Doctoral Degrees (Biochemistry)
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Browsing Doctoral Degrees (Biochemistry) by Author "Downing, T. G."
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- ItemThe role of nitrogen in the regulation of microcystin content in Microcystis aeruginosa(Stellenbosch : Stellenbosch University, 2005-12) Downing, T. G.; Hofmeyer, J. H. S.; Snoep, J.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.ENGLISH ABSTRACT: Several genera of cyanobacteria produce a range of toxins. The increased rate of eutrophication of surface fresh waters due to anthropogenic inputs has resulted in more frequent and severe cyanobacterial bloom events. Such bloom events make impoundments unsuitable for recreational use and increase the cost of production of potable water due to the necessity for removal of toxins released from cells during the purification process. Microcystis aeruginosa is the major freshwater bloom-forming toxic cyanobacterium. Concentrations of the hepatotoxin, microcystin, are highly variable in blooms. Published literature on environmental conditions leading to increased microcystin production was often contradictory and in many cases did not consider all relevant parameters. However, environmental nitrogen and phosphorus, temperature and light, and growth rate were implicated in regulation of toxin content. The purpose of this work was therefore to investigate environmental factors (specifically nitrogen and phosphorus) and cellular activities (specifically carbon fixation and nitrogen uptake rates and growth rate) involved in the modulation of microcystin production in M. aeruginosa in order to clarify the role of these parameters, and in an attempt to identify regulatory mechanisms for microcystin production. Environmental nitrogen, phosphorus and growth rate were shown to co-modulate microcystin production in M. aeruginosa. Adequate phosphorus is required for photosynthetic carbon fixation. Phosphorus uptake by M. aeruginosa is strongly correlated with carbon fixation rate. Although microcystin content increased with increasing nitrogen:phosphorus ratios in culture medium, under phosphorus limitation microcystin content was lower irrespective of nitrogen concentrations. This observation and the requirements for fixed carbon for nitrogen assimilation therefore prompted investigation of the effects of cellular carbon fixation and nitrogen uptake in the modulation of microcystin production. Microcystin production was found to be enhanced when nitrogen uptake rate relative to carbon fixation rate was higher than that required for balanced growth. The cellular nitrogen:carbon ratio above which microcystin concentrations increased substantially, corresponded to the Redfield ratio for balanced growth. Investigation of potential regulatory mechanisms involving the cyanobacterial nitrogen regulator, NtcA, yielded putative NtcA binding sites indicative of repression in the microcystin synthetase gene cluster. In culture, the polypeptide synthetase module gene, mcyA, and ntcA were inversely expressed as a function of carbon-fixation:nitrogen-uptake potential. However, no increase or decrease in microcystin production could be linked to either glutamine, glutamate or a-ketoglutarate, metabolites that are involved in regulation of ntcA. The role of NtcA in regulation of microcystin production could therefore not be confirmed. In conclusion, these data suggest that microcystin production is metabolically regulated by cellular C:N balance and specific growth rate. The primary importance of nitrogen and carbon was demonstrated by a simple model where only nitrogen uptake, carbon fixation and growth rate were used to predict microcystin levels. The model also explains results previously described in literature. Similarly, an artificial neural network model was used to show that the carbon fixation dependence on phosphorus allows accurate prediction of microcystin levels based on growth rate and environmental nitrogen and phosphorus.