Haloferax mediterranei is an extreme halophilic micro-organism belonging to the Archaea domain that was isolated from the Santa Pola solar salterns (Alicante, Spain) in 1983. The biochemistry of the proteins involved in nitrogen metabolism is being studied, but the knowledge of their regulation is very scarce at present. The PII superfamily is constituted by major regulators of nitrogen metabolism, which are widespread in prokaryotic and eukaryotic organisms. These trimeric proteins (12 kDa per subunit) have in Escherichia coli long been known to regulate GS (glutamine synthetase) activity via its adenylyltransferase/adenylyl-removing enzyme and, more recently, to be able to interact directly with this enzyme in methanogenic archaea. We have tested the possible role of PII proteins in the regulation of ammonium assimilation in our model organism and the results clearly indicate that the direct influence of GS by PII proteins can also take place in halophilic archaea, starting with the comprehension of nitrogen regulation in those organisms.
PII proteins are major regulators of nitrogen metabolism that are able to sense and integrate nitrogen, carbon and energy signals to enable the cell to give a proper combined response . They are the most widely distributed signalling proteins in Nature , being present in the three domains of life.
Proteins belonging to PII superfamily are small trimers with a highly conserved three-dimensional structure , which are organized in three major subgroups : glnB subgroup (formed by PII gene homologues either with monocistronic transcripts or linked to glnA or nadE); glnK subgroup (amtB-linked PII genes) and nifI subgroup (associated with the nitrogenase operon). Recently, a new group has been proposed, containing PII homologues with a highly conserved genetic linkage to heavy metal efflux pump-encoding genes .
The role of the PII proteins is accomplished in different organisms by protein–protein interactions with different sets of PII cellular targets . The first example described was Escherichia coli regulation of the GS (glutamine synthetase)-modifying enzyme ATase (adenylyltransferase) . More recently, a regulation of GS by direct interaction between GS and PII in Methanosarcina mazei has been found . Another well-studied PII role is the ability of GlnK proteins to block the membrane ammonium transporter channel AmtB as a response to an increase in the substrate concentration in the medium [9,10]; or the ability to induce membrane sequestration of DraG by the formation of the ternary complex DraG–GlnK–AmtB to avoid DraG-mediated nitrogenase switch-on and achieve a tight regulation of the dinitrogen fixation process . Meanwhile, in cyanobacteria a completely different set of protein targets has been shown: NAGK, PipX, PamA and NtcA .
Haloferax mediterranei (strain ATCC 33500) is an extreme halophilic archaeon isolated from Santa Pola solar salterns . It needs high NaCl concentrations (approx. 3 M), but also high amounts of Mg2+ compared with other similar organisms (0.37 M compared with 0.18 M in Haloferax volcanii) to grow under optimum conditions. Its nitrogen metabolism has been well studied from an enzymatic point of view [13,14]; this micro-organism is able to use not only ammonia as the nitrogen source for biosynthesis, but also nitrate and nitrite (via assimilatory nitrate reduction) and it can also use nitrate as a final electron acceptor under anoxic conditions (denitrification). However, investigation of the regulation of this essential part of metabolism in our model organism has just begun.
Results and discussion
PII genes in H. mediterranei
A draft of H. mediterranei genome has been recently obtained (Bonete, M. J. unpublished work), in which there can be found two genes encoding PII homologues. Both of them appear in the genome located downstream of ammonium transporter genes (amt). For this reason, they have been classified as glnK-like PII genes [4,17]. The two pairs of genes are located consecutively in the genome but separated by a 300 nt gap of non-coding sequence (Figure 1). Even though their sequences are almost identical (84% of amino acid identity, Figure 1), some differences can be found: in the first glnK–amt pair, both genes overlap by four nucleotides (and consequently they are coded in different open reading frames), while in the second pair a gap of three nucleotides exists between the stop codon of the amt gene and the initial ATG of glnK.
Genomic organization of H. mediterranei's PII genes and amino acid sequence alignment of the encoded proteins using MUSCLE 
The expression pattern of H. mediterranei's GlnK proteins under different nitrogen sources
To gain some information about the role of GlnK proteins in this extreme halophile, we tested whether they are differentially expressed depending on the type of nitrogen source in the growth medium. With this purpose, the micro-organism was grown in different conditions (Figure 2A): Mops-buffered salted water (25%) was supplemented with yeast extract as an organic nitrogen source or either ammonium or nitrate as inorganic nitrogen sources (glucose was added in excess as the carbon source for these synthetic media). Western-blot analysis of a SDS/12% PAGE of the cytoplasmic fractions (Figure 2B) was then performed using GlnK-specific antibodies (Genscript) that were able to detect both H. mediterranei's GlnK1 and GlnK2 proteins, because they are directed to a conserved peptide located in the predicted T-loop of the GlnK structure (Figure 1).
