A bentazone-resistant mutant of cyanobacterium, Synechococcus elongatus PCC7942 adapts different strategies to counteract on bromoxynil- and salt-mediated oxidative stress.

A bentazone-resistant mutant of Synechococcus elongatus PCC7942, called Mu2, tolerated elevated NaCl concentrations. As bentazone and bromoxynil exhibit similar mechanism of action, we investigated whether the mutant also toleratedbromoxynil and found it to be true. The line of investigation was then whether the acclimation strategy for the three stressors, bentazone, bromoxynil and NaCl was same or different. The cellular contents of malondialdehyde, hydrogen peroxide and superoxide increased in wild type strain following all the treatments suggesting their toxicities due to oxidative response. Notwithstanding, there were apparently different anti-oxidative measures pertaining to the herbicide and salinity stress. Glutathione contents and activities of superoxide dismutase, catalase-peroxidase, glutathione S-transferase and glutathione reductase decreased under NaCl, whereas bromoxynil affected only glutathione S-transferase reductase. Moreover, in-gel assays revealed that bromoxynil promoted appearance of isozymes of catalase-peroxidase, while NaCl induced such response only for superoxide dismutase. On the other hand, in Mu2, glutathione peroxidase-reductase and glutathione showed upward trend after bromoxynil exposure, whereas NaCl raised peroxidase and superoxide dismutase. Proteome comparison revealed peroxiredoxin Q to be highly expressed in wild type strain under bromoxynil, whereas NaCl favoured flavodoxin over-expression. Their amounts were already high in Mu2. We suggest that Mu2 acclimatized to bromoxynil in a manner similar to bentazone by upgrading peroxiredoxin Q and glutathione peroxidase-reductase. Conversely, for NaCl it devised another mechanism involving peroxidase and superoxide dismutase, and flavodoxin.

Sodium selenate effect on Synechococcus elongatus PCC 7942: appearance of novel enzymatic reaction, ATP:selenate adenylyltransferase, and variation in antioxidant enzyme activities.

Synechococcus elongatus PCC 7942 was grown with SO(4) (2-) or S(2)O(3) (2-) sulfur source with varying concentration of SeO(4) (2-). Up to 150 muM, SeO(4) (2-) was growth supportive and above this it was inhibitory. Selenate is believed to induce ROS production, and to counter, the cells produce increasing amounts of ROS quenching enzymes. To investigate the differential growth response of SeO(4) (2-) at the level of ROS production, the activities of antioxidant enzymes viz. glutathione peroxidase, catalase, pyrogallol-peroxidase, superoxide dismutase and glutathione reductase were determined. Regardless of SeO(4) (2-) concentration in the medium, except catalase, the activity of other enzymes increased over SeO(4) (2-)-free controls. To understand the fate of SeO(4) (2-) added at lower concentrations, the activities of key inorganic sulfur metabolizing enzymes, ATP sulfurylase in SO(4) (2-) medium and thiosulfate reductase in S(2)O(3) (2-) medium were measured. This was done with an assumption that these enzymes would consume the SO(4) (2-) analog SeO(4) (2-) as sulfur source. We found enzymatic SeO(4) (2-)/ATP dependent formation of inorganic pyrophosphate in a reaction, analogous to ATP sulfurylase (SO(4) (2-) + ATP --> pyrophosphate) carried out by an enzyme different from ATP sulfurylase, tentatively called ATP:selenate adenylyltransferase. We hypothesize that selenium could act as trace element for S. elongatus PCC 7942.

Catalytic and regulatory properties of sulphur metabolizing enzymes in cyanobacterium Synechococcus elongatus PCC 7942.

Synechococcus elongatus PCC 7942 was able to grow with several S sources. The sulphur metabolizing enzymes viz. ATP sulphurylase, cysteine synthase, thiosulphate reductase and L- and D-cysteine desulphydrases were regulated by sulphur sources, particularly by sulphur amino acids and organic sulphate esters. Sulphur starvation reduced ATP sulphurylase and cysteine synthase whereas reduced glutathione appreciated Cys degradation activity. With partially purified enzymes apparent Km values for sulphate, ATP, D- and L-Cys, thiosulphate, sulphide and O-acetyl serine were in a range of 12-50 microM. p-Nitrophenyl sulphate inhibited ATP sulphurylase competitively. Met was a feedback inhibitor of several key enzymes.

Characterization of proteases in guts of Daphnia magna and their inhibition by Microcystis aeruginosa PCC 7806.

Many cyanobacteria produce peptides that inhibit mammalian proteases. The hypothesis that inhibitors of mammalian proteases produced by cyanobacteria also interfere with digestive proteases of natural cladoceran grazers was tested by comparing the effects of cyanobacterial protease inhibitors on digestive proteases from Daphnia magna and on commercially available bovine proteases. The major digestive proteases of D. magna are trypsins and chymotrypsins, which differ from those of bovine origin in substrate specificity and susceptibility to synthetic inhibitors. An extract from Microcystis aeruginosa strain PCC 7806 inhibited both types of D. magna proteases. Subsequent fractionation of the extract by high-performance liquid chromatography indicated that several inhibitors are produced by M. aeruginosa that differ in their specificity for the trypsins and chymotrypsins of D. magna. Two fractions differed in their inhibitory effect on proteases of D. magna and bovine origin; therefore, assessment of the impact of cyanobacterial protease inhibitors on natural communities requires the use of digestive proteases from ecologically relevant grazers.

Cysteine and serine protease-mediated proteolysis in body homogenate of a zooplankter, Moina macrocopa, is inhibited by the toxic cyanobacterium, Microcystis aeruginosa PCC7806.

The paper describes the characterization of proteases in the whole body homogenate of Moina macrocopa, which can possibly be inhibited by the extracts of Microcystis aeruginosa PCC7806. With the use of oligopeptide substrates and specific inhibitors, we detected the activities of trypsin, chymotrypsin, elastase and cysteine protease. Cysteine protease, the predominant enzyme behind proteolysis of a natural substrate, casein, was partially purified by gel filtration. The substrate SDS-polyacrylamide gel electrophoresis of body homogenate revealed the presence of nine bands of proteases (17-72 kDa). The apparent molecular mass of an exclusive cysteine protease was 60 kDa, whereas of trypsin, it was 17-24 kDa. An extract of M. aeruginosa PCC7806 significantly inhibited the activities of trypsin, chymotrypsin and cysteine protease in M. macrocopa body homogenate at estimated IC(50) of 6- to 79-microg dry mass mL(-1). Upon fractionation by C-18 solid-phase extraction, 60% methanolic elute contained all the protease inhibitors, and these metabolites could be further separated by reverse-phase liquid chromatography. The metabolites inhibitory to M. macrocopa proteases also inhibited the corresponding class of proteases of mammalian/plant origin. The study suggests that protease inhibition may contribute to chemical interaction of cyanobacteria and crustacean zooplankton.