The DNA-binding Characteristics of the Streptomyces Reticuli Regulator FurS Depend on the Redox State of Its Cysteine Residues

Mol Gen Genet. 2000 Oct;264(3):341-53. doi: 10.1007/s004380000328.

Abstract

Streptomyces reticuli produces a mycelium-associated enzyme (CpeB) which exhibits heme-dependent catalase and peroxidase activity, as well as heme-independent manganese-peroxidase activity. The cpeB gene does not have a promoter of its own. It is co-transcribed together with the adjacent furS gene from at least one promoter, the position of which was deduced on the basis of high-resolution S1 mapping of transcriptional start sites. Physiological and transcriptional studies suggested that FurS acts as a transcriptional repressor in the presence of Mn2+ and Fe2+ ions. A FurS fusion protein was purified, after cloning of the corresponding gene, either from Escherichia coli or Streptomyces lividans transformants. The fusion protein from each host strain can be converted into a form that exhibits reduced electrophoretic mobility following treatment with thiol-reducing agents; in the presence of diamide, in contrast, the mobility of the protein is enhanced. Additional immunological studies have shown that the native S. reticuli FurS also shows these properties, which are due to the presence of redox-sensitive cysteine residues. As revealed by gel-shift and in vitro footprinting studies, only the reduced form of the FurS fusion protein and the reduced FurS protein (partially purified from S. reticuli) is able to bind to a motif upstream of the furS gene. In the absence of first-row divalent ions, the binding site encompasses 22 bp. In the presence of Mn2+, Fe2+, Co2+, Cu2+ or Zn2+, however, the region bound is extended by 18 bp. It is noteworthy that the region upstream of the furA gene in several mycobacteria contains a very similar motif. The predicted mycobacterial FurA shares a high degree of sequence identity with FurS, and the furA gene is linked to one that encodes a catalase-peroxidase (KatG). The implications of these findings are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Base Sequence
  • Blotting, Western
  • Cloning, Molecular
  • Cysteine*
  • DNA / metabolism*
  • DNA Footprinting
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism
  • Models, Genetic
  • Molecular Sequence Data
  • Oxidation-Reduction*
  • Peroxidases / genetics*
  • Plasmids / metabolism
  • Promoter Regions, Genetic
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / genetics
  • Sequence Analysis, DNA
  • Sequence Homology, Nucleic Acid
  • Streptomyces / genetics*
  • Transcription Factors / genetics*
  • Transcription, Genetic
  • Transformation, Genetic

Substances

  • Bacterial Proteins
  • FurS protein, Streptomyces reticuli
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Transcription Factors
  • ferric uptake regulating proteins, bacterial
  • DNA
  • Peroxidases
  • catalase-peroxidase, bacteria
  • catalase HPI
  • Cysteine