Functional proteomic discovery of Slr0110 as a central regulator of carbohydrate metabolism in Synechocystis species PCC6803

Abstract

The unicellular photosynthetic model-organism cyanobacterium Synechocystis sp. PCC6803 can grow photoautotrophically using CO2 or heterotrophically using glucose as the sole carbon source. Several pathways are involved in carbon metabolism in Synechocystis, and the concerted regulation of these pathways by numerous known and unknown genes is critical for the survival and growth of the organism. Here, we report that a hypothetical protein encoded by the open reading frame slr0110 is necessary for heterotrophic growth of Synechocystis. The slr0110-deletion mutant is defective in glucose uptake, heterotrophic growth, and dark viability without detectable defects in autotrophic growth, whereas the level of photosystem II and the rate of oxygen evolution are increased in the mutant. Quantitative proteomic analysis revealed that several proteins in glycolysis and the oxidative pentose phosphate pathway are down-regulated, whereas proteins in photosystem II and phycobilisome are significantly up-regulated, in the mutant. Among the down-regulated proteins are glucose transporter, glucokinase, glucose-6-phosphate isomerase, and glucose-6-phosphate dehydrogenase and its assembly protein OpcA, suggesting that glycolysis, oxidative pentose phosphate, and glycogen synthesis pathways are significantly inhibited in the mutant, which was further confirmed by enzymatic assays and quantification of glycogen content. These findings establish Slr0110 as a novel central regulator of carbon metabolism in Synechocystis, and shed light on an intricate mechanism whereby photosynthesis and carbon metabolism are well concerted to survive the crisis when one or more pathways of the system are impaired.

Fig. 1.

Generation of slr0110-deletion mutant of Synechocystis. A, the diagram depicts the plasmid construct used to disrupt the ORF of slr0110 on the Synechocystis genome. B, confirmation of the complete segregation of the mutant by PCR. The same pair of primers was used to amplify DNA fragments from the WT and the mutant. C, the genomic map shows the loci of slr0110 and its neighboring ORFs. D, transcription of slr0110 and its neighboring ORFs in WT and Δslr0110 was detected via RT-PCR. The gene rnpB was used as the internal loading control. E, transcription of the same set of ORFs except slr0110 in the WT and Δslr0110 was detected via qPCR. The dashed line indicates a ratio of 1 for the transcription level in Δslr0110 to that in the WT.

Fig. 2.

Δslr0110 is defective in heterotrophic growth with an increased level of PSII. A, growth curves of Δslr0110 and the WT were measured in the presence (+G) or absence of 5 mm glucose under medium light intensity (50 μmol m⁻² s⁻¹) (left-hand panel). The images of cell cultures were taken at the 72-h time point (right-hand panel). B, the growth curves of Δslr0110 and the WT cultured in the medium with 5 mm glucose and in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU). The images of cell cultures were taken at the 72-h time point. C, determination of glucose concentration in the culture medium for the mutant and the WT Synechocystis. The glucose concentration was measured at the time points as indicated after the cells were seeded in BG11 medium supplemented with 5 mm glucose. The bar graph is representative of the results from more than five repeated experiments performed at different times. D, low-temperature (77K) fluorescence emission spectra of Δslr0110 and the WT cells cultured under photoautotrophic conditions. E, the chlorophyll contents of Δslr0110 and the WT. The chlorophyll of 1 OD (730 nm) Synechocystis cells was extracted at the indicated time points after the cells were seeded in liquid culture. F, the growth curves of the WT and Δslr0110 in the presence of both glucose and B₁₂.

Fig. 3.

The experimental design and the iTRAQ labeling scheme for the quantitative identification of differentially expressed proteins in Δslr0110.

Fig. 4.

Quantitative identification of differentially expressed proteins in Δslr0110. A, the Venn diagram shows the overlapping and uniquely identified proteins across three experiments. B, distribution of TM-containing proteins identified in the current study or encoded by the whole Synechocystis genome. Bars represent the number of proteins containing a particular number of TMs as indicated in the x-axis. C, distribution of proteins that were confidently quantitated via iTRAQ. The protein list was sorted according to the mean iTRAQ ratios from all the experiments. The error bars represent the standard deviation of the mean. A 1.5-fold change was considered as significant and indicated as either up-regulated or down-regulated in Δslr0110, as shown within the dashed boxes.

Fig. 5.

Functional categorization of the up-regulated or down-regulated proteins in Δslr0110. A, all up-regulated or down-regulated proteins were categorized according to the first-class function annotated by CyanoBase. Bars represent the number of proteins in each functional category. B, the scatter plots of all confidently quantitated proteins highlighted with functional categories including translation (left-hand panel), photosynthesis and respiration (middle panel), and energy metabolism (right-hand panel). The second-class function in each category is shown with a different color as indicated. The relative abundance of each protein is indicated by the spectral count (y-axis).

Fig. 6.

Depletion of Slr0110 reduced protein but not mRNA levels of glucose transport and catabolism-related genes in Synechocystis. A, a diagram shows the glucose catabolism pathways and the involved proteins in Synechocystis. The relative abundance of each protein in Δslr0110 compared with the WT (the logarithm transformed mean iTRAQ ratio), if available, is shown in parentheses. A red asterisk indicates the corresponding reaction and proteins are also involved in the Calvin cycle; the dashed double-headed arrows indicate that the corresponding metabolites can be shared by more than one reaction. A red “X” indicates that the corresponding reaction or process is inhibited. B, confirmation of inhibited expression of sll0771 in Δslr0110 by Western blot. The knockin strains of Sll0771-His tag were generated from Δslr0110 and the WT, respectively, and subsequently cultured in the presence or absence of glucose. The cells were then lysed and the expression of Sll0771 was probed using anti-His tag antibody. C, the mRNA levels of the indicated ORFs involved in glucose catabolism in Δslr0110 and the WT were detected via RT-PCR. The cells used for RNA preparation were cultured in photoautotrophic conditions.

Fig. 7.

Confirmation of defective glucose transport and catabolism in Δslr0110. A, growth curves of the WT, Δslr0110, and Δsll0771 strains cultured in the presence of 5 mm glucose. The inset shows the complete segregation of Δsll0771 detected via PCR (right-hand panel). The images of the cell cultures were taken at 72 h post-seeding. B, determination of glucose concentration in the culture medium for the WT, Δslr0110, and Δsll0771 at the indicated time points. The cells were cultured in BG11 medium supplemented with 5 mm glucose. C, determination of GLK activities of the WT and Δslr0110. The cells were cultured in BG11 culture medium supplemented with or without 5 mm glucose under medium light intensity (50 μmol m⁻² s⁻¹). D, E, determination of G6PDH activity in WT, Δslr0110, Δsll0771, and Δsll0593. The cells were cultured in either the presence or the absence of glucose, as indicated.

Fig. 8.

Comparison of the viability and glycogen contents of the dark-incubated Δslr0110 and WT. A, WT and Δslr0110 cells were grown in liquid BG11 medium under medium light intensity (50 μmol m⁻² s⁻¹) without glucose to exponential phase. The cells were then harvested and transferred to solid BG11 agar medium with different dilutions as indicated. The cells on the plate were then either dark- or light-incubated (control) for 72 h. The dark-incubated cells were then re-exposed to light illumination, and the images of the cells were taken at the indicated time points of light incubation. B, determination of glycogen contents in light- and dark-grown cells. WT and Δslr0110 cells were cultured in an autotrophic condition to exponential phase and then incubated in the dark for 72 h. The glycogen contents of both strains were determined immediately before or after the dark incubation. Shown is a representative result from more than five independent measurements performed at different times.