Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Sat, 10 Nov 2018 22:40:16 Influence of fusaric acid on phenazine-1- carboxamide synthesis and gene expression of Pseudomonas chlororaphis strain PCL1391 E. Tjeerd van Rij,3 Genevie ` ve Girard,3 Ben J. J. Lugtenberg and Guido V. Bloemberg Correspondence Guido V. Bloemberg bloemberg@rulbim.leidenuniv.nl Leiden University, Institute of Biology, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Received 23 March 2005 Revised 27 May 2005 Accepted 6 June 2005 Production of the antifungal metabolite phenazine-1-carboxamide (PCN) by Pseudomonas chlororaphis strain PCL1391 is essential for the suppression of tomato foot and root rot caused by the soil-borne fungus F. oxysporum f. sp. radicis-lycopersici. The authors have shown previously that fusaric acid (FA), a phytotoxin produced by Fusarium oxysporum, represses the production of PCN and of the quorum-sensing signal N-hexanoyl-L-homoserine lactone (C 6 -HSL). Here they report that PCN repression by FA is maintained even during PCN-stimulating environmental conditions such as additional phenylalanine, additional ferric iron and a low Mg 2+ concentration. Constitutive expression of phzI or phzR increases the production of C 6 -HSL and abolishes the repression of PCN production by FA. Transcriptome analysis using P. chlororaphis PCL1391 microarrays showed that FA represses expression of the phenazine biosynthetic operon (phzABCDEFGH) and of the quorum-sensing regulatory genes phzI and phzR. FA does not alter expression of the PCN regulators gacS, rpoS and psrA. In conclusion, reduction of PCN levels by FA is due to direct or indirect repression of phzR and phzI. Microarray analyses identified genes of which the expression is strongly influenced by FA. Genes highly upregulated by FA are also upregulated by iron starvation in Pseudomonas aeruginosa. This remarkable overlap in the expression profile suggests an overlapping stress response to FA and iron starvation. INTRODUCTION Pseudomonas chlororaphis strain PCL1391 gives excellent biocontrol of tomato foot and root rot, a disease caused by the fungus Fusarium oxysporum f. sp. radicis-lycopersici (Chin-A-Woeng et al., 1998). In the tomato rhizosphere PCL1391 colonizes the same niche as occupied by F. oxy- sporum hyphae, and closely interacts with F. oxysporum by attaching to, and forming micro-colonies on, hyphae (Bolwerk et al., 2003). Secretion of toxic and growth- inhibiting compounds by both the fungus and the bacterial biocontrol strain is part of the complex interaction between fungi and bacteria. PCL1391 produces the antifungal metabolite phenazine-1- carboxamide (PCN), which inhibits hyphal growth (Chin- A-Woeng et al., 1998). Using a PCN biosynthetic mutant it was shown that PCN is essential for the biocontrol ability of PCL1391 (Chin-A-Woeng et al., 1998). PCN production is regulated by the quorum-sensing regulatory genes phzI and phzR, which are homologous to luxI and luxR (Chin- A-Woeng et al., 2001b). In addition, biosynthesis of PCN is dependent on the two-component regulatory system formed by GacS and GacA and is regulated by PsrA and the stationary-phase sigma factor encoded by rpoS (Chin-A- Woeng et al., 2005; G. Girard, unpublished). Plant-pathogenic and non-pathogenic Fusarium spp. pro- duce fusaric acid (FA; 5-butylpicolinic acid) (Bacon et al., 1996; Notz et al., 2002; Schouten et al., 2004). FA is toxic for eukaryotes and prokaryotes (Bochner et al., 1980; Wang & Ng, 1999). In addition, FA was shown to be involved in fungal defence against Pseudomonas spp. biocontrol strains by repressing the production of antifungal metabolites. FA represses the production of 2,4-diacetylphloroglucinol in Pseudomonas fluorescens CHA0 (Duffy & De ´fago, 1997) and the synthesis of PCN in P. chlororaphis PCL1391 (van Rij et al., 2004). The latter repression is correlated with a reduction of the level of the auto-inducer N-hexanoyl-L- homoserine lactone (C 6 -HSL) (van Rij et al., 2004). 3These authors contributed equally to this paper. Abbreviations: HSL, homoserine lactone; C 6 -HSL, N-hexanoyl-L- homoserine lactone; FA, fusaric acid; PCN, phenazine-1-carboxamide. Supplementary microarray data are available with the online version of this paper. 0002-8063 G 2005 SGM Printed in Great Britain 2805 Microbiology (2005), 151, 2805–2814 DOI 10.1099/mic.0.28063-0