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

Phenazine production byPseudomonas fluorescens 2-79 andP. 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 fromP. 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 ofP. chlororaphis strains 30-84 and PCL1391 have been reported to produce and respond toN -(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 byP. 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 clonedphzI 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 producedN -(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 accumulatedN -(3-OH-hexanoyl)-HSL to the highest levels, more than 100-fold greater than that ofN -(hexanoyl)-HSL. In titration assays, PhzR30-84 responded to bothN -(3-OH-hexanoyl)- andN -(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 useN -(3-OH-hexanoyl)-HSL to activate PhzR.