title: A direct screen for c-di-GMP modulators reveals a Salmonella Typhimurium periplasmic ʟ-arginine-sensing pathway.
abstract: Cyclic-di-GMP (c-di-GMP) is a bacterial second messenger that transduces internal and external signals and regulates bacterial motility and biofilm formation. Some organisms encode more than 100 c-di-GMP-modulating enzymes, but only for a few has a signal been defined that modulates their activity. We developed and applied a high-throughput, real-time flow cytometry method that uses a fluorescence resonance energy transfer (FRET)-based biosensor of free c-di-GMP to screen for signals that modulate its concentration within Salmonella Typhimurium. We identified multiple compounds, including glucose, N-acetyl-d-glucosamine, salicylic acid, and ʟ-arginine, that modulated the FRET signal and therefore the free c-di-GMP concentration. By screening a library of mutants, we identified proteins required for the c-di-GMP response to each compound. Furthermore, low micromolar concentrations of ʟ-arginine induced a rapid translation-independent increase in c-di-GMP concentrations and c-di-GMP-dependent cellulose synthesis, responses that required the regulatory periplasmic domain of the diguanylate cyclase STM1987. ʟ-Arginine signaling also required the periplasmic putative ʟ-arginine-binding protein ArtI, implying that ʟ-arginine sensing occurred in the periplasm. Among the 20 commonly used amino acids, S. Typhimurium specifically responded to ʟ-arginine with an increase in c-di-GMP, suggesting that ʟ-arginine may serve as a signal during S. Typhimurium infection. Our results demonstrate that a second-messenger biosensor can be used to identify environmental signals and define pathways that alter microbial behavior.
authors.affiliation: Departments of Microbiology, Medicinal Chemistry, Genome Sciences, Immunology, and Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Erez
authors.initials: E
authors.lastname: Mills
authors.affiliation: Departments of Microbiology, Medicinal Chemistry, Genome Sciences, Immunology, and Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Erik
authors.initials: E
authors.lastname: Petersen
authors.affiliation: Departments of Microbiology, Medicinal Chemistry, Genome Sciences, Immunology, and Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Bridget R
authors.initials: BR
authors.lastname: Kulasekara
authors.affiliation: Departments of Microbiology, Medicinal Chemistry, Genome Sciences, Immunology, and Medicine, University of Washington, Seattle, WA 98195, USA. millersi@uw.edu.
authors.forename: Samuel I
authors.initials: SI
authors.lastname: Miller
mesh_terms: D000596:Amino Acids / Q000378:metabolism / Q000494:pharmacology; D001120:Arginine / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000096:biosynthesis; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D009154:Mutation; D019897:Periplasm / Q000378:metabolism*; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015290:Second Messenger Systems / Q000187:drug effects; D015398:Signal Transduction
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C062025:bis(3',5')-cyclic diguanylic acid; D002482:Cellulose; D001120:Arginine; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP