INTRODUCTION

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Today it is widely recognized that there is probably no ecological niche on earth where bacteria have not evolved to exploit whatever nutrients are available to support life. Despite this enormous versatility of bacterial metabolism, certain fundamental mechanisms have to exist to regulate and integrate enzyme synthesis and enzyme activity. At the center of this metabolic control is the need to coordinate the catabolism and assimilation of carbon and nitrogen sources so as to maximize potential growth rates under any particular nutritional regime. There appear to be very few, if any, absolutely conserved mechanisms for achieving these ends among the major families of the eubacteria and the archaebacteria. However, the subject of this review, a small signal transduction protein commonly known as P_II, is undoubtedly one of the most conserved signal proteins in the bacterial world. From its initial identification in 1969 as the second peak eluting from a gel filtration column, hence the designation P_II (206), this protein family has since been found to play a significant role in the coordination of nitrogen metabolism in a wide variety of bacteria. The recent identification of members of the P_II family in plants extends the biological role of these proteins even further. In this review, we have tried to bring together information, old and new, to give a broad perspective of our current knowledge of this intriguing molecule and to suggest new areas where it may also play a pivotal role in nitrogen metabolism.

Nitrogen is one of the most important elements required for life, as it is necessary for the production of amino acids, nucleotides, amino sugars (required for the synthesis of lipopolysaccharides and peptidoglycan), NAD, and p-aminobenzoate (a precursor in folate biosynthesis). Consequently bacteria have developed a number of mechanisms by which nitrogen can be assimilated from a variety of sources, ranging from ammonium to atmospheric dinitrogen (N₂). Ammonium is almost always the preferred nitrogen source, as it can be assimilated directly into glutamine and glutamate, the key donors for biosynthetic reactions, and is therefore the least energetically expensive substrate to process. By contrast, organic sources such as amino acids must first be degraded to ammonium, and inorganic sources such as NO₃, NO₂, and N₂ must be reduced before assimilation (190).

There are two major assimilation pathways for ammonium, the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway, which is ubiquitous in bacteria, and the glutamate dehydrogenase pathway, which is an alternative route of assimilation in many bacteria, including the enterobacteria. While glutamate dehydrogenase is energetically more efficient than GS/GOGAT, it has a low affinity for ammonium (K_m of about 1 mM) and is consequently rather ineffective in cells growing in nitrogen-limited conditions.

Glutamine synthetase (GS) is a highly regulated enzyme at both the transcriptional and posttranslational level (for a review, see references 146, 155, 190, and 194). In enteric bacteria, GS is reversibly covalently modified by the bifunctional enzyme adenylyltransferase (ATase) in response to nitrogen availability, and the adenylylation state of GS regulates its catalytic activity. The mechanism of this regulation was determined by a series of elegant biochemical experiments initiated in the late 1960s. Initial studies showed that two protein components, P_I and P_II, were concerned with the adenylylation and deadenylylation process in Escherichia coli (206). The P_I fraction contained an adenylytransferase whose ability to adenylylate or deadenylylate GS was specified by the P_II protein and by the concentrations of P_I, ATP, UTP, glutamine, and 2-ketoglutarate (5). It was subsequently determined that the P_I fraction could be resolved into an ATase and a uridylyltransferase (UTase/UR) activity, the latter being capable of both uridylylation/uridylyl-removing and deuridylylation of P_II (30, 148). P_II stimulated adenylylation of GS by ATase, and P_II-UMP stimulated the reverse reaction. P_II was subsequently purified and proposed to be a tetramer with a molecular mass of about 44 kDa (1). The site of uridylylation on P_II was identified as one of the two tyrosine residues in the protein, and the sequence of a tryptic peptide containing the covalently bound nucleotide was also determined (1, 193). The uridylylation and deuridylylation reactions were demonstrated to be catalyzed by a single bifunctional protein when the enzyme was finally purified to homogeneity as a single polypeptide of 95 kDa (78, 117). Hence, by 1984 the biochemical characterization of P_II and the reactions that it regulated was well advanced, and a detailed model of the process of GS adenylylation had been proposed (1, 218).

In the meantime, genetic analysis of the regulation of GS and histidase in Klebsiella aerogenes had begun to shed light on the genes involved in these processes. Mutations in the gene encoding P_II (glnB) were first isolated in K. aerogenes in 1973 by selection for glutamine-requiring mutants (184). Two classes of mutant were identified, those in which GS levels were undetectable, which were designated as glnA mutants, and a second class, represented by a single mutant, with very low but detectable levels of GS, which was designated the glnB mutant. It was then correctly postulated that glnA was the structural gene for GS and that glnB encoded an activator required for the synthesis of GS and for regulation of synthesis in response to nitrogen availability. Significantly, the glnB mutant was also unable to induce histidase expression when cells were nitrogen limited, giving the first indication that P_II might have a role beyond the regulation of GS activity.

The product of glnB was finally identified as P_II by Foor et al., who showed that in the original K. aerogenes glnB mutant, P_II was altered so that it could not be uridylylated in nitrogen-limited conditions and consequently could not stimulate deadenylylation of GS by Atase (64, 65). A second class of glnB mutations resulted in loss of P_II, and these glnB null mutants were still able to adenylylate and deadenylylate GS, although the rates of these reactions were reduced. However, the absence of P_II also led to high levels of GS synthesis in the presence of ammonia, indicating a role of P_II in regulating GS expression as well as its activity. Further evidence for P_II having a role that is independent of its effect on ATase was provided by studies of glnB suppressor mutations which did not affect ATase activity but still affected levels of GS (191).

