The DNA-binding characteristics of the Streptomyces reticuli regulator FurS depend on the redox state of its cysteine re
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O R I GI N A L P A P E R
D. Ortiz de Orue Lucana á H. Schrempf
The DNA-binding characteristics of the Streptomyces reticuli regulator FurS depend on the redox state of its cysteine residues
Received: 28 December 1999 / Accepted: 20 July 2000 / Published online: 29 August 2000 Ó Springer-Verlag 2000
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 puri®ed, 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 puri®ed from S. reticuli) is able to bind to a motif upstream of the furS gene. In the absence of ®rst-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 ®ndings are discussed. Communicated by W. Goebel D. Ortiz de Orue Lucana á H. Schrempf (&) UniversitaÈt OsnabruÈck, FB Biologie/Chemie, Barbarastrasse 11, D-49069 OsnabruÈck, Germany E-mail: [email protected] Tel.: +49-541-9692895; Fax: +49-541-9692804
Key words Streptomycetes á Thiol-reduced FurS á Redox regulator protein á Metalloregulator á CpeB
Introduction Most microbial catalase-peroxidases contain two or four identical subunits, and they carry ferric protoporphyrin IX (0.5±1 molecule per subunit) as a prosthetic group. Monomeric catalase-peroxidases have been puri®ed from two halophilic archaebacteria (Fukumori et al. 1985; Cendrin et al. 1994). Proteins deduced from the few catalase-peroxidase gene sequences so far characterized show varying, but generally high, degrees of sequence identity, but exhibit little similarity to proteins derived from typical catalase genes
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