A transcriptional response to singlet oxygen, a toxic byproduct of photosynthesis

Abstract

The ability of phototrophs to convert light into biological energy is critical for life on Earth. However, there can be deleterious consequences associated with this bioenergetic conversion, including the production of toxic byproducts. For example, singlet oxygen (1O2) can be formed during photosynthesis by energy transfer from excited triplet-state chlorophyll pigments to O2. By monitoring gene expression and growth in the presence of 1O2, we show that the phototrophic bacterium Rhodobacter sphaeroides mounts a transcriptional response to this reactive oxygen species (ROS) that requires the alternative sigma factor, sigma(E). An increase in sigma(E) activity is seen when cells are exposed to 1O2 generated either by photochemistry within the photosynthetic apparatus or the photosensitizer, methylene blue. Wavelengths of light responsible for the generating triplet-state chlorophyll pigments in the photosynthetic apparatus are sufficient for a sustained increase in sigma(E) activity. Continued exposure to 1O2 is required to maintain this transcriptional response, and other ROS do not cause a similar increase in sigma(E)-dependent gene expression. When a sigma(E) mutant produces low levels of carotenoids, 1O2 is bacteriocidal, suggesting that this response is essential for protecting cells from this ROS. In addition, global gene expression analysis identified approximately 180 genes (approximately 60 operons) whose RNA levels increase > or = 3-fold in cells with increased sigma(E) activity. Gene products encoded by four newly identified sigma(E)-dependent operons are predicted to be involved in stress response, protecting cells from 1O2 damage, or the conservation of energy.

Fig. 1.

Conditions that generate ¹O₂ increase R. sphaeroides σ^E activity. Shown is β-galactosidase activity from a σ^E-dependent reporter gene in either steady-state cultures or after shifting cells from PS to aerobic conditions in the presence of light. The arrow indicates the time of shift. Shown are results from experiments where cells were exposed to white unfiltered light (light) or placed behind a filter to remove light ≥830 nm (>830-nm light).

Fig. 2.

Continued exposure to ¹O₂ is required for increased σ^E activity. Shown is β-galactosidase activity from the σ^E-dependent reporter gene when PS (PS) cells are shifted to aerobic conditions (Aero) in the presence or absence of light. Arrows indicate the time of each shift.

Fig. 3.

¹O₂ is bacteriocidal to a Δσ^E mutant when carotenoids are low. (A) Optical density measurements (OD_{500 nm}) and (B) viable plate counts (colony-forming units/ml) when aerobically grown WT cells or cells lacking σ^E (Δσ^E) were treated with methylene blue in the presence of light. The arrow indicates the time when methylene blue and light were added.

Fig. 4.

Identification of additional σ^E-dependent promoters. (A) Products of in vitro transcription reactions using reconstituted R. sphaeroides Eσ^E (17) and the indicated potential promoter. As an additional control to demonstrate the σ^E dependence of these transcripts, ChrR was added to indicated reactions (17-19). Note that the first four lanes were exposed to a phosphoscreen twice as long as the remainder of the gel to detect low-abundance transcripts from the cycA P3 and Rsp1409 promoters. Experiments were repeated at least three times, with a representative gel shown. The σ^E-dependent transcripts appear as two products due to termination at different bases within the SpoT 40 transcriptional terminator on the template used (32). (B) Activity of selected σ^E-dependent promoters in R. sphaeroides. Shown are β-galactosidase levels [Miller units (58)] from the indicated promoter fused to lacZ in WT cells (▪), ΔChrR cells (increased σ^E activity) (▧), or cells lacking both σ^E and ChrR (□). All assays were performed in triplicate, with bars denoting the standard deviation from the mean.