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. 2020 Oct 8;11:5080. doi: 10.1038/s41467-020-18866-y

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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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Fig. 3 — a top: the atomic model of VcPilQ is shown as one chain. The upper and lower beta hairpins of the gate are highlighted in a dashed line box (left) and magnified (right). Bottom: the atomic model of VcPilQ is shown as a multimer and a dashed line rectangle highlights the gate region (left), which is magnified (right) after a 90° rotation about the x-axis. The coordinate system is shown. The magnified regions from (a) are shown for (b) the VcPilQ S448C/S453C mutant, which locks the lower gate and for (c) the VcPilQ L445C/T493C mutant, which links the upper and lower gates. In (b, c) the residues mutated to cysteine are shown in red. d The transformation frequency of two cysteine pair VcPilQ mutants (left: S448C/S453C (Strain TND2169) and right: L445C/T493C (Strain TND2170)) is plotted. Data are normalized to the parental strain that expresses wild-type VcPilQ (Strain TND2140). Natural transformation assays were performed in the presence of varying concentrations of dithiothreitol (DTT) (0–1.0 mM). Data are from at least four independent biological replicates and shown as the mean ± standard deviation. The dashed line indicates the transformation frequency expected if mutants are equivalent to the parental strain expressing wild-type VcPilQ. Statistical comparisons were made by one way ANOVA with Tukey’s posttest. *p < 0.05, ***p < 0.001. The raw data are available in Supplementary Fig. 6. Representative phase contrast (top) and epifluorescence images (bottom) of V. cholerae in the hyperpiliated background expressing wild-type (WT) VcPilQ (Strain TND2244), or a cysteine mutant pair (S448C/S453C (Strain TND2242) or L445C/T493C (Strain TND2243)) grown in the presence of e 0 mM, f 1 mM, or g 2 mM dithiothreitol (DTT) prior to labeling with AlexaFluor 488-maleimide to visualize bacterial pili. More than 200 cells were imaged per condition and representative images of cells with pili are shown. In conditions where no cells analyzed exhibited an external pilus, a representative image of a non-piliated cell is shown.