Research ArticleMicrobiology

A direct screen for c-di-GMP modulators reveals a Salmonella Typhimurium periplasmic ʟ-arginine–sensing pathway

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Sci. Signal.  09 Jun 2015:
Vol. 8, Issue 380, pp. ra57
DOI: 10.1126/scisignal.aaa1796

Monitoring bacterial signaling

Certain environments trigger bacteria to form aggregates called biofilms, which contribute to antibiotic resistance of human pathogens. In Salmonella, increasing the second-messenger cyclic-di-GMP (c-di-GMP) reduces motility and promotes biofilm formation. Mills et al. expressed a biosensor for c-di-GMP in Salmonella Typhimurium and used flow cytometry to identify compounds that altered c-di-GMP concentration. Of the compounds tested, ʟ-arginine produced the greatest response at the lowest concentrations. Cellulose synthesis is a c-di-GMP–dependent process required for biofilm formation. Compounds that increased c-di-GMP concentration enhanced cellulose synthesis, whereas compounds that reduced c-di-GMP inhibited cellulose synthesis. This biosensor–flow cytometry screening method should aid in identifying compounds that inhibit bacterial c-di-GMP production and, therefore, reduce biofilm formation, and in exploring the pathways through which bacteria respond to signals in the environment.


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.

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