Susan H. Fisher. E-mail firstname.lastname@example.org; Tel. (+1) 617 638 5498; Fax (+1) 617 638 4286.
Nitrogen metabolism genes of Bacillus subtilis are regulated by the availability of rapidly metabolizable nitrogen sources, but not by any mechanism analogous to the two-component Ntr regulatory system found in enteric bacteria. Instead, at least three regulatory proteins independently control the expression of gene products involved in nitrogen metabolism in response to nutrient availability. Genes expressed at high levels during nitrogen-limited growth are controlled by two related proteins, GlnR and TnrA, which bind to similar DNA sequences under different nutritional conditions. The TnrA protein is active only during nitrogen limitation, whereas GlnR-dependent repression occurs in cells growing with excess nitrogen. Although the nitrogen signal regulating the activity of the GlnR and TnrA proteins is not known, the wild-type glutamine synthetase protein is required for the transduction of this signal to the GlnR and TnrA proteins. Examination of GlnR- and TnrA-regulated gene expression suggests that these proteins allow the cell to adapt to growth during nitrogen-limited conditions. A third regulatory protein, CodY, controls the expression of several genes involved in nitrogen metabolism, competence and acetate metabolism in response to growth rate. The highest levels of CodY-dependent repression occur in cells growing rapidly in a medium rich in amino acids, and this regulation is relieved during the transition to nutrient-limited growth. While the synthesis of amino acid degradative enzymes in B. subtilis is substrate inducible, their expression is generally not regulated in response to nitrogen availability by GlnR and TnrA. This pattern of regulation may reflect the fact that the catabolism of amino acids produced by proteolysis during sporulation and germination provides the cell with substrates for energy production and macromolecular synthesis. As a result, expression of amino acid degradative enzymes may be regulated to ensure that high levels of these enzymes are present in sporulating cells and in dormant spores.
When faced with an abundance of nutrients, microorganisms selectively use those compounds that allow optimal growth rates. Global regulatory systems that result in the preferential assimilation of certain carbon and nitrogen compounds have been identified in bacteria, yeast and fungi. By controlling the expression of permeases and degradative enzymes required for the catabolism of nutrients that can only support slower growth rates (non-preferred nutrients), these systems establish a hierarchy for nutrient utilization in cells growing in the presence of multiple food sources. The first comprehensive analysis of the physiology and regulation of nitrogen metabolism was performed in enteric bacteria. These findings will be discussed in the first part of this review, because they provide a useful framework for characterizing nitrogen metabolism in unrelated bacteria (see Merrick and Edwards, 1995; Magasanik, 1996; Reitzer, 1996a).
Regulation of nitrogen metabolism in enteric bacteria
Ammonium is the preferred source of nitrogen in enteric (and many other) bacteria by several criteria (Reitzer, 1996b). First, ammonium supports the fastest cell growth rate. Secondly, the synthesis of gene products needed for the utilization of non-preferred nitrogen-containing compounds is repressed in cells grown in the presence of ammonium. Finally, compared with cells grown with other nitrogen sources, ammonium-grown cells contain the lowest levels of glutamine synthetase (GS), a critical enzyme in ammonium assimilation. GS catalyses the formation of glutamine from ammonium and glutamate, the only pathway for glutamine synthesis in the cell. Glutamine serves both as an amino acid and as the nitrogen donor for the synthesis of approximately 15% of the nitrogenous molecules in the cell. As a result, the synthesis and activity of GS is tightly regulated in response to nitrogen availability to ensure that adequate supplies of glutamine are present in cells whose growth is limited by nitrogen availability (Reitzer, 1996b). Cells growing rapidly with preferred (excess) nitrogen sources contain low levels of GS, whereas high levels of GS are synthesized during nitrogen-limited growth. In enteric bacteria, the enzymatic activity of GS is regulated by a post-translational modification involving adenylylation in response to nitrogen availability. In addition, the activity of the Escherichia coli GS is subject to feedback inhibition by nine end-products of glutamine metabolism.
A second route for ammonium assimilation is provided by glutamate dehydrogenase, which synthesizes glutamate from ammonium and 2-ketoglutarate (Reitzer, 1996b). Because glutamate dehydrogenase enzymes generally have a high Km for ammonium, this pathway functions only in cells grown with high levels of ammonium. During ammonium-limited growth, glutamate must be synthesized from glutamine and 2-ketoglutarate by the enzyme glutamate synthase. As the amino group of glutamate is the nitrogen donor for the biosynthesis of approximately 85% of the cell's nitrogenous compounds, high-level expression of GS during nitrogen-limited growth ensures that glutamine is available for the synthesis of glutamate.
