Essential role of glutathione in acclimation to environmental and redox perturbations in the cyanobacterium Synechocystis sp. PCC 6803

Abstract

Glutathione, a nonribosomal thiol tripeptide, has been shown to be critical for many processes in plants. Much less is known about the roles of glutathione in cyanobacteria, oxygenic photosynthetic prokaryotes that are the evolutionary precursor of the chloroplast. An understanding of glutathione metabolism in cyanobacteria is expected to provide novel insight into the evolution of the elaborate and extensive pathways that utilize glutathione in photosynthetic organisms. To investigate the function of glutathione in cyanobacteria, we generated deletion mutants of glutamate-cysteine ligase (gshA) and glutathione synthetase (gshB) in Synechocystis sp. PCC 6803. Complete segregation of the ΔgshA mutation was not achieved, suggesting that GshA activity is essential for growth. In contrast, fully segregated ΔgshB mutants were isolated and characterized. The ΔgshB strain lacks reduced glutathione (GSH) but instead accumulates the precursor compound γ-glutamylcysteine (γ-EC). The ΔgshB strain grows slower than the wild-type strain under favorable conditions and exhibits extremely reduced growth or death when subjected to conditions promoting oxidative stress. Furthermore, we analyzed thiol contents in the wild type and the ΔgshB mutant after subjecting the strains to multiple environmental and redox perturbations. We found that conditions promoting growth stimulate glutathione biosynthesis. We also determined that cellular GSH and γ-EC content decline following exposure to dark and blue light and during photoheterotrophic growth. Moreover, a rapid depletion of GSH and γ-EC is observed in the wild type and the ΔgshB strain, respectively, when cells are starved for nitrate or sulfate.

Figure 1.

Disruption of glutathione biosynthesis in Synechocystis 6803. A, Diagram of the glutathione biosynthetic pathway. B, The entire open reading frame of the gshA gene was replaced with a kanamycin resistance cassette (Km^R) to generate the ΔgshA::Km^R strain. C, Segregation of ΔgshA::Km^R was tested by PCR using primers GshA5 and GshA6 shown in A. Lanes show wild-type genomic DNA (WT), pSL2083 (+), and ΔgshA::Km^R genomic DNA. D, The gshB gene was replaced with a gentamicin resistance cassette (Gm^R). E, Segregation of ΔgshB::Gm^R was confirmed by PCR using primers GshB5, GshB6, and GshB7 shown in D. Lanes show wild-type genomic DNA (WT), pSL2085 (+), and ΔgshB::Gm^R genomic DNA. F, Growth of wild-type cells in the presence of the GshA inhibitor BSO. G, Cellular GSH concentration after 96 h of growth in the presence of BSO. Primer sequences used in cloning and segregation analysis are shown in Table I.

Figure 2.

Genetic complementation of the ΔgshB::Gm^R strain and quantification of cellular thiols. A, The gshB gene was cloned into the pTCP2031V (Satoh et al., 2001; Muramatsu et al., 2009) vector under the control of the psbA2 promoter (top) and targeted to the slr2031 site (asterisk) in the ΔgshB::Gm^R mutant; the resulting strain is ΔgshB::Gm^R/T2086. B, Segregation of ΔgshB::Gm^R/T2086 was confirmed by PCR using primers shown in A and Figure 1D. Lanes show wild-type genomic DNA (WT), pSL2086 (lane 1), ΔgshB::Gm^R/T2086 genomic DNA (lanes 2 and 4), and pSL2085 (lane 3). C, HPLC elution profile of monobromobimane-derivatized thiols extracted from wild-type (solid line), ΔgshB::Gm^R (dashed line), and ΔgshB::Gm^R/T2086 (dotted line) cells. D to F, Quantification of cellular GSH (D), γ-EC (E), and Cys (F) levels. Data are means of three independent cultures ± se. Intracellular concentrations are based on an estimated cellular volume of Synechocystis 6803 equal to 4.4 × 10⁻¹⁵ L; for details, see “Materials and Methods.” n.d., Not detected.

Figure 3.