The Escherichia coli glnB gene was first sequenced by Son and Rhee (214). The deduced amino acid sequence agreed completely with that published earlier for the tryptic peptide containing the site of uridylylation (193) and showed that Tyr-51 was the modified residue. The isolation of further glutamine auxotrophs of both K. aerogenes and E. coli also led to identification of the structural genes for uridylytransferase (glnD) and adenylytransferase (glnE) that were finally cloned from E. coli and sequenced in 1993 (19, 25, 63, 64, 232).

Since the isolation of the first glnB mutant (184), there have been indications that P_II has a function beyond regulating ATase activity. Subsequent genetic studies with E. coli, Salmonella enterica serovar Typhimurium, K. aerogenes, and Klebsiella pneumoniae identified a two-component nitrogen regulatory (ntr) system encoded by the ntrBC genes that was responsible for the global transcriptional control of enzymes of nitrogen assimilation and catabolism (see reference 155 for a review). The link between NtrBC and P_II was demonstrated by further genetic studies in E. coli that led to the proposal that P_II modulated the activity of the histidine protein kinase sensor protein NtrB (31). This established P_II as the key link between changes in the intracellular nitrogen status and the activity of the transcriptional activator protein NtrC (Fig. 1).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Nitrogen regulation (Ntr) system of enteric bacteria. The activities of both GS and NtrC are regulated in response to the intracellular nitrogen status. UTase (glnD product) catalyzes the uridylylation and deuridylylation of PII (glnB product). ATase catalyzes the adenylylation and deadenylylation of GS. NtrB catalyzes the phosphorylation and dephosphorylation of NtrC.

Although by 1990 the molecular details of the enteric nitrogen regulation system seemed to be established, a number of inconsistencies were still apparent in the model. As the effects of UTase were predicted to be mediated through P_II, then in strains lacking P_II the glnD phenotype should be irrelevant, but this was not the case. E. coli cells lacking P_II are Ntr⁺, as defined by their ability to utilize arginine as the sole nitrogen source, whereas both glnD mutants and glnB glnD double mutants cannot utilize arginine (13, 31). Hence, UTase has a role in Ntr regulation that is independent of P_II. Similarly, cells lacking P_II regulate GS adenylylation normally, whereas in vitro experiments showed that deadenylylation of GS by ATase absolutely requires P_II-UMP (219, 233). A glnB glnD double mutant does not deadenylylate GS correctly in N limitation, indicating that UTase is also required for GS adenylylation in the absence of P_II. Taken together, these results suggested the existence of at least one more protein that was involved in both Ntr regulation and GS adenylylation and that was also a substrate for UTase. Indications that the model was incomplete were not limited to E. coli, as studies on nitrogen control of nitrogen fixation (nif) genes in K. pneumoniae had prompted similar conclusions (55, 95).

The presence of a second P_II gene in E. coli was confirmed in 1995 with the identification of a glnB homologue that was designated glnK (231, 233): ironically, this gene had been sequenced in 1993 but had gone unnoticed by researchers in nitrogen regulation (3). The E. coli GlnK protein is 67% identical to GlnB at the primary sequence level, and the protein is encoded in an operon with a second downstream gene, amtB, that is believed to encode a high-affinity ammonium transporter (231). Tyrosine 51 is conserved in GlnK, and indeed in nitrogen-limiting conditions, the protein is subject to uridylylation at this residue in a similar manner to GlnB (89, 231).

The subsequent publication of the E. coli genome sequence confirmed that the organism has just two glnB-like genes, a situation that has since been found in many bacterial species. Indeed, as we discuss below, some organisms carry up to four copies of this gene family. This mutiplicity of P_II-like proteins raises complications with respect to terminology that we will discuss later, but from this point onwards we will use the term P_II as a generic term to describe any member of the P_II protein family.

In recent years, the explosion in bacterial genomics together with recognition of the involvement of P_II proteins in a whole range of nitrogen regulation phenomena in bacteria led to the discovery that members of the P_II family are ubiquitous among prokaryotes. Genes encoding P_II proteins are found in the proteobacteria, the actinobacteria, the firmibacteria, the cyanobacteria, and the archaebacteria, and recently P_II-like proteins have also been found in higher plants. Within these different groups, P_II genes fall into a number of distinct classes with respect to both the primary amino acid sequences of their predicted products and their genetic linkage. Our current knowledge of the distribution of P_II-like proteins is summarized in Table 1. Information on expression of these genes varies considerably, with very detailed studies on some organisms and nothing at all to date on others.

The E. coli glnB gene is located at 50 min on the chomosome (139), between the hmpA gene (located at the 3' end of glnB and transcribed in the opposite direction) and yfhA (also called ORF-2 or OrfXB), located directly upstream of glnB and transcribed in the same direction (139, 232). This genetic organization is conserved in K. pneumoniae (96).

Four transcription starts involved in the transcription of E. coli glnB have been described, but none of these transcripts is regulated in response to nitrogen status (31, 87, 139, 232). Major promoters for glnB are located immediately upstream of the glnB gene (33 and 95 from the ATG), but transcription of glnB can also be initiated at a promoter located upstream of yfhA (139). The E. coli genome sequence reveals a potential operon of three genes (yfhKGA) upstream of glnB and transcribed in the same direction. The product of yfhA has a high degree of homology to the nitrogen-regulatory protein NtrC (232), and YfhK and YfhA constitute a potential two-component regulatory pair, the function of which is unknown (161). It is therefore conceivable that under some conditions glnB is transcribed from a promoter upstream of yfhK.

