Cyanophycin Synthesis Optimizes Nitrogen Utilization in the Unicellular Cyanobacterium Synechocystis sp. Strain PCC 6803.
ABSTRACT: Cyanophycin is a carbon/nitrogen storage polymer widely distributed in most cyanobacterial strains and in a few heterotrophic bacteria. It is a nonribosomal polypeptide consisting of equimolar amounts of aspartate and arginine. Here, we focused on the physiological function and cell biology of cyanophycin in the unicellular nondiazotrophic cyanobacterium Synechocystis sp. strain PCC 6803. To study the cellular localization of the cyanophycin-synthesizing enzyme CphA during cyanophycin synthesis and degradation, we fused it to green fluorescent protein. When CphA was inactive, it localized diffusely in the cytoplasm. When cyanophycin synthesis was triggered, CphA first aggregated into foci and later localized on the surface of cyanophycin granules. In the corresponding cell extracts, localization of CphA on the cyanophycin granule surface required Mg2+ During cyanophycin degradation, CphA dissociated from the granule surface and returned to its inactive form in the cytoplasm. To investigate the physiological role of cyanophycin, we compared wild-type cells with a CphA-deficient mutant. Under standard laboratory conditions, the ability to synthesize cyanophycin did not confer a growth advantage. To mimic the situation in natural habitats, cells were cultured with a fluctuating and limiting nitrogen supplementation and/or day/night cycles. Under all of these conditions, cyanophycin provided a fitness advantage to the wild type over the mutant lacking cyanophycin. During resuscitation from nitrogen starvation, wild-type cells accumulated cyanophycin during the night and used it as an internal nitrogen source during the day. This demonstrates that cyanophycin can be used as a temporary nitrogen storage to uncouple nitrogen assimilation from photosynthesis.IMPORTANCE We clarified the elusive biological function of cyanophycin in the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803. Cyanophycin is a dynamic carbon/nitrogen storage polymer (multi-arginyl-l-polyaspartate) that is conditionally present in most cyanobacteria and a few heterotrophic bacteria as cellular inclusion granules. Here, we show that the cyanophycin-synthesizing enzyme CphA in the nonactive state localizes diffusely in the cytoplasm. When cyanophycin synthesis is triggered, active CphA first aggregates into foci and then covers the surface of mature cyanophycin granules, which in vitro requires Mg2+ as a cofactor. Cyanophycin accumulation enables Synechocystis sp. to optimize nitrogen assimilation under nitrogen-poor conditions, in particular when the nitrogen supply fluctuates and during day/night cycles, by allowing continuous nitrogen assimilation and storage. Therefore, cyanophycin provides the wild-type cyanobacterium with a clear fitness advantage over non-cyanophycin-producing cells in natural environments with fluctuating nitrogen supply.
Project description:The thermophilic cyanobacterium Synechococcus sp. strain MA19 contained the structural genes for cyanophycin synthetase (cphA) and cyanophycinase (cphB), which were identified, cloned, and sequenced in this study. The translation products of cphA and cphB exhibited high levels of similarity to corresponding proteins of other cyanobacteria, such as Anabaena variabilis and Synechocystis sp. Recombinant cells of Escherichia coli harboring cphA colinear with lacPO accumulated cyanophycin that accounted for up to 25% (wt/wt) of the dry cell matter in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG). The cyanophycin synthetase was enriched 123-fold to electrophoretic homogeneity from the soluble fraction of the recombinant cells by anion-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified cyanophycin synthetase maintained the parental thermophilic character and was active even after prolonged incubation at 50 degrees C; in the presence of ectoine the enzyme retained 90% of its activity even after 2 h of incubation. The in vitro activity of the enzyme depended on ATP, primers, and both substrates, L-arginine and L-aspartic acid. In addition to native cyanophycin, the purified enzyme accepted a modified cyanophycin containing less arginine, alpha-arginyl aspartic acid dipeptide, and poly-alpha,beta-DL-aspartic acid as primers and also incorporated beta-hydroxyaspartic acid instead of L-aspartic acid or L-canavanine instead of L-arginine at a significant rate. The lack of specificity of this thermostable enzyme with respect to primers and substrates, the thermal stability of the enzyme, and the finding that the enzyme is suitable for in vitro production of cyanophycin make it an interesting candidate for biotechnological processes.
