Efficient preparation and metal specificity of the regulatory protein TroR from the human pathogen Treponema pallidum

TroR is a putative metal-dependent regulatory protein that has been linked to the virulence of the human pathogen Treponema pallidum. It shares high homology with the well-known iron-dependent regulatory protein DtxR from Corynebacterium diphtheriae, as well as the manganese-dependent MntR from Bacillus subtilis. However, it has been uncertain whether manganese or zinc is the natural cofactor of TroR to date. Herein, we established an efficient method named "double-fusion tagging" to obtain soluble TroR for the first time. A series of studies, including ICP, CD, fluorescence, ITC, and electrophoresis mobility shift assay (EMSA), were performed to resolve the discrepancies in its metal-binding specificity. In addition, bioinformatic analysis as well as mutation studies were carried out to find the genetic relationships of TroR with its homology proteins. In conclusion, our findings indicate that TroR is a manganese-dependent rather than a zinc-dependent regulatory protein.