This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bottiglieri, M. Articles by Keel, C. Search for Related Content PubMed PubMed Citation Articles by Bottiglieri, M. Articles by Keel, C. Agricola Articles by Bottiglieri, M. Articles by Keel, C.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, January 2006, p. 418-427, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.418-427.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Characterization of PhlG, a Hydrolase That Specifically Degrades the Antifungal Compound 2,4-Diacetylphloroglucinol in the Biocontrol Agent Pseudomonas fluorescens CHA0

Mélanie Bottiglieri and Christoph Keel^*

Department of Fundamental Microbiology, University of Lausanne, CH-1015 Lausanne, Switzerland

Received 21 July 2005/ Accepted 12 October 2005

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative phlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a phlA phlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a k_cat of 33 s^–1 and a K_m of 140 µM at 30°C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.

---

* Corresponding author. Mailing address: Département de Microbiologie Fondamentale, Btiment de Biologie, Université de Lausanne, CH-1015 Lausanne-Dorigny, Switzerland. Phone: 41 21 692 56 36. Fax: 41 21 692 56 05. E-mail: christoph.keel{at}unil.ch

---

Applied and Environmental Microbiology, January 2006, p. 418-427, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.418-427.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Moynihan, J. A., Morrissey, J. P., Coppoolse, E. R., Stiekema, W. J., O'Gara, F., Boyd, E. F. (2009). Evolutionary History of the phl Gene Cluster in the Plant-Associated Bacterium Pseudomonas fluorescens. Appl. Environ. Microbiol. 75: 2122-2131 [Abstract] [Full Text]