A binding site for the repressor protein PurR, which is responsible for repression of enzymes required for purine nucleotide biosynthesis, is located between the two major glnB transcriptional start sites (from 68 to 83 nucleotides), and PurR downregulates the transcription of glnB twofold in conditions of purine excess (87). The physiological significance of this regulation during nitrogen excess may be to apply an additional subtle regulation of GS activity when the glutamine requirement for purine synthesis is low and little glutamine is required for protein synthesis (230).

Like E. coli, other members of the  proteobacteria also have a second P_II gene, glnK, that is located upstream of the ammonium transporter gene amtB (101, 223, 231, 233). In both E. coli and K. pneumoniae, the region upstream of the glnK gene contains a ⁵⁴ binding site and a possible NtrC binding site, and expression of the operon is nitrogen regulated in an NtrC-dependent manner (13, 101, 231). Exceptions include Azotobacter vinelandii, which appears to have only one P_II gene that is linked to amtB and is expressed from a ⁷⁰-like promoter at a low constitutive level (153), and Haemophilus influenzae, which has a single glnB-like gene flanked by an upstream gene with similarity to E. coli mog and a downstream gene encoding a protein with 28% identity to E. coli YdgG and YhhT, which are both presumptive integral membrane proteins. A single P_II gene is located between glnA and amtB in Xylella fastidiosa (accession no. AAF84648 to -50), and a similar linkage of glnA and glnK has been reported in Xanthomonas citri (accession no. AF182395 and -96).

The majority of the  proteobacteria examined so far, including those in the genera Rhizobium, Bradyrhizobium, Azorhizobium, Acetobacter, Azospirillum, Rhodobacter, and Rhodospirillum, have two P_II genes. One gene encodes a P_II protein that is very similar to E. coli GlnB and is located upstream of and cotranscribed with glnA (the structural gene for glutamine synthetase I) (7, 27, 41, 94, 113, 127, 151, 158, 262). The second gene encodes a protein similar to GlnK and is linked to an amtB homologue (125, 157, 182, 185, 222, 254) (Y. Zhang and G. Roberts, personal communication). One exception to this pattern is Azospirillum brasilense, in which the glnK-like gene (termed glnZ) is not linked to amtB (46, 49, 228), but instead an open reading frame encoding a protein with 26% identity to the aspartate aminotransferase of Bacillus subtilis is found just 300 bp downstream of glnZ (47). The second exception is Acetobacter diazotrophicus, which has both a glnB-glnA operon and two glnK-amtB operons (D. Meletzus, personal communication).

Expression of the glnBA and glnK-amtB operons has been studied in some detail in the  proteobacteria. Transcription of the glnBA operon varies somewhat from one organism to another but shows a number of common features. Rhizobium leguminosarum and Sinorhizobium meliloti have promoters upstream of both glnB and glnA. The glnA promoter does not contain a consensus sequence characteristic of other previously described promoters, but the glnB promoter region contains a 24, 12 motif characteristic of ⁵⁴-dependent promoters. Expression of both glnB and glnA is partially NtrC dependent, but there are no clear NtrC binding sites upstream of glnB (6, 7, 36). In R. leguminosarum, the genes are cotranscribed, but neither the glnBA nor the glnA transcript is markedly nitrogen regulated (36, 195). However, expression of glnB is significantly downregulated during bacteroid differentiation (56).

Azospirillum brasilense also produces a glnBA and a glnA transcript, but in this case glnB is preceded by two promoters (48). The gene is expressed from a ⁷⁰-like promoter in nitrogen sufficiency, and transcription is elevated fivefold in nitrogen limitation as a second ⁵⁴-dependent promoter is activated. However, expression of glnB is not NtrC dependent (48, 49), and it has been proposed that glnB is activated by an NtrC homologue. The glnA promoter does not bear any similarity to the known consensus sites for  factors, and glnA transcription is downregulated when molecular nitrogen is the sole nitrogen source (48, 49).

In Bradyrhizobium japonicum, glnB and glnA expression is almost entirely separate, but glnB is transcribed from tandem promoters. The glnA gene is constitutively expressed from a ⁷⁰-like promoter, and although glnB is expressed from the ⁷⁰-like promoter in nitrogen sufficiency and from a ⁵⁴-dependent promoter in nitrogen limitation, there is very little variation in the amount of transcript in response to the change in nitrogen status (151).

The glnB and glnA genes are also cotranscribed from tandem promoters in Rhodobacter capsulatus. The first promoter, glnBp1, is repressed by NtrC, while the second, glnBp2, is activated. In R. capsulatus, NtrC does not function with an RNA polymerase containing ⁵⁴, and hence, not surprisingly, there are no ⁵⁴ binding sites upstream of glnB (43, 73). Borghese and Wall (26) studied glnB expression using a glnB::lacZ fusion and different growth conditions from those used by Foster-Hartnett and Kranz (73). In this case, glnB expression was found to occur at higher levels under nitrogen-rich conditions than under nitrogen-poor conditions even in an ntrC mutant background. Moreover, the data suggested a posttranscriptional processing event that resulted in unequal levels of expression of glnB and glnA (26). The glnBA promoter region of Rhodobacter sphaeroides is similar to that of R. capsulatus, suggesting similar regulation, including partial nitrogen control of a glnB expression (185, 262). Interestingly, in a ribulose bisphosphate carboxylase/oxygenase (RubisCO)-deficient mutant, glnB expression was repressed and GS activity was extremely low regardless of the nitrogen source, suggesting the presence of a weak constitutive glnA promoter (185).

