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要旨:
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 Forster resonance energy transfer and microfluidic measurements for 16 hybrid chemoreceptors that combine the periplasmic ligand binding domains of P. aeruginosa receptors and the cytoplasmic signaling domain of the Escherichia coli Tar receptor. Direct binding of putative ligands to chemoreceptors was further confirmed using thermal shift assay and microcalorimetry. Altogether, the combination of methods enabled us to assign several new attractants, including methyl 4-aminobutyrate, 5-aminovalerate, L-ornithine, 2-phenylethylamine, and tyramine, to previously characterized chemoreceptors and to annotate a novel purine-specific receptor PctP. Responses of hybrid receptors to changes in pH further revealed a complex bidirectional pH sensing mechanism in P. aeruginosa, which involves at least four chemoreceptors PctA, PctC, TlpQ, and PctP. Our screening strategy could be applied for the systematic characterization of unknown sensory domains in a wide range of bacterial species. IMPORTANCE Chemotaxis of motile bacteria has multiple physiological functions. It enables bacteria to locate optimal ecological niches, mediates collective behaviors, and can play an important role in infection. These multiple functions largely depend on ligand specificities of chemoreceptors, and the number and identities of chemoreceptors show high diversity between organisms. Similar diversity is observed for the spectra of chemoeffectors, which include not only chemicals of high metabolic value but also bacterial, plant, and animal signaling molecules. However, the systematic identification of chemoeffectors and their mapping to specific chemoreceptors remains a challenge. Here, we combined several in vivo and in vitro approaches to establish a systematic screening strategy for the identification of receptor ligands and we applied it to identify a number of new physiologically relevant chemoeffectors for the important opportunistic human pathogen P. aeruginosa. This strategy can be equally applicable to map specificities of sensory domains from a wide variety of receptor types and bacteria.
