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

Abstract

Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. 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 (C6-HSL). The two other autoinducers that were produced comigrate with N-butanoyl-L-homoserine lactone (C4-HSL) and N-octanoyl-L-homoserine lactone (C8-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 C6-HSL, but not by C4-HSL, C8-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 C6-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 C4-HSL or C8-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.

Regulatory roles of psrA and rpoS in phenazine-1-carboxamide synthesis by Pseudomonas chlororaphis PCL1391

“…Under various growth conditions the amounts of C 6 -HSL present in PCL1391 spent culture medium were shown to be correlated with the amounts of PCN produced (Chin-A-Woeng et al, 2001bvan Rij et al, 2004). In our study, the correlation of PCN and C 6 -HSL levels among the various PCL1391 derivatives (Fig.…”

“…Sequencing showed that this fragment shared high homologies (see Results) with rpoS genes of other Pseudomonas strains. Subsequently, primers oMP770 and oMP771 were designed based on the partial rpoS sequence and used in PCR on a pBlueScript chromosomal library of PCL1391 (Chin-A-Woeng et al, 2001b) in combination with oMP49 and oMP50, which anneal close to the multi-cloning site of pBlueScript. oMP770 in combination with oMP49 produced a fragment of 1?2 kb containing the flanking region upstream of rpoS.…”

“…In Pseudomonas chlororaphis PCL1391, the production of the antifungal metabolite phenazine-1-carboxamide (PCN) (Chin-A-Woeng et al, 2003) is synthesized through expression of the biosynthetic phzABCDEFGH operon (Chin-A-Woeng et al, 1998). Previous work led to a model of regulation of PCN production involving three different groups of genes: the phzI/ phzR quorum-sensing system (Chin-A-Woeng et al, 2001b), gacS/gacA (global antibiotic and cyanide control), and the regulatory psrA gene (Pseudomonas sigma regulator) (Chin-A-Woeng et al, 2005).…”

“…The phzI gene is responsible for the synthesis of autoinducers, of which N-hexanoyl-L-homoserine lactone (C 6 -HSL) is the main product (Chin-A-Woeng et al, 2001b). C 6 -HSL is believed to bind to PhzR, thereby activating it.…”

The ppuI-rsaL-ppuR quorum-sensing system regulates cellular motility, pectate lyase activity, and virulence in potato opportunistic pathogen Pseudomonas sp. StFLB209

“…11) When AHL concentrations increase and reach a threshold, AHL receptor proteins belonging to the LuxR protein family bind AHL and regulate the expression of numerous genes. AHL-mediated quorum sensing regulates various phenotypes in plants, including the production of exoenzymes 12) and antimicrobial substances, 13) motility, 14−15) biofilm formation, 16) epiphytic fitness, 17) and pathogenicity. 12,18) Although many AHL-producing bacteria have been isolated from the phyllosphere, 4−7) the role of quorum sensing in adaptation to survival on the plant surface and its contribution to shaping certain community structures has not yet been elucidated.…”