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Applied and Environmental Microbiology, September 2002, p. 4301-4306, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4301-4306.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Isolation of Antibiotics Turbomycin A and B from a Metagenomic Library of Soil Microbial DNA

Doreen E. Gillespie,1 Sean F. Brady,2 Alan D. Bettermann,1 Nicholas P. Cianciotto,3 Mark R. Liles,1 Michelle R. Rondon,1,{dagger} Jon Clardy,2 Robert M. Goodman,1 and Jo Handelsman1*

Department of Plant Pathology, University of Wisconsin—Madison, Madison, Wisconsin 53706,1 Department of Chemistry and Chemical Biology, Cornell University, Baker Lab, Ithaca, New York 14853,2 Department of Microbiology and Immunology, Northwestern University, Chicago, Illinois 606113

Received 8 February 2002/ Accepted 28 May 2002

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.

* Corresponding author. Mailing address: Department of Plant Pathology, University of Wisconsin—Madison, Madison, WI 53706. Phone: (608) 263-8783. Fax: (608) 262-8643. E-mail: joh{at}plantpath.wisc.edu.

{dagger} Present address: Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706.

Applied and Environmental Microbiology, September 2002, p. 4301-4306, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4301-4306.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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