Traits of Bathy Phytochromes and Application to Bacterial Optogenetics

Abstract

Phytochromes are photoreceptors sensitive to red and far-red light, found in a wide variety of organisms, including plants, fungi, and bacteria. Bacteriophytochromes (BphPs) can be switched between a red light-sensitive Pr state and a far-red light-sensitive Pfr state by illumination. In so-called prototypical BphPs, the Pr state functions as the thermally favored resting state, whereas Pfr is more stable in bathy BphPs. The prototypical DrBphP from Deinococcus radiodurans has been shown to be compatible with different output module types. Even though red light-regulated optogenetic tools are available, like the pREDusk system based on the DrBphP photosensory module, far-red light-modulated variants are still rare. Here, we study the underlying contributors to bathy over prototypical BphP behavior by way of various chimeric constructs between pREDusk and representative bathy BphPs. We pinpoint shared traits of the otherwise heterogeneous subgroup of bathy BphPs and highlight the importance of the sensor-effector linker in light modulation of histidine kinase activity. Informed by these data, we introduce the far-red light-activated system "pFREDusk", based on a histidine kinase activity governed by a bathy photosensory module. With this tool, we expand the optogenetic toolbox into wavelengths of increased sample and tissue penetration.

Keywords: histidine kinase; optogenetics; photoreceptor; phytochrome; protein chimera; two-component system.

1

Structure of bacteriophytochrome histidine kinases. (A) Structural model of a bacteriophytochrome with a histidine kinase (HK) output module. The structural model is assembled from the cryo-EM structure of DrBphP in the Pr state (PDB code: 8AVW) and the HK module crystal structure of TM0853 (PDB code: 2C2A). (B-C) Structure of the DrBphP PHY-tongue (PTG) in its Pr (B, PDB code: 8AVV) and Pfr state (C, PDB code: 8AVX). Selected residues and biliverdin chromophore (BV, orange) are indicated (Trp451 and Arg466 are part of the WAG and PRXSF motifs, respectively).

2

Features conserved more strictly in bathy phytochromes than in BphPs in general. Comparison of PHY-tongue lengths in representative bona fide BphPs counted from WAG–5 (DrBphP R446) to PRXSF+8 (DrBphP R477). Residues are colored according to Jalview sequence ID (hues of blue correspond to ≥84%, ≥68%, ≥40%, <40% residue conservation) based on an alignment of 404 sequences, numbering corresponds to DrBphP. HWE-HKs are marked with *, bathy BphPs in orange. PRXSF–8 glycine and PRXSF–3 arginine are marked with arrows. XccBphP, though initially classified as “bathy-like”, is also considered a bathy BphP due to the full Pfr resting state of its PSM truncation. See Table S1 for a list of represented BphPs, and supplementary files for the full alignment and details on all sequences.

3

DrF1 bathy chimaeras and their absorption spectra. (A) Schematic presentation of DrF1 chimeras containing structural elements from three different bathy BphPs. Origins of the derived domain exchanges are color coded as indicated, see Table S2 for domain boundary definitions. (C) Absorption spectra of DrF1 chimaeras where the PHY-tongue (PTG) was exchanged for the corresponding bathy sequence. (D) DrF1 chimaeras where the entire bathy photosensory module (PSM) was introduced.

4

Histidine kinase activity analysis. (A) In vivo HK activity assay, where DsRed fluorescence (normalized to DrF1, dark) corresponds to net kinase activity of the DrF1 variant. Results are shown as mean ± SD of three biological repeats, see Figure S7A for individual biological repeats. pREDusk with the DsRed reporter gene replaced by a multiple cloning site functions as negative control (−). (B) Phos-tag analysis of DrF1 variant activity in dark (D), in red light (R), or in far-red light (F). In the assay, phosphorylated BjFixJ (p-BjFixJ) migrates more slowly than the unphosphorylated one. See Figure S7B for full gels.

5

PaPSM linker length variants and PaF1. (A) Linker length comparison of DrF1 and PaPSM linker deletions and PATCHY products. Colored according to Jalview sequence ID (hues of blue correspond to ≥84%, ≥68%, ≥40%, <40% residue conservation), numbering corresponds to DrBphP. (B) In vivo HK activity assay visualized by DsRed fluorescence, normalized to DrF1 (dark). Results are shown as mean ± SD of three biological repeats, see Figure S9B for individual biological repeats. pREDusk with the DsRed gene replaced by a multiple cloning site functions as negative control (−). (C) Phos-tag analysis of the PaF1 variant activity in dark (D) or in far-red light (F). Phosphorylated BjFixJ (p-BjFixJ) migrates more slowly in comparison. See Figure S9C for full gels. (D) Spectral properties of PaF1 and PaF1₊₃₄, both of which appear bathy.

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pFREDusk performance. (A) Schematic representation describing cellular pREDusk function (red light-repressed) compared to pFREDusk function (far-red light-activated). (B) PaF1-mediated in vivo HK activity. DsRed fluorescence is shown as mean ± SD of three biological repeats, normalized to KanR in far-red. See Figure S10B for individual biological repeats. pREDusk with the DsRed gene replaced by a multiple cloning site functions as negative control (−). (C) DsRed production in bacteria harboring pFREDusk versions with different antibiotic resistance in response to varied far-red light (800 nm) intensities, normalized to KanR endpoint. See Figure S10C for three biological repeats and Figure S10D for pFREDusk-KanR response to red light. Light intensities are averaged over the duty cycle as marked by angled brackets.