H. mediterranei growth curves with different nitrogen sources (A) and analysis of GlnK expression by SDS/12% PAGE and Western blot (B)
The results showed that only when cells are grown with nitrate are GlnK proteins expressed and that no signal can be detected in those samples corresponding to cytoplasmic extracts obtained from cells grown either with ammonium or in complex medium (Figure 2B).
Proposal of a GlnK role in nitrogen metabolism regulation in H. mediterranei
As was previously mentioned, our model organism is able to use nitrate as nitrogen source for anabolism as a result of the assimilatory nitrate reduction pathway [19,20]. The newly formed ammonium can be further incorporated into carbon skeletons by two pathways. GDH (glutamate dehydrogenase) [13,21,22], which catalyses the incorporation of ammonium to 2-oxoglutarate and gives glutamate as the product in a process with low affinity for ammonium but with no need for ATP consumption, or the GS/GOGAT (glutamine oxoglutarate aminotransferase) pathway [13,23,24], where GS mediates the incorporation of an ammonium molecule to glutamate to produce glutamine. In the latter reaction, the enzyme has a very high affinity for ammonium (low Km) but needs ATP.
As it has been observed in previous studies, GS is active in H. mediterranei cells grown with nitrate . The first and most studied role described for PII proteins  was GS activity control in E. coli through covalent modification. This reaction is catalysed by an ATase enzyme that was seen to be controlled by PII. Taken into account that GlnK proteins can be detected (Figure 2B) and GS activity can be measured  in H. mediterranei cells grown with nitrate, one could think about the possibility of a relation between the two proteins in which the regulator PII would activate GS. Even more, when comparing the activity measurements of GS during growth with 75 mM nitrate (Figure 1 in ) with the expression pattern of GlnK under the same conditions, we can appreciate that, in exponential phase, no high GS activity is detected and at the same time no intense GlnK signal appears (Figure 2B, lane 8). Meanwhile, in the stationary phase of the same culture, a very high GS activity is measured and a GlnK signal is then clearly observed (Figure 2B, lane 9). This suggests an activating role for GlnK in ammonium assimilation via GS/GOGAT in H. mediterranei.
A hypothetical mechanism of GlnK control in H. mediterranei
The physiological experiments described above suggest an activating effect of H. mediterranei PII proteins in GS. Some proof for this statement was obtained by experimental procedures using purified recombinant GS from our model organism. It folded correctly as a dodecamer giving an active enzyme that enabled in vitro biosynthetic activity measurements to be made to test these effects. Also both GlnKs were obtained as recombinant proteins by heterologous overexpression, incorporating a His tag to facilitate the purification process.
The ability of PII proteins from H. mediterranei to control ammonium assimilation by activating the GS/GOGAT pathway via GS activity modification was proved in vitro. Biosynthetic activity measurements were performed in the presence and absence of GlnK, and an increase of GS enzymatic activity up to 50% as a result of purified recombinant GlnK presence in the assays was obtained. Both His6–GlnK1 and His6–GlnK2 gave equivalent results.
It must be mentioned that another case of the direct influence of GlnK in GS activity has been reported to date: in the methanogenic archaeon M. mazei, a direct interaction between GlnK1 and GS (GlnA1) has been shown using recombinant proteins as well . Nevertheless, this interaction has opposite consequences compared with H. mediterranei: in the methanogenic model, interaction between the two proteins results in a decrease in the enzyme activity, whereas, as mentioned above, in the halophilic model, it has an activating effect. As these are the first published data of this kind in halophiles, no further comparison can be made with members of the Halobacteriaceae family.
Also it must be taken into account that no details about possible covalent modifications in GlnK and GS proteins from H. mediterranei have been obtained to date, and so other regulatory mechanisms in GS activity remains possible that would complement this reported direct functional relation.
Enzymology and Ecology of the Nitrogen Cycle: A Biochemical Society Focused Meeting held at University of Birmingham, U.K., 15–17 September 2010. Organized and Edited by Jeff Cole (University of Birmingham, U.K.), Rosa María Martínez-Espinosa (University of Alicante, Spain), David Richardson (University of East Anglia, Norwich, U.K.) and Nick Watmough (University of East Anglia, Norwich, U.K.).
This work was funded by research grants from the Ministerio de Ciencia e Innovación from Spain [grant number AP2007-02932] and research projects from the same institution [grant number BIO2008-00082].