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. 2022 Jul 23;13:4258. doi: 10.1038/s41467-022-31925-w

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© The Author(s) 2022

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 Agar overlay assays using PvCL10 as the overlay strain with 450 ng His-BcpT, or His-BcpT cleaved with Factor Xa, or Factor Xa alone. b Coomassie-stained polyacrylamide gel of His-BcpT cleaved with Factor Xa for various periods of time showing cleavage products a, b, and c. c Western immunoblot of supernatants of overnight cultures of the indicated strain or mutant probed with α-BcpT showing a representative of three independent blots. d Sequence of BcpT with the native signal sequence showing the SpII cleavage site and the sequence of the construct with the His-tag. The N-terminal sequences of the a, b, and c fragments of panel B determined by amino acid sequencing are boxed. Fragment a is colored blue and fragment c is colored green. Fragment b is the combination of the beige and green sequences. e Coomassie-stained gels of recombinant BcpT with mutants at LTR sites, R65N and R199N, cleaved with FXa. The (*) indicates the presence of a probable protein from E. coli in that lane and the following three lanes. f Agar overlay assays of PvCL10 were spotted with purified BcpT WT and activation site mutants. The horizontal and vertical hashmarks are from the polystyrene plate grids. g Western blot of supernatants of overnight cultures of the reconstructed BcpT site mutants R65N, R199N, R65RN199N in the PdCL02 ΔbcpT background probed with α-BcpT. h Agar overlay assays with spotted WT PdCL02 and each of the PdCL02 reconstructed BcpT site mutants in the PdCL02 ΔbcpT background overlaid with strain PvCL10. The (*) indicates reconstructed plasmid. All overlay assays, Coomassie-stained gels and western blots were performed at least twice. Source data are provided as a Source Data file.