The CyAbrB transcription factor CalA regulates the iron superoxide dismutase in Nostoc sp. strain PCC 7120 (original) (raw)
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Journal of Biological Chemistry, 2004
The nitrogen-fixing filamentous cyanobacterium Nostoc PCC 7120 (formerly named Anabaena PCC 7120) possesses two genes for superoxide dismutase, a unique membrane-associated manganese superoxide dismutase (MnSOD) and a soluble iron superoxide dismutase (FeSOD). A phylogenetic analysis of FeSODs shows that cyanobacterial enzymes form a well separated cluster with filamentous species found in one subcluster and unicellular species in the other. Activity staining, inhibition patterns, and immunogold labeling show that FeSOD is localized in the cytosol of vegetative cells and heterocysts (nitrogenase containing specialized cells formed during nitrogen-limiting conditions). The recombinant Nostoc FeSOD is a homodimeric, acidic enzyme exhibiting the characteristic iron peak at 350 nm in its ferric state, an almost 100% occupancy of iron per subunit, a specific activity using the ferricytochrome assay of (2040 ؎ 90) units mg ؊1 at pH 7.8, and a dissociation constant K d of the azide-FeSOD complex of 2.1 mM. Using stopped flow spectroscopy it was shown that the decay of superoxide in the presence of various FeSOD concentrations is first-order in enzyme concentration allowing the calculation of the catalytic rate constants, which increase with decreasing pH: 5.3 ؋ 10 9 M ؊1 s ؊1 (pH 7) to 4.8 ؋ 10 6 M ؊1 s ؊1 (pH 10). FeSOD and MnSOD complement each other to keep the superoxide level low in Nostoc PCC 7120, which is discussed with respect to the fact that Nostoc PCC 7120 exhibits oxygenic photosynthesis and oxygen-dependent respiration within a single prokaryotic cell and also has the ability to form differentiated cells under nitrogen-limiting conditions.
Proceedings of the National Academy of Sciences, 1992
The enzyme superoxide dismutase is ubiquitous in aerobic organisms where it plays a major role in alleviating oxygen-radical toxicity. An insertion mutation introduced into the iron superoxide dismutase locus (designated sodE) of the cyanobacterium Synechococcus sp. PCC 7942 created a mutant strain devoid of detectable iron superoxide dismutase activity. Both wild-type and mutant strains exhibited similar photosynthetic activity and viability when grown with 17 jmolm-2-s'1 illumination in liquid culture supplemented with 3% carbon dioxide. In contrast, the sodB mutant exhibited significantly greater damage to its photosynthetic system than the wild-type strain when grown under increased oxygen
Diversity, Prevalence and Role of Superoxide Dismutase in Cyanobacteria
Superoxide dismutase (SOD), the antioxidant enzyme exists in four diverse forms in cyanobacteria i.e. FeSOD, MnSOD, Cu/ZnSOD and NiSOD. FeSOD has an ancestral origin from the GSB ancestor of PSI while MnSOD has protobacterial ancestor to PSII & mitochondria. Cu/ZnSOD shows lateral gene transfer while NiSOD is rarely found. Fe & Mn forms share similarity while Cu/Zn form does not. SOD is prevalent in all subcellular locations where O 2-(superoxide) radicals are formed. SOD plays a defensive role at the time of environmental stress like chilling, dessication, light stress, metatoxicity etc and its activity is usually seen higher at the time of stress. Inactivation of SOD gene results in oxidative damage to the cell.
Isolation and in silico analysis of Fe-superoxide dismutase in the cyanobacterium Nostoc commune
Gene, 2014
Cyanobacteria are known to endure various stress conditions due to the inbuilt potential for oxidative stress alleviation owing to the presence of an array of antioxidants. The present study shows that Antarctic cyanobacterium Nostoc commune possesses two antioxidative enzymes viz., superoxide dismutase (SOD) and catalase that jointly cope with environmental stresses prevailing at its natural habitat. Native-PAGE analysis illustrates the presence of a single prominent isoform recognized as Fe-SOD and three distinct isoforms of catalase. The protein sequence of Fe-SOD in N. commune retrieved from NCBI protein sequence database was used for in silico analysis. 3D structure of N. commune was predicted by comparative modeling using MODELLER 9v11. Further, this model was validated for its quality by Ramachandran plot, ERRAT, Verify 3D and ProSA-web which revealed good structure quality of the model. Multiple sequence alignment showed high conservation in N and C-terminal domain regions along with all metal binding positions in Fe-SOD which were also found to be highly conserved in all 28 cyanobacterial species under study, including N. commune. In silico prediction of isoelectric point and molecular weight of Fe-SOD was found to be 5.48 and 22,342.98 Da respectively. The phylogenetic tree revealed that among 28 cyanobacterial species, Fe-SOD in N. commune was the closest evolutionary homolog of Fe-SOD in Nostoc punctiforme as evident by strong bootstrap value. Thus, N. commune may serve as a good biological model for studies related to survival of life under extreme conditions prevailing at the Antarctic region. Moreover cyanobacteria may be exploited for biochemical and biotechnological applications of enzymatic antioxidants.