In Rhodospirillum rubrum, glnB and glnA are cotranscribed from a weak ⁷⁰-like promoter (glnBp1) and a strong ⁵⁴-like promoter (glnBp2) in either nitrogen-rich or nitrogen-fixing conditions (34, 113). However, glnBp2 activity is enhanced by NtrC in nitrogen-fixing conditions. Although Northern blotting analysis revealed the presence of two transcripts, no glnA promoter was detected, suggesting that the glnA mRNA is produced by processing (34, 113). As in Rhodospirillum rubrum and Rhodobacter capsulatus, Azorhizobium caulinaudans glnB and glnA appear to be cotranscribed (158). In both nitrogen excess and nitrogen limitation, the glnBA operon is transcribed from two overlapping promoters having the same start site, one of them ⁵⁴ and NtrC dependent and the other uncharacterized. Again, no promoter was detected upstream of glnA, and an mRNA processing event was proposed (158).

Regulation of glnK expression is similar in all the  proteobacteria so far studied, Azospirillum brasilense (glnZ), Azorhizobium caulinodans, and Rhizobium etli. In each case, the primary transcript is expressed from a ⁵⁴-dependent promoter that is active in nitrogen-limiting conditions and is NtrC dependent (47, 157, 222). In Rhodobacter sphaeroides, glnK is also expressed only under nitrogen-limiting conditions, and in a RubisCO-deficient mutant, glnK expression is partially derepressed in the presence of ammonium (185).

Two representatives of the  proteobacteria, Herbasprillum seropedicae and an Azoarcus sp. have been studied in detail. H. seropedicae has two P_II-like genes, one of which is linked to a homologue of E. coli nadE (a gene encoding ammonia-dependent NAD synthetase) (21). The Azoarcus sp. has three P_II-like genes, one glnB homologue and two glnK homologues, each of which is linked to an amtB homologue (glnK to amtB and glnY to amtY) (119, 150, 188). Two of the Azoarcus P_II genes, glnK and glnY, are preferentially transcribed under conditions of nitrogen fixation (150).

Within the  proteobacteria, there is evidence for one glnB-like gene in the form of an open reading frame downstream of the nitrogenase structural gene nifH of Desulfovibrio gigas (accession no. U68183). This situation is very similar to that seen in the diazotrophic methanogens (see below).

Among the gram-positive bacteria, there are relatively few reports of P_II genes to date. Bacillus subtilis has a single P_II gene that was originally designated nrgB and is located downstream of an amtB homologue (nrgA) (246). The upstream region contains a B. subtilis ^A-dependent promoter, and characterization of two transcripts separated by a single nucleotide suggests a common promoter origin. The operon is markedly induced in nitrogen limitation, but regulation of an nrgA fusion is not altered in either a nrgB background or a nrgAB background, suggesting that these gene products are not required for their nitrogen regulation; rather, expression is activated by the Bacillus global nitrogen-regulatory protein TnrA (246-249).

Searches of the DNA sequence databases also reveal evidence for glnB-like genes in Clostridium acetobutylicum (http://www.genomecorp.com/genesequences/clostridium/clospage.html), Clostridium cellobioparum (accession no. U59414), Clostridium longisporum (29), and Listeria monocytogenes (accession no. AF104224).

P_II genes have been identified in a number of actinomycetes. Mycobacterium tuberculosis has a single gene, with an amtB homologue upstream and a glnD homologue downstream, forming a potential amtB-glnK-glnD operon (38). A similar organization is found in Corynebacterium glutamicum (104) and in Streptomyces coelicolor.

The presence of a GlnB protein in cyanobacteria was first recognized with the N-terminal sequencing of a phosphorylated 13-kDa protein from Synechococcus sp. strain PCC6301 (85). Subsequent investigations of the occurrence of glnB genes in cyanobacteria included analysis of Synechococcus sp. strain PCC7942, Calothrix sp. strain PCC7601, Pseudanabena sp. strain PCC6901, Microcystis sp. strain PCC7813, and Nostoc sp. strain PCC8009 and suggested that glnB is likely to be present in all cyanobacteria (225). Since then, the glnB genes from Synechococcus sp. strain PCC7942, Synechocystis sp. strain PCC6803, Nostoc punctiforme, and Anabaena sp. strain PCC7120 have been cloned and sequenced (76, 80, 84, 225). In all cases glnB appears to be monocistronic, and its transcription is enhanced by nitrogen limitation.

In both Synechococcus sp. strain PCC7942 and Synechocystis sp. strain PCC6803, glnB is expressed from two tandem promoters, a ⁷⁰ E. coli-type promoter, leading to constitutive expression of glnB, and a nitrogen-regulated promoter activated by the nitrogen-regulatory protein NtcA. Despite similarities, the physical organization of the promoters differs, and in Synechocystis sp. strain PCC6803 the regulated promoter is functional only in cells starved for nitrogen, whereas in Synechococcus sp. strain PCC7942 it is active both in the absence of nitrogen and in the presence of nitrate (76, 132). Moreover, in Synechocystis sp. strain PCC6803, glnB mRNA levels decrease when cells are either transferred to the dark or incubated in the presence of photosynthesis inhibitors, indicating that glnB transcription is under the control of the redox state of the cell (76).

