Activation of thephzOperon ofPseudomonas fluorescens2-79 Requires the LuxR Homolog PhzR,N-(3-OH-Hexanoyl)-l-Homoserine Lactone Produced by the LuxI Homolog PhzI, and acis-ActingphzBox

Abstract

The phz operon of Pseudomonas fluorescens 2-79, which produces phenazine-1-carboxylate, is preceded by two genes, phzR and phzI, that are homologs of quorum-sensing gene pairs of the luxR-luxI family. Deleting phzR and phzI from strain 2-79 led to loss of production of the antibiotics, as well as a suite of six acyl-homoserine lactones (acyl-HSLs) that includes four 3-hydroxy- derivatives and two alkanoyl-HSLs. Strain 2-79 accumulates N-(3-hydroxy-hexanoyl)-L-HSL to levels 20 and 30 times those of N-(hexanoyl)-L-HSL and N-(3-hydroxy-octanoyl)-HSL, the next most abundant species produced by this isolate. Expression of a clone of phzI in Escherichia coli and P. fluorescens 1855 resulted in the synthesis of all six acyl-HSLs. Maximal activation of phzA and phzR fused to lacZ and uidA reporters, respectively, required PhzR and the acyl-HSL signals. PhzR-mediated expression of the phzA::lacZ fusion responded with highest sensitivity and greatest magnitude to pure N-(3-hydroxy-hexanoyl)-L-HSL. When exposed to organic extracts of culture supernatants containing the six acyl-HSLs at their normal levels, the reporter responded strongly to N-(3-hydroxy-hexanoyl)-L-HSL but did not respond to any of the other five acyl-HSLs. The transcriptional start sites for the divergently oriented phzA and phzR genes were mapped by primer extension analysis. An 18-bp almost perfect inverted repeat, the phz box, is located between the phzI and phzR promoters. Disrupting this repeat abolished PhzR-dependent activation of phzA and phzR. We conclude that PhzI of strain 2-79 synthesizes 3-OH acyl-HSLs and that P. fluorescens 2-79 uses N-(3-hydroxy-hexanoyl)-HSL as its quorum-sensing signal. We also conclude that PhzR, with its quormone, activates expression of phzA and phzR and that this activation requires an intact phz box sequence located in the divergent promoter region.