Overexpression of oxyR Increases Phenazine-1-Carboxylic Acid Biosynthesis via Small RNA phrS in the Rhizobacterium Strain Pseudomonas PA1201

Abstract

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.

H-NS family protein MvaU downregulates phenazine-1-carboxylic acid (PCA) biosynthesis via binding to an AT-rich region within the promoter of the phz2 gene cluster in the rhizobacterium Pseudomonas strain PA1201

“…OxyR is a LysR-type transcriptional regulator that is involved in H 2 O 2 sensing and the regulation of antioxidant defense systems in many bacterial species [ 33 ]. In our previous study, the deletion or overexpression of oxyR in PA1201 led to significant increases in PCA biosynthesis [ 28 ]. To investigate the role of OxyR in MvaU-dependent repression of PCA biosynthesis, it was deleted in strains MSH and ΔmvaU.…”

“…Phenazines are one of the most important metabolites produced by P. aeruginosa and play diverse roles in cellular growth, development, and virulence. The regulation of the phenazine PYO in P. aeruginosa has been thoroughly studied in the clinically isolated PAO1 strain [ 19 , 20 , 28 , 29 , [34] , [35] , [36] , [37] , [38] ] and both MvaT and MvaU have been demonstrated to negatively regulate PYO production in PAO1 [ 30 ]. Both mvaT and mvaU are present in PA1201.…”

“…The LysR-type transcriptional regulator OxyR is involved in H 2 O 2 sensing and the regulation of antioxidant defense systems in many bacterial species [ 33 ]. In PA1201, deletion or overexpression of oxyR was shown to increase PCA biosynthesis [ 28 ]. QscR is a third AHL-responsive LuxR homologue and regulates global quorum sensing gene expression and PYO biosynthesis in P. aeruginosa [ 26 ].…”

“…OxyR is a LysR-family transcriptional regulator and is responsible for H 2 O 2 tolerance in PA1201. Overexpression of oxyR increases PCA biosynthesis via regulation by the small RNA phrS [ 28 ]. QslA is an anti-activator, which negatively regulates PCA biosynthesis by interacting with the QS regulator MvfR in PA1201 [ 29 ].…”

Identification of a Strong Quorum Sensing- and Thermo-Regulated Promoter for the Biosynthesis of a New Metabolite Pesticide Phenazine-1-carboxamide in Pseudomonas strain PA1201

“…Two duplicated gene clusters, phzA 1 B 1 C 1 D 1 E 1 F 1 G 1 ( phz1 ) and phzA 2 B 2 C 2 D 2 E 2 F 2 G 2 ( phz2 ), are responsible for PCA biosynthesis in both Pseudomonas strains . Over the last 10 years, PCA biosynthesis in M18 or PA1201 has been shown to be regulated by an untranslated region (UTR) in the phz1 promoter region, by three quorum sensing (QS) systems (LasI/LasR, RhlI/RhlR, and PQS/PQSR), and by the transcription factors RsaL, QslA, and OxyR. − On the basis of these findings, at least five rounds of genetic engineering have been conducted in both PA1201 and M18, and the PCA fermentation titer in the engineered strain PA-IV was reported to reach 9.8 g/L . PCA was commercially rebranded as Shenqinmycin in 2005.…”