Low-carbon acclimation in carboxysome-less and photorespiratory mutants of the cyanobacterium Synechocystis sp. strain PCC 6803

Abstract

Using metabolic and transcriptomic phenotyping, we studied acclimation of cyanobacteria to low inorganic carbon (LC) conditions and the requirements for coordinated alteration of metabolism and gene expression. To analyse possible metabolic signals for LC sensing and compensating reactions, the carboxysome-less mutant DccmM and the photorespiratory mutant DglcD1/D2 were compared with wild-type (WT) Synechocystis. Metabolic phenotyping revealed accumulation of 2-phosphoglycolate (2PG) in DccmM and of glycolate in DglcD1/D2 in LC-but also in high inorganic carbon (HC)-grown mutant cells. The accumulation of photorespiratory metabolites provided evidence for the oxygenase activity of RubisCO at HC. The global gene expression patterns of HC-grown DccmM and DglcD1/D2 showed differential expression of many genes involved in photosynthesis, high-light stress and N assimilation. In contrast, the transcripts of LC-specific genes, such as those for inorganic carbon transporters and components of the carbon-concentrating mechanism (CCM), remained unchanged in HC cells. After a shift to LC, DglcD1/D2 and WT cells displayed induction of many of the LC-inducible genes, whereas DccmM lacked similar changes in expression. From the coincidence of the presence of 2PG in DccmM without CCM induction and of glycolate in DglcD1/D2 with CCM induction, we regard a direct role for 2PG as a metabolic signal for the induction of CCM during LC acclimation as less likely. Instead, our data suggest a potential role for glycolate as a signal molecule for enhanced expression of CCM genes. Abbreviations: CBB, Calvin-Benson-Bassham; CCM, carbon-concentrating mechanism; C i , inorganic carbon; HC, high inorganic carbon; HCR, high-carbon-requiring; LC, low inorganic carbon; 2OG, 2-oxoglutarate; OPP, oxidative pentose phosphate; PEP, phosphoenolpyruvate; 2PG, 2-phosphoglycolate; 3PGA, 3-phosphoglycerate; RbcL, large RubisCO subunit; Ru5P, ribulose 5-phosphate; TCA, tricarboxylic acid; WT, wild-type.The microarray data discussed in this paper have been submitted to the NCBI Gene Expression Omnibus (GEO) database under accession number GSE31672.Two supplementary figures, showing mutant genotypes and growth and global changes in transcriptomic patterns, and three supplementary tables listing the complete metabolomic and transcriptomic datasets, and the distribution of differentially regulated genes in different metabolic categories in cells of the WT and DccmM and DglcD1/D2 mutants, are available with the online version of this paper. Microbiology

Integrated analysis of engineered carbon limitation in a quadruple CO2/HCO3--uptake mutant of Synechocystis sp. PCC 6803

“…A, The Δ4 mutant is a quadruple carbon transporter mutant that lacks four of the five HCO 3 2 /CO 2 -uptake systems known in Synechocystis 6803 (Shibata et al, 2002;Xu et al, 2008). The Δ4 mutant is compared with the ΔndhR mutant (Klähn et al, 2015) and the DccmM mutant (Hackenberg et al, 2012). NdhR is a central regulator of carbon metabolism in Synechocystis 6803 and, among others, a repressor of ndhD3 and sbtA.…”

“…CmpA is part of the cmpABCD operon, which is controlled by the transcriptional regulator CmpR. The DccmM mutant does not express CcmM, one of the shell proteins of carboxysomes (CBX) that is encoded in the ccmKLMNO operon and serves as a reference mutant with a carboxysomeless phenotype (Hackenberg et al, 2012). (This scheme was modified from Daley et al [2012] and Rae et al [2013] LC conditions, indicating an attenuated photorespiratory burst in the Δ4 mutant (Fig.…”

“…The D4 mutant compensates Ci limitation caused by the lack of NdhD3, NdhD4, CmpA, and SbtA by BicA activity and grows in ambient air, although at a reduced rate compared with the Synechocystis 6803 wild type. Here, we present a functional systems analysis of engineered intracellular Ci limitation in the D4 mutant and compare it with the wild type, with the regulatory mutant ΔndhR (Δsll1594; Klähn et al, 2015), and with the carboxysomeless ΔccmM (Δsll1031) mutant (Hackenberg et al, 2012). We compare HC-and LC-grown cells and focus on the integration of primary metabolic pathways, including photorespiration and global transcriptional changes.…”

Functions, Compositions, and Evolution of the Two Types of Carboxysomes: Polyhedral Microcompartments That Facilitate CO 2 Fixation in Cyanobacteria and Some Proteobacteria

“…Studies using quantitative PCR analysis of transcript changes upon transfer of cells from 2 to 5% CO 2 to air (Ci limitation) have found little change or transient small changes (2-to 5-fold increases) in the ccmKLMNO, ccaA, and rbcLXS genes (56,188,189). Changes found using microarray analysis have generally been small (190,191). In comparison, low-CO 2 -responsive genes (coding for CO 2 -and HCO 3 Ϫ -uptake systems) such as bicA (except in PCC 6803), sbtAB, cmpABCD, porB (in PCC 7002), and ndhF3-ndhD3-chpY undergo large changes in transcript abundance, although sometimes these changes are transiently maximal over the first 2 h from the onset of induction (56,(188)(189)(190)(191).…”

“…Changes found using microarray analysis have generally been small (190,191). In comparison, low-CO 2 -responsive genes (coding for CO 2 -and HCO 3 Ϫ -uptake systems) such as bicA (except in PCC 6803), sbtAB, cmpABCD, porB (in PCC 7002), and ndhF3-ndhD3-chpY undergo large changes in transcript abundance, although sometimes these changes are transiently maximal over the first 2 h from the onset of induction (56,(188)(189)(190)(191). An electron microscopic investigation found that carboxysome numbers per cell for cells grown under severe Ci limitation can be up to 4-fold higher than those for high-CO 2 -grown cells of Synechococcus UTEX 625 (equivalent to PCC 6301) (192).…”