Light-mediated control of gene expression in the anoxygenic phototrophic bacterium Rhodobacter capsulatus using photocaged inducers

Abstract

Photocaged inducer molecules, especially photocaged isopropyl-β-d-1-thiogalactopyranoside (cIPTG), are well-established optochemical tools for light-regulated gene expression and have been intensively applied in Escherichia coli and other bacteria including Corynebacterium glutamicum, Pseudomonas putida or Bacillus subtilis. In this study, we aimed to implement a light-mediated on-switch for target gene expression in the facultative anoxygenic phototroph Rhodobacter capsulatus by using different cIPTG variants under both phototrophic and non-phototrophic cultivation conditions. We could demonstrate that especially 6-nitropiperonyl-(NP)-cIPTG can be applied for light-mediated induction of target gene expression in this facultative phototrophic bacterium. Furthermore, we successfully applied the optochemical approach to induce the intrinsic carotenoid biosynthesis to showcase engineering of a cellular function. Photocaged IPTG thus represents a light-responsive tool, which offers various promising properties suitable for future applications in biology and biotechnology including automated multi-factorial control of cellular functions as well as optimization of production processes.

Keywords: Rhodobacter capsulatus; caged compounds; light-controlled gene expression; optogenetics; purple non-sulfur photosynthetic bacteria.

FIGURE 1

Establishment of optochemical control over gene expression in R. capsulatus under non-phototrophic and phototrophic growth conditions. (A) Light-controlled expression of the reporter gene eyfp in R. capsulatus using photocaged IPTG (cIPTG; red circle with blue frames). Upon illumination with UV-A light (purple flash symbol), the protection group is cleaved off, the previously inactive IPTG molecule is released and induces LacI/P _tac -mediated eyfp expression. (B) For analyzing light-dependent control of gene expression, R. capsulatus cells were either cultivated under non-phototrophic conditions (i.e., aerobic or microaerobic conditions in Round Well Plates with filling volumes of 0.8 and 1.5 mL, respectively) or anaerobic, phototrophic growth conditions using NIR light LED as sole light source (red LED diodes; λ _max = 850 nm, small scale screw neck vials). (C) Photocaged IPTG variants NP-cIPTG, BC-cIPTG and BEC-cIPTG used in this study as well as the respective cleavage sites addressed by photolysis and subsequent enzymatic hydrolysis (purple circles).

FIGURE 2

Application of cIPTG derivatives for light-mediated control of gene expression in R. capsulatus under non-phototrophic conditions. Light-controlled eyfp reporter gene expression and corresponding cell densities in aerobically (A,B) and microaerobically (C,D) grown R. capsulatus SB1003 cultures carrying pRholHi-2-eYFP using the three cIPTG derivatives NP-, BC- and BEC-cIPTG. Biomass-normalized eYFP in vivo fluorescence (λ _ex = 508 nm, λ _em = 532 nm) and cell growth represented by the scattered light intensity (λ = 620 nm) of cultures supplemented with 1 mm of each cIPTG variant is shown in relation to a control culture (1 mm IPTG) after 48 h of cultivation (RCV medium, 30°C; for aerobic cultures 800 rpm and 800 µL filling volume and for microaerobic cultures 400 rpm and 1,500 µL filling volume). Induction of reporter gene expression was performed after 9 h via UV-A light exposure at 365 nm (∼1 mW/cm²) for 30 min or the addition of 1 mm IPTG. (B,D). Values are means of individual biological triplicates. Error bars indicate the respective standard deviations.

FIGURE 3

Application of cIPTG derivatives for light-controlled induction of gene expression in phototrophically grown R. capsulatus cells. Light-controlled eyfp reporter gene expression (A) and corresponding cell densities (B) of phototrophically grown R. capsulatus SB1003 cells carrying pRholHi-2-eYFP using the cIPTG derivatives NP-, BC- and BEC-cIPTG. Biomass-normalized eYFP in vivo fluorescence (λ _ex = 508 nm, λ _em = 532 nm) and cell growth represented by the scattered light intensity of cultures supplemented with 1 mm of each cIPTG variant are shown in relation to a control culture (1 mm IPTG) after 48 h of cultivation [30°C, screw neck vials, constant NIR light illumination (λ _max = 850 nm, 1.7 mW/cm²)]. Induction of reporter gene expression was performed after 6 h via UV-A light exposure (365 nm; ∼2 mW/cm²) for 30 min. Values are means of individual biological triplicates. Error bars indicate the respective standard deviations.

