Characterization of TrxC, an Atypical Thioredoxin Exclusively Present in Cyanobacteria

Abstract

Cyanobacteria form a diverse group of oxygenic photosynthetic prokaryotes considered to be the antecessor of plant chloroplast. They contain four different thioredoxins isoforms, three of them corresponding to m, x and y type present in plant chloroplast, while the fourth one (named TrxC) is exclusively found in cyanobacteria. TrxC has a modified active site (WCGLC) instead of the canonical (WCGPC) present in most thioredoxins. We have purified it and assayed its activity but surprisingly TrxC lacked all the classical activities, such as insulin precipitation or activation of the fructose-1,6-bisphosphatase. Mutants lacking trxC or over-expressing it were generated in the model cyanobacterium Synechocystis sp. PCC 6803 and their phenotypes have been analyzed. The ΔtrxC mutant grew at similar rates to WT in all conditions tested although it showed an increased carotenoid content especially under low carbon conditions. Overexpression strains showed reduced growth under the same conditions and accumulated lower amounts of carotenoids. They also showed lower oxygen evolution rates at high light but higher Fv'/Fm' and Non-photochemical-quenching (NPQ) in dark adapted cells, suggesting a more oxidized plastoquinone pool. All these data suggest that TrxC might have a role in regulating photosynthetic adaptation to low carbon and/or high light conditions.

Keywords: cyanobacteria; photosynthesis; thioredoxin.

Figure 1

TrxC is an atypical thioredoxin. (A) Sequence logo of TrxC proteins form cyanobacteria. TrxC proteins were identified using blast at NCBI and manually curated to retain only those with a WCGL/V/IC sequence (269 sequences). This sequences were aligned using muscle and the alignment was submitted to weblogo3 to generate the consensus sequence shown. (B) Insulin reduction assay. 3 µM of recombinant TrxA (■), TrxC (●) and TrxCL32P (▲) were incubated with insulin in the presence of 1 mM of DTT. Insulin precipitation was measured as an increase in absorbance at 650 nm. Three independent purification were assayed for TrxC and two for TrxCL32P with identical results to the one shown. (C) FBPase activation assay. Oxidased pea FBPase was preincubated for 30 min with 100 µM DTT (control), 10 mM DTT or 100 µM DTT and 3 µM of TrxA, TrxC, TrxCL32P or 30 µM GST-TrxC. Data are the mean and standard error of 3 independent assays.

Figure 2

Construction of WT, WT_OE, STXC and STXCOE strains. (A) Schematic representation of the trxC and glnN loci in the WT and mutant strains. (B) PCR analysis of the mutant strains using the oligonucleotides indicated in (A). (C) Western-blot analysis of TrxC protein levels in the WT, WTOE, STXC and STXCOE strains. Cells were grown in BG11 supplemented with 1% CO₂ to mid-log growth phase and 1 OD_750nm was collected before and after 2 h of 0.3 µM Cu addition. The pellet was resuspended in 1× Laemmli buffer and boiled for 10 min, then 20 µL of the boiled cell suspension were loaded on and gel and analyzed by western blot.

Figure 3

Redox related proteins in WT, WTOE, STXC2 and STXCOE strains. Western-blot analysis of TrxA, TrxB, TrxQ, GrxA, GrxC, DDOR and 2-cysprx in the WT, WTOE, STXC2 and STXCOE strains. Cells were grown in BG11 supplemented with 1% CO₂ to mid-log growth phase and collected. Cells were broken and 20 μg of total protein from soluble extracts were separated by SDS PAGE and analysed by western blot to detect the different proteins using specific antibodies. Experiments were repeated at least two (for TrxQ antibody) or three (all other antibodies) times with biological independent samples. SDS PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 4

Overexpression of trxC slows growth in low carbon conditions. (A) Growth of trxC mutant strains in high carbon conditions. WT (□), STXC2 (○), WTOE (■) and STXCOE (●) were grown in BG11 pH 7.5 under low light until the exponential phase, diluted to 0.2 OD_750nm and shifted to 180 μmol m⁻² s⁻¹ light intensity bubbled with air + 1% CO₂. Growth was monitored by measuring OD_750nm. Data represented are the mean and standard error of 3–4 (depending on the time point) biological independent cultures. (B) Whole cell spectra of WT (blue solid line), STXC2 (green solid line), WTOE (blue dashed line) and STXCOE (green dashed line) grown as in (A). (C) Growth of trxC mutant strains in low carbon conditions. WT (□), STXC2 (○), WTOE (■) and STXCOE (●) were grown in BG11 pH 7.5 under low light until the exponential phase, diluted to 0.2 OD_750nm and shifted to 180 μmol m⁻² s⁻¹ light intensity and bubbled with air. Growth was monitored by measuring OD_750nm. Data represented are the mean and standard error of 3–4 (depending on the time point) biological independent cultures. (D) Whole cell spectra of WT (blue solid line), STXC2 (green solid line), WTOE (blue dashed line) and STXCOE (green dashed line) grown as in (C). (E) Photograph of WT, STXC2, WTOE and STXCOE cultures grown in BG11 pH 7.5 bubbled with air + 1% CO₂ or air.

Figure 5

Overexpression of trxC causes lower photosynthetic efficiency. Oxygen evolution was measured in a Clark electrode at increasing light intensities in exponential growing cultures (OD_750nm = 0.5–1) of WT (□), STXC2 (○), WTOE (■) and STXCOE (●) grown in BG11 pH 7.5 at 180 μmol m⁻² s⁻¹ light intensity and bubbled with air.