DNA-binding characteristics of the regulator SenR in response to phosphorylation by the sensor histidine autokinase SenS from Streptomyces reticuli.
The two-component system SenS-SenR from Streptomyces reticuli has been shown to influence the production of the redox regulator FurS, the mycelium-associated enzyme CpeB, which displays heme-dependent catalase and peroxidase activity as well as heme-independent manganese peroxidase activity, and the extracellular heme-binding protein HbpS. In addition, it was suggested to participate in the sensing of redox changes. In this work, the tagged cytoplasmic domain of SenS (SenS(c)), as well as the full-length differently tagged SenR, and corresponding mutant proteins carrying specific amino acid exchanges were purified after heterologous expression in Escherichia coli. In vitro, SenS(c) is autophosphorylated to SenS(c) approximately P at the histidine residue at position 199, transfers the phosphate group to the aspartic acid residue at position 65 in SenR, and acts as a phosphatase for SenR approximately P. Bandshift and footprinting assays in combination with competition and mutational analyses revealed that only unphosphorylated SenR binds to specific sites upstream of the furS-cpeB operon. Further specific sites within the regulatory region, common to the oppositely orientated senS and hbpS genes, were recognized by SenR. Upon its phosphorylation, the DNA-binding affinity of this area was enhanced. These data, together with previous in vivo studies using mutants lacking functional senS and senR, indicate that the two-component SenS-SenR system governs the transcription of the furS-cpeB operon, senS-senR and the hbpS gene. Comparative analyses reveal that only the genomes of a few actinobacteria encode two-component systems that are closely related to SenS-SenR.
Bogel
Gabriele
G
FB Biologie/Chemie, Universität Osnabrück, Osnabrück, Germany.
Schrempf
Hildgund
H
Ortiz de Orué Lucana
Darío
D
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D004247:DNA; D011494:Protein Kinases