The Ntr system
In enteric bacteria, the Ntr nitrogen-regulatory system controls the expression of GS, glutamate dehydrogenase and several gene products involved in the catabolism of nitrogenous compounds (Merrick and Edwards, 1995; Magasanik, 1996; Reitzer, 1996a). Central to the Ntr system is a two-component regulatory system that activates transcription of σ54-dependent promoters. Phosphorylation of the response regulator NtrC by the NtrB kinase is controlled by the PII signal transduction protein in response to the intracellular ratio of glutamine and 2-ketoglutarate. During nitrogen-limited growth, the intracellular levels of glutamine decline, NtrC is phosphorylated and high-level expression of Ntr-regulated genes occurs (Ikeda et al., 1996). Not all nitrogen-regulated genes are controlled directly by NtrC. Regulation of some genes in the Ntr regulon requires the secondary transcriptional regulator Nac, whose expression is activated by NtrC (Magasanik, 1996).
Nitrogen metabolism in Bacillus subtilis
Bacillus subtilis is a Gram-positive, sporulating soil bacterium often found associated with decaying organic matter. Because sporulation in B. subtilis is initiated by nutrient depletion, this developmental process provides a survival mechanism when nutrients become scarce in the environment (Hoch, 1993). Despite the different ecological habitats and life cycles of B. subtilis and enteric bacteria, there are only minor differences in the physiology of ammonium assimilation between these bacteria (see Schreier, 1993). Because B. subtilis has no assimilatory glutamate dehydrogenase activity, ammonium assimilation occurs solely by the GS–glutamate synthase pathway. The synthesis of GS is raised during nitrogen-limited growth in B. subtilis, but the activity of the B. subtilis GS enzyme is not regulated by any known post-translational protein modification. Instead, the enzymatic activity of the B. subtilis GS enzyme is thought to be feedback inhibited by glutamine in vivo. As judged by growth rates and GS levels present in cells growing with various nitrogen compounds, glutamine is the best (preferred) nitrogen source for B. subtilis, followed by arginine (Atkinson and Fisher, 1991). Ammonium is also a good nitrogen source for B. subtilis, because the expression of GS synthesis is partially repressed in ammonium-grown cells.
Regulation of gene products involved in nitrogen metabolism
The first striking difference noted between B. subtilis and enteric bacteria with respect to nitrogen metabolism was that the expression of several amino acid catabolic enzymes is not subject to nitrogen regulation in B. subtilis. (In this review, the term nitrogen regulation is used to refer to regulatory mechanisms that elevate gene expression during nitrogen-limited growth.) Expression of the arginine, proline and histidine degradative enzymes is substrate inducible in both B. subtilis and enteric bacteria (Fisher, 1993). However, in contrast to the case for enteric bacteria, the presence of a good nitrogen source (such as ammonium) does not repress the expression of these amino acid catabolic enzymes in B. subtilis. These observations raised the possibility that B. subtilis does not contain any global nitrogen-regulatory system. This view was reinforced by the discovery that transcription of GS (glnA) is not regulated by any Ntr-like regulatory system in B. subtilis. In enteric bacteria, high-level glnA expression during nitrogen-limited growth occurs from a σ54-dependent promoter whose transcription is activated by the phosphorylated form of NtrC (Magasanik, 1996). The B. subtilis GS is encoded within a dicistronic operon (glnRA) that is transcribed from a vegetative (σA-dependent) promoter (Schreier et al., 1989; Schreier, 1993). During growth with excess nitrogen, GlnR represses transcription of the glnRA promoter. As glnR mutants have no defects in growth or sporulation, GlnR was initially thought to be an operon-specific regulator rather than a global regulatory protein. Furthermore, although no nitrogen-regulated genes (other than the glnRA operon) had been identified in B. subtilis, higher levels of sporulation occur in nitrogen-limited cultures than in cultures growing rapidly in the presence of preferred nitrogen sources (Sonenshein, 1989). All these observations led to the generally held belief that B. subtilis has no global nitrogen-regulatory system, because this bacterium preferentially initiates sporulation during nitrogen-limited growth.
