Biocontrol strain Pseudomonas PA1201 produces pyoluteorin (Plt), which is an antimicrobial secondary metabolite. Plt represents a promising candidate pesticide due to its broad-spectrum antifungal and antibacterial activity. Although PA1201 contains a complete genetic cluster for Plt biosynthesis, it fails to produce detectable level of Plt when grown in media typically used for Pseudomonas strains. In this study, minimum medium (MM) was found to favor Plt biosynthesis. Using the medium M, which contains all the salts of MM medium except for mannitol, as a basal medium, we compared 10 carbon sources for their ability to promote Plt biosynthesis. Fructose, mannitol, and glycerol promoted Plt biosynthesis, with fructose being the most effective carbon source. Glucose or succinic acid had no significant effect on Plt biosynthesis, but effectively antagonized fructose-dependent synthesis of Plt. Promoter-lacZ fusion reporter strains demonstrated that fructose acted through activation of the pltLABCDEFG (pltL) operon but had no effect on other genes of plt gene cluster; glucose or succinic acid antagonized fructose-dependent pltL induction. Mechanistically, fructose-mediated Plt synthesis involved carbon catabolism repression. The two-component system CbrA/CbrB and small RNA catabolite repression control Z (crcZ) were essential for fructose-induced Plt synthesis. The small RNA binding protein Hfq and Crc negatively regulated fructose-induced Plt. Taken together, this study provides a new model of fructose-dependent Plt production in PA1201 that can help improve Plt yield by biosynthetic approaches.
Phenazine-1-carboxylic acid (PCA) is the primary active component in the newly registered, commercial biopesticide Shenqinmycin and is produced during fermentation by the engineered rhizobacterium strain Pseudomonas PA1201. Both phz1 and phz2 gene clusters contribute to PCA biosynthesis. In this study, we evaluated the role of OxyR in the regulation of PCA biosynthesis in PA1201. We first showed a functional link between oxyR expression and PCA biosynthesis. Deletion of oxyR and overexpression of oxyR both increase PCA biosynthesis. The molecular mechanisms underlying OxyR regulation of PCA production were investigated using several approaches. OxyR acts divergently in phz1 and phz2. Overexpression of oxyR activated the expression of phz1 and phz1-dependent PCA production. However, overexpression of oxyR had little effect on phz2-dependent PCA biosynthesis, while deletion of oxyR promoted phz2-dependent PCA production and exerted a negative effect on phz2 expression. Further, OxyR directly bound to the phz2 promoter region. In addition, the regulation of PCA biosynthesis by OxyR was associated with quorum sensing (QS) systems. Overexpression of OxyR positively regulated pqs QS system. Finally, transcriptomic analysis and subsequent genetic analysis revealed the small RNA phrS plays a key role in OxyR-dependent PCA accumulation. Specifically, OxyR directly binds to the phrS promoter region to positively regulate phrS expression wherein PhrS regulates the PCA positive regulator MvfR in order to control PCA biosynthesis.