Overexpression of the EspM N-terminus or EspE negatively impacts ESX-1 transcription in the absence of EspM. (A) Schematic of transcriptional regulation by EspM, EspN, and WhiB6. EspE and EspF are ESX-1 substrates that negatively regulate the WhiB6 transcription factor (28). (B) Schematic of the predicted domains of the EspM protein. (C) sRBC lysis measuring the hemolytic activity of M. marinum. Outliers were identified using ROUT analysis, Q = 0.05%. Statistical analysis was performed using one-way ANOVA (P < 0.0001) followed by Dunnett’s multiple comparisons test (**P = 0.0034, ****P < 0.0001). The data include at least three independent biological replicates each in technical triplicate. (D) Relative qRT-PCR analysis of whiB6 compared to sigA transcript levels. Significance was determined using ordinary one-way ANOVA (P < 0.0001), followed by Dunnett's multiple comparisons test (****P < 0.0001) relative to the ΔespM strain. The qRT-PCR data include at least three independent biological replicates each in technical triplicate. (E) sRBC lysis measuring hemolytic activity of M. marinum. Outliers were identified using ROUT analysis, Q = 0.05%. Statistical analysis was performed using ordinary one-way ANOVA (P = 0.9639), which did not indicate significant differences. The data include at least three independent biological replicates each in technical triplicate. (F) Relative qRT-PCR analysis of whiB6 compared to sigA transcript levels. Outliers were identified using ROUT analysis (Q = 0.5%). Significance was determined using ordinary one-way ANOVA (P < 0.0001), followed by Dunnett's multiple comparisons test (**P = 0.0070, ****P < 0.0001) relative to the ΔespM strain. The qRT-PCR data include at least three independent biological replicates each in technical triplicate. FHA, forkhead-associated domain; HTH, helix-turn-helix; NT, N-terminus.
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