Functional analysis of the GlnK1 protein of Methanosarcina mazei strain Gö1: Aspects of nitrogen regulation

PII-proteins like GlnB and GlnK are ubiquitous distributed small regulator proteins that play a key role in regulation of nitrogen metabolism by sensing and signalling the intracellular nitrogen status of the cell. The intention of this thesis was to get an insight into the regulation of nitrogen metabolism of M. mazei strain Go1 by focussing on the contribution of the PII-like protein GlnK1. One major goal was the characterization of the GlnK1 protein concerning transcriptional regulation, synthesis and modification. The second goal was to identify potential interaction partners of GlnK1 and to determine its potential regulatory function.The GlnK1 protein of M. mazei contains the conserved tyrosine residue (Tyr 51) which in bacteria is post-translationally modified dependent on the nitrogen availability. However, both in vitro and in vivo experiments showed that GlnK1 of M. mazei is not modified dependent on nitrogen supplementation. Further structural differences to bacterial PII-like proteins were demonstrated by heterotrimer formation experiments. Despite these distinct differences M. mazei GlnK1 was able to complement growth of an E. coli glnK mutant indicating that the archaeal GlnK protein is involved in regulation of nitrogen metabolism in M. mazei as well. In order to assign a function to M. mazei GlnK1 in nitrogen metabolism, a chromosomal glnK1 deletion mutant was constructed. Therefore genetic methods, e. g. effective plating and transformation protocols had to be established. M. mazei was finally genetical tractable by (i) selecting a potential spontaneous cell wall mutant of M. mazei that showed a higher plating efficiency and (ii) using the optimized liposome-mediated transformation protocol specifically developed for M. mazei strain Go1. Growth experiments showed that under nitrogen limitation a partial reduced growth rate was observed in the mutant strain compared to the wild type. However, quantitative reverse transcription-PCR analysis of selected genes of the nitrogen regulon excluded a regulatory function of GlnK1 in transcriptional activation or repression.Biochemical experiments and in vivo data clearly demonstrated that GlnK1 interacts with glutamine synthetase (GlnA) thereby inhibiting its enzyme activity. In contrast, an activating effect on GlnA enzyme activity was observed in the presence of 2-oxoglutarate that reflects the internal nitrogen status of the cell. On the basis of these results we postulate following model: M. mazei perceives external nitrogen limitation by sensing the increase in the internal 2-oxoglutarate pool. Upon direct binding of 2-oxoglutarate, apparently resulting in a conformational change of GlnA, GlnA activity is highly stimulated and complex formation with GlnK1 is inhibited. After a shift to nitrogen sufficiency, however, the internal 2-oxoglutarate level decreases resulting in reduction of glutamine synthetase activity. It is therefore likely to propose that the nitrogen sensor GlnK1 allows fine tuning control of the glutamine synthetase activity under changing nitrogen availabilities.