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

Universität Rostock, Institut für Biowissenschaften, Pflanzenphysiologie, Albert-Einstein-Str. 3, D-18059 Rostock, Germany.
Microbiology (Impact Factor: 2.84). 11/2011; 158(Pt 2):398-413. DOI: 10.1099/mic.0.054544-0
Source: PubMed

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 ΔccmM and the photorespiratory mutant ΔglcD1/D2 were compared with wild-type (WT) Synechocystis. Metabolic phenotyping revealed accumulation of 2-phosphoglycolate (2PG) in ΔccmM and of glycolate in ΔglcD1/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 ΔccmM and ΔglcD1/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, ΔglcD1/D2 and WT cells displayed induction of many of the LC-inducible genes, whereas ΔccmM lacked similar changes in expression. From the coincidence of the presence of 2PG in ΔccmM without CCM induction and of glycolate in ΔglcD1/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.

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Available from: Claudia Hackenberg, Apr 23, 2015
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    • "Conversion of bicarbonate inside the carboxysomes by carbonic anhydrases raises the local CO 2 concentration, near RuBisCO, which can then efficiently fix CO 2 (Price et al., 2008). However, photorespiration and Mehler-like reactions have also been identified in cyanobacteria, signifying that cyanobacterial photosynthesis can still be limited by a low C i availability despite their CCM (Helman et al., 2005; Zhang et al., 2009; Hackenberg et al., 2012). Up to four flavodiiron proteins (Flv1-4) are involved in the low C i adaptation process, acting as an electron sink at photosystem I (PSI, for Flv1,3) and photosystem II (PSII, for Flv2,4) (Allahverdiyeva et al., 2011; Zhang et al., 2012; Bersanini et al., 2014). "
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    ABSTRACT: Rising CO2 concentrations may have large effects on aquatic microorganisms. In this study, we investigated how elevated pCO2 affects the harmful freshwater cyanobacterium Microcystis aeruginosa. This species is capable of producing dense blooms and hepatotoxins called microcystins. Strain PCC 7806 was cultured in chemostats that were shifted from low to high pCO2 conditions. This resulted in a transition from a C-limited to a light-limited steady state, with a ~2.7-fold increase of the cyanobacterial biomass and ~2.5-fold more microcystin per cell. Cells increased their chlorophyll a and phycocyanin content, and raised their PSI/PSII ratio at high pCO2. Surprisingly, cells had a lower dry weight and contained less carbohydrates, which might be an adaptation to improve the buoyancy of Microcystis when light becomes more limiting at high pCO2. Only 234 of the 4691 genes responded to elevated pCO2. For instance, expression of the carboxysome, RuBisCO, photosystem and C metabolism genes did not change significantly, and only a few N assimilation genes were expressed differently. The lack of large-scale changes in the transcriptome could suit a buoyant species that lives in eutrophic lakes with strong CO2 fluctuations very well. However, we found major responses in inorganic carbon uptake. At low pCO2, cells were mainly dependent on bicarbonate uptake, whereas at high pCO2 gene expression of the bicarbonate uptake systems was down-regulated and cells shifted to CO2 and low-affinity bicarbonate uptake. These results show that the need for high-affinity bicarbonate uptake systems ceases at elevated CO2. Moreover, the combination of an increased cyanobacterial abundance, improved buoyancy, and higher toxin content per cell indicates that rising atmospheric CO2 levels may increase the problems associated with the harmful cyanobacterium Microcystis in eutrophic lakes.
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    • "Particularly, we observed no changes in metabolites, such as the photorespiratory metabolites 2PG or glycolate, characteristic of the LC-shift response of wild-type cells under HC conditions (Fig. 6). These compounds were accumulated under HC conditions in the ccmM mutant of Synechocystis 6803 (Hackenberg et al., 2012). Among the few differences between the DndhR mutant and wild-type cells under HC conditions was a significant decrease in Glc-6-P, indicating a decrease in soluble sugar metabolism (Fig. 6). "
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    ABSTRACT: The acquisition and assimilation of inorganic carbon (Ci) represents the largest flux of inorganic matter in photosynthetic organisms; hence, this process is tightly regulated. We examined the Ci-dependent transcriptional and metabolic regulation in wild-type Synechocystis sp. PCC 6803 compared with a ∆ndhR mutant defective in the main transcriptional repressor for Ci acquisition genes, NdhR (CcmR). The analysis revealed that many protein-coding transcripts that are normally repressed in the presence of high CO2 concentrations (HC) were strongly expressed in ∆ndhR, whereas other mRNAs were strongly down-regulated in mutant cells, suggesting a potential activating role for NdhR. A conserved NdhR-binding motif was identified in the promoters of de-repressed genes. Interestingly, the expression of some NdhR-regulated genes remained further inducible under low CO2 conditions (LC), indicating the involvement of additional NdhR-independent Ci-regulatory mechanisms. Intriguingly, we also observed that the abundance of 52 antisense RNAs and 34 potential non-coding RNAs was affected by Ci supply, although most of these molecules were not regulated through NdhR. Thus, antisense and non-coding RNAs could contribute to NdhR-independent carbon regulation. In contrast to the transcriptome, the metabolome in ∆ndhR cells was similar to that of wild-type cells under HC conditions. This observation and the delayed metabolic responses to the LC-shift in ∆ndhR, specifically the lack of transient increases in the photorespiratory pathways intermediates 2-phosphoglycolate, glycolate and glycine, suggest that the deregulation of gene expression in the ∆ndhR mutant successfully pre-acclimates cyanobacterial cells to lowered Ci supply under HC conditions. Copyright © 2015, American Society of Plant Biologists.
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    • "This variant interacts with RbcL to create an internal network of protein interactions that organizes multiple Rubisco molecules into an ordered array within the core of the carboxysome. The CcmM/CcmN/CcaA complex also appears capable of interacting with CcmK2 in the shell through either the N-terminal domain of CcmM (Cot et al. 2008), or the extended C-terminus of CcmN (Kinney et al. 2012).The absence of carboxysomes and concomitant high CO 2 requiring phenotype of DccmM mutants attest to the importance of this multi-domain protein to the structure and function of b-carboxysomes (Berry et al. 2005; Hackenberg et al. 2012; Ludwig et al. 2000; Marcus et al. 1992). As mentioned, the N-terminal domain of CcmM has detectable sequence homology to c-CAs, including 38 and 29 % identity, respectively, to the Methanosarcina thermophila Cam (MtCam) and CamH enzymes (MtCamH) (Alber et al. 1999; Alber and Ferry 1994, 1996; Zimmerman et al. 2010). "
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    ABSTRACT: Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO3 (-) dehydration and the subsequent fixation of CO2. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein-protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k cat of 2.0 × 10(4) s(-1) and k cat/K m of 4.1 × 10(6) M(-1) s(-1) at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25-35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO2 analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.
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