Regulatory Targets of the Response Regulator RR_1586 from Clostridioides difficile Identified Using a Bacterial One-Hybrid Screen

RR_1586 binds direct repeats in vitro. The presence of RR_1586 shifted direct repeats of the B1H-derived motif (arrow) in EMSAs. Inverting or substituting (cross) one or both of the repeats diminished the shift, suggesting a weakened interaction. The oligonucleotide sequences are listed in Table S2 in the supplemental material. The presence (+) or absence (−) of RR_1586 protein is indicated above each lane.

In vitro validation of conserved binding sites. Titrations of RR_1586 against predicted genomic binding sites upstream of CDR20219_3145 (A) and CDR20291_3121 (B) with 0 or 2 nucleotides, respectively, mismatching the search model were tested. Each gel shows 500 nM DNA alone and in the presence of 1×, 5×, 10×, and 20× molar equivalents of RR_1586. The oligonucleotide sequences are listed in Table S2 in the supplemental material.

Expression of GFP from C. difficile R20291 promoters in response to RR_1586. Cell density-normalized fluorescence (relative fluorescence units/optical density at 600 nm, plotted on the y axis) of GFP was observed in E. coli Rosetta cells transformed with a reporter plasmid and/or an RR_1586 expression vector (indicated below each graph). Samples were recorded in the absence (orange) and presence (blue) of 40 μM IPTG, used to induce production of RR_1586. IPTG had no effect on fluorescence in the absence of the RR_1586-harboring vector (A), but a decrease in fluorescence was observed for vectors reporting transcription from the CDR20291_0610 (B) and CDR20291_3145 (C) promoters.

Phosphorylation dependence of oligomeric state analyzed by SEC-MALS. SEC elution (curves) and light-scattering (dots) profiles are shown. Addition of the small-molecule phosphodonor phosphoramidate shifts the molecular weight of wild-type RR_1586 (red) from 57.5 to 119 kDa. In contrast, the apparent molecular weight of nonphosphorylatable RR_1586^D50G (blue) shifts the molecular weight only from 59.4 to 52.6 kDa. Monomeric RR_1586 is expected to be 28 kDa.

Phosphorylation dependence of DNA binding also depends on DNA sequence. The presence (*) of phosphoramidate has only minor effects on RR_1586 binding to a site upstream of CDR20291_2142 (A) but disrupts binding to sites with a mismatch to the B1H-derived motif, such as the one upstream of CDR20291_1583 (B). In both cases, use of the phosphorylation mutant RR_1586^D50G reverses these effects. The oligonucleotide sequences are listed in Table S2 in the supplemental material.