Journal of Bacteriology
Superoxide dismutases (Sods) play very important roles in preventing oxidative damages in aerobic organisms. The nitrogen-fixing heterocystous cyanobacterium Anabaena sp. strain PCC 7120 has two Sod-encoding genes: a sodB, encoding a soluble iron-containing Sod (FeSod), and a sodA, encoding a manganese-containing Sod (MnSod). The FeSod was purified and characterized. A recombinant FeSod was also obtained by overproduction in Escherichia coli. Immunoblot study of the FeSod during induction of heterocyst differentiation showed that the cells produced six-to eightfold more FeSod 8 h after a shift from a nitrogen-replete condition to a nitrogen-depleted condition. However, the amount of FeSod protein in filaments with mature heterocysts was the same as that in filaments grown with combined nitrogen. Superoxide anion-generating chemicals such as methyl viologen did not induce upregulation of the sodB gene expression. The predicted preprotein of the sodA gene has a leader peptide and a motif for membrane attachment at the N terminus of the mature protein. Activity staining after gel electrophoresis of the purified thylakoid membranes showed that most of the MnSod in Anabaena sp. strain PCC 7120 was located on thylakoids toward the lumenal side. Expression of the sodA gene in E. coli shows that the leader peptide was required for its activity and the membrane localization of the MnSod. Northern hybridization detected one 0.82-kb transcript of sodA. The sodA gene was upregulated by methyl viologen, whereas its amount was unchanged during heterocyst differentiation. Immunoblotting and activity staining showed that isolated heterocysts contained a lower but still significant amount of FeSod, suggesting that its function is required in heterocysts. No MnSod was observed in isolated heterocysts. These results show that the two different Sod proteins have differentiated roles in Anabaena sp. strain PCC 7120.
A strain ofSynechococcus sp. strain PCC 7942 with no functional Fe superoxide dismutase (SOD), designatedsodB −, was characterized by its growth rate, photosynthetic pigments, and cyclic photosynthetic electron transport activity when treated with methyl viologen or norflurazon (NF). In their unstressed conditions, both thesodB − and wild-type strains had similar chlorophyll and carotenoid contents and catalase activity, but the wild type had a faster growth rate and higher cyclic electron transport activity. The sodB − was very sensitive to methyl viologen, indicating a specific role for the FeSOD in protection against superoxide generated in the cytosol. In contrast, thesodB − mutant was less sensitive than the wild type to oxidative stress imposed with NF. This suggests that the FeSOD does not protect the cell from excited singlet-state oxygen generated within the thylakoid membrane. Another up-regulated antioxidant, possibly the MnSOD, may confer protection against NF in the sodB − strain. These results support the hypothesis that different SODs have specific protective functions within the cell.
Physiological Roles of Superoxide Dismutases in the Cyanobacterium, Synechococcus sp. Strain PCC7942
Superoxide dismutases detoxify O2 - produced as a byproduct of normal aerobic metabolism. They are essential parts of a cell's antioxidant system. A strain of Synechococcus sp. strain PCC7942 with no functional iron superoxide dismutase, designated sodB-, was characterized by its growth rate, photosynthetic pigments, and cyclic photosynthetic electron transport activity when treated with methyl viologen or norflurazon. In their unstressed conditions, both the sodB- and wild type strains had similar chlorophyll and carotenoid contents and catalase activity, but the wild type had a slightly faster growth rate and higher cyclic photosynthetic electron transport activity. The sodB- was very sensitive to methyl viologen, indicating a specific role for the iron superoxide dismutase in protection against superoxide generated in the cytosol. In contrast, the sodB- mutant was less sensitive than the wild type to oxidative stress imposed with norflurazon. This suggests that the iron superoxide dismutase does not protect the cell from 1O2* generated within the thylakoid membrane. Another upregulated antioxidant, possibly the manganese superoxide dismutase, may confer protection against NF in the sodB- strain. The two strains were also compared under regimes of temperature stress (0°, 10°, 17°, 27°, 32°, 37° and 42°C). The sodB- strain was found to be sensitive to both chilling and heat stress. These results support the hypothesis that different SODs have specific protective functions within the cell. In addition, a method for the detection of oxidative damage to DNA was developed for use in cyanobacteria, and a partial sequence for the sodA gene was obtained.
