Heme is an essential metabolite for most life on earth. Bacterial pathogensalmost universally require iron to infect a host, often acquiring this nutrient inthe form of heme. The Gram-negative pathogen Pseudomonas aeruginosa is noexception, where heme acquisition and metabolism are known to be crucial for bothchronic and acute infections. To unveil unknown genes and pathways that couldplay a role with heme metabolic flux in this pathogen, we devised an omic-basedapproach we dubbed “Met-Seq,” for metabolite-coupled transposon sequencing.Met-Seq couples a biosensor with fluorescence-activated cell sorting (FACS) andmassively parallel sequencing, allowing for direct identification of genes associatedwith metabolic changes. In this work, we first construct and validate a heme biosensorfor use with P. aeruginosa and exploit Met-Seq to identify 188 genes that potentiallyinfluence intracellular heme levels. Identified genes largely consisted of metabolicpathways not previously associated with heme, including many secretedvirulence effectors, as well as 11 predicted small RNAs (sRNAs) and riboswitcheswhose functions are not currently understood. We verify that five Met-Seq hits affectintracellular heme levels; a predicted extracytoplasmic function (ECF) factor, a phospholipidacquisition system, heme biosynthesis regulator Dnr, and two predicted antibioticmonooxygenase (ABM) domains of unknown function (PA0709 and PA3390).Finally, we demonstrate that PA0709 and PA3390 are novel heme-binding proteins.Our data suggest that Met-Seq could be extrapolated to other biological systemsand metabolites for which there is an available biosensor, and provides a new templatefor further exploration of iron/heme regulation and metabolism in P. aeruginosaand other pathogens.