Characterization of the GbdR regulon in Pseudomonas aeruginosa

Pseudomonas aeruginosa displays tremendous metabolic diversity, controlled in part by the abundance of transcription regulators in the genome. We have been investigating P. aeruginosa’s response to the host, particularly changes regulated by the host-derived quaternary amines choline and glycine betaine (GB). We previously identified GbdR as an AraC-family transcription factor that directly regulates choline acquisition from host phospholipids (via binding to plcH and pchP promoters), is required for catabolism of the choline metabolite GB, and is an activator that induces transcription in response to GB or dimethylglycine. Our goal was to characterize the GbdR regulon in P. aeruginosa using genetics and chemical biology in combination with transcriptomics and in vitro DNA-binding assays. Here we show that GbdR activation regulates transcription of 26 genes from 12 promoters; 11 of which have measureable binding to GbdR in vitro. The GbdR regulon includes the genes encoding GB, dimethylglycine, sarcosine, glycine, and serine catabolic enzymes, and the BetX and CbcXWV quaternary amine transport proteins. . Additionally, identification of two uncharacterized regulon members suggests roles for these proteins in response to choline metabolites. The conditions for inclusion in the gbdR regulon were: (i) called present in the array, (ii) changed more than 2.5-fold in signal in a statistically significant manner, (iii) altered in all three 'inducing' conditions (ethylcholine, choline+propargylcholine, and choline in the dgcA mutant), but (iv) not induced in the wild-type choline or diethylcholine treatments. Wild type and dgcA mutant cells were pre-grown in MOPS minimal media and then split and resuspended in MOPS pyruvate, MOPS pyruvate + choline, MOPS pyruvate + ethylcholine, MOPS pyruvate + diethylcholine, or MOPS pyruvate & choline + propargylcholine.