Spectroscopic characterization of bacteriophytochrome photosensory core modules (PCM) from Azorhizobium caulinodans (AcPCM), Agrobacterium vitis (AvPCM), and Pseudomonas aeruginosa (PaPCM).

(a) UV/vis absorbance spectra of AcPCM at pH 8 in darkness (black line), and after illumination with near-infrared (NIR, purple line), red (red), and blue light (blue). Data are normalized to the absorbance at 403 nm within the Soret band. The lower panel shows the NIR-dark difference absorbance spectrum with maxima and minima marked. (b) and (c) as in (a) but for AvPCM and PaPCM. (d) Absorbance spectra of AcPCM in darkness (solid lines) and after NIR illumination (dashed lines) at different pH values as indicated by color. (e) and (f) as in (d) but for AvPCM and PaPCM. (g) Absorbance at 700 nm for AcPCM (light purple circles), AvPCM (dark green squares), and PaPCM (orange triangles) upon NIR-light exposure at different pH values. Data were fitted to the Henderson-Hasselbalch equation to determine apparent pKa values (dashed lines, AcPCM: 7.2 ± 0.1, AvPCM: 8.0 ± 0.1, PaPCM: 9.9 ± 0.2). (h) Calculated Pfr fractions of AcPCM (light purple circles), AvPCM (dark green square), and PaPCM (orange triangles) after red-light exposure at different pH values. The underlying absorbance spectra are shown in Suppl. Fig. 1a-c. (i) Dark-recovery kinetics at 25°C and pH 8 after NIR-illumination as followed by absorbance at 760 nm. Time constants for AcPCM (light purple), AvPCM (dark green), and PaPCM (orange) were determined by single-exponential fits.

Engineering of light-regulated sensor histidine kinases (SHK) based on photosensory core modules (PCM) from bathy-bacteriophytochromes (BphP).

(a) Schematic of the pNIRusk plasmids to control gene expression in bacteria by NIR light. The LacIq promoter constitutively expresses a tricistronic operon consisting of heme oxygenase (HO1), the light-regulated SHK (chimera of Ac/AvPCM and FixL), and the response regulator FixJ. When phosphorylated, FixJ binds to the FixK2 promoter and triggers the expression of a gene of interest (GOI). (b) Response of select AcNIRusk (left) and AvNIRusk (right) systems to different light conditions (grey: darkness; purple: NIR light; red: red light). DsRed reporter fluorescence was normalized to the optical density of the bacterial cultures. Individual pNIRusk variants are denoted by the relative lengths of the linkers within their underlying SHKs (see Suppl. Fig. 3), with the variants chosen for further analyses highlighted in bold. As control, an empty-vector construct (MCS) is shown, and data are normalized to the maximal fluorescence. (c) Flow-cytometric analyses of bacteria containing AcNIRusk-1a (top) or AvNIRusk+17a (bottom) upon incubation in darkness (black), moderate NIR (brown), or strong NIR light (purple) (also see Suppl. Fig. 5). An empty-vector control (MCS) is shown in green. (d) Light-dose response of bacteria containing AcNIRusk-1a incubated in NIR (purple circles), red (red squares), and blue light (blue pentagons). (e) as in (d) but for AvNIRusk+17a.

Engineering of orthogonal light-regulated two-component systems (TCS). (a) Schematic of the DmTtr-REDusk circuit based on the TtrSR TCS from Shewanella baltica combined with the photosensory core module (PCM) from the Deinococcus maricopensis bacteriophytochrome (BphP) (top).

Reporter fluorescence of bacteria bearing circuits with different linker lengths of their underlying sensor histidine kinases (SHK) upon cultivation in darkness (grey), NIR (purple), and red light (red) (bottom). An empty-vector control is denoted as ‘MCS’, and data are normalized to the maximal fluorescence. Variants are marked by the relative lengths of the linkers within their underlying SHKs compared to pREDusk (see Suppl. Fig. 6). Variants characterized further are highlighted in bold. (b) as in (a) but for the AvTod-NIRusk circuit based on the TodST TCS from Pseudomonas putida combined with the BphP PCM from Agrobacterium vitis. (c) Multiplexing of bacteria containing two optogenetic circuits, as noted below the graph, to separately control the expression of DsRed (top) and YPet (bottom) reporters. After incubation in darkness (gray), NIR (purple), red (red), and combined NIR and red light (red and purple stripes), fluorescence was measured and normalized to the maximal value.