Journal of Bacteriology, 2006
To investigate the roles of these proteins further, single and double null-mutant strains of Synechococcus sp. strain PCC 7002 were constructed by insertional inactivation of genes homologous to sufA, iscA, and nfu. Demonstrating the nonessential nature of their products, the sufA, iscA, and sufA iscA mutants grew photoautotrophically with doubling times that were similar to the wild type under standard growth conditions. In contrast, attempts to inactivate the nfu gene only resulted in stable merodiploids. These results imply that Nfu, but not SufA or IscA, is the essential Fe/S scaffold protein in cyanobacteria. When cells were grown under iron-limiting conditions, the iscA and sufA mutant strains exhibited less chlorosis than the wild type. Under iron-sufficient growth conditions, isiA transcript levels, a marker for iron limitation in cyanobacteria, as well as transcript levels of genes in both the suf and isc regulons were significantly higher in the iscA mutant than in the wild type. Under photosynthesis-induced redox stress conditions, the transcript levels of the suf genes are notably higher in the sufA and the sufA iscA mutants than in the wild type. The growth phenotypes and mRNA abundance patterns of the mutant strains contradict the proposed scaffold function for the SufA and IscA proteins in generalized Fe/S cluster assembly and instead suggest that they play regulatory roles in iron homeostasis and the sensing of redox stress in cyanobacteria.
Plant Physiology, 1999
A strain of Synechococcus sp. PCC7942 lacking functional Fe superoxide dismutase (SOD), designated sodB ؊ , was characterized by its growth rate, photosynthetic pigments, inhibition of photosynthetic electron transport activity, and total SOD activity at 0°C, 10°C, 17°C, and 27°C in moderate light. At 27°C, the sodB ؊ and wild-type strains had similar growth rates, chlorophyll and carotenoid contents, and cyclic photosynthetic electron transport activity. The sodB ؊ strain was more sensitive to chilling stress at 17°C than the wild type, indicating a role for FeSOD in protection against photooxidative damage during moderate chilling in light. However, both the wild-type and sodB ؊ strains exhibited similar chilling damage at 0°C and 10°C, indicating that the FeSOD does not provide protection against severe chilling stress in light. Total SOD activity was lower in the sodB ؊ strain than in the wild type at 17°C and 27°C. Total SOD activity decreased with decreasing temperature in both strains but more so in the wild type. Total SOD activity was equal in the two strains when assayed at 0°C.
Annales de Limnologie - International Journal of Limnology, 2002
Fur (Ferric uptake regulator) is a DNA-binding protein which represses the transcription of iron-regulated promoters using Fe 2+ as a cofactor. In Gram negative bacteria, fur controls the expression of toxins and virulence factors when facing iron-stress. Iron is a key element limiting the growth and proliferation of phytoplankton. We have cloned the fur-gene from the cyanobacterium Synechococcus PCC 7942, applying the polymerase chain reaction (PCR) technique. The DNA fragments amplified encodes a Fur-type protein, which conserves the main motifs found in the family of Fur proteins. Recombinant Fur protein was successfully overproduced, reaching about 15 % of the total protein content. Also, a Fur protein was identified in the filamentous, nitrogen-fixing cyanobacterium Anabaena PCC 7120. Réponse génétique au stress ferrique chez les Cyanobactéries : les gènes fur de Synechococcus PC 7942 et Anabaena PCC 7120 Mots-clés : ferrie uptake regulator (fur), fer, cyanobactérie. Fur (Ferrie uptake regulator) est une protéine d'union à l'ADN qui réprime la transcription des promoteurs régulés par le fer, utilisé sous forme Fe 2+ comme cofacteur. Chez les bactéries Gram négatif, fur controle l'expression des toxines et des facteurs de virulence lors de stress ferrique. Le fer est un élément fondamental qui limite la croissance et la prolifération du phytoplancton. Nous avons cloné le gène fur de la cyanobactérie Synechococcus PCC 7942, en appliquant la technique de la réaction en chaîne de la polymerase (PCR). Le fragment d'ADN exprime une protéine de la famille de Fur qui conserve les motifs principaux trouvés dans cette famille. La protéine Fur de Synechococcus a été produite avec succès, avec un rendement important de l'ordre de 15 % du contenu total des protéines. De plus, une autre protéine Fur a été identifiée dans une cyanobactérie filamenteuse fixative d'azote : Anabaena PCC 7120.