Growth of wild-type, ΔgshB::Gm^R, and ΔgshB::Gm^R/T2086 strains. Treatments are as follows: photoautotrophic (A), 1.5 mm H₂O₂ (B), 5 μm RB (C), 1 μm MV (D). Strains are wild type (circles, solid line), ΔgshB::Gm^R (squares, solid line), and ΔgshB::Gm^R/T2086 (diamonds, dashed line). Growth was monitored as turbidity at 730 nm. Error bars represent se of three independent cultures.

Figure 4.

Changes in cellular thiol content after redox perturbations. GSH (black bars) and γ-EC (gray bars) were measured in untreated wild-type (WT; A) and ΔgshB::Gm^R (B) cells (Ctrl) or cells exposed to 1 μm MV, 5 μm RB, or 1 mm H₂O₂ for 3 h under continuous illumination at 30 μmol photons m⁻² s⁻¹. Each bar represents the mean of three independent cultures, and error bars represent se.

Figure 5.

Influence of light quality and intensity on glutathione metabolism. The major cellular thiol was measured in the wild type (WT; GSH; black bars; A) and ΔgshB::Gm^R (γ-EC; gray bars; B) after exposure to different light conditions. Low light (LL; 20 μmol photons m⁻² s⁻¹)-grown cells were transferred to low light, dark (D), blue (B), orange-red (R), or high light (HL; 150 μmol photons m⁻² s⁻¹) for 3 h. Thiols were then analyzed by HPLC. Each bar represents the mean of three independent cultures, and error bars represent se. For further details, see “Materials and Methods.”

Figure 6.

Cellular thiol content after addition of Glc. The major cellular thiol was measured in the wild type (WT; GSH; black bars; A) and ΔgshB::Gm^R (γ-EC; gray bars; B) grown photoautotrophically (PA), photomixotrophically (PM) in the presence of 5 mm Glc, or photoheterotrophically (PH) in the presence of 5 mm Glc and 10 μm DCMU for 24 h. Values for preculture cells used as the inoculum for the experiment are also shown (C). Error bars represent se of two separate measurements each from two independent cultures.

Figure 7.

Effect of nutrient depletion on glutathione metabolism. The major primary thiol was measured in the wild type (GSH; solid line, circles) and ΔgshB::GmR (γ-EC; dashed line, squares) over the course of 12 d in deplete (shaded) and replete (white) conditions. Cells were precultured in BG11 medium (0 d) prior to transfer to BG11 (A) or BG11 lacking nitrate (B), sulfate (C), or phosphate (D) for 6 d. After 6 d of growth in deplete conditions, cells were transferred to fresh BG11 medium and grown for an additional 6 d. Error bars represent se of two measurements each from two independent cultures.

Figure 8.

Visual comparison of wild-type (WT) and ΔgshB::Gm^R cultures. Photographs show cultures after 24 h of growth in BG11 medium lacking nitrate (−N), sulfate (−S), or phosphate (−P) or with the addition of 5 mm Glc with or without 10 μm DCMU.

Figure 9.

Characterization of sulfate (S) depletion in ΔgshB::Gm^R cells. A, Growth of the wild type (circles) and ΔgshB::Gm^R (squares) in BG11 (black symbols) or BG11 containing 10% sulfate (30.3 μm; white symbols). Values represent means ± se (n = 3). B, Growth of the wild type (circles) and ΔgshB::Gm^R (squares) in BG11 after 264 h of growth in sulfate-deplete (white symbols) or BG11 (black symbols) medium. Values are means ± se of three cultures. C, The major cellular thiol in the wild type (GSH; black bars) and ΔgshB::Gm^R (γ-EC; gray bars) was quantified by HPLC prior to sulfate depletion (0 h) after 264 h of growth in BG11 or in sulfate-deplete medium. Each value represents a single measurement and is consistent with the results of at least three independent experiments. D, ROS accumulation after 264 h of growth in BG11 or sulfate-deplete medium and after 144 h of recovery in BG11 medium. Error bars represent se of three measurements each from three independent cultures.