Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Characterization of a Gene Encoding $\gamma-Butyrolactone$ Autoregulator Receptor from Saccharopolyspora erythraea

  • LEE YONG-JIK (International Center for Biotechnology, Osaka University) ;
  • YEO SOO-HWAN (The Center for Traditional Microorganism Resources, Keimyung University) ;
  • LEE IN SEON (The Center for Traditional Microorganism Resources, Keimyung University) ;
  • LEE SAM-PIN (The Center for Traditional Microorganism Resources, Keimyung University) ;
  • KITANI SHIGERU (International Center for Biotechnology, Osaka University) ;
  • NIHIRA TAKUYA (International Center for Biotechnology, Osaka University) ;
  • KIM HYUN SOO (The Center for Traditional Microorganism Resources, Keimyung University, Department of Microbiology, Keimyung University)
  • Published : 2006.01.01
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Abstract

A gene encoding a $\gamma-butyrolactone$ autoregulator receptor was cloned from Saccharopolyspora erythraea, and the biochemical characteristics, including the autoregulator specificity, were determined with the purified recombinant protein. Using primers designed for the conserved amino acid sequence of Streptomyces $\gamma-butyrolactone$ autoregulator receptors, a 120 bp S. erythraea DNA fragment was obtained by PCR. Southern and colony hybridization with the 120 bp fragment as a probe allowed to select a genomic clone of S. erythraea, pESG, harboring a 3.2 kb SacI fragment. Nucleotide sequencing analysis revealed a 615 bp open reading frame (ORF), showing moderate homology (identity, $31-34\%$; similarity, $45-47\%$) with the $\gamma-butyrolactone$ autoregulator receptors from Streptomyces sp., and this ORF was named seaR (Saccharopolyspora erythraea autoregulator receptor). The seaR/pET-3d plasmid was constructed to overexpress the recombinant SeaR protein (rSeaR) in Escherichia coli, and the rSeaR protein was purified to homogeneity by DEAE-Sephacel column chromatography, followed by DEAE-ion-exchange HPLC. The molecular mass of the purified rSeaR protein was 52 kDa by HPLC gel-filtration chromatography and 27 kDa by SDS-polyacrylamide gel electrophoresis, indicating that the rSeaR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that rSeaR has clear binding activity with a VB-C-type autoregulator as the most effective ligand, demonstrating for the first time that the erythromycin producer S. erythraea possesses a gene for the $\gamma-butyrolactone$autoregulator receptor.

Keywords

References

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