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Journal of Bacteriology, November 1999, p. 6958-6968, Vol. 181, No. 22
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Characterization of the Pathway-Specific Positive Transcriptional Regulator for Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2) as a DNA-Binding Protein

Paloma Arias, Miguel A. Fernández-Moreno,dagger and Francisco Malpartida*

Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 24 June 1999/Accepted 7 September 1999

The ActII-ORF4 protein has been characterized as a DNA-binding protein that positively regulates the transcription of the actinorhodin biosynthetic genes. The target regions for the ActII-ORF4 protein were located within the act cluster. These regions, at high copy number, generate a nonproducer strain by in vivo titration of the regulator. The mutant phenotype could be made to revert with extra copies of the wild-type actII-ORF4 gene but not with the actII-ORF4-177 mutant. His-tagged recombinant wild-type ActII-ORF4 and mutant ActII-ORF4-177 proteins were purified from Escherichia coli cultures; both showed specific DNA-binding activity for the actVI-ORF1-ORFA and actIII-actI intergenic regions. DNase I footprinting assays clearly located the DNA-binding sites within the -35 regions of the corresponding promoters, showing the consensus sequence 5'-TCGAG-3'. Although both gene products (wild-type and mutant ActII-ORF4) showed DNA-binding activity, only the wild-type gene was capable of activating transcription of the act genes; thus, two basic functions can be differentiated within the regulatory protein: a specific DNA-binding activity and a transcriptional activation of the act biosynthetic genes.

* Corresponding author. Mailing address: Centro Nacional de Biotecnología, CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: 34-915854548. Fax: 34-915854506. E-mail: fmalpart{at}cnb.uam.es.

dagger Present address: Departamento de Bioquímica, Facultad de Medicina de la UAM e Instituto de Investigaciones Biomédicas, 28029 Madrid, Spain.

Journal of Bacteriology, November 1999, p. 6958-6968, Vol. 181, No. 22
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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