Differential catalase activity and tolerance to hydrogen peroxide in the filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Anabaena sp. PCC 7120.

Photoautotrophic cyanobacteria often confront hydrogen peroxide (H2O2), a reactive oxygen species potentially toxic to cells when present in sufficiently high concentrations. In this study, H2O2 tolerance ability of filamentous cyanobacteria Nostoc punctiforme ATCC 29133 (Nostoc 29133) and Anabaena sp. PCC 7120 (Anabaena 7120) was investigated at increasing concentrations of H2O2 (0-0.5 mM). In Nostoc 29133, 0.25 and 0.5 mM H2O2 caused a reduction in chlorophyll a content by 12 and 20%, respectively, whereas with similar treatments, a total loss of chlorophyll a was detected in Anabaena 7120. Further, Nostoc 29133 was able to maintain its photosystem II performance in the presence of H2O2 up to a concentration of 0.5 mM, whereas in Anabaena 7120, 0.25 mM H2O2 caused a complete reduction of photosystem II performance. The intracellular hydroperoxide level (indicator of oxidative status) did not increase to the same high level in Nostoc 29133, as compared to in Anabaena 7120 after H2O2 treatment. This might be explained by that Nostoc 29133 showed a 20-fold higher intrinsic constitutive catalase activity than Anabaena 7120, thus indicating that the superior tolerance of Nostoc 29133 to H2O2 stems from its higher ability to decompose H2O2. It is suggested that difference in H2O2 tolerance between closely related filamentous cyanobacteria, as revealed in this study, may be taken into account for judicious selection and effective use of strains in biotechnological applications.

Molecular Cloning and Biochemical Characterization of the Iron Superoxide Dismutase from the Cyanobacterium Nostoc punctiforme ATCC 29133 and Its Response to Methyl Viologen-Induced Oxidative Stress.

Superoxide dismutase (SOD) detoxifies cell-toxic superoxide radicals and constitutes an important component of antioxidant machinery in aerobic organisms, including cyanobacteria. The iron-containing SOD (SodB) is one of the most abundant soluble proteins in the cytosol of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133, and therefore, we investigated its biochemical properties and response to oxidative stress. The putative SodB-encoding open reading frame Npun_R6491 was cloned and overexpressed in Escherichia coli as a C-terminally hexahistidine-tagged protein. The purified recombinant protein had a SodB specific activity of 2560 ± 48 U/mg protein at pH 7.8 and was highly thermostable. The presence of a characteristic iron absorption peak at 350 nm, and its sensitivity to H2O2 and azide, confirmed that the SodB is an iron-containing SOD. Transcript level of SodB in nitrogen-fixing cultures of N. punctiforme decreased considerably (threefold) after exposure to an oxidative stress-generating herbicide methyl viologen for 4 h. Furthermore, in-gel SOD activity analysis of such cultures grown at increasing concentrations of methyl viologen also showed a loss of SodB activity. These results suggest that SodB is not the primary scavenger of superoxide radicals induced by methyl viologen in N. punctiforme.

A high constitutive catalase activity confers resistance to methyl viologen-promoted oxidative stress in a mutant of the cyanobacterium Nostoc punctiforme ATCC 29133.

A spontaneous methyl viologen (MV)-resistant mutant of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133 was isolated and the major enzymatic antioxidants involved in combating MV-induced oxidative stress were evaluated. The mutant displayed a high constitutive catalase activity as a consequence of which, the intracellular level of reactive oxygen species in the mutant was lower than the wild type (N. punctiforme) in the presence of MV. The superoxide dismutase (SOD) activity that consisted of a SodA (manganese-SOD) and a SodB (iron-SOD) was not suppressed in the mutant following MV treatment. The mutant was, however, characterised by a lower peroxidase activity compared with its wild type, and its improved tolerance to externally added H2O2 could only be attributed to enhanced catalase activity. Furthermore, MV-induced toxic effects on the wild type such as (1) loss of photosynthetic performance assessed as maximal quantum yield of photosystem II, (2) nitrogenase inactivation, and (3) filament fragmentation and cell lysis were not observed in the mutant. These findings highlight the importance of catalase in preventing MV-promoted oxidative damage and cell death in the cyanobacterium N. punctiforme. Such oxidative stress resistant mutants of cyanobacteria are likely to be a better source of biofertilisers, as they can grow and fix nitrogen in an unhindered manner in agricultural fields that are often contaminated with the herbicide MV, also commonly known as paraquat.

Thermodynamical property of entanglement entropy for excited states.