Light-regulated gene expression in Escherichia coli Nissle (EcN) and Agrobacterium tumefaciens.

(a) Light-dose response of EcN harboring AcNIRusk (light purple circles) and AvNIRusk (dark green squares) after incubation at varying NIR-light intensities. (b) Schematic of the AvNIRlux and DmREDlux circuits based on AvPCM and DmPCM. The tricistronic cassette comprising heme oxygenase (HO1), the sensor histidine kinase (chimera of Av/DmPCM and FixL), and FixJ is controlled by the constitutive PmamDC promoter from Magnetospirillum gryphiswaldense, whereas the FixK2 promoter regulates the expression of the luxABCDE operon. (c) A. tumefaciens bacteria bearing AvNIRlux (left) and DmREDlux (right) were spotted on plates and incubated at different light conditions (darkness, NIR, red, or blue light), followed by luminescence (Lumi.) measurements (see Suppl. Fig. 8a). (d) A. tumefaciens bacteria carrying AvNIRlux and DmREDlux were incubated in liquid culture for 20 h under different light conditions (gray: darkness, purple: NIR light, red: red light), followed by recording of reporter luminescence (see Suppl. Fig. 8c-d).

Photoreception by select bathy-bacteriophytochromes (BphP) at different pH values.

Throughout the entire tested pH range, the photosensory core modules (PCM) of the bathy-BphPs from Azorhizobium caulinodans (AcPCM), Agrobacterium vitis (AvPCM), and Pseudomonas aeruginosa (PaPCM) adopt a pure Pfr state in darkness with fully protonated biliverdin (BV) chromophore. NIR light drives the complete conversion to the Pr state in which BV can be either protonated or deprotonated as governed by pH and the respective pKa value of the bathy-BphP. Red light populates a photostationary mixture with Pr and Pfr proportions strongly depending on pH. At high pH values, red light can drive the bathy-BphP nearly completely to the Pr state for AcPCM and AvPCM. Owing to a higher pKa value for BV protonation, PaPCM is less affected but follows the same trend.

Additional spectroscopic characterization of bacteriophytochrome photosensory core modules (PCM) from Azorhizobium caulinodans (AcPCM), Agrobacterium vitis (AvPCM), and Pseudomonas aeruginosa (PaPCM).

(a) Absorbance spectra of AcPCM at different pH values as indicated below the graph in darkness (solid line) and after red-light-illumination (dotted line). Data are normalized to the absorbance at 403 nm within the Soret band. (b) and (c) as in (a) but for AvPCM and PaPCM. (d) Dark-recovery kinetics of AcPCM (light purple circles), AvPCM (dark green circles), and PaPCM (orange circles) monitored at 760 nm, 25°C, and pH 8 after illumination with red light. Time constants determined by single-exponential fits are (147 ± 1) s, (71 ± 1) s and (599 ± 2) s for AcPCM, AvPCM, and PaPCM, respectively. (e) Absorbance spectra recorded in darkness (solid lines) and after dark recovery (dotted lines) for AcPCM (light purple), AvPCM (dark green), and PaPCM (orange) at pH 8. (f) Ratio of the quantum yields for Pr→Pfr over Pfr→Pr photoconversion as a function of pH. (g) Dark-recovery kinetics of AcPCM recorded at 760 nm after illuminating with NIR light at 25°C and different pH as indicated below the graph. Time constants were determined by single-exponential fits, with the results shown in the table. (h) and (i) as in (g) but for AvPCM and PaPCM. (j) Absorbance spectra of AcPCM recorded in darkness (solid lines) and after dark recovery (dotted lines) at different pH as indicated below the graph. (k) and (l) as in (j) but for AvPCM and PaPCM.

Multiple sequence alignment of the sensor histidine kinases within DmREDusk, AcNIRusk+0, and AvNIRusk+0.

