On the circadian organization of nitrogen and sulphur metabolism in Neurospora crassa
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Original versionOn the circadian organization of nitrogen and sulphur metabolism in Neurospora crassa av Karène Jacques Jensen. Stavanger ; Universitetet i Stavanger 2012 (PhD thesis UiS, no. 157)
The purpose of this thesis was to study the circadian organization of the nitrogen and sulphur metabolism in the filamentous fungi Neurospora crassa. Neurospora is an important model organism used in genetics and circadian rhythm research. Its sporulation rhythm is an easily assayed output of the circadian clock, and is generally used to study properties of the clock. The period length of the sporulation rhythm is temperature compensated and is approximately 22 h when Neurospora is grown in constant darkness at 15-30°C. The sporulation rhythm in Neurospora has been shown to be dependent on the frequency (frq) gene, which encodes a negative acting element in the so-called FRQ/WCC (Frequency/White Collar Complex) feedback loop. The FRQ/WCC loop is a negative feedback in which the FRQ protein inhibits its own transcription, and is the assumed core oscillator in the Neurospora circadian clock. Nitrogen is an essential nutrient in all organisms as it is an integral part of proteins and nucleic acids. As nitrogen is often a limiting factor in the environment, many organisms possess complex control systems for its regulation. Nitrate is a secondary source of nitrogen in Neurospora. Nitrate assimilation is repressed when preferable nitrogen sources such as ammonium and glutamine are available. In the absence of preferable nitrogen sources, activation of the nitrate assimilation pathway requires the de-repression of the assimilatory pathway. The assimilation of nitrate requires the enzymes nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS). The Neurospora NR enzyme is highly regulated, and the NR system can be considered as an autonomous negative feedback oscillator. Endogenous oscillations in NR activity with a period length of approximately 24 h have been found in the Neurospora wild-type (wt) strain, as well as in several mutants in which putative key components VI of the FRQ/WCC core circadian oscillator were knocked out. In order to further study the nature of the NR activity rhythm, Neurospora luciferase (luc) reporter strains were constructed. The luc gene from the firefly P. pyralis had been codon-optimized for Neurospora, and was used in the construction of reporter strains in which the promoter of the NR structural gene, nit-3, drove the luc activity. The NR activity assay and quantitative real-time PCR (qPCR) was used to study the oscillations in NR activity and in nit-3 mRNA levels. The luc activity in the nit-3-luc reporter strains was shown to oscillate. However neither the period lengths, nor the phase of the oscillations coincided with activity and transcript measurements obtained from qPCR/activity experiments. Moreover, the luc reporter signal was observed in a negative control strain in which the luc gene was expressed in the absence of a promoter. Results indicate that nitrogen, molecular oxygen, and metabolic intermediates from intracellular processes appear to modulate the luc reporter activity. The NMR protein has been implicated in the repression of NR activity. NR activity levels were measured for the nmr-1 mutant, in which the negative feedback of the NR system is removed. As expected, the overall NR activity levels were elevated, but, surprisingly, an oscillatory response with a period length of approximately 24 h was also observed. The oscillations in NR activity levels had been shown to be independent of frq, and it was therefore hypothesized that the oscillations in NR activity would be abolished in frq and nmr double knock-out (KO) strain. Because an oscillatory response in NR could in principle still be mediated via FRQ, ΔfrqΔnmr KO strains were constructed and the NR activity levels and nit-3 mRNA expression measured. Surprisingly, oscillations in NR activity with period of approximately 24 h were still observed, suggesting additional control mechanisms other than repression by NMR. The sporulation rhythm in a ΔfrqΔnmr KO strain was assayed at 20°, 25°, and 30°C, both under sole nitrate conditions and in the presence of VII ammonium. The rhythm persisted at all temperatures under nitrate conditions, but showed poor temperature compensation. This was also found to be the case in a frq single KO strain (frq10), and in the wt strain. In both cases, a difference in the period length of the sporulation rhythm in nitrate and ammonium was observed. Results therefore indicate that nitrate may exert an effect on the sporulation rhythm of Neurospora, and that frq appears to be important for the temperature compensation of the sporulation rhythm. The regulation of the Neurospora crassa sulphur circuit is similar to that of nitrate. It is assumed that transcriptional/translational feedback loops involving the positive acting transcription factor CYS-3 as well as the negative acting protein SCON-2, ensure transcription of genes needed for the uptake of sulphur in the form of sulphate. It was therefore hypothesized that CYS-3 and SCON-2 would show periodic oscillations on a circadian time scale. A reaction kinetic model of the Neurospora sulphur circuit was tested, and results indicated that CYS-3 and SCON-2 protein concentrations oscillated with a period length of approximately 22 ½ h. To further study the regulation of the sulphur circuit, cys-3- and scon-2 luc-reporter strains were constructed. Oscillations in luc activity were observed for both cys-3 and scon-2, both under nitrate and ammonium conditions. qPCR showed that cys-3 mRNA was expressed in a rhythmic manner in nitrate. However, a difference in period length was observed when qPCR and the luc reporter data were compared. Interestingly, the oscillations observed in the cys-3-luc reporters under ammonium conditions, had the same phase and period length (22 h) as cys-3 levels determined by qPCR.