A high-throughput method for identifying novel genes that influence metabolic pathways reveals new iron and heme regulation in Pseudomonas aeruginosa

The ability to simultaneously, more directly correlate genes with metabolite levels on a global level would provide novel information for many biological platforms, yet has thus far been challenging. Here we describe a method to help address this problem, which we dub Met-Seq (Metabolite-coupled Tn-Sequencing). Met-Seq uses the powerful combination of fluorescent biosensors, fluorescence-activated cell sorting (FACS) and next generation sequencing (NGS) to rapidly identify genes that influence the levels of specific intracellular metabolites. For proof of concept we create and test a heme biosensor, and then exploit Met-Seq to identify novel genes involved in the regulation of heme in the pathogen Pseudomonas aeruginosa. Met-Seq-generated data were largely comprised of genes which have not previously been reported to influence heme levels in this pathogen, two of which we verify as novel heme binding proteins. As heme is a required metabolite for host infection in P. aeruginosa and most other pathogens, our studies provide a new list of targets for potential antimicrobial therapies and sheds additional light on the balance between infection, heme uptake and heme biosynthesis.