The different shading marks the fusion of the photosensory core modules (PCM) of the bacteriophytochromes from Deinococcus maricopensis (DmPCM), Azorhizobium caulinodans (AcPCM), and Agrobacterium vitis (AvPCM) with the catalytic domains of the sensor histidine kinase FixL from Bradyrhizobium japonicum. The linker region is shown in pink, and the active-site histidine is marked with a red triangle.

AcNIRusk (a) and AvNIRusk (b) PATCHY start constructs (sc) and derived linker variants.

The linker between the Ac/AvPCM and FixL was adopted from the parental proteins and is defined as the region between the PCM (residues VLRHN) and the active-site histidine at position 291. At the junction, a KspAI restriction site and a frameshift were inserted. Underlined amino acids originate from a cloning artifact. PATCHY primers are listed in Suppl. Table 1.

Setup for illumination with near-infrared (NIR) light at different intensities in a microtiter-plate (MTP) format.

(a) The programmable 8-by-8 matrix of NIR light-emitting diodes (LED) is based on a previous setup (Hennemann et al., 2018; Stüven et al., 2019) and integrated into a 3D-printed adapter. NIR-light intensities can be adjusted by an Arduino microcontroller. (b) Emission spectrum of the NIR LED matrix with a maximum at 800 nm and a full width at half-maximum of 18 nm.

Analyses by flow cytometry of DsRed production of bacteria containing AcNIRusk (a) or AvNIRusk (b) when incubated in darkness (black), or under different NIR-light intensities.

An empty-vector control (MCS) is shown in green.

DmTtr-REDusk (a) and AvTod-NIRusk (b) PATCHY start constructs (sc) and derived linker variants.

The linker between the Dm/AvPCM and the TtrS/TodS two-component system was adopted from the natural proteins. At the junction, a Eco32I or KspAI restriction site and a frameshift were inserted. Underlined amino acids highlighted derive from a cloning artifact. PATCHY primers are listed in Suppl. Table 1.

Characterization of constructs used in multiplexing experiments.

(a) Schematic of DmTtr+7b (top) and associated light-dose response (bottom). (b) and (c) as in (a) but for AvTod+16 and AvTod+21. Data in panels a-c are normalized to the maximal fluorescence obtained for AvTod+21. (d) Schematic of DmDERusk-StrR-YPet (top) and the associated light-dose response (bottom). (e) as in (d) but for AvNIRusk-StrR-YPet. Data in panels d-f are normalized to the maximal fluorescence obtained for of AvNIRusk-StrR-YPet. (f) The schematic summarizes the results of the multiplexing experiments from Fig. 3c. The production of DsRed and YPet under different light conditions are shown by pink pentagons and green heptagons, respectively.

Gene expression regulated by AvNIRlux and DmREDlux in Agrobacterium tumefaciens.

(a) Quantified luminescence from the spotting assay in Fig. 4b. Bar colors represent incubation in darkness (gray), NIR (purple), red (red), or blue (blue) light. (b) Optical density at 650 nm of A. tumefaciens bacteria bearing AvNIRlux or DmREDlux grown in liquid culture under different light conditions. (c) Normalized reporter luminescence of the bacteria from panel (b) harboring AvNIRlux and grown in darkness (grey) or under NIR light (purple). The vertical black line denotes a time point of 20 h on which the data in Fig. 4d are based. (d) as for (c) but for A. tumefaciens bacteria containing DmREDlux and illumination with red instead of NIR light (red).

Biliverdin binding pocket in the photosensory core modules (PCM) of (a) Azorhizobium caulinodans bacteriophytochrome (BphP), (b) Agrobacterium vitis BphP, (c) Pseudomonas aerugionsa BphP, and (d) Agrobacterium tumefaciens Agp2 BphP.

Whereas experimental structures are available for the PCMs of AgP2 [PDB entry 6g1y (Schmidt et al., 2018)] and PaBphP [3c2w (Yang et al., 2008)], for AcBphP and AvBphP, PCM models were calculated with AlphaFold3 (Abramson et al., 2024). Tyrosine 190 and serine 275 are highlighted in the PaPCM structure by magenta color. The other three PCMs feature less polar phenylalanine and alanine residues, respectively, in the structurally equivalent positions.

Oligonucleotides used in this study. Overhang regions are indicated in bold, and restriction enzyme recognition sites are underlined.

Plasmids used in this study.