DNA Distortions Imposed by the Transcriptional Regulator PecS from Pectobacterium atrosepticum are Attenuated on Protein Oxidation

Abstract

The phytopathogen Pectobacterium atrosepticum is responsible for causing soft rot and blackleg disease in potato. Soft rot is characterized by maceration of plant cell wall and middle lamella by enzymes such as pectate lyases, cellulases, and proteases. Pathogenicity of the bacterium depends on responding to environmental cues for efficient colonization of host plants. MarR family transcriptional regulators modulate virulence and adaptation to hostile environments. Previous studies have shown that in the related bacterium Dickeya dadantii, the MarR transcription factor PecS negatively controls expression of the pectinase gene and secretion of the antioxidant indigoidine. PecS downregulates the expression of both pecS and pecM genes; PecM is an efflux pump, responsible for secretion of indigoidine, which is involved in neutralization of reaction oxygen species. Here, we report that P. atrosepticum PecS binds pecS‐pecM intergenic DNA with very high affinity (Kd = 1.0 ± 0.1 nM) and specificity at pH 7.4. DNase I footprinting analysis reveals that PecS binds two palindromic sites in the pecS‐pecM intergenic region and significantly distorts the DNA upon binding, as evidenced by presence of hypersensitive sites. Structure based modeling shows cysteine at the dimer interface, raising the possibility that oxidizing agents can modulate DNA binding by disulfide bond formation between redox‐active cysteines. PecS forms dimers and multimers in presence of organic and inorganic peroxides, as evidenced by SDS‐PAGE analysis. Thermal shift assays suggest that oxidants change the protein conformation since thermal stability of reduced PecS (Tm ~ 50 °C) is significantly lower in presence of oxidants. DNase I footprinting of oxidized PecS shows identical protection pattern as reduced PecS, however hypersensitive sites are no longer present, indicating that the DNA distortion imposed by reduced PecS is absent on binding oxidized protein. This is reminiscent of our previous observation that an increase in pH from 7.4 to 8.3 is associated with PecS failing to induce hypersensitive DNase I cleavage and in protein‐DNA complexes dissociating during electrophoresis, conditions under which PecS is unable to repress the pecS promoter in vivo. Likewise, we observed reduced DNA binding by oxidized PecS by electrophoretic mobility shift assay. We propose that cysteine oxidation induces conformational changes in the DNA binding domain, which modulate binding to cognate DNA. We further propose that PecS controls gene expression by sensing oxidants.Support or Funding InformationNational Science Foundation(MCB‐1515349)