Engineering NIR-sighted bacteria
- Department of Biochemistry, University of Bayreuth, Germany
- Department of Biological and Environmental Science, Nanoscience Center, University of Jyvaskyla, Finland
- Institute of Biochemistry, Graz University of Technology, Austria
- Department of Microbiology, University of Bayreuth, Germany
- Bayreuth Center for Biochemistry and Molecular Biology, Universität Bayreuth, Germany
- North-Bavarian NMR Center, Universität Bayreuth, Germany
Figures
Figure 1 with 1 supplement
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 8.0 in darkness (black line), and after illumination with near-infrared (NIR, purple line), red (red), and blue light (blue). Data are normalized by 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 Figure 1—figure supplement 1a–c. (i) Dark-recovery kinetics at 25°C and pH 8.0 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.
Figure 1—figure supplement 1
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 by 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.
Figure 2 with 4 supplements
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 (gray: darkness; purple: NIR light; red: red light). DsRed reporter fluorescence was normalized by the optical density of the bacterial cultures. Individual pNIRusk variants are denoted by the relative lengths of the linkers within their underlying SHKs (see Figure 2—figure supplement 2), with the variants chosen for further analyses highlighted in bold. As control, an empty-vector construct (MCS) is shown, and data are normalized by the reference fluorescence value determined for the Av+17a variant. (c) Flow-cytometric analyses of bacteria containing AcNIRusk-1a (top) or AvNIRusk+17a (bottom) upon incubation in darkness (black), blue (blue, 70 µW cm–2), red (red, 100 µW cm–2), or NIR light (purple, 200 µW cm–2). 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. Data in panels b, d, and e represent mean ± s.d. of three biologically independent samples.
Figure 2—figure supplement 1
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.
Figure 2—figure supplement 2
PATCHY library generation for AcNIRusk and AvNIRusk.
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 Supplementary file 1A.
Figure 2—figure supplement 3
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.
Figure 2—figure supplement 4
Additional characterization of the AcNIRusk and AvNIRusk circuits.
(a–b) Analyses by flow cytometry of DsRed production in 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. (c) Growth kinetics of bacteria bearing AcNIRusk-DsRed (circles) or empty-vector controls (MCS, squares) in darkness (gray) or NIR light (purple). (d) As in (c) but for AvNIRusk. (e) DsRed production induced by NIR light in bacteria containing AcNIRusk (light purple circles) or AvNIRusk (dark green circles) over time. The background fluorescence of bacteria carrying the empty-vector control is indicated by the dashed line. Fluorescence readings are normalized as in Figure 2b. Data were fitted by logistic functions. Data in panels c-e represent mean ± s.d. of three biologically independent samples.
Figure 3 with 2 supplements
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 (gray), NIR (purple), and red light (red) (bottom). An empty-vector control is denoted as ‘MCS’, and data are normalized as in Figure 2. Variants are marked by the relative lengths of the linkers within their underlying SHKs compared to pREDusk (see Figure 3—figure supplement 1). 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 bathy-BphP PCM from Agrobacterium vitis. (c) Multiplexing of bacteria containing two optogenetic circuits, as noted below the graphs, 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, in the case of DsRed, normalized as in Figure 2. YPet fluorescence was normalized by the reference value determined for DmDERusk-YPet under red light (this panel). Data in panels a-c represent mean ± s.d. of three biologically independent samples.
Figure 3—figure supplement 1
PATCHY library generation for DmTtr-REDusk and AvTod-NIRusk.
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, an Eco32I or KspAI restriction site and a frameshift were inserted. Underlined amino acids highlighted derive from a cloning artifact. PATCHY primers are listed in Supplementary file 1A.
Figure 3—figure supplement 2
Additional 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 as in Figure 2. (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 as in Figure 3. Data in panels a-e represent mean ± s.d. of three biologically independent samples. (f) The schematic summarizes the results of the multiplexing experiments from Figure 3c. The production of DsRed and YPet under different light conditions is shown by pink pentagons and green heptagons, respectively.
Figure 4 with 2 supplements
Light-regulated gene expression in Escherichia coli Nissle (EcN) and Agrobacterium tumefaciens.
(a) Light-dose response of EcN harboring AvNIRusk after incubation at varying NIR- (purple circles), red- (red squares), and blue-light (blue pentagons) intensities. Corresponding data for AcNIRusk are shown in Figure 4—figure supplement 1. (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 Figure 4—figure supplement 2a). (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 Figure 4—figure supplement 2c and d). Data in panels a and d represent mean ± s.d. of three biologically independent samples.
Figure 4—figure supplement 1
Gene expression regulated by AcNIRusk in Escherichia coli Nissle.
Light-regulated gene expression in E. Nissle (EcN) containing AcNIRusk after incubation at varying NIR-light (purple circles), red-light (red squares), and blue-light (blue pentagons) intensities. Data represent mean ± s.d. of three biologically independent samples.
Figure 4—figure supplement 2
Gene expression regulated by AvNIRlux and DmREDlux in Agrobacterium tumefaciens.
(a) Quantified luminescence from the spotting assay in Figure 4c. 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 (gray) or under NIR light (purple). The vertical black line denotes a time point of 20 h on which the data in Figure 4d are based. (d) As for (c) but for A. tumefaciens bacteria containing DmREDlux and illumination with red instead of NIR light (red). Data in panels a-d represent mean ± s.d. of at least three biologically independent samples.
Figure 5 with 1 supplement
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) chromophores. 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.
Figure 5—figure supplement 1
Molecular environment of the bilin chromophore in bathy-bacteriophytochromes.
Biliverdin binding pocket in the photosensory core modules (PCM) of (a) Azorhizobium caulinodans bacteriophytochrome (BphP), (b) Agrobacterium vitis BphP, (c) Pseudomonas aeruginosa 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.
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/107069/elife-107069-mdarchecklist1-v1.pdf
-
Supplementary file 1
Information on oligonucleotide primers (A) and plasmids (B) used in this study.
- https://cdn.elifesciences.org/articles/107069/elife-107069-supp1-v1.pdf
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Engineering NIR-sighted bacteria
eLife 14:RP107069.
https://doi.org/10.7554/eLife.107069.3
