FIG 4.

MrpC positively regulates the expression of pmxA. (A) Schematic of the pmxA locus. The direction of transcription is indicated by the arrows. +1 indicates TSC of pmxA. Numbers above indicate the distance between start and stop codons of flanking genes. MXAN_2063 encodes a FecR domain-containing protein with a lipoprotein signal peptide and MXAN_2062 encodes a protein with a type I signal peptide, an N-terminal LysM domain, and a C-terminal extracellular fibronectin type III domain. The function of these two proteins is not known. (B) RNA-seq (bottom) and Cappable-seq (top) data at different time points for genes at pmxA locus. For each time point, data for both biological replicates are shown in blue and orange. For Cappable-seq, the RRS is indicated for each TSS on a log₂ scale; for RNA-seq, RPKM values were calculated for each nucleotide position. The data from RNA-seq and Cappable-seq were obtained from different samples. Left, +1 indicates TSCs of MXAN_2064-_2060; right, zoom of region indicated in the hatched box in left panels immediately upstream of pmxA and where +1 indicates the TSC of pmxA. In both sets of panels, TSSs as mapped by Cappable-seq are indicated in purple relative to the nearest TSC. The center of the MrpC ChIP-seq peak is indicated in brown. (C) Feature map of pmxA promoter region. +1 and color code is as in panel B. Green boxes labeled BS1 to 3 indicate potential MrpC binding sites based on the consensus sequence as defined by reference 50; sequences of BS1 to 3 are shown below and in which underlined text indicates a mismatch. Red indicates the sequence used to generate the mutant binding sites. The gray line indicates the EMSA probe and contains all three predicted MrpC binding sites. (D and E) MrpC binds to the pmxA promoter region using BS1 and BS2. The indicated Hex-labeled probes were mixed with the indicated concentrations of His₆-MrpC EMSA and analyzed by EMSA. (F) MrpC activates pmxA promoter(s). Total cell lysates from the indicated strains expressing mCherry from P_pmxA^WT were harvested from cells developed in MC7 submerged cultures at the indicated time points and then analyzed as in Fig. 3F. (G) BS1 and BS2 are important for MrpC-dependent activation of the pmxA promoter(s). Total cell lysates from the indicated WT strains expressing mCherry from the indicated promoters were prepared and analyzed as in Fig. 3F. (H) PmxA accumulates at reduced levels in the ΔmrpC mutant. Total cell lysates of the indicated strains were harvested from cells developed in MC7 submerged conditions at indicated time points and analyzed as in Fig. 3F except that the accumulation of PmxA-mVenus relative to PilC was calculated.