PII-like proteins GlnB and GlnK belong to the family of small signalling proteins identified in all three domains of life (Ninfa and Atkinson, 2000; Arcondeguy et al., 2001). They are known to play an important role in sensing and transducing cellular nitrogen signals and therefore being involved in the regulation of nitrogen metabolism (reviewed by Arcondeguy et al., 2001; Kessler et al., 2001; Ehlers et al., 2002; Forchhammer, 2004). Regulatory mechanisms mediated by PII-like proteins are well studied in bacteria, for which a variety of different receptor proteins have been identified and characterized (Kamberov et al., 1994; Jaggi et al., 1997; de Zamaroczy, 1998; Nolden et al., 2001; Coutts et al., 2002; Heinrich et al., 2004; Javelle et al., 2004). In general, bacterial PII-like proteins are covalently modified and demodified in response to changes in nitrogen availability; however, the modification differs: uridylylation of PII-like proteins has been demonstrated for enteric bacteria (Jiang et al., 1998a; Atkinson and Ninfa, 1999), adenylylation for the actinomycetes Streptomyces coelicolor and Corynebacterium glutamicum (Hesketh et al., 2002; Strösser et al., 2004) and phosphorylation for the cyanobacterium Synechococcus elongatus (Forchhammer and Tandeau de Marsac, 1995). Exception is the GlnY protein of Azoarcus sp. BH72, which exists exclusively in one modification state, in its uridylylated form, independently of the actual nitrogen availability (Martin et al., 2000). Furthermore, in various systems PII-like proteins seem not to be subject to covalent modification, such as in Prochlorophytes (Palinska et al., 2002), Bacillus subtilis (Detsch and Stülke, 2003), and in plant PII-like proteins (Smith et al., 2004). It has been recently shown that external nitrogen limitation is perceived as internal glutamine limitation in case of Escherichia coli (Ikeda et al., 1996; Jiang et al., 1998a). However, in unicellular cyanobacteria the cellular 2-oxoglutarate level is the internal nitrogen signal, which determines the modification state of the PII protein in response to nitrogen availability (Irmler et al., 1997; reviewed by Forchhammer, 1999). In addition to covalent modification, allosteric binding of small effector molecules, in particular ATP and 2-oxoglutarate to PII is an important signal input into the PII system, thus allowing the integration of various signals to generate a coherent response. Depending on their modification and ligand binding states, bacterial PII-like proteins modulate the activity of several receptor proteins involved in nitrogen assimilation. (i) E. coli GlnB effects the activity of the transcriptional activator NtrC by interacting with the histidine kinase NtrB (Jiang et al., 1998b) and regulates the activity of the adenylyltransferase (ATase), which controls the glutamine synthetase activity (Jiang et al., 1998c; Reitzer, 2003). (ii) The second PII-like protein in E. coli, GlnK, has been shown to act as a backup system and as a fine control regulator for the GlnB regulatory cascade (Atkinson et al., 2002). In addition, GlnK appears to regulate the activity of the ammonium transporter AmtB by direct protein interaction after a shift to nitrogen sufficiency (Coutts et al., 2002; Javelle et al., 2004). (iii) In nitrogen fixing bacteria, GlnK has been shown to transduce the internal nitrogen status towards the regulatory proteins NifA or NifL by direct interaction (Liang et al., 1992; Arsene et al., 1996; 1999; Little et al., 2000; Rudnick et al., 2002; Drepper et al., 2003; Stips et al., 2004). Besides those receptor proteins, a new target protein was recently discovered for the PII protein of S. elongatus. Under nitrogen excess conditions, the non-phosphorylated PII-protein forms stable complexes with the key enzyme of the arginine biosynthesis pathway, N-acetylglutamate kinase, and thereby enhances its enzyme activity by an order of magnitude (Heinrich et al., 2004).
In contrast to bacterial PII-like proteins, until now only one potential receptor protein has been identified for archaeal PII-like proteins. The gene products of the glnB-like genes nifI1 and nifI2 located within the nif gene operon in Methanococcus maripaludis have been shown to be essential for the ammonia switch-off of nitrogenase activity in response to a shift to nitrogen sufficiency (Kessler et al., 2001). Characterizing nifI mutant strains demonstrated that both NifI proteins are essential for modulating nitrogenase activity (Kessler et al., 2001). However, as no biochemical data are available, the regulatory mechanism is still not completely understood. Recently, we characterized the archaeal PII-like protein GlnK1 of Methanosarcina mazei strain Gö1 and demonstrated that the archaeal GlnK1 protein structurally differs significantly from bacterial PII-like proteins. Nevertheless, M. mazei GlnK1 was able to complement an E. coli ΔglnK mutant strain, strongly indicating that the archaeal GlnK protein is involved in nitrogen regulation (Ehlers et al., 2002). The goal of this work was to elucidate the regulatory function of GlnK1 in nitrogen metabolism of M. mazei by identifying potential interacting partners. During our studies, we identified glutamine synthetase (GlnA1) as the first receptor protein of GlnK1 and characterized the effect of complex formation between GlnK1 and GlnA1 on glutamine synthetase activity.