New Orange Ligand-Dependent Fluorescent Reporter for Anaerobic Imaging

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Abstract
Bilin-binding fluorescent proteins like UnaG–bilirubin are noncovalent ligand-dependent reporters for oxygen-free microscopy but are restricted to blue and far-red fluorescence. Here we describe a high-throughput screening approach to provide a new UnaG–ligand pair that can be excited in the 532 nm green excitation microscopy channel. We identified a novel orange UnaG–ligand pair that maximally emits at 581 nm. Whereas the benzothiazole-based ligand itself is nominally fluorescent, the compound binds UnaG with high affinity (Kd = 3 nM) to induce a 2.5-fold fluorescence intensity enhancement and a 10 nm red shift. We demonstrated this pair in the anaerobic fluorescence microscopy of the prevalent gut bacterium Bacteroides thetaiotaomicron and in Escherichia coli. This UnaG–ligand pair can also be coupled to IFP2.0-biliverdin to differentiate cells in mixed-species two-color imaging. Our results demonstrate the versatility of the UnaG ligand-binding pocket and extend the ability to image cells at longer wavelengths in anoxic environments.
Funding Information
  • Division of Chemistry (CHE-1608553, CHE-1904759)
  • University of Michigan
  • Army Research Office (W911NF1810339)
  • Division of Graduate Education (DGE-1256260)

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