Fig. 3: Thiaminase I CcThi1 from the cell membrane of strain EGB inhibits P. sojae growth by decomposition of essential thiamine.
a Schematic diagram of the purification process with corresponding gel chromatograms. IEC ion exchange chromatography, HC hydroxyapatite chromatography, GFC gel filtration chromatography. Blue curve, protein concentration; red curve, salt concentration; green circle, active range. The cartoon image was drawn with Biorender. b Growth inhibition by the purified proteins from GFC and the corresponding SDS-PAGE electropherograms. The P6497 growth was observed using a stereomicroscope (Nikon SMZ-10). Scale bar, 5 mm. c Inhibition of mycelial growth by various concentrations of CcThi1. The aerial mycelia of P6497 were observed, and the growth inhibition was assessed by measuring the colony diameter (up). The data represent the means ± SEM (n = 3 biological independent replicates). CcThi1 inhibited the elongation of zoospore germ tubes of P6497. Microscopic observation of mycelia after CcThi1 treatment was performed to measure the hyphal length (down). Scale bars: 100 μm. The data represent the means ± SEM (n = 5 biological independent replicates). d Diagram of the reaction catalyzed by thiaminase I. Nu− represents a nucleophile. e, f Measurement of thiamine content in the reaction system containing thiamine and H2O (e) or 10% V8 medium (f) with different concentrations of CcThi1. The data represent the means ± SEM (n = 3 biological independent replicates). g, h Addition of thiamine restored the CcThi1-mediated growth inhibition of P6497 (g), and the growth state was assessed by measuring the colony diameter (h). The data represent the means ± SEM (n = 3 biological independent replicates). i WebLogo depicting conserved residues of CcThi1. The black arrows indicate the predicted critical active-site residues C148 and E269. j Thiaminase I activity and growth inhibitory effect of CcThi1 and the mutant CcThi1C148A; E269A (Supplementary Fig. 11). The data represent the means ± SEM (n = 3 biological independent replicates). Means within columns followed by different letters are significantly different in c, e, f, h, j (p < 0.05, one-way ANOVA, Duncan’s multiple range test). Source data are provided as a Source Data file.