In all the archaebacteria for which complete genome sequences are available, glnB-like genes are found linked to amtB homologues and are usually present in mutiple copies (223). However, in the diazotrophic methanogens, two additional glnB-like genes are located between nifH and nifD within the nif gene cluster (122, 123, 208, 210, 215). These genes have been anlayzed in most detail in Methanococcus maripaludis, in which they are apparently transcribed in a 7.6-kb nif mRNA transcript from a single promoter with a transcritional start site located 80 nucleotides upstream of the translational start site of nifH (122). Interestingly, a comparable gene organization is found in nitrogen-fixing microorganisms inhabiting the gut of the termite Neotermes koshunensis, in which two glnB-like genes are located between anfH and anfD (174).

The genome sequence of the extreme thermophile Aquifex aeolicus revealed two glnB-like genes (45). One of these is in a glnB-glnA operon similar to that found in the  proteobacteria, and the other is the first gene in a cluster comprising glnB_i, nasA, and narB. This latter glnB-like gene apparently encodes a totally novel P_II polypeptide of 205 amino acids. The gene comprises a tandem duplication, so that the C terminus of the first "copy" is fused directly to the N terminus of the second copy. Both copies are predicted to have all the characteristic features known from structural studies of P_II (see later), with the exception that the N-terminal copy is truncated prior to the last two -sheets that comprise the C-loop. This sequence would suggest that if the gene is expressed, it could encode a trimeric protein that comprises six P_II-like domains with three T-loops exposed on one face and another three similar but not identical T-loops on the opposite face. In alignments of P_II amino acid sequences, the predicted product of Aguifex aeolicus glnB_i is most similar to the glnB-like proteins encoded downstream of nifH in the diazotrophic archaebacteria.

The first discovery of a P_II gene outside the bacteria was the identification of a glnB-like gene in the chloroplast genome of the red alga Porphyra purpurea (189), and a second such sequence has since been found in the chloroplast of Cyanidium caldarium (accession no. AF022186). However, more recent studies indicate that the P_II protein is probably quite widely distributed among higher plants. Genes encoding P_II-like proteins have been cloned from Arabidopsis thaliana and Ricinus communis (castor bean) (97), and similar genes have been sequenced in Lycopersicon esculentum (tomato) (accession no. AI773079) and Glycine max (soybean) (accession no. AW153272). In all cases, the predicted protein sequences show significant homology to the bacterial GlnB proteins but have an additional N-terminal domain of about 70 amino acids.

The P_II protein in Arabidopsis thaliana is encoded by the GLB1 gene, mapped at 10.8 centimorgans from the top of chromosome IV (97). When extracted from leaves, this nucleus-encoded chloroplast protein exhibits a molecular mass of 18 kDa. However, its predicted molecular mass of 21.4 kDa (196 amino acids) suggests that P_II may be processed inside the plant cell. The transcription of GLB1 mRNA is induced by light or sucrose and is repressed by the amino acids asparagine, glutamine, and glutamate (97).

With the dramatic increase in availability of prokaryotic genome sequences, conservation of genetic linkage is increasingly being recognized as offering predictions of functional relationships between gene products (44, 177). Multiple sequence alignments can also provide information on sequence similarity, which, at least in some cases, may reflect similarity of function.

Multiple sequence alignments using Clustal W and analysis of the resultant dendrograms show, perhaps not surprisingly, that P_II sequences cluster into groups that reflect almost completely the taxonomic relationships of the originating organism, and furthermore, within certain groups, e.g., the archaebacteria, clear subgroups occur. We have combined this information with knowledge of gene linkage to define just three major groups of prokaryotic P_II proteins (Table 1). Based on this subdivision, we consider that it is now appropriate to review the current gene nomenclature, and we suggest a revised format that we hope will aid gene description in the future. This nomenclature is not meant to imply a strict link between gene designation and function, particularly given the recognized overlapping functions that can occur, e.g., with GlnB and GlnK in some organisms (see later). However, it is hoped that these suggestions will provide a rational framework for describing members of the P_II family.

The designation glnK was originally adopted for the amtB-linked gene of E. coli (231). It has since become apparent that the ammonium transporter gene amtB is almost invariably linked to a P_II gene, a phenomenon that may imply a functional relationship between GlnK and AmtB (223). We suggest that the glnK nomenclature be retained for all amtB-linked P_II genes. In some cases, e.g., Methanobacterium thermoautotrophicum and Archaeglobus fulgidus, organisms have multiple amtB-glnK operons, and in these cases the genes should be designated amtB₁-glnK₁, amtB₂-glnK₂, etc., to follow standard nomenclature for such situations. The GlnK proteins are often, but not invariably, distinguished from GlnB proteins in having a hydrophobic residue (leucine, isoleucine, methionine, or phenylalanine) at position 3 and isoleucine, threonine, or methionine at position 5. We suggest extending the glnK designation to include the exceptional glnZ gene of A. brasilense, which is not linked to amtB but encodes a P_II protein that is very similar to the GlnK proteins of Rhizobium etli and Acetobacter diazotrophicus. Likewise, Azoarcus sp. has two glnK-like genes, one of which has been designated glnY and is linked to an amtB homologue designated amtY (150). On the basis of amino acid sequence, GlnY clusters quite closely with other GlnK proteins and could therefore perhaps more appropriately be designated GlnK₂ rather than GlnY.

