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Journal of Bacteriology, August 1999, p. 5075-5080, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Characterization of Binding Sequences for Butyrolactone Autoregulator Receptors in Streptomycetes

Hiroshi Kinoshita, Tomohiro Tsuji, Hiroomi Ipposhi, Takuya Nihira,* and Yasuhiro Yamada

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

Received 16 February 1999/Accepted 24 May 1999

BarA of Streptomyces virginiae is a specific receptor protein for a member of butyrolactone autoregulators which binds to an upstream region of target genes to control transcription, leading to the production of the antibiotic virginiamycin M1 and S. BarA-binding DNA sequences (BarA-responsive elements [BAREs]), to which BarA binds for transcriptional control, were restricted to 26 to 29-nucleotide (nt) sequences on barA and barB upstream regions by the surface plasmon resonance technique, gel shift assay, and DNase I footprint analysis. Two BAREs (BARE-1 and BARE-2) on the barB upstream region were located 57 to 29 bp (BARE-1) and 268 to 241 bp (BARE-2) upstream from the barB translational start codon. The BARE located on the barA upstream region (BARE-3) was found 101 to 76 bp upstream of the barA start codon. High-resolution S1 nuclease mapping analysis revealed that BARE-1 covered the barB transcription start site and BARE-3 covered an autoregulator-dependent transcription start site of the barA gene. Deletion and mutation analysis of BARE-2 demonstrated that at least a 19-nt sequence was required for sufficient BarA binding, and A or T residues at the edge as well as internal conserved nucleotides were indispensable. The identified binding sequences for autoregulator receptor proteins were found to be highly conserved among Streptomyces species.

* Corresponding author. Mailing address: Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-7433. Fax: 81-6-6879-7432. E-mail: nihira{at}biochem.bio.eng.osaka-u.ac.jp.

Journal of Bacteriology, August 1999, p. 5075-5080, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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