Effects of deficiency and overdose of group 2 sigma factors in triple inactivation strains of Synechocystis sp. strain PCC 6803

Abstract

Acclimation of cyanobacteria to environmental changes includes major changes in the gene expression patterns partly orchestrated by the replacement of a particular σ subunit with another in the RNA polymerase holoenzyme. The cyanobacterium Synechocystis sp. strain PCC 6803 encodes nine σ factors, all belonging to the σ(70) family. Cyanobacteria typically encode many group 2 σ factors that closely resemble the principal σ factor. We inactivated three out of the four group 2 σ factors of Synechocystis simultaneously in all possible combinations and found that all triple inactivation strains grow well under standard conditions. Unlike the other strains, the ΔsigBCD strain, which contains SigE as the only functional group 2 σ factor, did not grow faster under mixotrophic than under autotrophic conditions. The SigB and SigD factors were important in low-temperature acclimation, especially under diurnal light rhythm. The ΔsigBCD, ΔsigBCE, and ΔsigBDE strains were sensitive to high-light-induced photoinhibition, indicating a central role of the SigB factor in high-light tolerance. Furthermore, the ΔsigBCE strain (SigD is the only functional group 2 σ factor) appeared to be locked in the high-fluorescence state (state 1) and grew slowly in blue but not in orange or white light. Our results suggest that features of the triple inactivation strains can be categorized as (i) direct consequences of the inactivation of a particular σ factor(s) and (ii) effects resulting from the higher probability that the remaining group 2 σ factors associate with the RNA polymerase core.

FIG. 1.

Triple inactivation strains of group 2 σ factors in Synechocystis. (A) PCR analysis of the ΔsigBCD, ΔsigBCE, ΔsigBDE, and ΔsigCDE inactivation strains. Genomic DNA was isolated from the inactivation strains, and each sig gene was amplified by PCR. The expected size of each PCR product is shown under each gel. std, standard. (B) Growth of strains. The A₇₃₀ of the cultures was set to 0.1, and the cells were grown in BG-11 medium, pH 7.5, at a PPFD of 40 μmol m⁻² s⁻¹ in continuous light (solid lines) or in a 12-h light/12-h dark rhythm (dashed lines) at 32°C. Each data point represents the mean of at least three biological replicates with independent liquid cultures, and the error bars indicate standard errors (SE). CS, control strain. (C) Doubling times of the strains in the presence and absence of 5 mM glucose at a PPFD of 40 μmol m⁻² s⁻¹. The doubling times were calculated on the basis of growth during the first 24 h. Each data point represents the mean of at least four biological replicates with independent liquid cultures, and the error bars indicate SE. The asterisks denote significant differences between the growth rate in the absence and in the presence of glucose (P < 0.01; Student's t test).

FIG. 2.

Growth of single and triple inactivation strains at low temperature. The cell cultures were grown at 22°C in continuous light (A and B) or in a 12-h light/12-h dark rhythm (C and D) at a PPFD of 40 μmol m⁻² s⁻¹. Each data point represents the mean of at least three biological replicates with independent liquid cultures, and the error bars denote SE.

FIG. 3.

Photoinhibition of the triple inactivation strains. Cell cultures were illuminated at a PPFD of 1,500 μmol m⁻² s⁻¹, and light-saturated PSII activity was measured after 0, 15, 30, and 45 min with a Clark-type oxygen electrode at 32°C using 0.7 mM 2.6-dichlorobentzoquinone as an artificial electron acceptor. PSII activity is expressed as a percentage of the activity measured from untreated control samples. Each data point represents an average of five independent experiments, and the error bars denote SE. After 45 min of illumination, the difference between the control strain and the ΔsigBCD, ΔsigBCE, and ΔsigBDE strains was statistically significant (P < 0.05; Student's t test).

FIG. 4.

Growth of the control strain and the triple inactivation strains in blue light, preferentially absorbed by PSI (A), or in orange light, preferentially absorbed by PSII (B). The PPFD was 40 μmol photons m⁻² s⁻¹. Each data point represents the mean of three independent cultures, and the error bars denote SE.

FIG. 5.

Orange carotenoid protein content and nonphotochemical quenching in the control and ΔsigBCE strains. (A) Western blot showing the amounts of the orange carotenoid protein under standard growth conditions (standard), after 1 h of illumination at a PPFD of 1,000 μmol m⁻² s⁻¹ (high light), and after 1 h of illumination with blue light at 40 μmol photons m⁻² s⁻¹ (blue light). (B and C) Chlorophyll a fluorescence yield measured with the PAM fluorometer from dark-adapted cells of the control strain (B) or the ΔsigBCE strain (C) during illumination with blue-green light (PPFD, 1,000 μmol m⁻² s⁻¹). Each peak corresponds to a saturating flash. rel, relative.

FIG. 6.

State transitions and PSII/PSI ratios in the control and ΔsigBCE strains. Emission spectra were measured at 77 K from control (A) and ΔsigBCE (B) cells taken directly from growth light conditions or from cells illuminated first for 5 min with blue light (blue 1), then incubated for 5 min in the dark, and finally illuminated for 5 min with blue light (blue 2). Light at 590 nm was used for excitation, and the data were normalized by dividing by the height of the photosystem I emission peak at 723 nm. Each spectrum represents an average of four independent experiments. (C) Absorption spectra of control and ΔsigBCE cells grown under standard conditions. The spectra were normalized to the Chl a absorption peak at 440 nm. (D) Amounts of PSII reaction center protein D1 and PSI reaction center protein PsaA. Proteins of isolated thylakoids (0.5 μg of Chl a) were separated by SDS-PAGE, and the amounts of the reaction center proteins were determined by Western blotting using D1 and PsaA protein-specific antibodies. (E and F) Fluorescence yields from dark-adapted control (E) and ΔsigBCE (F) cells were measured with a PAM-2000 fluorometer. The illumination protocol consisted of darkness, blue-green light (PPFD, 80 μmol m⁻² s⁻¹), orange light (PPFD, 20 μmol m⁻² s⁻¹), and finally darkness, as indicated. A saturating pulse was fired every 30 s to measure F_M′.

FIG. 7.

Summary of light and low-temperature acclimation properties of different inactivation strains. The results concerning photoinhibition of the ΔsigBD, ΔsigD, and ΔsigB strains were published previously (24); other data are from this study. Symbols indicate no phenotype (−) or a slight (+/−), clear (+), or prominent (++) phenotype. ns, not studied.