FIGURE 4

Optimization of expression plasmid pRholHi-2-eYFP. To improve the properties of the expression plasmid pRholHi-2-eYFP (A) several engineering steps were conducted (A,B). First, a defective lacI gene (lacI variant) was exchanged by the corresponding wildtype lacI gene from E. coli K12. Secondly, a 519-bp lacI-homologous DNA fragment approximately 1.5 kb upstream of the lacI gene was deleted (lacI duplicate region). Additionally, the ribosome binding site (RBS) consisting of the Shine-Dalgarno (SD) sequence, and its corresponding downstream spacer was replaced by an in silico optimized version. The induction factors of the respective expression systems were calculated as the ratio of the normalized eYFP fluorescence values of induced and non-induced cultures. (C) After construction, the three plasmid variants were evaluated regarding their resulting basal expression levels and induction factors in phototrophically grown R. capsulatus SB1003 cultures. Biomass-normalized eYFP in vivo fluorescence (λ _ex = 508 nm, λ _em = 532 nm) of cultures supplemented with 1 mm IPTG is shown in comparison to uninduced cultures and the R. capsulatus wildtype control strain after 48 h of cultivation (30°C, screw neck vials, NIR light: λ _max = 850 nm, 1.7 mW/cm²). Induction of reporter gene expression was performed after 6 h. (D) Light-controlled eyfp reporter gene expression in phototrophically grown R. capsulatus SB1003 cells carrying the best performing expression plasmid pRholtHi-eYFP using NP-cIPTG. Biomass-normalized eYFP in vivo fluorescence (λ _ex = 508 nm, λ _em = 532 nm) of cultures supplemented with 1 mm NP-cIPTG is shown in relation to a 1 mm IPTG and a R. capsulatus wildtype control after 48 h of cultivation (30°C, screw neck vials, NIR light: λ _max = 850 nm, 1.7 mW/cm²). Induction of reporter gene expression was performed after 6 h via UV-A light exposure (365 nm; ∼2 mW/cm², 30 min). Values are means of individual biological triplicates. Error bars indicate the respective standard deviations.

FIGURE 5

Light-controlled induction of carotenoid production in phototrophically grown R. capsulatus SB1003 ΔcrtEF cells carrying pRholtHi-crtE-crtF. (A) Intrinsic carotenoid synthesis of R. capsulatus starting from the terpene C₅ building blocks IPP and DMAPP. Deletion of genes encoding the relevant carotenoid pathway enzymes CrtE and CrtF results in a carotenoid-deficient strain (Hage-Hülsmann et al., 2019). The genes crtE and crtF were cloned in a plasmid and placed under control of the P _tac promoter to facilitate IPTG-based control. Light-mediated induction of crt gene expression was achieved by using NP-cIPTG. IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; FPP, farnesyl pyrophosphate; IspA, FPP synthase; GGPP, geranylgeranyl pyrophosphate; CrtE, GGPP synthase; CrtB, phytoene synthase; CrtI, phytoene desaturase; CrtC, hydroxyneurosporene synthase; CrtD, hydroxyneurosporene desaturase; CrtF, demethylspheroidene O-methyltransferase. (B) Pigmentation of phototrophically grown R. capsulatus strains SB1003 ΔcrtEF (ΔcrtEF), SB1003 ΔcrtEF/pRholtHi-crtE-crtF supplemented with 1 mm NP-cIPTG under non-illuminated (-UV, L-crtEF) or UV-A illuminated conditions (+UV, L-crtEF) and SB1003 wildtype strain (SB1003). Besides liquid cultures, cell pellets corresponding to an optical density at 660 nm of 2.5 are shown. (C) Carotenoid absorption at 484 nm of R. capsulatus SB1003 ΔcrtEF/pRholtHi-crtE-crtF (L-crtEF) cultures supplemented with 1 mm NP-cIPTG (+NP-cIPTG + UV and -UV) are shown in comparison to the respective absorption of extracts from R. capsulatus SB1003 wildtype (SB1003) cells. In addition, cell cultures supplemented with 1 mm IPTG (positive control, +IPTG) or without IPTG (negative control, -IPTG) were likewise analyzed after 48 h of phototrophic cultivation [RCV medium, 30°C, screw neck vials, NIR light (λ _max = 850 nm, 1.7 mW/cm²)]. R. capsulatus SB1003 ΔcrtEF cultures were used to determine the background absorption at 484 nm and thus corresponding values were subtracted from all other values. Induction was performed after a cultivation time of six or 18 h via UV-A light exposure at 365 nm (∼2 mW/cm²) for 30 min. Values are means of individual biological triplicates. Error bars indicate the respective standard deviations.