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. 2020 Feb 20;10:3077. doi: 10.1038/s41598-020-59536-9

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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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∆dgcP mutant presents defects related to surface behaviors. Cells were stabbed into the bottom of an agar plate by using a toothpick and incubated upright at 37 °C overnight, followed by 48 h of incubation at room temperature. The medium was discarded, and adherent cells stained with crystal violet. (A) Diameter of the twitching colonies was measured in triplicates. Data are the means ± SD. (B) Light microscopy images of PA14 and ΔdgcP twitching colonies. Interstitial biofilms formed at the interface between a microscope slide coated in solidified nutrient media (Gelzan Pad) after four hours of colony expansion. The ΔfimV mutant was used as a negative control of twitching. (C) A silicon slide was placed upright in a culture tube and after the different time points cells at the air-liquid interface were washed, fixed and the spread of cells during the initial stages of biofilm formation was observed by FESEM. (D) *p < 0.05; **p < 0.01; ***p < 0.001.