We argue that the entanglement entropy for a very small subsystem obeys a property which is analogous to the first law of thermodynamics when we excite the system. In relativistic setups, its effective temperature is proportional to the inverse of the subsystem size. This provides a universal relationship between the energy and the amount of quantum information. We derive the results using holography and confirm them in two-dimensional field theories. We will also comment on an example with negative specific heat and suggest a connection between the second law of thermodynamics and the strong subadditivity of entanglement entropy.

Inactivation of the extrinsic subunit of photosystem II, PsbU, in Synechococcus PCC 7942 results in elevated resistance to oxidative stress.

PsbU is a subunit of the extrinsic complex attached to the core of photosystem II. A PsbU-mutant of Synechococcus PCC 7942 was isolated based on its elevated resistance to externally applied oxidative stress. PsbU-mutant exhibits fast rates of degradation of the photosystem II core protein, D1, under sub-saturating as well as high-light conditions. While forward electron transfer is not affected, back electron flow is severely impaired in the mutant. We suggest that impairment of psbU results in production of reactive-oxygen-species, which trigger antioxidative mechanisms even under standard growth conditions. Accordingly, when challenged with external oxidative stress, these cells are more resistant than wild type cells.

Synechocystis Fe superoxide dismutase gene confers oxidative stress tolerance to Escherichia coli.

The superoxide dismutase (SOD) gene (slr 1516) from the cyanobacterium Synechocystis sp. PCC 6803 was cloned and overexpressed in Escherichia coli BL 21 (DE3) using the pET-20b(+) expression vector. E. coli cells transformed with pET-SOD overexpressed the protein in cytosol, upon induction by isopropyl beta-D-thiogalactopyranoside (IPTG). The recombinant protein was purified to near homogeneity by gel filtration and ion-exchange chromatography. The SOD activity of the recombinant protein was sensitive to hydrogen peroxide and sodium azide, confirming it to be FeSOD. The pET-FeSOD transformed E. coli showed significantly higher SOD activity and tolerance to paraquat-mediated growth inhibition compared to the empty vector transformed cells. Based on these results it is suggested that overexpression of FeSOD gene from a heterologous source like Synechocystis sp. PCC 6803 may provide protection to E. coli against superoxide radical-mediated oxidative stress mediated by paraquat.

Isolation and characterization of a chlorate-resistant mutant (Clo- R) of the symbiotic cyanobacterium Nostoc ANTH: heterocyst formation and N(2)-fixation in the presence of nitrate, and evidence for separate nitrate and nitrite transport systems.

Nostoc ANTH is a filamentous, heterocystous cyanobacterium capable of N(2)-fixation in the absence of combined nitrogen. A chlorate-resistant mutant (Clo- R) of Nostoc ANTH was isolated that differentiates heterocysts and fixes N(2) in the presence of nitrate, but not in the presence of nitrite or ammonium. The mutant lacks nitrate uptake and thereby also lacks induction of nitrate reductase activity by nitrate. However, this mutant is able to transport and assimilate nitrite, indicating that there is a transport system for nitrite that is distinct from that for the nitrate. The lack of inhibitory effect of nitrate on N(2)-fixation was owing to lack of nitrate uptake and not to lack of enzymes for its assimilation (nitrate reductase and glutamine synthetase) or the lack of an ammonium transport system for retention of ammonia. The mutant has potential for use as a biofertilizer supplementing chemical nitrate fertilizer in rice fields, without N(2)-fixation being adversely affected.

Nitrogen nutrition in the cyanobacterium Nostoc ANTH, a symbiotic isolate from Anthoceros: uptake and assimilation of inorganic-n and amino acids.

Amino acid uptake and utilization of various nitrogen sources (amino acids, nitrite, nitrate and ammonia) were studied in Nostoc ANTH and i ts mu tant (Het(-)Nif(-)) isolate defective in heterocyst formation and N2-fixation. Both parent and its mutant grew at the expense of glutamine, asparagine and arginine as a source of fixed-nitrogen. Growth was better in glutamine-and asparagine-media as compared to that in arginine media. Glutamine and asparagine repressed heterocyst formation, N2-fixation and nitrate reduction in Nostoc ANTH, but arginine did so only partially. The poor growth in arginine-medium was not due to poor uptake rates, since the uptake rates were not significantly different from those for glutamine or asparagine. The glutamine synthetase activity remained unaffected during cultivation in media containing any one of the three amino acids tested. The uptake of amino acids was substrate-inducible, energy-dependent and required de novo protein synthesis. Nitrate and ammonium repressed ammonium uptake, but did not repress uptake of amino acids. In N2-medium (BG-11(0)), the uptake of ammonium and amino acids in the mutant was significantly higher than its parent strain. This was apparently due to nitrogen limitation since the mutant was unable to fix N2 and the growth medium lacked combined-N.