Several genera of cyanobacteria produce microcystin, a monocyclic peptide, with a unique chemical structure. To date, there have been over a 100 different structural variants of microcystin which have been identified. Microcystin production is affected by numerous environmental factors. However, the primary modulating factor for intracellular microcystin quota is the intracellular N:C ratio. No clearly defined biological role has been described for microcystin. Proposed roles for microcystin include defence against plankton grazers, metal chelation, an infochemical and a protectant against oxidative stress. There is sufficient evidence to support a biological role for microcystin in photosynthesis: microcystin is predominantly located in the thylakoid membranes, the microcystin gene cluster is differentially expressed as a function of light and a growth advantage for the microcystin producer in saturating light intensities. The purpose of this study is to investigate a possible biological role for microcystin in preventing photoinhibition and thus explaining the growth advantage observed in toxin-producers over non-toxin-producers. The uptake of exogenous microcystin was observed in Synechocystis PCC 6803 which was internalized and located in the thylakoid membranes and caused the inhibition of photosynthesis. Microcystin variants and increasing concentrations of microcystin-LR had no effect on the fluidity of the thylakoid membranes. The exposure of thylakoid membranes from Synechocystis PCC 6803 to physiologically relevant concentrations of different microcystin variants resulted in the inhibition of photosystem II activity but not photosystem I activity. The inhibition of photosystem II was variant dependent and concentration dependent for microcystin-LR and microcystin-RR. Chlorophyll a fluorescence data showed that photosystem II inhibition was caused by the inhibition of the oxygen evolving complex. Furthermore, a completion study revealed that the microcystin-producing Microcystis PCC 7806 had a competitive advantage over the non-microcystin producing ΔmcyA mutant of Microcystis PCC 7806 at high light intensities. The data indicates that microcystin protects the toxin-producer by either retarding or preventing photoinhibition and thus identifying the first data supported function for microcystin in cyanobacteria.

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A recombinant phage that produced a polypeptide possessing the characteristics of pneumolysin, the membrane damaging toxin of the pneumococcus, was isolated from a bank of pneumococcal sequences in ?gt10. Subclones carrying the pneumolysin gene in various plasmids were haemolytic regardless of the orientation of the insert. The nucleotide sequence of a 5 kb fragment carrying the pneumolysin gene was determined. An open reading frame 1413 bp long was identified that when translated encoded a polypeptide with 471 amino acids and a molecular weight 52.8 kD. The N-terminal amino acid sequence of the predicted protein was identical to that of native pneumolysin. A single cysteine residue was present at position 428 in the amino acid sequence. Comparison of the DNA and amino acid sequences of pneumolysin with streptolysin O (SLO) revealed extensive homology in the amino acid sequence. The longest region of identity was a sequence of 12 amino acids surrounding the unique cysteine. A hybrid gene consisting of the 5' region of the pneumolysin gene and the 3' end of the SLO gene was constructed. The fusion polypeptide was made in E. coli, but possessed a very low haemolytic activity. Using the technique of oligonucleotide-mediated site-directed mutagenesis, two mutant genes were constructed in which the cysteine codon was changed to either a glycine or serine codon. Modified toxins when purified from E. coli had a specific activity of about 1-2 % that of wild type pneumolysin.

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M.Sc. (Biochemistry) / Please refer to full text to view abstract

Blooms of cyanobacteria have been known to cause illness in humans and death in wild and domestic animals. One of the toxins produced by cyanobacteria is microcystin, which is a potent hepatotoxin. Microcystin is taken up by bile acid transporters in the intestine and transported into the liver. After exposure to acute doses of microcystin, severe haemorrhage has been observed along with apoptotic and necrotic hepatocytes. The cytoskeletal structure of the hepatocytes is disrupted and oxidative stress is induced. Selenium, a known anti-oxidant, has been shown to induce increased activity of glutathione peroxidase. Glutathione peroxidase removes peroxides from cells protecting them from oxidative stress. This study set out to determine if selenium could play a role in preventing the damage to mice livers due to microcystin toxin. The protective role of selenium was explored in three main studies: in the first study, the ability of selenium to increase the survival time of mice exposed to a lethal dose of toxin was determined. In the second study the mice were exposed to sublethal chronic doses of toxin over 30 days. The ability of selenium to minimise liver damage under these conditions was determined. The final study investigated the mechanism of the protective effect of selenium. The results of the first study suggested that selenium could extend survival time. In the second study the selenium supplemented mice showed a reduction in the extent of the increase in liver weight and a decrease in the amount of lipid peroxidation induced compared to the mice that received only toxin. The histology of the selenium supplemented mice also showed a decrease in the severity and amount of morphological changes in the liver. The third study indicated that the protection shown by selenium might be mediated by an increase in the glutathione peroxidase (GPX) activity in selenium supplemented mice. This increase in GPX activity would increase the removal of the lipid hydroperoxides and prevent the damage they would cause in the cell. A further result indicated an increase in glutathione S-transferase in only the toxin control mice when compared to the selenium supplemented and control mice. ii In conclusion selenium offers protection against microcystin but further studies need to be done to provide statistically valid results to clarify the level of protection.

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