A quite distinct group of P_II proteins are encoded by the nifH-linked genes found in the diazotrophic methanogens. These genes always occur in pairs and fall into two discrete subgroups, the nifH-proximal gene, encoding a polypeptide of about 105 amino acids, and the nifH-distal gene, encoding a polypeptide of 120 to 130 amino acids. At present these genes have been designated glnB, with a variety of subdivisions. In M. maripaludis, the products of these genes have now been shown to function in ammonia switch-off of nitrogen fixation (123, 123a). For this reason, we propose that this pair of genes should be renamed nifI₁ and nifI₂, respectively, to reflect both their distinction from glnB and glnK and their specific biological role. The nifI genes are not restricted to the archaebacteria, as sequences of nifI₁ genes have also been identified downstream of nifH genes in Desulfovibrio gigas, Clostridium acetobutylicum, and Clostridium cellobioparum. The novel glnB-like gene in Aguifex aeolicus that is located upstream of nasA and narB and currently designated glnB_i is also most similar to this group of P_II proteins.

Initial characterization of E coli P_II by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation centrifugation indicated that the E. coli protein formed a tetramer (1). However, subsequent crystallization studies and sedimentation equilibrium experiments have shown that it is in fact a trimer (33, 46, 236). The molecular mass of the P_II monomer was elucidated by matrix-assisted laser desorption mass spectrometry and shown to be 12,435 Da, which agrees very well with the prediction from the DNA sequence of 12,427 Da (235, 236).

An X-ray crystal structure of E. coli GlnB was initially achieved at a resolution of 2.7 Å and subsequently refined to a resolution of 1.9 Å (32, 33, 46) (Fig. 2). Each monomer contains two -helices and six -strands arranged so that the two -helices and -strands 1 to 4 form a double motif connected by a large loop stretching from Gly-37 to Phe-55. At the apex of this loop is the site of uridylylation, Tyr-51, and consequently this is referred to as the T loop. A smaller loop (the B loop) stretching from Gln-82 to Asp-88 separates the second -helix from the fourth -sheet, and a third loop (the C loop) containing -strands 5 and 6 is found at the C terminus. Analysis of the refined structure has revealed that hydrogen bonds are formed between Gly-27 and Val-64, stabilizing -sheets 2 and 3, and between Glu-106 and Arg-101 and between Thr-104 and Gly-105, stabilizing the C loop.

View larger version (42K): [in this window] [in a new window]   FIG. 2.   Structure of the E. coli GlnB trimer viewed (top) from above and (bottom) from the side. Individual monomers are colored red, green, and blue.

The trimer is arranged so that the four major -strands of each monomer are flanked on one side by the T loop from the second monomer and on the other side by the C-terminal loop of the third. The arrangement of the monomers relative to one another causes a cleft, which may have functional significance, to form between the T and B loops of one monomer and the C loop of the next. Three six-stranded antiparallel -sheets are arranged as a concave surface in the center of the trimer. The majority of the protein therefore packs into a squat barrel 30 Å high. The diameter of the cavity ranges from approximately 24 Å at the ends to 5.5 Å about 7.0 Å from either end, the diameter at the center of the cavity being about 12.0 Å. The cavity contains several ordered water molecules, and it has also been suggested that the cavity could contain metals (32). Each monomer is arranged so that an eight-residue stretch of the loop containing Tyr-51 at its apex is exposed at the same surface of the trimer about 13 Å above the surface of the barrel.

The structure of E. coli GlnK has also been solved to a resolution of 2.0 Å (145, 252). GlnK also forms a trimer, with a core structure that is very similar to that of GlnB (Fig. 3). However, the GlnK protein was found to be present in two conformations in the crystals. They had core structures similar to each other's and to GlnB, but the structure of the T loop in each case was significantly different. It was only possible to solve the structure for one T loop which was stabilized by lattice contacts; the T loop of the other protein was disordered. The structure of the T loop differed from that of GlnB in that residues 47 to 49 of GlnK form a 3₁₀ helix and the apex of the T loop was much closer to the top of the core of the molecule than seen with GlnB. It should be noted that the T loop of GlnK was only resolvable when it was constrained, and in the GlnB crystals the T loop was also latticed to an adjacent molecule. Nevertheless, the fact that the T loops form different conformations in the crystals might suggest that they behave differently in solution and that the T loop might be flexible rather than rigid. The T loop of E. coli GlnB is known to be essential for interaction with all three of its known targets, ATase, UTase, and NtrB (103, 112). These different targets may require different T-loop conformations to facilitate specific high-affinity interactions.

View larger version (30K): [in this window] [in a new window]   FIG. 3.   Comparison of the structures of E. coli GlnB and GlnK. Superimposition of the C traces for GlnB (black) and GlnK (red) with the position of the Tyr-51 residues in each molecule is indicated. Adapted from reference 252.

Both the B and C loops of GlnK also differ from those of GlnB. These loops form part of the cleft thought to be the important in the binding of effector molecules and proteins. The apex of the B loop of GlnK differs from that of GlnB by 4.3 Å, and the C loop contains a single rather than two -strands and a 3₁₀ helix which causes the end of the peptide to turn back into the bottom of the cleft. The structure of ATP-bound GlnK was also elucidated and showed that ATP makes contacts along the length of the cleft and bonds with residues from both subunits. In particular, the nitrogen and carbonyl oxygen of Ala-64 (which is not a conserved residue and is valine in GlnB) are thought to form hydrogen bonds with the adenine group. The B loop has a consensus sequence of Thr-Gly-X-X-Gly-Asp-Gly-Lys-Ile-Phe, which is very similar to that of mononucleotide-binding proteins such as adenylate kinase. The architecture of the phosphate-binding loop is different from that of other nucleotide-binding proteins, and there is no evidence for Mg²⁺ binding as seen in adenylate kinase and p21^ras. The motif is one of the most highly conserved regions in all the P_II proteins from eubacteria, archaebacteria, and plants. This suggests that ATP binding is probably a common property of P_II, and indeed it has also been demonstrated for the GlnB protein of Synechococcus sp. (67). The precise role of ATP is presently unknown, although biochemical data suggest that high-affinity binding of 2-ketoglutarate requires ATP (67, 118). The binding of ATP may also cause alterations in the structure and mobility of the T loop and in the structure of the B loop, both of which could affect molecular recognition.

