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Title: CHARACTERIZATION OF A GENOMIC LOCUS REQUIRED FOR SYNTHESIS OF THE ANTIBIOTIC 2,4-DIACETYLPHLOROGLUCINOL BY THE BIOLOGICAL CONTROL AGENT PSEUDOMONAS FLUORESCENS Q2-87

Author
item BANGERA, M - WASHINGTON STATE UNIV
item Thomashow, Linda

Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/2/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: The antibiotic 2,4-diacetylphloroglucinol (Phl) is produced by many fluorescent Pseudomonas species that colonize and protect crop plants from root diseases caused by soilborne pathogens. Production of Phl requires the expression of biosynthetic genes that encode the enzymes that catalyze Phl synthesis, as well as regulatory genes that allow the biosynthetic genes to be expressed. Pseudomonas fluorescens Q2-87 produces Phl and protects wheat against take-all disease. In this report, a previously- cloned, 6.5-kb fragment of DNA from Q2-87 is shown to transfer the ability to produce Phl to 13 Phl-nonproducing Pseudomonas strains into which it was introduced, suggesting that the fragment contains biosynthetic genes encoding a complete Phl biosynthetic pathway. Characterization of the 6.5- kb fragment by transposon mutagenesis showed that at least 5 kb are required for Phl production, and that the DNA is expressed as at least two divergently transcribed units. Mutations within these units caused loss of Phl production but did not detectably alter other traits, supporting the conclusion that the genes encode biosynthetic enzymes, rather than regulatory proteins. Knowledge of the structure and function of these genes will aid in optimizing the use of strain Q2-87 as a biocontrol agent, and in developing novel recombinant agents into which the cloned genes have been introduced.

Technical Abstract: The antibiotic 2,4-diacetylphloroglucinol (Phl) is an important factor in the biological control by fluorescent Pseudomonas spp. of many soilborne diseases including take-all disease of wheat. A 6.5-kb genomic DNA fragment from Pseudomonas fluorescens Q2-87 conferred production of Phl and of a red pigment distinct from Phl, but which typically is present when Phl is produced, upon all of 13 Phl- nonproducing recipient Pseudomonas strains into which it was introduced. Larger fragments that included flanking DNA sequences did not transfer this capability, suggesting that they contain negative regulatory element(s). Analysis of the 6.5-kb fragment by Tn3HoHo1 mutagenesis further localized the sequences required for Phl production to a segment of approximately 5 kb and revealed the presence of at least two divergently oriented transcriptional units. Insertions within the smaller unit or within about 3 kb of the 5' end of the larger unit caused loss of production of both Phl and the red pigment. Other insertions within the distal 1.5 kb of the larger transcriptional unit abolished production of only the red pigment. Pleiotropic changes in secondary metabolism or colony morphology were not observed in Pseudomonas strains containing the 6.5-kb fragment, although some Phl- producing derivatives grew more slowly and gave rise to smaller colonies than did the wild-type parental strains. The size of the genomic region involved in Phl production, and the consistency and specificity with which these sequences transferred Phl biosynthetic capability, support the conclusion that the 6.5-kb fragment contains the Phl biosynthetic locus.