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.

Histone-like nucleoid-structuring (H-NS) proteins are key regulators in gene expression silencing and in nucleoid compaction. The H-NS family member proteins MvaU in Pseudomonas aeruginosa are thought to bind the same AT-rich regions of chromosomes and function to coordinate the control of a common set of genes. Here, we explored the molecular mechanism by which MvaU controls PCA biosynthesis in P. aeruginosa PA1201. We present evidence suggesting that MvaU is self-regulated. Deletion of mvaU significantly increased PCA production, and PCA production sharply decreased when mvaU was over-expressed. MvaU transcriptionally repressed phz2 cluster expression and consequently reduced PCA biosynthesis. ß-galactosidase assays confirmed that base pairing near the -35 box is required when MvaU regulates PCA production in PA1201. Electrophoretic mobility shift assays (EMSA) and additional point mutation analysis demonstrated that MvaU directly bound to an AT-rich motif within the promoter of the phz2 cluster. Chromatin immunoprecipitation (ChIP) analysis also indicated that MvaU directly bound to the P5 region of the phz2 cluster promoter. MvaU repression of PCA biosynthesis was independent of QscR and OxyR in PA1201 and neither PCA or H2O2 were the environmental signals that induced mvaU expression. These findings detail a new MvaU-dependent regulatory pathway of PCA biosynthesis in PA1201 and provide a foundation to increase PCA fermentation titer by genetic engineering.

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

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.

The Anti-activator QslA Negatively Regulates Phenazine-1-Carboxylic Acid Biosynthesis by Interacting With the Quorum Sensing Regulator MvfR in the Rhizobacterium Pseudomonas aeruginosa Strain PA1201.

Two almost identical gene clusters (phz1 and phz2) are responsible for phenazine-1-carboxylic acid (PCA) production in Pseudomonas aeruginosa (P. aeruginosa) strain MSH (derived from strain PA1201). Here, we showed that the anti-activator QslA negatively regulated PCA biosynthesis and phz1 expression in strain PA1201 but had little effect on phz2 expression. This downregulation was mediated by a 56-bp region within the 5'-untranslated region (5'-UTR) of the phz1 promoter and was independent of LasR and RsaL signaling. QslA also negatively regulated Pseudomonas quinolone signal (PQS) production. Indeed, QslA controlled the PQS threshold concentration needed for PQS-dependent PCA biosynthesis. The quorum sensing regulator MvfR was required for the QslA-dependent inhibition of PCA production. We identified a direct protein-protein interaction between QslA and MvfR. The ligand-binding domain of MvfR (residues 123-306) was involved in this interaction. Our results suggested that MvfR bound directly to the promoter of the phz1 cluster. QslA interaction with MvfR prevented the binding of MvfR to the phz1 promoter regions. Thus, this study depicted a new mechanism by which QslA controls PCA and PQS biosynthesis in P. aeruginosa.

Characterization of the multiple molecular mechanisms underlying RsaL control of phenazine-1-carboxylic acid biosynthesis in the rhizosphere bacterium Pseudomonas aeruginosa PA1201.

Phenazines are important secondary metabolites that have been found to affect a broad spectrum of organisms. Two almost identical gene clusters phz1 and phz2 are responsible for phenazines biosynthesis in the rhizobacterium Pseudomonas aeruginosa PA1201. Here, we show that the transcriptional regulator RsaL is a potent repressor of phenazine-1-carboxylic acid (PCA) biosynthesis. RsaL negatively regulates phz1 expression and positively regulates phz2 expression via multiple mechanisms. First, RsaL binds to a 25-bp DNA region within the phz1 promoter to directly repress phz1 expression. Second, RsaL indirectly regulates the expression of both phz clusters by decreasing the activity of the las and pqs quorum sensing (QS) systems, and by promoting the rhl QS system. Finally, RsaL represses phz1 expression through the downstream transcriptional regulator CdpR. RsaL directly binds to the promoter region of cdpR to positively regulate its expression, and subsequently CdpR regulates phz1 expression in a negative manner. We also show that RsaL represents a new mechanism for the turnover of the QS signal molecule N-3-oxododecanoyl-homoserine lactone (3-oxo-C12-HSL). Overall, this study elucidates RsaL control of phenazines biosynthesis and indicates that a PA1201 strain harboring deletions in both the rsaL and cdpR genes could be used to improve the industrial production of PCA.