Insight into the regulatory mechanism of the MFS transporter, SCO4121 by the MarR regulator, SCO4122

MarR group of transcriptional regulators are ubiquitous in bacteria and found to be involved in regulation of efflux pumps that confer multidrug resistance phenotype. While most characterized MarR regulators act as transcriptional repressors, we earlier identified a MarR regulator SCO4122 in Streptomyces coelicolor , playing an essential role in transcriptional activation of the MFS transporter SCO4121 in response to multiple substrates of the latter, including streptomycin, ciprofloxacin and chloramphenicol. In this study, using Surface Plasmon Resonance, we demonstrate that SCO4122 interacts directly with the diverse substrates of SCO4121 with the highest affinity for streptomycin with a KD of 0.73 μM. Further, in-vitro and in-vivo studies reveal that SCO4122 also binds to the intergenic region between sco4121 and sco4122, where the interaction is dependent on the cooperative binding of SCO4122 to three motifs in this region. A conserved Methionine, M93, in SCO4122 is identified to be an integral amino acid residue that is involved in activation of SCO4121 in response to ciprofloxacin, streptomycin and EtBr but not chloramphenicol. Furthermore, our studies also indicate that upon binding to different substrates, the affinity of SCO4122 to the sco4121 promoter increases 50-1000 fold, thereby leading to enhanced expression of the transporter, SCO4121. This study thus highlights that SCO4122 is a novel MarR regulator that functions as a strong transcriptional activator of an efflux pump, SCO4121, through intricate molecular mechanisms in presence of structurally dissimilar substrates.