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Applied and Environmental Microbiology, December 2002, p. 6371-6382, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.6371-6382.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of an N-Acylhomoserine Lactone-Dependent Quorum-Sensing System in Pseudomonas putida Strain IsoF

Anette Steidle,1 Marie Allesen-Holm,2 Kathrin Riedel,1 Gabriele Berg,3 Michael Givskov,2 Søren Molin,2 and Leo Eberl1*

Department of Microbiology, TUM, D-85350 Freising,1 Department of Microbiology, University of Rostock, D-18051 Rostock, Germany,3 Department of Microbiology, DTU, DK-2800 Lyngby, Denmark2

Received 5 June 2002/ Accepted 8 September 2002

Recent reports have shown that several strains of Pseudomonas putida produce N-acylhomoserine lactones (AHLs). These signal molecules enable bacteria to coordinately express certain phenotypic traits in a density-dependent manner in a process referred to as quorum sensing. In this study we have cloned a genomic region of the plant growth-promoting P. putida strain IsoF that, when present in trans, provoked induction of a bioluminescent AHL reporter plasmid. Sequence analysis identified a gene cluster consisting of four genes: ppuI and ppuR, whose predicted amino acid sequences are highly similar to proteins of the LuxI-LuxR family, an open reading frame (ORF) located in the intergenic region between ppuI and ppuR with significant homology to rsaL from Pseudomonas aeruginosa, and a gene, designated ppuA, present upstream of ppuR, the deduced amino acid sequence of which shows similarity to long-chain fatty acid coenzyme A ligases from various organisms. Using a transcriptional ppuA::luxAB fusion we demonstrate that expression of ppuA is AHL dependent. Furthermore, transcription of the AHL synthase ppuI is shown to be subject to quorum-sensing regulation, creating a positive feedback loop. Sequencing of the DNA regions flanking the ppu gene cluster indicated that the four genes form an island in the suhB-PA3819 intergenic region of the currently sequenced P. putida strain KT2440. Moreover, we provide evidence that the ppu genes are not present in other AHL-producing P. putida strains, indicating that this gene cluster is so far unique for strain IsoF. While the wild-type strain formed very homogenous biofilms, both a ppuI and a ppuA mutant formed structured biofilms with characteristic microcolonies and water-filled channels. These results suggest that the quorum-sensing system influences biofilm structural development.

* Corresponding author. Mailing address: Department of Microbiology, TUM, Am Hochanger 4, D-85350 Freising, Germany. Phone: 49 8161 715446. Fax: 49 8161 715475. E-mail: EBERL{at}mikro.biologie.tu-muenchen.de.

Applied and Environmental Microbiology, December 2002, p. 6371-6382, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.6371-6382.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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