However, sporulation is not the only strategy used by B. subtilis for survival during nutritional limitation. The frequency at which sporulation occurs is also increased in carbon- and phosphate-limited cultures (Sonenshein, 1989), but B. subtilis contains global phosphate and carbon regulatory systems that allow cells to continue to grow vegetatively under phosphate- and carbon-restricted conditions (Hueck and Hillen, 1995; Hulett, 1996). In fact, sporulation appears to be the last resort for the nutritionally challenged cell. Before initiating sporulation, B. subtilis attempts a number of adaptive approaches (motility, chemotaxis, synthesis of extracellular degradative enzymes and antibiotics), any of which, if successful, would allow vegetative growth to resume (Fisher and Sonenshein, 1991). A more extensive analysis of the expression of permeases and degradative enzymes involved in nitrogen metabolism revealed that these gene products are indeed regulated by multiple global regulatory systems, each of which functions under different nutritional conditions.
The TnrA/GlnR regulon
TnrA, a global nitrogen-regulatory protein
Expression of asparaginase, γ-aminobutyric acid permease (gabP ), urease (ureABC ), a putative ammonium permease (nrgA) and the nitrate assimilatory enzymes (nasABCDEF ) is elevated during nitrogen limitation in B. subtilis (Atkinson and Fisher, 1991; Wray and Fisher, 1994; Nakano et al., 1995; 1998; Ferson et al., 1996). Transcription of the gabP, ureABC, nrgAB and nas genes is not controlled by pathway-specific inducers. Instead, their expression is activated by the global nitrogen-regulatory protein TnrA in response to nitrogen limitation (Table 1) (Wray et al., 1996; Nakano et al., 1998). In addition, TnrA positively regulates its own synthesis (L. V. Wray, Jr, J. M. Zalieckas and S. H. Fisher, unpublished) as well as that of some gene products not directly involved in nitrogen metabolism. For instance, expression of KipI, an inhibitor of the sporulation kinase A, is activated by TnrA (Wang et al., 1997). TnrA also functions as a negative regulatory protein, repressing the expression of the ammonium assimilatory enzymes, GS and glutamate synthase, during nitrogen-limited growth (Table 1) (Wray et al., 1996; B. R. Belitsky and A. L. Sonenshein, personal communication).
Table 1. . Transcription factors regulating genes involved in nitrogen metabolism in B. subtilis. References for genes cited in this table can be found in the text.
TnrA is a homologue of GlnR, the negative regulatory protein for the glnRA operon (Wray et al., 1996). These two proteins belong to the MerR family of DNA-binding regulatory proteins. GlnR and TnrA are very similar except in their C-terminal signal-transducing domains. Their proposed N-terminal DNA-binding domains are nearly identical, and both proteins bind to similar DNA sequences (TGTNAN7TNACA; Gutowski and Schreier, 1992; Nakano et al., 1995; Brown and Sonenshein, 1996; Wray et al., 1996; 1998). The two GlnR binding sites in the glnRA promoter region contain the same inverted repeat sequence (GlnR/TnrA site) found in the cis-acting sites required for TnrA-dependent activation of gapP, nrgAB, nas, tnrA and ureABC expression. As all the promoters positively regulated by TnrA contain a GlnR/TnrA site located upstream of their −35 region, TnrA most probably activates their expression by facilitating the binding of RNA polymerase to the promoter region (Wray et al., 1996; 1997). TnrA negatively regulates glnRA expression by binding to the glnRA O2 operator, which overlaps the −35 region of the glnRA promoter (J. M. Zalieckas, L. V. Wray, Jr and S. H. Fisher, unpublished). In contrast, GlnR binds co-operatively to both the glnRA O2 operator and a second operator, glnRA O1, that lies upstream of the −35 region for the glnRA promoter (Gutowski and Schreier, 1992; Brown and Sonenshein, 1996).
The mechanism by which TnrA negatively regulates glutamate synthase (gltAB ) expression is not completely understood. Glutamate synthase levels are reduced in cells grown with nitrogen compounds that are sources of glutamate (e.g. glutamine, glutamate and arginine) (Pan and Coote, 1979; Bohannon et al., 1985). Transcription of gltAB is activated by GltC, a member of the LysR family of regulatory proteins (Bohannon and Sonenshein, 1989). However, GltC is not required for regulation of gltAB expression by TnrA (B. R. Belitsky and A. L. Sonenshein, personal communication). Because the gltAB promoter region does not contain an obvious GlnR/TnrA site, TnrA regulation of the gltAB operon may be mediated indirectly.
