The role of nitrogen in the regulation of microcystin content in Microcystis aeruginosa

[摘要] ENGLISH ABSTRACT:Several genera of cyanobacteria produce a range of toxins. The increasedrate of eutrophication of surface fresh waters due to anthropogenic inputs hasresulted in more frequent and severe cyanobacterial bloom events. Suchbloom events make impoundments unsuitable for recreational use andincrease the cost of production of potable water due to the necessity forremoval of toxins released from cells during the purification process.Microcystis aeruginosa is the major freshwater bloom-forming toxiccyanobacterium. Concentrations of the hepatotoxin, microcystin, are highlyvariable in blooms. Published literature on environmental conditions leading toincreased microcystin production was often contradictory and in many casesdid not consider all relevant parameters. However, environmental nitrogenand phosphorus, temperature and light, and growth rate were implicated inregulation of toxin content. The purpose of this work was therefore toinvestigate environmental factors (specifically nitrogen and phosphorus) andcellular activities (specifically carbon fixation and nitrogen uptake rates andgrowth rate) involved in the modulation of microcystin production in M.aeruginosa in order to clarify the role of these parameters, and in an attemptto identify regulatory mechanisms for microcystin production. Environmentalnitrogen, phosphorus and growth rate were shown to co-modulate microcystinproduction in M. aeruginosa. Adequate phosphorus is required forphotosynthetic carbon fixation. Phosphorus uptake by M. aeruginosa isstrongly correlated with carbon fixation rate. Although microcystin contentincreased with increasing nitrogen:phosphorus ratios in culture medium,under phosphorus limitation microcystin content was lower irrespective ofnitrogen concentrations. This observation and the requirements for fixedcarbon for nitrogen assimilation therefore prompted investigation of the effectsof cellular carbon fixation and nitrogen uptake in the modulation of microcystinproduction. Microcystin production was found to be enhanced when nitrogenuptake rate relative to carbon fixation rate was higher than that required forbalanced growth. The cellular nitrogen:carbon ratio above which microcystinconcentrations increased substantially, corresponded to the Redfield ratio forbalanced growth. Investigation of potential regulatory mechanisms involvingthe cyanobacterial nitrogen regulator, NtcA, yielded putative NtcA bindingsites indicative of repression in the microcystin synthetase gene cluster. Inculture, the polypeptide synthetase module gene, mcyA, and ntcA wereinversely expressed as a function of carbon-fixation:nitrogen-uptake potential.However, no increase or decrease in microcystin production could be linked toeither glutamine, glutamate or a-ketoglutarate, metabolites that are involved inregulation of ntcA. The role of NtcA in regulation of microcystin productioncould therefore not be confirmed. In conclusion, these data suggest thatmicrocystin production is metabolically regulated by cellular C:N balance andspecific growth rate. The primary importance of nitrogen and carbon wasdemonstrated by a simple model where only nitrogen uptake, carbon fixationand growth rate were used to predict microcystin levels. The model alsoexplains results previously described in literature. Similarly, an artificial neuralnetwork model was used to show that the carbon fixation dependence onphosphorus allows accurate prediction of microcystin levels based on growthrate and environmental nitrogen and phosphorus.

[发布机构] Stellenbosch University