Thesis (PhD)--University of Stellenbosch, 2005. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/50523 |
| Date | 12 1900 |
| Creators | Downing, T. G. |
| Contributors | Hofmeyer, J. H. S., Snoep, J., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 129 p. : ill. |
| Rights | Stellenbosch University |
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