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Phenazine-1-Carboxamide Production in the Biocontrol Strain Pseudomonas chlororaphis PCL1391 Is Regulated by Multiple Factors Secreted into the Growth Medium

¹Leiden University, Institute of Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands; ²Department of Biomolecular Mass Spectrometry, Faculty of Chemistry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands; ³Michael Barber Centre for Mass Spectrometry, Department of Chemistry, University of Science and Technology in Manchester (UMIST), P.O. Box 88, Manchester M60 1QD, U.K.

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. The production of phenazine-1-carboxamide (PCN) is crucial for this biocontrol activity. In vitro production of PCN is observed only at high-population densities, suggesting that production is under the regulation of quorum sensing. The main autoinducer molecule produced by PCL1391 was identified structurally as N-hexanoyl-l-homoserine lactone (C_6^-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-l-homoserine lactone (C_4^-HSL) and N-octanoyl-l-homoserine lactone (C_8^-HSL). Two PCL1391 mutants lacking production of PCN were defective in the genes phzI and phzR, respectively, the nucleotide sequences of which were determined completely. Production of PCN by the phzI mutant could be complemented by the addition of exogenous synthetic C_6^-HSL, but not by C_4^-HSL, C_8^-HSL, or any other HSL tested. Expression analyses of Tn5luxAB reporter strains of phzI, phzR, and the phz biosynthetic operon clearly showed that phzI expression and PCN production is regulated by C_6^-HSL in a population density-dependent manner. The introduction of multiple copies of the regulatory genes phzI and phzR on various plasmids resulted in an increase of the production of HSLs, expression of the PCN biosynthetic operon, and consequently, PCN production, up to a sixfold increase in a copy-dependent manner. Surprisingly, our expression studies show that an additional, yet unidentified factor(s), which are neither PCN nor C_4^-HSL or C_8^-HSL, secreted into the growth medium of the overnight cultures, is involved in the positive regulation of phzI, and is able to induce PCN biosynthesis at low cell densities in a growing culture, resulting in an increase of PCN production.

Additional keywords: biofungicide, luxI, phenazine, plant growth-promoting rhizobacteria, signal molecules.