E. coli GlnB and GlnK can form heterotrimers in vivo when glnB and glnK are coexpressed from a plasmid or when the wild-type strain is grown in nitrogen-limiting conditions (68, 234). GS adenylylation experiments in vitro demonstrate that the fully uridylylated heterotrimer is still capable of stimulating the deadenylylation activity of ATase, albeit to a lower extent than homotrimeric P_II-UMP. It has been proposed that heterotrimers could facilitate fine-tuning of the signal transduction cascade (234). While the physiological consequences of heterotrimer formation have yet to be fully established, heterotrimer formation is possibly not restricted to E. coli because it is also observed between Synechococcus GlnB and E. coli GlnB or GlnK when the cyanobacterial protein is expressed in E. coli (68). Given this observation, one might expect heterotrimer formation to occur in other organisms which express more than one form of P_II concurrently. The factors controlling such events could be of considerable interest and certainly complicate the interpretation of mutant phenotypes.

Analysis of sequence comparisons revealed that residues that are either involved in the formation of the trimer (residues 30, 32, 34, 93, 95, and 98) or interact in the interior of the central cavity (residues 3, 5, 60, and 62) are not always conserved in P_II proteins (252). In particular, residues 3 and 5 vary between the different subgroups of P_II proteins (101). In E. coli GlnB, Lys-3 and Asp-5 form a ring of alternating charge in the central cavity of the trimer, but in GlnK these residues are replaced by neutral Leu-3 and Thr-5 residues (252). The distinction between the nature of residues 3 and 5 in GlnB and GlnK extends to other members of the P_II family, e.g., NifI₁ polypeptides are characterized by Met-3 and Arg-5 or Lys-5, while NifI₂ is characterized by Glu-3 and Ile-5. The significance of this signature is not clear.

A considerable number of mutations have been studied in E. coli glnB, and with the available crystal structure, these can help to shed some light on structure-function relationships in the protein. Uridylylation of GlnB is prevented by mutation of Tyr-51 to Phe, Asn, or Ser (14, 89, 103, 111). It is also severely affected by other alterations in the exposed part of the T loop, including E50Q, the same alteration as that produced by the K. pneumoniae glnB502 mutation (96, 111). Residue Tyr-46 is highly conserved in P_II proteins, and a Y46F variant is uridylylated at a much lower rate than the wild type (103). The mutation does not alter the structure of the protein or binding of ATP or 2-ketoglutarate, suggesting that the residue may play an important role in recognition and binding by UTase (103, 111). Removal of the apex of the T loop (47-53) also prevents uridylylation. Indeed, it alters interaction with all three known receptors but not the binding of small effector molecules, implying that the T loop is necessary for interaction with GlnB targets (111).

Mutations of different residues within the T loop of GlnB affect inteaction with all targets to some degree, but two of them, G41A and A49P, are notable exceptions dramatically eliminating interaction with UTase or NtrB, respectively, without having any dramatic effect on their other targets (111). A number of other alterations have also been made to residues found in the conserved region comprising the B loop and the fourth -sheet of E. coli GlnB. The effects of these changes on the interaction of GlnB with 2-ketoglutarate, ATP, and the signal-transducing enzymes were then studied. Mutations around the B loop and the base of the T loop tend to affect the binding of the small-molecule ligands and some or all of the three protein receptors UTase/UR, ATase, and NtrB (111). It may be that the effect on enzyme interactions is a result of poor ligand binding. This second group of mutations cluster around the cleft formed by the T and B loops of one monomer and the C loop of the neighboring monomer, adding weight to the theory that this structure plays an important role in the interaction with receptor proteins and ligands.

Finally, two other alterations with marked effects on GlnB function fall outside these regions. A Thr29Met mutation was identified by the mutant's inability to activate glnA transcription, but further investigation revealed that the protein was affected in all its interactions. As this mutation destroys the hydrogen bond link with Glu-62 (found at the base of the third -sheet), it may result in an overall change in protein conformation. A Gly89Ala mutation causes an almost total loss of function, probably linked to the inability to bind small-molecule effectors. Gly-89 is at the base of the B loop at a sharp turn, which allows the Lys-90 of -4 to line the cleft. It is possible that this mutation causes a conformational change that prevents this turn.

Experiments investigating the functionality of heterotrimers comprising 47-53 GlnB and wild-type GlnB showed that the T loops can act independently of one another and a single loop is sufficient to allow interaction with the receptor proteins. Heterotrimers of Gly89Ala and wild-type monomers had different effects on interaction with different protein receptors, indicating that changes at the base of the B loop probably affect intersubunit interactions and thereby the activity of the wild-type subunit. Heterotrimers of Gly89Ala (containing a normal T loop but unable to bind small-molecule effectors) and 47-53 (missing the T loop) remained inactive, suggesting that there is no trans complementation between subunits (112).

