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Applied and Environmental Microbiology, November 2007, p. 7443-7455, Vol. 73, No. 22
0099-2240/07/$08.00+0     doi:10.1128/AEM.01354-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

N-(3-Hydroxyhexanoyl)-L-Homoserine Lactone Is the Biologically Relevant Quormone That Regulates the phz Operon of Pseudomonas chlororaphis Strain 30-84

Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801,¹ Department of Pharmacology, Program in Biomolecular Structure, The University of Colorado at Denver Health Sciences Center, Aurora, Colorado 80045,² Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan³

Received 18 June 2007/ Accepted 24 September 2007

Phenazine production by Pseudomonas fluorescens 2-79 and P. chlororaphis isolates 30-84 and PCL1391 is regulated by quorum sensing through the activator PhzR and acyl-homoserine lactones (acyl-HSLs) synthesized by PhzI. PhzI from P. fluorescens 2-79 produces five acyl-HSLs that include four 3-hydroxy species. Of these, N-(3-hydroxyhexanoyl)-HSL is the biologically relevant ligand for PhzR. The quorum-sensing systems of P. chlororaphis strains 30-84 and PCL1391 have been reported to produce and respond to N-(hexanoyl)-HSL. These differences were of interest since PhzI and PhzR of strain 2-79 share almost 90% sequence identity with orthologs from strains 30-84 and PCL1391. In this study, as assessed by thin-layer chromatography, the three strains produce almost identical complements of acyl-HSLs. The major species produced by P. chlororaphis 30-84 were identified by mass spectrometry as 3-OH-acyl-HSLs with chain lengths of 6, 8, and 10 carbons. Heterologous bacteria expressing cloned phzI from strain 30-84 produced the four 3-OH acyl-HSLs in amounts similar to those seen for the wild type. Strain 30-84, but not strain 2-79, also produced N-(butanoyl)-HSL. A second acyl-HSL synthase of strain 30-84, CsaI, is responsible for the synthesis of this short-chain signal. Strain 30-84 accumulated N-(3-OH-hexanoyl)-HSL to the highest levels, more than 100-fold greater than that of N-(hexanoyl)-HSL. In titration assays, PhzR_30-84 responded to both N-(3-OH-hexanoyl)- and N-(hexanoyl)-HSL with equal sensitivities. However, only the 3-OH-hexanoyl signal is produced by strain 30-84 at levels high enough to activate PhzR. We conclude that strains 2-79, 30-84, and PCL1391 use N-(3-OH-hexanoyl)-HSL to activate PhzR.

Published ahead of print on 5 October 2007.

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