Wolfgang Lockau

The Metabolic Network of Synechocystis sp. PCC 6803: Systemic Properties of Autotrophic Growth

Unicellular cyanobacteria have attracted growing attention as potential host organisms for the production of valuable organic products and provide an ideal model to understand oxygenic photosynthesis and phototrophic metabolism. To obtain insight into the functional properties of phototrophic growth, we present a detailed reconstruction of the primary metabolic network of the autotrophic prokaryote Synechocystis sp. PCC 6803. The reconstruction is based on multiple data sources and extensive manual curation and significantly extends currently available repositories of cyanobacterial metabolism. A systematic functional analysis, utilizing the framework of flux-balance analysis, allows the prediction of essential metabolic pathways and reactions and allows the identification of inconsistencies in the current annotation. As a counterintuitive result, our computational model indicates that photorespiration is beneficial to achieve optimal growth rates. The reconstruction process highlights several obstacles currently encountered in the context of large-scale reconstructions of metabolic networks.

Flux balance analysis of cyanobacterial metabolism: the metabolic network of Synechocystis sp. PCC 6803.

Cyanobacteria are versatile unicellular phototrophic microorganisms that are highly abundant in many environments. Owing to their capability to utilize solar energy and atmospheric carbon dioxide for growth, cyanobacteria are increasingly recognized as a prolific resource for the synthesis of valuable chemicals and various biofuels. To fully harness the metabolic capabilities of cyanobacteria necessitates an in-depth understanding of the metabolic interconversions taking place during phototrophic growth, as provided by genome-scale reconstructions of microbial organisms. Here we present an extended reconstruction and analysis of the metabolic network of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Building upon several recent reconstructions of cyanobacterial metabolism, unclear reaction steps are experimentally validated and the functional consequences of unknown or dissenting pathway topologies are discussed. The updated model integrates novel results with respect to the cyanobacterial TCA cycle, an alleged glyoxylate shunt, and the role of photorespiration in cellular growth. Going beyond conventional flux-balance analysis, we extend the computational analysis to diurnal light/dark cycles of cyanobacterial metabolism.

Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803

The biosynthesis of glycogen or starch is one of the main strategies developed by living organisms for the intracellular storage of carbon and energy. In phototrophic organisms, such polyglucans accumulate due to carbon fixation during photosynthesis and are used to provide maintenance energy for cell integrity, function and viability in dark periods. Moreover, it is assumed that glycogen enables cyanobacteria to cope with transient starvation conditions, as observed in most micro-organisms. Here, glycogen accumulates when an appropriate carbon source is available in sufficient amounts but growth is inhibited by lack of other nutrients. In this study, the role of glycogen in energy and carbon metabolism of phototrophic cyanobacteria was first analysed via a comparative physiological and metabolic characterization of knockout mutants defective in glycogen synthesis. We first proved the role of glycogen as a respiratory substrate in periods of darkness, the role of glycogen as a reserve to survive starvation periods such as nitrogen depletion and the role of glycogen synthesis as an ameliorator of carbon excess conditions in the model organism Synechocystis sp. PCC 6803. We provide striking new insights into the complex carbon and nitrogen metabolism of non-diazotrophic cyanobacteria: a phenotype of sensitivity to photomixotrophic conditions and of reduced glucose uptake, a non-bleaching phenotype based on an impaired acclimation response to nitrogen depletion and furthermore a phenotype of energy spilling. This study shows that the analysis of deficiencies in glycogen metabolism is a valuable tool for the identification of metabolic regulatory principles and signals.

Cyanophycin synthetase-like enzymes of non-cyanobacterial eubacteria: characterization of the polymer produced by a recombinant synthetase of Desulfitobacterium hafniense.

Some bacterial genomes were found to contain genes encoding putative proteins with considerable sequence homology to cyanophycin synthetase CphA of cyanobacteria. Such a gene from the Gram-positive, spore-forming anaerobe Desulfitobacterium hafniense was cloned. Expression in Escherichia coli resulted in the formation of a polydispers copolymer of aspartic acid and arginine, with a minor amount of lysine, of about 30 kDa molecular mass. In contrast to cyanophycin, this polymer was water-soluble. The structure of the polymer formed by the synthetase from Desulfitobacterium hafniense was studied by enzymatic degradation with the cyanophycin-specific hydrolase cyanophycinase, and by chemical and mass-spectroscopic analyses. Despite of the differences in solubility, indicating that both polymers cannot be completely identical, the chemical structure was found to be very similar to that of cyanophycin. The results suggest that the use of cyanophycin-like polymers as a nitrogen- rich reserve material is not restricted to cyanobacteria, and that such polymers may not necessarily be stored in granules.

Calcium-dependent protease of the cyanobacterium Anabaena : molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis

SummaryIt has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N2. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca2+-dependent protease activity, were not impaired in heterocyst formation and grew on N2 as sole nitrogen source.

