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

Downing, T. G. (2005-12)

Thesis (PhD)--University of Stellenbosch, 2005.

Thesis

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.

AFRIKAANSE OPSOMMING: Verskeie genera van sianobakterieë produseer 'n verskeidenheid van toksiene. Die toename in die tempo van eutrofikasie van varswater oppervlaktes as gevolg van antropogeniese insette veroorsaak al hoe meer en al hoe erger sianobakteriële infestasies. Dit veroorsaak probleme vir ontspanninggebruik van hierdie waters en verhoog die koste van produksie van drinkbare water as gevolg van die noodsaak om die toksiene wat deur die selle gedurende die suiweringsproses vrygelaat word te verwyder. Microcystis aeruginosa is die belangrikste varswater bloeisel-vormende toksiese sianobakterium. Die konsentrasie van die hepatotoksien mikrosistien is hoogs varieerbaar in sulke bloeisels. Gepubliseerde literatuur oor die omgewingskondisies wat lei na verhoogde mikrosistienproduksie is dikwels weersprekend en neem in vele gevalle nie al die relevante parameters in ag nie. Desnieteenstaande word omgewingstikstof, fosfor, temperatuur en lig, asook groeisnelheid, geïmpliseer in die regulering van toksieninhoud. Die doel van hierdie navorsing was dus om omgewingsfaktore (spesifiek stikstof en fosfor) en sellulêre aktiwiteite (spesifiek koolstoffiskering en die snelheid van stikstofopname en van groei) betrokke by die modulering van mikrosistienproduksie in M. aeruginosa te ondersoek in 'n poging om die rol van hierdie parameters te verstaan en om regulatoriese meganismes vir mikrosistienproduksie te identifiseer. In hierdie studie is aangetoon dat omgewingstikstof en fosfor sowel as groeisnelheid mikrosistienproduksie in M. aeruginosa ko-moduleer. Genoegsame fosfor word benodig vir fotosintetiese koolstoffiksering. Fosforopname deur M. aeruginosa korreleer sterk met die snelheid van koolstoffiksering. Alhoewel mikrosistieninhoud toegeneem het met 'n toename in die stikstof:fosfor verhouding in die kultuurmedium, was die mikrosistieninhoud onder kondisies van fosforlimitering laer ongeag die stikstofkonsentrasie. Hierdie waarneming, tesame met die noodsaak van gefikseerde koolstof vir stikstofassimilering, het gelei na 'n studie van die effekte van sellulêre koolstoffiksering and stikstofopname op die modulering van mikrosistienproduksie. Dit is gevind dat mikrosistienproduksie verhoog was wanneer die snelheid van stikstofopname relatief tot die snelheid van koolstoffiksering hoër was as die waarde wat benodig word vir gebalanseerde groei. Die sellulêre stikstof:koolstof verhouding waarbo mikrosistienkonsentrasies beduidend verhoog is stem ooreen met die Redfield verhouding vir gebalanseerde groei. 'n Ondersoek na potensiële reguleringsmeganismes waarby die sianobakteriële stikstofreguleerder NtcA betrokke is het gelei na die ontdekking van moontlike NtcA bindingseteis; dit kan dui op die repressie van die mikrosistiensintetase geengroepering. Onder kultuurkondisies is gevind dat die geen vir die polipeptiedsintetase module, mcyA, en ntcA omgekeerd uitgedruk word as 'n funksie van koolstofopname:stikstofopname potensiale. Geen toename of afname in mikrosistienproduksie kon egter gekoppel word aan óf glutamien, óf glutamaat, óf a-ketoglutaraat nie, metaboliete wat betrokke is by die regulering van ntcA. Die rol van NtcA in die regulering van mikrosistienproduksie kon dus nie bevestig word nie. Die gevolgtrekking is dus gemaak dat mikrosistienproduksie metabolies gereguleer word deur die C:N balans en die spesifieke groeisnelheid. Die primêre belang van stikstof en koolstof is gedemonstreer deur 'n eenvoudige model waarin slegs stikstofopname, koolstoffiksering en groeisnelheid gebruik word om mikrosistienvlakke te voorspel. Die model verklaar ook resultate wat tevore in die literatuur beskryf is. Soortgelyk is 'n artifisiële neurale netwerkmodel gebruik om te toon dat die afhanklikheid van koolstoffiksering van fosfor akkurate voorspelling van mikrosistienvlakke gebaseer of groeisnelheid en omgewingstikstof en fosfor moontlik maak.

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