DdaR (PA1196) Regulates Expression of Dimethylarginine Dimethylaminohydrolase for the Metabolism of Methylarginines in Pseudomonas aeruginosa PAO1

Abstract

Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield L-citrulline and methylamines as products. DDAHs and their central roles in methylarginine metabolism have been characterized for eukaryotic cells. While DDAHs are known to exist in some bacteria, including Streptomyces coelicolor and Pseudomonas aeruginosa, the physiological importance and genetic regulation of bacterial DDAHs remain poorly understood. To provide some insight into bacterial methylarginine metabolism, this study focused on identifying the key elements or factors regulating DDAH expression in P. aeruginosa PAO1. First, results revealed that P. aeruginosa can utilize N G ,N Gdimethyl-L-arginine (ADMA) as a sole source of nitrogen but not carbon. Second, expression of the ddaH gene was observed to be induced in the presence of methylarginines, including N G -monomethyl-L-arginine (L-NMMA) and ADMA. Third, induction of the ddaH gene was shown to be achieved through a mechanism consisting of the putative enhancer-binding protein PA1196 and the alternative sigma factor RpoN. Both PA1196 and RpoN were essential for the expression of the ddaH gene in response to methylarginines. On the basis of the results of this study, PA1196 was given the name DdaR, for dimethylarginine dimethylaminohydrolase regulator. Interestingly, DdaR and its target ddaH gene are conserved only among P. aeruginosa strains, suggesting that this particular Pseudomonas species has evolved to utilize methylarginines from its environment.IMPORTANCE Methylated arginine residues are common constituents of eukaryotic proteins. During proteolysis, methylarginines are released in their free forms and become accessible nutrients for bacteria to utilize as growth substrates. In order to have a clearer and better understanding of this process, we explored methylarginine utilization in the metabolically versatile bacterium Pseudomonas aeruginosa PAO1. Our results show that the transcriptional regulator DdaR (PA1196) and the sigma factor RpoN positively regulate expression of dimethylarginine dimethylaminohydrolases (DDAHs) in response to exogenous methylarginines. DDAH is the central enzyme of methylarginine degradation, and its transcriptional regulation by DdaR-RpoN is expected to be conserved among P. aeruginosa strains.

RpoN-Dependent Direct Regulation of Quorum Sensing and the Type VI Secretion System in Pseudomonas aeruginosa PAO1

“…Taken together, the present results showed that RpoN directly regulates QS and T6SS by directly binding to promoters of lasI, rhlI, rhlA, rhlR, pqsA, pqsH, pqsR, hcpA, and hcpB. Previous studies show that RpoN affects the expression of nitrogenregulated genes (glnK-amtB, nirBD, nasA, nasST, and nosRZDFYL) (16) and interacts with EBPs to regulate diverse metabolic pathways, including glycine catabolism, (R)-3hydroxybutyrate catabolism, ethanolamine metabolism, methylarginine metabolism, flagellum biosynthesis, dicarboxylate transport, aromatic amino acid catabolism, and acetoin catabolism (24)(25)(26)(27)(28)(29) (Fig. 8).…”

“…The potential regulatory pathways and interplays of RpoN are proposed according to our observations and previous studies. RpoN affects the expression of nitrogen-regulated genes (glnK-amtB, nirBD, nasA, nasST, and nosRZDFYL) (16) and regulates dicarboxylates transport, amino acid catabolism, ethanolamine catabolism, (R)-3-hydroxybutyrate, methylarginines, and acetoin catabolism (24)(25)(26)(27)(28)(29). The typical QS systems, including the las, rhl, and pqs systems and their interactions, were summarized based on previous reports (8).…”

“…A group of the enhancer-binding proteins (EBPs) interact with RpoN to activate the transcription of target genes (see Table S1 in the supplemental material). For example, DdaR responds to aromatic amino acids (24). RpoN regulates the glyoxylate pathway and ethanolamine catabolism via EatR (25).…”

Metabolic Engineering of Pseudomonas putida for Fermentative Production of l-Theanine

“…P. aeruginosa PAO1 can convert methylarginines resulting in l -citrulline and methylamines by use of dimethylarginine dimethylaminohydrolases, coded by ddaH gene, which is induced in the presence of the substrate …”

The Regulatory Functions of σ54 Factor in Phytopathogenic Bacteria

“…Different stress-related signals regulate these EBPs through their N-terminal regulatory domains [ 39 ]. Two EBPs, DdaR and MifR, interact with the σ 54 factor to induce the transcription of dimethylarginine dimethylaminohydrolase and PA5530 genes by sensing aromatic amino acids and extracellular C 5 -dicarboxylates, respectively [ 40 , 41 ]. In addition, the σ 54 factor regulates the glyoxylate pathway, ethanolamine catabolism, ( R )-3-hydroxybutyrate, the glycine cleavage system, and pyocyanin biosynthesis via EatR, HbcR, and GcsR, respectively [ 42 , 43 , 44 , 45 ].…”