Project description:Nitrogen limitation imposes a major transition in the lifestyle of nondiazotrophic cyanobacteria that is controlled by a complex interplay of regulatory factors involving the pervasive signal processor P<sub>II</sub> Immediately upon nitrogen limitation, newly fixed carbon is redirected toward glycogen synthesis. How the metabolic switch for diverting fixed carbon toward the synthesis of glycogen or of cellular building blocks is operated was so far poorly understood. Here, using the nondiazotrophic cyanobacterium <i>Synechocystis</i> sp. PCC 6803 as model system, we identified a novel P<sub>II</sub> interactor, the product of the <i>sll0944</i> gene, which we named PirC. We show that PirC binds to and inhibits the activity of 2,3-phosphoglycerate-independent phosphoglycerate mutase (PGAM), the enzyme that deviates newly fixed CO<sub>2</sub> toward lower glycolysis. The binding of PirC to either P<sub>II</sub> or PGAM is tuned by the metabolite 2-oxoglutarate (2-OG), which accumulates upon nitrogen starvation. In these conditions, the high levels of 2-OG dissociate the PirC-P<sub>II</sub> complex to promote PirC binding to and inhibition of PGAM. Accordingly, a PirC-deficient mutant showed strongly reduced glycogen levels upon nitrogen deprivation, whereas polyhydroxybutyrate granules were overaccumulated compared to wild-type. Metabolome analysis revealed an imbalance in 3-phosphoglycerate to pyruvate levels in the <i>pirC</i> mutant, confirming that PirC controls the carbon flux in cyanobacteria via mutually exclusive interaction with either P<sub>II</sub> or PGAM.
Project description:Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-L-arginyl-poly-L-aspartic acid [CGP]) synthesis and to explore the applicability of this industrially widely used microorganism for the production of this polyamide. Therefore, the CGP synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA(6308)) was expressed under the control of the alcohol oxidase 1 promoter, yielding CGP contents of up to 10.4% (wt/wt), with the main fraction consisting of the soluble form of the polymer. To increase the polymer contents and to obtain further insights into the structural or catalytic properties of the enzyme, site-directed mutagenesis was applied to cphA(6308) and the mutated gene products were analyzed after expression in P. pastoris and Escherichia coli, respectively. CphA(6308)Delta1, which was truncated by one amino acid at the C terminus; point mutated CphA(6308)C595S; and the combined double-mutant CphA(6308)Delta1C595S protein were purified. They exhibited up to 2.5-fold higher enzyme activities of 4.95 U/mg, 3.20 U/mg, and 4.17 U/mg, respectively, than wild-type CphA(6308) (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA(6308)Delta2) or three (CphA(6308)Delta3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA(6308). In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA(6308)Delta1 in P. pastoris. For stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae was cloned into the expression vector used and the constructs were transferred to histidine auxotrophic P. pastoris strain GS115. Parallel fermentations at a one-to-one scale revealed 26 degrees C and 6.0 as the optimal temperature and pH, respectively, for CGP synthesis. After optimization of fermentation parameters, medium composition, and the length of the cultivation period, CGP contents could be increased from 3.2 to 13.0% (wt/wt) in cells of P. pastoris GS115 expressing CphA(6308) and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA(6308)Delta1.
Project description:Unicellular nitrogen fixing cyanobacteria (UCYN) are abundant members of phytoplankton communities in a wide range of marine environments, including those with rapidly changing nitrogen (N) concentrations. We hypothesized that differences in N availability (N<sub>2</sub> vs. combined N) would cause UCYN to shift strategies of intracellular N and C allocation. We used transmission electron microscopy and nanoscale secondary ion mass spectrometry imaging to track assimilation and intracellular allocation of <sup>13</sup>C-labeled CO<sub>2</sub> and <sup>15</sup>N-labeled N<sub>2</sub> or NO<sub>3</sub> at different periods across a diel cycle in <i>Cyanothece</i> sp. ATCC 51142. We present new ideas on interpreting these imaging data, including the influences of pre-incubation cellular C and N contents and turnover rates of inclusion bodies. Within cultures growing diazotrophically, distinct subpopulations were detected that fixed N<sub>2</sub> at night or in the morning. Additional significant within-population heterogeneity was likely caused by differences in the relative amounts of N assimilated into cyanophycin from sources external and internal to the cells. Whether growing on N<sub>2</sub> or NO<sub>3</sub>, cells prioritized cyanophycin synthesis when N assimilation rates were highest. N assimilation in cells growing on NO<sub>3</sub> switched from cyanophycin synthesis to protein synthesis, suggesting that once a cyanophycin quota is met, it is bypassed in favor of protein synthesis. Growth on NO<sub>3</sub> also revealed that at night, there is a very low level of CO<sub>2</sub> assimilation into polysaccharides simultaneous with their catabolism for protein synthesis. This study revealed multiple, detailed mechanisms underlying C and N management in <i>Cyanothece</i> that facilitate its success in dynamic aquatic environments.