GlnR is a global nitrogen-regulatory protein
As GlnR and TnrA bind to similar DNA sequences, it is not surprising that GlnR also represses the expression of several TnrA-regulated genes. In addition to the glnRA promoter, GlnR represses the expression of the tnrA and ureABC P3 promoters during growth with excess nitrogen (Wray et al., 1997; L. V. Wray, Jr, J. M. Zalieckas and S. H. Fisher, unpublished). Thus, GlnR and TnrA not only regulate their own synthesis, but also cross-regulate each other's expression. A somewhat analogous nitrogen-regulatory system is present in yeast, in which the expression of nitrogen catabolic genes is governed by a cross-regulatory system involving four members of the GATA family of transcriptional regulators (Coffman et al., 1997). While TnrA and GlnR are homologues, there is no evidence that the formation of GlnR–TnrA heterodimers is required for nitrogen regulation. TnrA-dependent regulation is not defective in glnR mutants, and no defect in GlnR-dependent repression is observed in tnrA mutants (Wray et al., 1996; 1997).
The transcription of the nas, gabP and nrgAB genes is activated by TnrA, but the expression of these genes is not regulated by GlnR (Atkinson and Fisher, 1991; Nakano et al., 1995). Several different possibilities may explain why some TnrA-activated genes are not subject to GlnR-mediated regulation. First, although GlnR and TnrA bind to similar DNA sequences, there may be unrecognized differences between the sequence of an optimal GlnR binding site and an optimal TnrA binding site. In this scenario, GlnR would not bind with high affinity to all the GlnR/TnrA sites at which TnrA regulation occurs. Alternatively, because two potential GlnR/TnrA sites are present in the promoter regions of the GlnR-repressed glnRA, ureP3 and tnrA genes (Gutowski and Schreier, 1992; Brown and Sonenshein, 1996; Wray et al., 1997; L. V. Wray, Jr and S. H. Fisher, unpublished), the GlnR protein may bind with high affinity only to DNA containing two appropriately spaced GlnR/TnrA sites. This hypothesis is consistent with in vitro studies showing that GlnR binds simultaneously to both operators in the glnRA promoter, whereas TnrA binds to an nrgAB promoter DNA fragment that contains only one GlnR/TnrA site (Brown and Sonenshein, 1996; Wray et al., 1998). Finally, the GlnR/TnrA sites for the nas, gabP and nrgAB genes are all positioned upstream of the −35 promoter region for these genes (Wray et al., 1996). It is possible that, during growth with excess nitrogen, GlnR binds to these upstream GlnR/TnrA sites but does not activate transcription, because the GlnR protein lacks a transcription activation domain that is present in the TnrA protein.
How are the activities of TnrA and GlnR regulated?
Although all genes regulated by GlnR and TnrA are expressed at high levels during nitrogen limitation, the GlnR and TnrA proteins are active during different nutritional conditions. Examination of the expression of genes regulated by GlnR in glnR mutants indicates that GlnR represses gene expression in cells growing on excess (preferred) nitrogen sources (Schreier et al., 1989; Wray et al., 1997). In contrast, TnrA was found to regulate gene expression only during nitrogen-limited growth (Wray et al., 1996; 1997). This type of regulatory arrangement is advantageous to the cell, because it allows more flexible and responsive regulation from a single DNA site.
The nitrogen signal(s) regulating the activity of GlnR and TnrA has not been identified. It is also not known whether GlnR and TnrA respond to the same or different signals. Regardless, the nitrogen signal pathways for GlnR and TnrA must overlap, because two experimental observations indicate that GS is required for transduction of the signal for nitrogen availability to both proteins. First, all known GlnR- and TnrA-regulated genes are expressed constitutively in B. subtilis glnA mutants that have no or little GS activity (Dean et al., 1977; Schreier and Sonenshein, 1986; Atkinson and Fisher, 1991; Nakano, 1995; Ferson et al., 1996; Wray et al., 1996). Secondly, when the expression of the glnRA promoter was examined in E. coli glnA mutants, glnRA was expressed constitutively in E. coli cells containing only GlnR (Schreier et al., 1985; 1989). When the E. coli cells contained both GlnR and the B. subtilis GS, glnRA transcription was repressed during growth with excess nitrogen. The simplest model to explain these observations is that, during growth on excess nitrogen, GS is required for the generation and/or transduction of a nitrogen-regulatory signal that controls the activity of both GlnR and TnrA (Fig. 1) (Schreier et al., 1985; 1989; Wray et al., 1996).
