RsaL is a self-regulatory switch that controls alternative biosynthesis of two AHL-type quorum sensing signals in Pseudomonas aeruginosa PA1201.

Pseudomonas aeruginosa is a ubiquitous and metabolically versatile microorganism naturally found in soil and water. It is also an opportunistic pathogen in plants, insects, animals, and humans. In response to increasing cell density, P. aeruginosa uses two acyl-homoserine lactone (AHL) quorum-sensing (QS) signals (i.e., N-3-oxo-dodecanoyl homoserine lactone [3-oxo-C12-HSL] and N-butanoyl-homoserine lactone [C4-HSL]), which regulate the expression of hundreds of genes. However, how the biosynthesis of these two QS signals is coordinated remains unknown. We studied the regulation of these two QS signals in the rhizosphere strain PA1201. PA1201 sequentially produced 3-oxo-C12-HSL and C4-HSL at the early and late growth stages, respectively. The highest 3-oxo-C12-HSL-dependent elastase activity was observed at the early stage, while the highest C4-HSL-dependent rhamnolipid production was observed at the late stage. The atypical regulator RsaL played a pivotal role in coordinating 3-oxo-C12-HSL and C4-HSL biosynthesis and QS-associated virulence. RsaL repressed lasI transcription by binding the -10 and -35 boxes of the lasI promoter. In contrast, RsaL activated rhlI transcription by binding the region encoding the 5'-untranslated region of the rhlI mRNA. Further, RsaL repressed its own expression by binding a nucleotide motif located in the -35 box of the rsaL promoter. Thus, RsaL acts as a molecular switch that coordinates the sequential biosynthesis of AHL QS signals and differential virulence in PA1201. Finally, C4-HSL activation by RsaL was independent of the Las and Pseudomonas quinolone signal (PQS) QS signaling systems. Therefore, we propose a new model of the QS regulatory network in PA1201, in which RsaL represents a superior player acting at the top of the hierarchy.

Fructose promotes pyoluteorin biosynthesis via the CbrAB-CrcZ-Hfq/Crc pathway in the biocontrol strain Pseudomonas PA1201.

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.

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.

Phenazine-1-carboxamide (PCN) produced by multifarious Pseudomonas strains represents a promising candidate as a new metabolite pesticide due to its broad-spectrum antifungal activity and capacity to induce systemic resistance in plants. The rice rhizosphere Pseudomonas strain PA1201 contains two reiterated gene clusters, phz1 and phz2, for phenazine-1-carboxylic acid (PCA) biosynthesis; PCA is further converted into PCN by this strain using a functional phzH-encoding glutamine aminotransferase. However, PCN levels in PA1201 constitute approximately one-fifth of PCA levels and the optimal temperature for PCN synthesis is 28 °C. In this study, the phzH open reading frame (ORF) and promoter region were investigated and reannotated. phzH promoter PphzH was found to be a weak promoter, and PhzH levels were not sufficient to convert all of the native PCA into PCN. Following RNA Seq and promoter-lacZ fusion analyses, a strong quorum sensing (QS)- and thermo-regulated promoter PrhlI was identified and characterized. The activity of PphzH is approximately 1% of PrhlI in PA1201. After three rounds of promoter editing and swapping by PrhlI, a new PCN-overproducing strain UP46 was generated. The optimal fermentation temperature for PCN biosynthesis in UP46 was increased from 28 to 37 °C and the PCN fermentation titer increased 179.5-fold, reaching 14.1 g/L, the highest ever reported.

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.

Coenzyme Q biosynthesis in the biopesticide Shenqinmycin-producing Pseudomonas aeruginosa strain M18.

Coenzyme Q (ubiquinone) is a redox-active isoprenylated benzoquinone commonly found in living organisms. The biosynthetic pathway for this lipid has been extensively studied in Escherichia coli and Saccharomyces cerevisiae; however, little is known in Pseudomonas aeruginosa. In this study, we observed that CoQ9 is the predominant coenzyme Q synthesized by the Shenqinmycin-producing strain M18. BLASTP and domain organization analyses identified 15 putative genes for CoQ biosynthesis in M18. The roles of 5 of these genes were genetically and biochemically investigated. PAM18_4662 encodes a nonaprenyl diphosphate synthase (Nds) and determines the number of isoprenoid units of CoQ9 in M18. PAM18_0636 (coq7PA) and PAM18_5179 (ubiJPA) are essential for aerobic growth and CoQ9 biosynthesis. Deletion of ubiJPA, ubiBPA and ubiKPA led to reduced CoQ biosynthesis and an accumulation of the CoQ9 biosynthetic intermediate 3-nonaprenylphenol (NPP). Moreover, we also provide evidence that the truncated UbiJPA interacts with UbiBPA and UbiKPA to affect CoQ9 biosynthesis by forming a regulatory complex. The genetic diversity of coenzyme Q biosynthesis may provide targets for the future design of specific drugs to prevent P. aeruginosa-related infections.

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.