A principal characteristic of the P_II proteins is their ability to be switched between two forms by covalent modification of a residue in the T loop. This modification was initially recognized as uridylylation of Tyr-51 in the T loop of E. coli GlnB. This residue is highly conserved, being present in the GlnB and GlnK proteins of the proteobacteria and the actinobacteria. Uridylylation of P_II proteins has been demonstrated in Rhizobium leguminosarum (40), Klebsiella pneumoniae (55), Azospirillum brasilense (47, 50), Azotobacter vinelandii (196), Herbaspirillum seropedicae (20), and Azoarcus sp. strain BH72 (150) and suggested in Sinorhizobium meliloti (8), Rhodospirillum rubrum (114), and Corynebacterium glutamicum (104). While this process is widespread, it is not universal; P_II proteins in the cyanobacteria are phosphorylated on a serine residue, Ser-49, in the T loop, and in other organisms, e.g., the archaebacteria and higher plants, modification of P_II proteins has yet to be investigated.

Uridylylation of P_II proteins. Uridylylation of E. coli GlnB in vitro is a noncooperative reaction (15). The transferase reaction shows linear kinetics, with GlnB binding before ATP and pyrophosphate being released before GlnB-UMP. By contrast, the uridylyl-removing reaction proceeds with rapid equilibrium binding of substrate and random release of products. Both reactions are activated by ATP and 2-ketoglutarate, while glutamine inhibits the transferase reaction. The kinetic data are consistent with a single binding site on GlnD for GlnB and GlnB-UMP and with a single catalytic center on the enzyme, which could be located at the conserved nucleotidyl transferase site (108). In vitro, GlnK is uridylylated as effectively as GlnB, but deuridylylation of GlnK-UMP is slower than that of GlnB-UMP (14).

Phosphorylation of P_II proteins. Although Tyr-51 is present in GlnB from the cyanobacterium Synechococcus sp. strain PCC6803, this protein is not uridylylated but is phosphorylated at the nearby Ser-49 residue (69, 71). This is consistent with the absence of a glnD homologue. Despite this difference, heterologous expression of Synechococcus sp. strain PCC7942 GlnB in E. coli results in uridylylation of this protein, albeit with low efficiency (67, 68). P_II was originally identified in cyanobacteria as a 13-kDa protein in Synechococcus sp. strain PCC7942 that exhibited a posttranslational modification dependent on the nitrogen source and the spectral light quality (225). However, the spectral light quality dependency is only seen in the presence of nitrate, and hence, as in enterics, the primary role of GlnB appears to be in sensing nitrogen status (69). In the presence of ammonium, GlnB is unmodified, and in nitrate GlnB is predominantly modified. Moreover, in cells grown in ammonium but in the presence of L-methionine sulfoximine (a GS inhibitor), GlnB exhibits the modified pattern, indicating that ammonium assimilation is required to elicit dephosphorylation.

Other possible modifications. P_II modification has not yet been studied in the archaebacteria. A Tyr-51 residue (or its equivalent) is present in the P_II proteins encoded by the amtB-linked genes of Methanococcus jannaschii and Methanobacterium thermoautotrophicum, but there is no glnD homologue in these organisms, suggesting that in these cases modification, if it occurs at all, is not by uridylylation (35). The archebacterial nif cluster GlnB homologues fall into two classes, NifI₁, with a predicted T loop of only 15 residues, and NifI₂, with a predicted T loop of 27 or more residues. Both contain conserved tyrosine residues, but they are not predicted to be at the apex of the T loop. Tyr-51 is also absent in Bacillus subtilis GlnK (246) and Clostridium longisporum GlnK (29), and consistent with this, there is no glnD homologue in the Bacillus subtilis genome. Both organisms have a serine residue in the T loop which could constitute a potential modification site.

Although P_II was originally discovered as a consequence of studies on factors controlling the activity of GS in E. coli, P_II proteins are now known to have many complex roles in the regulation of microbial nitrogen metabolism. The mutiplicity of possible roles for P_II proteins is presented schematically in Fig. 4. The recognition that many organisms have multiple P_II genes has added a further layer of complexity, and it is extremely likely that there are still new roles for P_II waiting to be discovered. In the following sections we discuss those functions of P_II that have already been recognized and consider the evidence for other possible targets.

View larger version (23K): [in this window] [in a new window]   FIG. 4.   Schematic representation of the potential mutiplicity of roles for PII proteins. This cartoon summarizes information (both definitive and predicted) obtained from many different organisms.

The most common form of GS in bacteria is termed GSI. It is encoded by glnA and consists of 12 identical subunits of 55 kDa, which form two superimposed hexagonal rings (256; for a review, see reference 155). In a number of species, including E. coli, GSI is posttranslationally modified by the adenylylation of a tyrosine residue (Tyr-397) on each subunit by the ATase enzyme encoded by glnE (64, 207). This allows the progressive inactivation of GSI as each subunit is independently inactivated in response to increasing levels of intracellular nitrogen. It has been proposed that the physiological significance of the posttranslational regulation of GS is to prevent a sustained decrease in the ATP concentration and to protect the cellular glutamate pool, allowing rapid growth (129, 204).

ATase is one of the best characterized of the known P_II targets, but there is presently no information on the precise site of interaction of GlnB with ATase. The gene for ATase (glnE) has been cloned from E. coli and sequenced, and the protein has been shown to be a monomer of 115 kDa (232). Like UTase, ATase is a bifunctional enzyme catalyzing either the addition of AMP to Tyr-397 of GS using ATP as substrate or the removal of the adenylyl group, which results in the release of ADP. Separation of the two antagonistic activities of the protein, adenylylation and deadenylylation, has been demonstrated by the formation of two truncated proteins. The N-terminal domain of ATase deadenylylates GS, and this activity is regulated by GlnB-UMP and 2-ketoglutarate but is not affected by glutamine, whereas the C-terminal domain adenylylates GS in a glutamine-dependent manner. GlnB is required for the latter activity