthesized peptidoglycan in the cell wall. This chase was followed by a short expression of SlpA_R20291 which was specifically immunolabeled with Cy5 (White). After merging HADA and Cy5 channels (righthand panels, duplicated for clarity), cell sides (yellow bars) were selected for signal intensity analysis. For this example cell the intensity plots for cell sides (i) and (ii) are shown in panel C. Scale bar indicates 6 µm. (c) Intensity plot depicting signal from HADA (Blue) and Cy5 (Grey) along the yellow bars illustrated in (b). Trace (i) was calculated to have a significant positive correlation and (ii) a significant negative correlation.