Activation of the phz Operon of Pseudomonas fluorescens 2-79 Requires the LuxR Homolog PhzR, N-(3-OH-Hexanoyl)-l-Homoserine Lactone Produced by the LuxI Homolog PhzI, and a cis-Acting phz Box

Many pseudomonads, including the generalist Pseudomonas aeruginosa and fluorescent soil forms, such as Pseudomonas fluorescens, Pseudomonas aureofaciens, and Pseudomonas chlororaphis, produce phenazine-1-carboxylic acid (PCA) or its derivatives (7, 11, 49). These secondary metabolites exhibit antimicrobial activity against a wide range of prokaryotic and eukaryotic microbes, and the synthesis and secretion of these compounds is thought to confer an advantage on the producer bacteria when in competition with other microflora in the soil habitat (29). Production of substituted phenazines is also required by these bacteria to fully protect plants against diseases caused by fungal and bacterial pathogens that are susceptible to the antibiotics (2, 7, 9, 33, 49). Such biocontrol properties are of considerable interest, since their application in the field could reduce reliance on the use of environmentally toxic antimicrobial chemicals. However, for such strategies to be successful, the antibiotics must be synthesized reliably in the appropriate environmental sites. Hence, it is important to identify the genetic determinants required for synthesis of the phenazines, as well as the mechanisms by which expression of these genes is regulated. The phz cluster is organized as an operon of seven genes, the products of which catalyze the synthesis of chorismate and its conversion to PCA (9, 27, 28, 35). This parent compound can be substituted at several positions, depending upon the producer strain, and the genes coding for the enzymes involved in such substitutions are restricted to those strains producing the modified compounds. While gene nomenclature differs with the isolate (for an example, see reference 27), the order of the seven genes and the amino acid sequences of their products are conserved in all phz operons examined to date (9, 27, 28, 35). Although regulation is complex and multifactorial, the phz operons of P. aureofaciens 30-84 and P. chlororaphis PCL1391 are controlled at the transcriptional level by a ligand-dependent transcription factor called PhzR (8, 34). This activator is a member of the LuxR family and, as such, requires a low-molecular-weight signal, an acyl-homoserine lactone (acyl-HSL), for activity (8, 56). Signal production in both pseudomonads is catalyzed by PhzI, an acyl-HSL synthase of the LuxI family (8, 56). The acyl-HSL most probably transits out of and back into the cell by diffusion and thus accumulates within the environment as the population size of the dividing bacteria increases. When the concentration of the signal reaches a critical level, which corresponds to a particular population size of the bacteria, it interacts with and activates PhzR, thus initiating expression of the phz operon. In P. aureofaciens 30-84 and P. chlororaphis PCL1391, phzR and phzI are adjacent and convergently oriented, with phzR being located just upstream of and in opposite orientation to the phz operon (8, 56). Similarly, the phz operon of P. fluorescens 2-79 is preceded by phzR and phzI homologs organized in identical manners (Fig. ​(Fig.1).1). Moreover, the three phzR genes and the three phzI genes, as well as their encoded protein products, are all strongly orthologous (8). FIG. 1. Gene structure of the phz locus from P. fluorescens 2-79 and its mutants. (A) The organizations of the phz operon and the two regulatory genes phzR and phzI are shown by arrows. Alterations in the three mutants—strain 2-79IR, in which phzR and ... Strain 2-79 has served as a model for the genetics, biochemistry, and enzymology of phenazine biosynthesis. We previously reported that this isolate produces at least six acyl-HSLs, including the 3-hydroxy forms, N-(3-hydroxy-hexanoyl)-l-homoserine lactone (3-OH-C6-HSL), N-(3-hydroxy-octanoyl)-l-homoserine lactone (3-OH-C8-HSL), and N-(3-hydroxy-decanoyl)-l-homoserine lactone (3-OH-C10-HSL); the alkanoyl forms hexanoyl-homoserine lactone (C6-HSL) and octanoyl-homoserine lactone (C8-HSL); and an active signal compound of unknown structure (45). However, P. aureofaciens 30-84 and P. chlororaphis PCL1391 both have been reported to produce and utilize the alkanoyl signal, C6-HSL (8, 57). Moreover, the last two strains have not been reported to produce 3-hydroxy-acyl-HSLs. Given the very close amino acid sequence relatedness of the PhzI and PhzR proteins from these three strains, it became important to determine what acyl-HSL signals are produced by PhzI of P. fluorescens 2-79 and which of these signals activates PhzR of this strain. In this report, we show that PhzI catalyzes synthesis of all of the 3-OH- and alkanoyl-acyl-HSLs produced by P. fluorescens 2-79. We also show that 3-OH-C6 accumulates to the highest concentration in culture supernatants and that PhzR from strain 2-79 responds with greatest sensitivity to this signal. In addition, we show that activation of the phz operon by PhzR is dependent upon an intact inverted repeat, the phz box, located in the divergent phzA-phzR promoter region and that PhzR weakly autoactivates its own transcription, also in a phz box-dependent manner.