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GEMM-I riboswitch-based fluorescent biosensors for live cell analysis of cyclic dinucleotide signaling

  • Author(s): Wang, Xin
  • Advisor(s): Hammond, Ming C
  • et al.
Abstract

The bacterial second messenger cyclic di-GMP (c-di-GMP) holds a prominent position within the repertoire of bacterial signaling molecules. With its presence established in over 75% of sequence bacteria, c-di-GMP responds to primary environmental signals by affecting the bacterial lifestyle transition between the motile and sessile states, regulating biofilm formation, host colonization, and bacterial virulence. Over the past 30 years, many aspects of the c-di-GMP signaling pathway have become well characterized in no small part due to the plethora of tools that can quickly and conveniently detect c-di-GMP. Despite these triumphs, we have only begun to contend with the enormous scope of c-di-GMP signaling, an endeavor that would be greatly aided by new tools that are harder, better, faster, and stronger.

The four fluorescent biosensors introduced herein bring us towards that goal. These second-generation RNA biosensors were designed based on a natural c-di-GMP riboswitch aptamer fused to the Spinach dye-binding aptamer, producing a fluorescent signal upon c-di-GMP binding. Their speed, sensitivity, and selectivity secure their place as a valuable tool for studying c-di-GMP signaling, with demonstrated efficacy in monitoring c-di-GMP in vivo in E. coli in a variety of conditions, including anaerobic and zinc-exposed environments. Furthermore, these sensors were adapted towards studying the related cyclic dinucleotide cyclic GMP-AMP (cGAMP), a recently discovered second messenger known for roles in bacterial intestinal colonization and surface sensing. Both c-di-G and cGAMP-specific biosensors were used to uncover components of the cGAMP signaling pathway in organisms not previously known to have cGAMP signaling. It is envisioned that these biosensors can be used to further understand c-di-GMP and cGAMP signaling in a variety of organisms, in vivo, in real time.

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