Phenotypic and transcriptional analysis of secondary metabolites production in Pseudomonas corrugata CFBP5454

Abstract

Natural products have received a great deal of attention as sustainable alternative for management of plant diseases. In this study the potential and actual arsenal of secondary antimicrobial metabolites of the bacterium Pseudomonas corrugata, an ubiquitous bacterium, was dissected by phenotypic assays, genome mining and transcriptional studies. Pseudomonas corrugata strain CFBP 5454 was shown to produce diffusible compounds that inhibit the growth of plant pathogenic fungi and bacteria and antifungal volatile compounds and to produce cyanide. Mutant derivatives in LuxR transcriptional regulators, i.e. P. corrugata GL2 (pcoR mutant) and GLRFIA (rfiA mutant) impaired in cyclic lipopeptide (CLP) production, showed a diffusible compound-mediated reduced activity, depending on the biocontrol strain, challenge microorganism and culture medium but clearly indicate the production of additional antimicrobial substances. PcoR is the cognate receptor of the N-acyl-homoserine lactones, synthetised by PcoI in P. corrugata quorum sensing (QS) system. The QS system also directly activates the transcriptional regulator gene rfiA. In order to define the complete regulon of PcoR and RfiA, deep sequencing of cDNA library (RNA-seq) was used to analyse the whole transcriptomes of GL2 (pcoR-) and GLRFIA (rfiA-) mutants at early stationary phase of growth on a minimal medium. Differential expression analysis between the Wt and the mutants showed that the CLP cormycin and corpeptins biosynthetic clusters as well as that of an additional putative bioactive peptide and the exopolisaccharide alginate are under the control of the PcoR-RfiA regulon. Quantitative Real-time PCR assays demonstrated that CLP genes are upregulated in minimal medium and plant growth conditions as compared to reach medium cultures, whereas alginate production and biosynthesis gene expression were upregulated in rich growth conditions. In addition this study shows that citronellol is able to inhibit the growth of plant pathogenic bacteria but also could interfere with Quorum sensing system mediated by medium chain AHL in the biosensor strain Chromobacterium violaceum and in P. corrugata with QS system and the expression of genes contributing to the production of CLPs.