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Applied and Environmental Microbiology, July 2009, p. 4753-4761, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.00575-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Diversity and Functional Analysis of LuxR-Type Transcriptional Regulators of Cyclic Lipopeptide Biosynthesis in Pseudomonas fluorescens ^,

I. de Bruijn and J. M. Raaijmakers^*

Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands

Received 10 March 2009/ Accepted 11 May 2009

Cyclic lipopeptides (CLPs) are produced by many Pseudomonas species and have several biological functions, including a role in surface motility, biofilm formation, virulence, and antimicrobial activity. This study focused on the diversity and role of LuxR-type transcriptional regulators in CLP biosynthesis in Pseudomonas species and, specifically, viscosin production by Pseudomonas fluorescens strain SBW25. Phylogenetic analyses showed that CLP biosynthesis genes in Pseudomonas strains are flanked by LuxR-type regulators that contain a DNA-binding helix-turn-helix domain but lack N-acylhomoserine lactone-binding or response regulator domains. For SBW25, site-directed mutagenesis of the genes coding for either of the two identified LuxR-type regulators, designated ViscAR and ViscBCR, strongly reduced transcript levels of the viscABC biosynthesis genes and resulted in a loss of viscosin production. Expression analyses further showed that a mutation in either viscAR or viscBCR did not substantially (change of <2.5-fold) affect transcription of the other regulator. Transformation of the viscAR mutant of SBW25 with a LuxR-type regulatory gene from P. fluorescens strain SS101 that produces massetolide, a CLP structurally related to viscosin, restored transcription of the viscABC genes and viscosin production. The results further showed that a functional viscAR gene was required for heterologous expression of the massetolide biosynthesis genes of strain SS101 in strain SBW25, leading to the production of both viscosin and massetolide. Collectively, these results indicate that the regulators flanking the CLP biosynthesis genes in Pseudomonas species represent a unique LuxR subfamily of proteins and that viscosin biosynthesis in P. fluorescens SBW25 is controlled by two LuxR-type transcriptional regulators.

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* Corresponding author. Mailing address: Laboratory of Phytopathology, Droevendaalse Steeg 1, 6708 PD Wageningen, The Netherlands. Phone: 31 317 483427. Fax: 31 317 483412. E-mail: jos.raaijmakers{at}wur.nl

Published ahead of print on 15 May 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, July 2009, p. 4753-4761, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.00575-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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