Figure 1

Construction of conditional EmbA/GlfT2_KD strains and validation of impaired arabinogalactan synthesis in Mycobacterium marinum cell wall. (A) qPCR proved the corresponding relative expression of embA or glfT2 of embA knock-down (EmbA_KD) or glfT2 knock-down (GlfT2_KD) strains with or without anhydrotetracycline (ATc) induction at logarithmic growth stage. Data are plotted as mean ± standard deviation (SD), unpaired t-test: ****p < 0.0001, *p = 0.0236. (B, C) Growth curves of EmbA/GlfT2_KD strain with or without ATc induction. Absorbance (OD₆₀₀) measured in the bacterial liquid reflects the growth. (D) High-performance gel permeation chromatography was used to analyze the polysaccharide molecular weights of knock-down strain cell walls. The linear regression equation of retention time (t_R) and relative molecular weight (MW) of polysaccharide is logMW = -0.5042 t_R + 11.884. Relative molecular weights corresponding to peaks are marked. (E) High-performance liquid chromatography was used to analyze the monosaccharide contents in the cell walls of control and knock-down strains. The peak area integral was used to calculate the proportion of each monosaccharide, and the ratios of arabinose–galactose/mannose or galactose/arabinose were calculated. The calculation results are presented in the table.