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.

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 P phzH 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 P rhlI was identified and characterized. The activity of P phzH is approximately 1% of P rhlI in PA1201. After three rounds of promoter editing and swapping by P rhlI , a new PCNoverproducing 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.

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.

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.

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