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Cloning and characterization of a gene encoding the γ-butyrolactone autoregulator receptor from Streptomyces clavuligerus

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Abstract

With primers designed for the conserved region of the γ-butyrolactone autoregulator receptor proteins from Streptomyces species, PCR using the Streptomyces clavuligerus genome DNA as a template gave a clear band of 100 bp, the sequence of which revealed high similarity to the expected region of a receptor gene. By Southern blot and colony hybridization with the 100-bp insert as a probe, plasmid pSCA, harboring a 4.2 kb-SalI fragment, was obtained. Sequence analysis on the insert revealed a 702-bp ORF encoding a protein with a moderate similarity (identity, 33–43%; similarity, 51–62%) to known γ-butyrolactone autoregulator receptor proteins from Streptomyces sp. The ORF was named scaR (S. clavuligerus autoregulator receptor). The scaR/pET-3d plasmid was constructed for overexpression of the recombinant ScaR protein (rScaR) in Escherichia coli, and the rScaR protein was purified to homogeneity by DEAE-ion-exchange HPLC. The molecular mass of the purified rScaR protein was determined to be 27 kDa as determined by SDS-PAGE, and 54 kDa by gel filtration HPLC under nondenatured conditions at a low protein concentration, indicating that the majority of the native ScaR is present in the form of a dimer, although rScaR tended to aggregate into a higher molecular form of 230 kDa at a high protein concentration. A binding assay with tritium-labeled autoregulators indicated that IM-2 type compounds with a long C2 side chain were the most effective ligands for rScaR, demonstrating for the first time that the β-lactam producer S. clavuligerus contains a gene for the γ-butyrolactone autoregulator receptor.

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Acknowledgments

This work was supported in part by a grant (KRF-2002-042-C00086) from the Korea Research Foundation, and by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan.

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Authors and Affiliations

  1. Department of Microbiology, Keimyung University, 1000 Shindang-dong, Daegu, 704-701, South Korea

    Hyun Soo Kim, Yong Jik Lee & Tae Shick Yu

  2. PrimBioR&Ds, Institute for Genome Reconstruction, Kyungnam University, 449 Wolyong-dong, Masan, 631-701, South Korea

    Chang Kwon Lee

  3. International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871, Japan

    Sun Uk Choi, Hiroshi Kinoshita & Takuya Nihira

  4. Traditional Microorganism Resources Center, Keimyung University, 1000 Shindang-dong, Daegu, 704-701, South Korea

    Soo-Hwan Yeo

  5. Division of Life Science, Kyungnam University, 449 Wolyong-dong, Masan, 631-701, South Korea

    Yong Il Hwang

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  1. Hyun Soo Kim
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Correspondence to Takuya Nihira.

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Kim, H.S., Lee, Y.J., Lee, C.K. et al. Cloning and characterization of a gene encoding the γ-butyrolactone autoregulator receptor from Streptomyces clavuligerus. Arch Microbiol 182, 44–50 (2004). https://doi.org/10.1007/s00203-004-0697-x

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  • DOI: https://doi.org/10.1007/s00203-004-0697-x

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