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 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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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.Photosynthesis Research 06/2014; · 3.15 Impact Factor
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ABSTRACT: Thin, filamentous, non-heterocystous, benthic cyanobacteria (Subsection III) from some marine, lacustrine and thermal environments aggregate into macroscopic cones and conical stromatolites. We investigate the uptake and storage of inorganic carbon by cone-forming cyanobacteria from Yellowstone National Park using high-resolution stable isotope mapping of labeled carbon (H(13)CO3 (-)) and immunoassays. Observations and incubation experiments in actively photosynthesizing enrichment cultures and field samples reveal the presence of abundant cyanophycin granules in the active growth layer of cones. These ultrastructurally heterogeneous granules rapidly accumulate newly fixed carbon and store 18% of the total particulate labeled carbon after 120 mins of incubation. The intracellular distribution of labeled carbon during the incubation experiment demonstrates an unexpectedly large contribution of PEP carboxylase to carbon fixation, and a large flow of carbon and nitrogen toward cyanophycin in thin filamentous, non-heterocystous cyanobacteria. This pattern does not occur in obvious response to a changing N or C status. Instead, it may suggest an unusual interplay between the regulation of carbon concentration mechanisms and accumulation of photorespiratory products that facilitates uptake of inorganic C and reduces photorespiration in the dense, surface-attached communities of cyanobacteria from Subsection III.PLoS ONE 01/2014; 9(2):e88142. · 3.53 Impact Factor
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ABSTRACT: Our knowledge on cyanobacterial molecular biology increased tremendously by the application of the "omics" techniques. Only recently, metabolomics was applied systematically to model cyanobacteria. Metabolomics, the quantitative estimation of ideally the complete set of cellular metabolites, is particularly well suited to mirror cellular metabolism and its flexibility under diverse conditions. Traditionally, small sets of metabolites are quantified in targeted metabolome approaches. The development of separation technologies coupled to mass-spectroscopy- or nuclear-magnetic-resonance-based identification of low molecular mass molecules presently allows the profiling of hundreds of metabolites of diverse chemical nature. Metabolome analysis was applied to characterize changes in the cyanobacterial primary metabolism under diverse environmental conditions or in defined mutants. The resulting lists of metabolites and their steady state concentrations in combination with transcriptomics can be used in system biology approaches. The application of stable isotopes in fluxomics, i.e. the quantitative estimation of carbon and nitrogen fluxes through the biochemical network, has only rarely been applied to cyanobacteria, but particularly this technique will allow the making of kinetic models of cyanobacterial systems. The further application of metabolomics in the concert of other "omics" technologies will not only broaden our knowledge, but will also certainly strengthen the base for the biotechnological application of cyanobacteria.Metabolites. 01/2013; 3(1):72-100.