NblA, a Key Protein of Phycobilisome Degradation, Interacts with ClpC, a HSP100 Chaperone Partner of a Cyanobacterial Clp Protease

When cyanobacteria are starved for nitrogen, expression of the NblA protein increases and thereby induces proteolytic degradation of phycobilisomes, light-harvesting complexes of pigmented proteins. Phycobilisome degradation leads to a color change of the cells from blue-green to yellow-green, referred to as bleaching or chlorosis. As reported previously, NblA binds via a conserved region at its C terminus to the α-subunits of phycobiliproteins, the main components of phycobilisomes. We demonstrate here that a highly conserved stretch of amino acids in the N-terminal helix of NblA is essential for protein function in vivo. Affinity purification of glutathione S-transferase-tagged NblA, expressed in a Nostoc sp. PCC7120 mutant lacking wild-type NblA, resulted in co-precipitation of ClpC, encoded by open reading frame alr2999 of the Nostoc chromosome. ClpC is a HSP100 chaperone partner of the Clp protease. ATP-dependent binding of NblA to ClpC was corroborated by in vitro pull-down assays. Introducing amino acid exchanges, we verified that the conserved N-terminal motif of NblA mediates the interaction with ClpC. Further results indicate that NblA binds phycobiliprotein subunits and ClpC simultaneously, thus bringing the proteins into close proximity. Altogether these results suggest that NblA may act as an adaptor protein that guides a ClpC·ClpP complex to the phycobiliprotein disks in the rods of phycobilisomes, thereby initiating the degradation process.

Plastid targeting strategies for cyanophycin synthetase to achieve high-level polymer accumulation in Nicotiana tabacum

The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the cyanophycin synthetase gene (cphA(Te)) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphA(Te), pCP24-cphA(Te), pFNR-cphA(Te) and pPsbY-cphA(Te). These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphA(Te) led to cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphA(Te) and pFNR-cphA(Te) constructs, water-soluble and water-insoluble forms of cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphA(Te) produced predominantly the water-insoluble form of cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T(0)) transformants. Specific T(2) plants produced 6.8% of dry weight as cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest cyanophycin-producing line showed reduced seed production compared with control plants.

PII-Regulated Arginine Synthesis Controls Accumulation of Cyanophycin in Synechocystis sp. Strain PCC 6803

Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a nitrogen storage polymer found in most cyanobacteria and some heterotrophic bacteria. The cyanobacterium Synechocystis sp. strain PCC 6803 accumulates cyanophycin following a transition from nitrogen-limited to nitrogen-excess conditions. Here we show that the accumulation of cyanophycin depends on the activation of the key enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase, by signal transduction protein PII.

Crystal Structure of NblA from Anabaena sp. PCC 7120, a Small Protein Playing a Key Role in Phycobilisome Degradation

Cyanobacterial light-harvesting complexes, the phycobilisomes, are proteolytically degraded when the organisms are starved for combined nitrogen, a process referred to as chlorosis or bleaching. Gene nblA, present in all phycobilisome-containing organisms, encodes a protein of about 7 kDa that plays a key role in phycobilisome degradation. The mode of action of NblA in this degradation process is poorly understood. Here we presented the 1.8-Å crystal structure of NblA from Anabaena sp. PCC 7120. In the crystal, NblA is present as a four-helix bundle formed by dimers, the basic structural units. By using pull-down assays with immobilized NblA and peptide scanning, we showed that NblA specifically binds to the α-subunits of phycocyanin and phycoerythrocyanin, the main building blocks of the phycobilisome rod structure. By site-directed mutagenesis, we identified amino acid residues in NblA that are involved in phycobilisome binding. The results provided evidence that NblA is directly involved in phycobilisome degradation, and the results allowed us to present a model that gives insight into the interaction of this small protein with the phycobilisomes.

Degradation of Phycobilisomes in Synechocystis sp. PCC6803 EVIDENCE FOR ESSENTIAL FORMATION OF AN NblA1/NblA2 HETERODIMER AND ITS CODEGRADATION BY A Clp PROTEASE COMPLEX

Background: In cyanobacteria, starvation-induced phycobilisome degradation is caused by NblA. Results: Synechocystis expresses two NblA proteins that form a heterodimer. The heterodimer binds phycobiliproteins and ClpC and is degraded by a ClpC-ClpP1ClpR protease in vitro. Conclusion: NblA1/NblA2 is an adaptor protein that mediates degradation of phycobilisomes by the ATP-dependent protease ClpC-ClpP1ClpR. Significance: These findings improve our understanding of the mechanisms by which cyanobacteria adapt to changing environmental conditions. When cyanobacteria acclimate to nitrogen deficiency, they degrade their large (3–5-MDa), light-harvesting complexes, the phycobilisomes. This massive, yet specific, intracellular degradation of the pigmented phycobiliproteins causes a color change of cyanobacterial cultures from blue-green to yellow-green, a process referred to as chlorosis or bleaching. Phycobilisome degradation is induced by expression of the nblA gene, which encodes a protein of ∼7 kDa. NblA most likely acts as an adaptor protein that guides a Clp protease to the phycobiliproteins, thereby initiating the degradation process. Most cyanobacteria and red algae possess just one nblA-homologous gene. As an exception, the widely used “model organism” Synechocystis sp. PCC6803 expresses two such genes, nblA16803 and nblA26803, both of whose products are required for phycobilisome degradation. Here, we demonstrate that the two NblA proteins heterodimerize in vitro and in vivo using pull-down assays and a Förster energy-transfer approach, respectively. We further show that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease, a finding that supports a proposed mechanism of the degradation process. Expression of the single nblA gene encoded by the genome of the N2-fixing, filamentous cyanobacterium Nostoc sp. PCC7120 in the nblA1/nblA2 mutant of Synechocystis sp. PCC6803 induced phycobilisome degradation, suggesting that the function of the NblA heterodimer of Synechocystis sp. PCC6803 is combined in the homodimeric protein of Nostoc sp. PCC7120.