Sll1717 affects the redox state of the plastoquinone pool by modulating quinol oxidase activity in thylakoids

Abstract

A Synechocystis sp. strain PCC 6803 mutant lacking CtaI, a main subunit of cytochrome c oxidase, is not capable of growing at light intensities below 5 micromol photons m(-2) s(-1), presumably due to an overreduced plastoquinone pool in the thylakoid membrane. Upon selection for growth at light intensities below 5 micromol photons m(-2) s(-1), a secondary mutant was generated that retained the CtaI deletion and had fully assembled photosystem II complexes; in this secondary mutant (pseudorevertant), oxygen evolution and respiratory activities were similar to those in the wild type. Functional complementation of the original CtaI-less strain to low-light tolerance by transformation with restriction fragments of genomic DNA of the pseudorevertant and subsequent mapping of the pseudoreversion site showed that the point mutation led to a Ser186Cys substitution in Sll1717, a protein of as-yet-unknown function and with a predicted ATP/GTP-binding domain. This mutation caused a decrease in the plastoquinone pool reduction level of thylakoids compared to that observed for the wild type. Based on a variety of experimental evidence, the most plausible mechanism to cause this effect is an activation of plastoquinol oxidation in thylakoids by the quinol oxidase CydAB that occurs without upregulation of the corresponding gene and that may be caused by an increased CydAB activity in thylakoids, conceivably due to altered CydAB sorting between cytoplasmic and thylakoid membranes. Sll1717 appears to be unique to Synechocystis sp. strain PCC 6803 and has a close homologue encoded in the genome of this organism. The transcript level of sll1717 is low, which suggests that the corresponding protein is regulatory rather than structural.

FIG. 1.

Growth curve of the Synechocystis sp. strain PCC 6803 wild type (triangles), the CtaI-less strain (squares), and a pseudorevertant of the CtaI-less strain that is tolerant to low light intensity (circles). Cells were grown photomixotrophically at a light intensity of 2 μmol photons m⁻² s⁻¹. The growth curves of the wild type and pseudorevertant partially overlap. Values were averaged from 10 independent experiments.

FIG. 2.

77K fluorescence emission spectra of intact cells of the Synechocystis sp. strain PCC 6803 wild type (top), the CtaI-less strain (middle), and the CtaI-less pseudorevertant (bottom). Excitation was at 435 nm. Cells were grown at a light intensity of 40 μmol photons m⁻² s⁻¹. Fluorescence intensities were normalized to the fluorescence intensity at 725 nm, and curves were offset relative to each other. a.u., arbitrary units.

FIG. 3.

Primary structure of Sll1717 and its homologue Sll1273 in Synechocystis sp. strain PCC 6803. The predicted ATP/GTP-binding motif of Sll1717 and Sll1273 is in boldface type. The arrow indicates Ser186, which is mutated in the CtaI-less pseudorevertant.

FIG. 4.

Fluorescence yield of the PSI-less strain (○) and the PSI-less/Sll1717S186C strain (x) measured over time in the presence of 1 mM KCN and 60 μM DBMIB with flashes of weak measuring light that did not have an actinic effect. To prevent fluorescence quenching by oxidized DBMIB, 5 mM sodium ascorbate was added to the sample. The duration of each flash was 8 s, and the interval between the flashes was of similar length. The rise and decay kinetics of the signal upon turning on or off the light are artifacts, due to the time constant of the instrument that was set to a high value to be able to monitor fluorescence at very low, non-actinic intensities of measuring light. The upper curve reflects the maximum fluorescence yield recorded in actinic light in the presence of 10 μM DCMU in both the PSI-less strain (▴) and the PSI-less/Sll1717S186C strain (○) and reflects accumulation of reduced Q_A when electron transfer to Q_B is blocked. a.u., arbitrary units.

FIG. 5.

Accumulation of RT-PCR products generated from slr1379 (cydA) transcripts using total RNA isolated from the wild-type strain, the CtaI-less strain, or the pseudorevertant of the CtaI-less strain (Δslr1137/Sll1717 S186C) as a template. The following dilutions of the total RNA preparation were used as templates: 1:20 (1), 1:2,000 (2), and 1:200,000 (3). ΔRn is baseline-subtracted fluorescence from the PCR product normalized to an internal dye (ROX). The C_T value is the number of PCR cycle at which the increase of fluorescence (and therefore the cDNA amplification) is maximally logarithmic. The C_T values for the slr1379 amplification in all three strains were very close to each other at each dilution of the RNA preparation and were 16.80 ± 0.14 (1), 23.58 ± 0.34 (2), and 31.00 ± 0.18 (3).

FIG. 6.

Effect of the sll1717 deletion on photomixotrophic growth of Synechocystis sp. strain PCC 6803 at elevated temperatures. Cultures of the wild type (closed squares) and the CtaI-less mutant (closed circles) were grown at 40 μmol photons m⁻² s⁻¹ and at a temperature of 39°C. Open symbols represent growth curves of the corresponding strains with a sll1717 deletion.

FIG. 7.

(A) Accumulation of sll1717 RT-PCR products using total RNA isolated from the wild type strain, the CtaI-less strain, and the low-light-tolerant pseudorevertant of the CtaI-less strain as a template. Primers 5′ sll1717 (1) and 3′ sll1717 (1) and the sll1717 probe (Table 1) were used for the PCR. The following dilutions of the total RNA preparation were used as a template: 1:100 (1), 1:2,000 (2), and 1:40,000 (3). ΔRn is baseline-subtracted fluorescence from the PCR product normalized to an internal dye (ROX). The C_T value is the number of PCR cycles at which the increase of fluorescence (and therefore the cDNA amplification) is maximally logarithmic. The C_T values for sll1717 amplification in all three strains were very close to each other at each dilution of the RNA preparation and were 26.24 ± 0.09 (1), 31.37 ± 0.02 (2), and 34.89 ± 0.14 (3). (B) Accumulation of RT-PCR products corresponding to sll1717 (lanes 1 to 4) and psbDI (lanes 5 to 8) transcripts using total RNA isolated from wild-type Synechocystis sp. strain PCC 6803 as a template. Primers 5′ sll1717 (2) and 3′ sll1717 (2) were used to amplify sll1717 (Table 1). The number of PCR cycles to which the samples have been exposed has been indicated. Molecular weight markers (M) are in the left lane.