Pseudomonas aeruginosa gbdR gene is transcribed from a s 54-dependent promoter under the control of NtrC / CbrB , IHF and BetI Diego Germ

Pseudomonasaeruginosa uses choline as a source of carbon and nitrogen, and also for the synthesis of glycine betaine, an osmoprotectant under stress conditions such as drought and salinity. The transcription factor GbdR is the specific regulator of choline metabolism and it belongs to the Arac/XylS family of transcriptional regulators. Despite the link between choline catabolism and bacterial pathogenicity, gbdR regulation has not been explored in detail. In the present work, we describe how gbdR transcription can be initiated from a s-dependent promoter. gbdR transcription can be activated by NtrC in the absence of a preferential nitrogen source, by CbrB in the absence of a preferential carbon source, and by the integration host factor favouring DNA bending. In addition, we found that BetI negatively regulates gbdR expression in the absence of choline. We identified two overlapping BetI binding sites in the gbdR promoter sequence, providing an additional example of s -promoter down-regulation. Based on our findings, we propose a model for gdbR regulation and its impact on choline metabolism. INTRODUCTION Pseudomonas aeruginosa is found in a wide range of environments, from hyperosmotic dry soils to aqueous hyposmotic media. It is an opportunistic pathogen with high nutritional versatility that is controlled by numerous transcriptional regulators (nearly 9% of its genome) [1]. P. aeruginosa uses many substrates as carbon (C) and nitrogen (N) sources, including amino acids, polyamines, agmatine and quaternary amines, such as choline (cho) [2–6]. In addition to its use as a source of C and N, cho is the main source for the synthesis and accumulation of glycine betaine (GB), an osmoprotectant under stress caused by drought and salinity. Most of the available cho is present in macromolecules such as phosphatidylcholine, a major component of the plasma membrane of eukaryotic cells and the mammalian pulmonary surfactant [7, 8], and acetylcholine, which is abundant in the cornea [9]. Several studies performed on this issue have suggested a relationship between cho metabolism and virulence factors in P. aeruginosa [4, 6, 10–12] and P. syringae [13]. P. aeruginosa synthesizes several enzymes, such as hemolytic phospholipase C (PlcH), phosphorylcholine phosphatase (PchP) and cholinesterase (ChoE), to acquire cho from phosphatidylcholine, phosphocholine and acetylcholine, respectively [14–18]. The uptake of cho in P. aeruginosa occurs through various specific and non-specific transporter systems [16, 19]. When cho enters the cell, it is first converted – via a two-step oxidation process – into GB by the action of BetAB enzymes. betA and betB genes encode choline dehydrogenase and betaine aldehyde dehydrogenase, respectively. GB can be accumulated as an osmoprotectant, or it can be metabolized to obtain C and/or N [20]. GB, dimethylglycine (DMG) or cho itself induce the synthesis of cho catabolic enzymes in the absence of other C and/or N preferential sources [6, 21]. Furthermore, the enzymes involved in the initial steps of cho conversion into GB (BetA–BetB) are inducible at the transcriptional level by cho, but not by osmotic stress [22]. The betA and betB genes, together with betI, belong to the betIBA operon [22]. BetI, a 21.8 kDa protein that is a transcriptional repressor of Received 3 March 2017; Accepted 16 June 2017 Author affiliations: Department of Molecular Biology, FCEFQN, National University of Río Cuarto, Route 36-Km 601, 5800, Argentina; Laboratory of Phagocytosis and Intracellular Transport, School of Medicine, University of Cuyo, IHEM-CONICET, Mendoza 5500, Argentina. *Correspondence: Diego Germ an S anchez, dgsanch@yahoo.com. ar