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. 2023 Apr 5;19(4):e1011306. doi: 10.1371/journal.ppat.1011306

Fig 2. ChiA expression is strictly regulated by the chitinase regulator ChiR (STM0017).

Fig 2

(A) Schematic overview of the chitinase A operon in S. Typhimurium. (B) chiA promoter activity assay of wild-type S. Typhimurium and clean deletion mutants Δstm0014 and Δstm0017 each carrying a plasmid expressing sfGFP under the control of the chiA promoter (chiA-P_sfGFP). As a negative control, S. Typhimurium wild-type carrying a promoterless_sfGFP plasmid was used. The strains were grown in standard LB rich medium for 6 h and harvested for fluorescence intensitity analyses. Statistical analysis was implemented with ordinary one-way ANOVA and Bonferroni´s multiple comparisons test P>0.05: ns. (C) Detection of ChiA expression by Western blot analysis in S. Typhimurium wild-type, Δstm0014, and Δstm0017 mutant strains, carrying chromosomally encoded 3xFLAG tagged ChiA. To overexpress stm0014 and stm0017, genes were cloned in a rhamnose inducible plasmid pTG0034 or pTG0035 respectively. Strains were grown for 6 h in standard LB rich medium. To induce the plasmids, 0.1% rhamnose was added to the medium for 3 h. (D) chiA promoter activity assay with S. Typhimurium carrying a chromosomal replacement of chiA::sfGFP. In this strain, plasmids pTG0034 (stm0014) or pTG0035 (stm0017) were introduced for overexpression analyses under LB rich medium growth conditions. Statistical analysis was implemented with ordinary one-way ANOVA and Bonferroni´s multiple comparisons test P>0.05: ns; P≤0.05: *; P≤0.01: **. (E) ChiA expression analysis by Western blot. Indicated S. Typhimurium strains (wild-type and Δstm0017), carrying chromosomally encoded 3xFLAG tagged ChiA and plasmid pTG0069 (RecA-3xFLAG), were incubated for 3 h with polarized Caco-2 and HT29-MTX cells, cell-free supernatants of polarized Caco-2 and HT29-MTX cells, and 1% porcine mucin extract (Sigma) and harvested for Western blot analysis. RecA was used as a loading control. (F) Quantitative RT-PCR analysis of stm0017 mRNA levels of S. Typhimurium before and after 3 h contact with polarized and non-polarized Caco-2 and HT29-MTX cells. Statistical analysis was implemented with unpaired student`s t-test P>0.05: ns; P≤0.001: ***.