Fig 1

Fig 1

EspN binds the whiB6 promoter and activates whiB6 expression in the absence of EspM. (A) Mass spectrometry analysis of the DNA affinity chromatography showing enrichment of the MMAR_1626 and HupB proteins. The HupB protein binds non-specifically to both DNA probes. The scale represents the log2 intensity of Mass Spectral peak area (MS peak areas). The data were published in Sanchez et al. (22) and adapted in Data set S1. (B) The predicted domain structure of MMAR_1626, which we renamed EspN. Modeled using RoseTTAFold from Robetta (29). Model confidence: 0.80. (C) (Top) Western blot analysis of M. marinum cell-associated proteins. RpoB is a loading control. All strains include a whiB6-3xFl allele at the whiB6 locus (20). (Bottom) Relative qRT-PCR analysis of M. marinum strains compared to sigA transcript levels. Statistical analysis was performed using one-way ANOVA (P = 0.0359), followed by a Dunnett’s multiple comparison test, which revealed no significant differences relative to the WT strain. (D) (Top) Western blot analysis of 10 µg of M. marinum whole-cell lysates. RpoB serves as a loading control. All strains include a whiB6-3xFl allele at the whiB6 locus (20); (bottom) qRT-PCR of the whiB6 transcript relative to sigA. Significance was determined using ordinary one-way ANOVA (P = 0.0001), followed by Tukey’s multiple comparisons test. Significance shown is relative to the ΔespM strain, with additional statistics of interest discussed in the text. ****P < 0.0001, ***P = 0.0002 for ΔeccCb₁, **P = 0.0010, ***P = 0.0002 for ΔespM/pespN, ***P = 0.0009 for ΔwhiB6. Western blots are representative of three independent biological replicates. All qRT-PCRs include at least three independent biological replicates, each in technical triplicate. ANOVA, analysis of variance; au, arbitrary units; qRT-PCR, quantitative reverse transcription PCR; SCP2, sterol carrier protein 2; wHTH, winged helix-turn-helix; WT, wild type.