Project description:Heterocyst-forming cyanobacteria are multicellular organisms in which growth requires the activity of two metabolically interdependent cell types, the vegetative cells that perform oxygenic photosynthesis and the dinitrogen-fixing heterocysts. Vegetative cells provide the heterocysts with reduced carbon, and heterocysts provide the vegetative cells with fixed nitrogen. Heterocysts conspicuously accumulate polar granules made of cyanophycin [multi-L-arginyl-poly (L-aspartic acid)], which is synthesized by cyanophycin synthetase and degraded by the concerted action of cyanophycinase (that releases ?-aspartyl-arginine) and isoaspartyl dipeptidase (that produces aspartate and arginine). Cyanophycin synthetase and cyanophycinase are present at high levels in the heterocysts. Here we created a deletion mutant of gene all3922 encoding isoaspartyl dipeptidase in the model heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. The mutant accumulated cyanophycin and ?-aspartyl-arginine, and was impaired specifically in diazotrophic growth. Analysis of an Anabaena strain bearing an All3922-GFP (green fluorescent protein) fusion and determination of the enzyme activity in specific cell types showed that isoaspartyl dipeptidase is present at significantly lower levels in heterocysts than in vegetative cells. Consistently, isolated heterocysts released substantial amounts of ?-aspartyl-arginine. These observations imply that ?-aspartyl-arginine produced from cyanophycin in the heterocysts is transferred intercellularly to be hydrolyzed, producing aspartate and arginine in the vegetative cells. Our results showing compartmentalized metabolism of cyanophycin identify the nitrogen-rich molecule ?-aspartyl-arginine as a nitrogen vehicle in the unique multicellular system represented by the heterocyst-forming cyanobacteria.
Project description:<h4>Unlabelled</h4>Heat-resistant endospore formation plays an important role in Clostridium perfringens-associated foodborne illnesses. The spores allow the bacterium to survive heating during normal cooking processes, followed by germination and outgrowth of the bacterium in contaminated foods. To identify proteins associated with germination and other spore functions, a comparative spore membrane proteome analysis of dormant and germinated spores of C. perfringens strain SM101 was performed by using gel-based protein separation and liquid chromatography coupled with matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) mass spectrometry. A total of 494 proteins were identified, and 117 of them were predicted to be integral membrane or membrane-associated proteins. Among these membrane proteins, 16 and 26 were detected only in dormant and germinated spores, respectively. One protein that was detected only in germinated spore membranes was the enzyme cyanophycinase, a protease that cleaves the polymer cyanophycin, which is composed of l-arginine-poly(l-aspartic acid), to ?-Asp-Arg. Genes encoding cyanophycinase and cyanophycin synthetase have been observed in many species of Clostridium, but their role has not been defined. To determine the function of cyanophycin in C. perfringens, a mutation was introduced into the cphA gene, encoding cyanophycin synthetase. In comparison to parent strain SM101, the spores of the mutant strain retained wild-type levels of heat resistance, but fewer spores were made, and they were smaller, suggesting that cyanophycin synthesis plays a role in spore assembly. Although cyanophycin could not be extracted from sporulating C. perfringens cells, an Escherichia coli strain expressing the cphA gene made copious amounts of cyanophycin, confirming that cphA encodes a cyanophycin synthetase.<h4>Importance</h4>Clostridium perfringens is a common cause of food poisoning, and germination of spores after cooking is thought to play a significant role in the disease. How C. perfringens controls the germination process is still not completely understood. We characterized the proteome of the membranes from dormant and germinated spores and discovered that large-scale changes occur after germination is initiated. One of the proteins that was detected after germination was the enzyme cyanophycinase, which degrades the storage compound cyanophycin, which is found in cyanobacteria and other prokaryotes. A cyanophycin synthetase mutant was constructed and found to make spores with altered morphology but normal heat resistance, suggesting that cyanophycin plays a different role in C. perfringens than it does in cyanobacteria.
