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The chemotaxis network, one of the most prominent prokaryotic sensory systems, is present in most motile bacteria and archaea. Although the conserved signaling core of this network is well characterized, ligand specificities of a large majority of diverse chemoreceptors encoded in bacterial genomes remain unknown. Here, we performed a systematic identification and characterization of new chemoeffectors for the opportunistic pathogen Pseudomonas aeruginosa, which has 26 chemoreceptors possessing most of the common types of ligand binding domains. By performing capillary chemotaxis assays for a library of growth-promoting compounds, we first identified a number of novel chemoattractants of varying strength. We subsequently mapped specificities of these ligands by performing Förster resonance energy transfer and microfluidic measurements for 16 hybrid chemoreceptors that combine the …