Research ArticleMicrobiology

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

See allHide authors and affiliations

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

You are currently viewing the abstract.

View Full Text

Log in to view the full text

Log in through your institution

Log in through your institution

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.

View Full Text