Project description:Nitrogen is often a limiting nutrient in natural habitats. Therefore, cyanobacteria have developed multiple responses, which are controlled by transcription factor NtcA and the PII-signaling protein, to adapt to nitrogen deficiency. Transcriptional analyses of Synechocystis sp. strain PCC 6803 under nitrogen-deficient conditions revealed a highly induced gene (sll0783) which is annotated as encoding a conserved protein with an unknown function. This gene is part of a cluster of seven genes and has potential NtcA-binding sites in the upstream region. Homologues of this cluster occur in some unicellular, nondiazotrophic cyanobacteria and in several Alpha, Beta-, and Gammaproteobacteria, as well as in some Gram-positive bacteria. Most of the heterotrophic bacteria harboring this gene cluster are able to fix nitrogen and to produce polyhydroxybutyrate (PHB), whereas of the cyanobacteria, only Synechocystis sp. strain PCC 6803 can accumulate PHB. In this work, a Synechocystis sp. strain PCC 6803 sll0783 gene knockout mutant is characterized. This mutant is unable to accumulate PHB, a carbon and energy storage compound. In contrast, the levels of the carbon storage compound glycogen and the PHB precursor acetyl coenzyme A were similar to those of the wild type, indicating that the PHB-deficient phenotype does not likely result from a global deficiency in carbon metabolism. A specific deficiency in PHB synthesis was implied by the fact that the mutant exhibits impaired PHB synthase activity during prolonged nitrogen starvation. However, the expression of PHB synthase-encoding genes was not strongly affected in the mutant, suggesting that the impaired PHB synthase activity observed depends on a posttranscriptional process in which the product of sll0783 is involved.
Project description:During phases of nitrogen starvation, the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 produces polyhydroxybutyrate (PHB). This polymer is of high biotechnological relevance because of its potential as biodegradable plastic. Analysis of the Synechocystis genome revealed an operon (slr0058-slr0061) containing several genes, which are putatively related to the PHB metabolism. While Slr0058 show similarities with the regulatory phasin PhaF, the protein Slr0060 could serve as an intracellular PHB depolymerase. Investigation of respective knock-out mutants showed no distinct phenotype for the strain lacking Slr0060, whereas the ?slr0058 mutant displayed a growth impairment as well as a change in pigmentation. During nitrogen starvation, the ?slr0058 mutant produced in average more than twice the amount of PHB granules per cell, while the overall amount of PHB remained the same. This indicates that Slr0058 plays a role in PHB granule formation and controls it surface-to-volume ratio. GFP-tagged Slr0058 did not co-localize with PHB granules during nitrogen starvation but aggregated in distinct foci during vegetative growth. This work helps to better understand the PHB metabolism of Synechocystis sp. PCC 6803, coming closer to a sustainable, industrial production of PHB.
Project description:The introduction of alternative CO2-fixing pathways in photoautotrophic organism may improve the efficiency of biological carbon fixation such as minimizing the carbon loss due to photorespiration. Here, we analyzed the effects of creating a formate entry point into the primary metabolism of the cyanobacterium Synechocystis sp. PCC 6803. The formate-tetrahydrofolate ligase (FTL) from Methylobacterium extorquens AM1 was expressed in Synechocystis to enable formate assimilation and reducing the loss of fixed carbon in the photorespiratory pathway. Transgenic strains accumulated serine and 3-phosphoglycerate, and consumed more 2-phosphoglycolate and glycine, which seemed to reflect an efficient utilization of formate. However, labeling experiments showed that the serine accumulation was not due to the expected incorporation of formate. Subsequent DNA-microarray analysis revealed profound changes in transcript abundance due to ftl expression. Transcriptome changes were observed in relation to serine and glycine metabolism, C1-metabolism and particularly nitrogen assimilation. The data implied that ftl expression interfered with the signaling the carbon/nitrogen ratio in Synechocystis. Our results indicate that the expression of new enzymes could have a severe impact on the cellular regulatory network, which potentially hinders the establishment of newly designed pathways.
Project description:Nitrogen is essential for the biosynthesis of various molecules in cells, such as amino acids and nucleotides, as well as several types of lipids and sugars. Cyanobacteria can assimilate several forms of nitrogen, including nitrate, ammonium, and urea, and the physiological and genetic responses to these nitrogen sources have been studied previously. However, the metabolic changes in cyanobacteria caused by different nitrogen sources have not yet been characterized. This study aimed to elucidate the influence of nitrate and ammonium on the metabolic profiles of the cyanobacterium <i>Synechocystis</i> sp. strain PCC 6803. When supplemented with NaNO<sub>3</sub> or NH<sub>4</sub>Cl as the nitrogen source, <i>Synechocystis</i> sp. PCC 6803 grew faster in NH<sub>4</sub>Cl medium than in NaNO<sub>3</sub> medium. Metabolome analysis indicated that some metabolites in the CBB cycle, glycolysis, and TCA cycle, and amino acids were more abundant when grown in NH<sub>4</sub>Cl medium than NaNO<sub>3</sub> medium. <sup>15</sup>N turnover rate analysis revealed that the nitrogen assimilation rate in NH<sub>4</sub>Cl medium was higher than in NaNO<sub>3</sub> medium. These results indicate that the mechanism of nitrogen assimilation in the GS-GOGAT cycle differs between NaNO<sub>3</sub> and NH<sub>4</sub>Cl. We conclude that the amounts and biosynthetic rate of cyanobacterial metabolites varies depending on the type of nitrogen.