Characterization of an N-acyl-L-homoserine lactone-mediated regulatory system controlling phenazine biosynthesis in Pseudomonas aureofaciens 30-84: In vitro and in situ analysis

Abstract

Pseudomonas aureofaciens 30-84 is a soilborne bacterium that colonizes the wheat rhizosphere. This strain produces three phenazine antibiotics which are responsible for both suppression of take-all disease of wheat caused by Gaeumannomyces graminis var. tritici and enhanced survival of 30-84 within the wheat rhizosphere in competition with other organisms. A gene (phzR) was identified just prior to the start of this work that is required for phenazine production by 30-84. PhzR was identified as a positive regulator of the phenazine biosynthetic operon. During the course of this dissertation it was discovered that PhzR belongs to the LuxR family of N-acyl- scL-homoserine lactone-responsive transcriptional regulators and that phenazine production in P. aureofaciens 30-84 is mediated by a diffusible signal molecule. The gene responsible for production of this signal (phzI) was identified. Both phzI and phzR are required for the production of phenazines in vitro. Together these two proteins (PhzR/PhzI) comprise a N-acyl- scL-homoserine lactone (AHL) response system that controls phenazine antibiotic production in P. aureofaciens 30-84. Classic AHL-mediated regulatory systems consist of two proteins, a LuxR homolog (PhzR) which transcriptionally activates target gene expression in response to AHL produced by the second protein, the LuxI homolog (PhzI). Using HPLC coupled with high resolution mass spectroscopy, the specific AHL produced via PhzI has been identified as N-hexanoyl- scL-homoserine lactone (HHL). It has been determined that PhzR activates phenazine production in conjunction with HHL produced by PhzI via transcriptional activation of the phenazine biosynthetic gene phzB. A variety of synthetic AHLs restore transcription of phzB and phenazine production in phzI mutants suggesting that phzI mutants can be used to detect the presence of exogenous AHLs. This ability was exploited to show that HHL is required for phenazine expression in situ and is an effective interpopulation signal molecule in the wheat rhizosphere. The work presented in this dissertation is the first to show that AHL-mediated regulation, previously only examined in vitro, can operate within the natural habitat of a bacterium.