The nitrogen-regulatory signal transduced by GS affects the activity of GlnR and TnrA differently (Fig. 1). As GlnR-dependent repression is observed in vivo only under growth conditions in which the signal is produced, i.e. wild-type cells growing in the presence of excess nitrogen, this signal is required for GlnR activity (Schreier et al., 1989). In contrast, the nitrogen-regulatory signal must inhibit the activity of TnrA activity, because TnrA-dependent regulation does not occur in cells growing with excess nitrogen (Wray et al., 1996). Thus, TnrA is only active during nitrogen-limited growth in which the excess nitrogen-regulatory signal is not present. Moreover, the GlnR and TnrA proteins have different sensitivities to the GS-dependent nitrogen-regulatory signal (or may respond to different signals). When wild-type cells are grown with ammonium as sole nitrogen source, the GlnR-dependent repression of the glnRA promoter is partially relieved, whereas the TnrA-dependent activation of the nrgAB and gabP promoters is completely inhibited (Atkinson and Fisher, 1991). This suggests that, during the transition from nitrogen excess to nitrogen-limited growth conditions, GS transduces sufficient levels of the nitrogen-regulatory signal to inhibit TnrA activity, but not to activate GlnR repression. The differential response to the nitrogen-regulatory signal by the GlnR and TnrA proteins allows the expression of GS (and other GlnR-regulated genes) to derepress before TnrA regulation occurs under these growth conditions. Interestingly, the enteric Ntr system is similarly fine-tuned, so that the activation of GS expression has precedence over the activation of expression for other Ntr-regulated genes (Magasanik, 1996; Reitzer, 1996b).
It is possible that GS activity is required for the synthesis of a metabolite that regulates the activity of GlnR and TnrA directly. Because changes in the intracellular level of glutamine are not sufficient for regulation of the activity of GlnR and TnrA, this metabolite is unlikely to be glutamine (the product of GS enzymatic activity). During growth with excess nitrogen, GlnR- and TnrA-regulated genes are expressed constitutively in the B. subtilis glnA22 mutant (Schreier and Sonenshein, 1986; Atkinson and Fisher, 1991; Nakano, 1995; Ferson et al., 1996; Wray et al., 1996). As intracellular glutamine pools are sixfold higher in the glnA22 mutant than in wild-type cells, reduced intracellular levels of glutamine are not required for high-level expression of nitrogen-regulated genes in B. subtilis (Fisher and Sonenshein, 1984). Furthermore, the in vitro sequence-specific binding of GlnR and TnrA with DNA is not altered by the presence of glutamine (Nakano and Kimura, 1991; Brown and Sonenshein, 1996; Wray et al., 1998).
An alternative model for the role of GS in nitrogen regulation is that GS functions as a sensor of nitrogen availability and transmits this signal to GlnR and TnrA by a signal transduction pathway containing one or more other proteins. However, the observation that only the B. subtilis GS is required for the GlnR-dependent nitrogen regulation of glnRA expression in E. coli cells suggests that no additional B. subtilis proteins are required for the transmission of the nitrogen-regulatory signal from GS to GlnR (Schreier et al., 1985; 1989). A third possibility is that the GS protein interacts with and regulates the activity of the GlnR and TnrA proteins directly. This model is supported by in vitro experiments showing that the binding of GlnR to its operators is weakly stimulated by purified wild-type B. subtilis GS (Nakano and Kimura, 1991; Brown and Sonenshein, 1996). Because the mechanism responsible for the GS stimulation of GlnR binding in vitro has not been identified, the physiological significance of this observation is unclear. Interestingly, purified GS from Thermus thermophilus 111 has been shown to bind preferentially to curved DNA regions in vitro (Mary and Révet, 1999). This raises the possibility that the in vitro stimulation of GlnR binding by B. subtilis GS could involve an interaction of GS with DNA.
Paradoxically, although all in vivo experimental evidence indicates that wild-type GS (and the nitrogen-regulatory signal) is required for GlnR activity, purified GlnR protein binds tightly (KD = 10−11 M) to the glnRA operators in vitro in the absence of any cofactors. It is possible that the in vitro affinity of GlnR for the glnRA operators would be increased in the presence of the nitrogen-regulatory signal. Alternatively, the binding of GlnR to DNA in the absence of any cofactor may indicate that the in vitro binding conditions do not accurately reflect the in vivo conditions.
The CodY regulon
The expression of several genes involved in nitrogen metabolism, including the histidine degradative operon (hut ), the dipeptide transport operon (dpp), the isoleucine and valine degradative operon (bkd ), ureABC and gabP, are negatively regulated by the CodY repressor protein (Slack et al., 1995; Ferson et al., 1996; Fisher et al., 1996; Wray et al., 1997; M. Débarbouillé, personal communication). CodY also represses the expression of several gene products not involved in nitrogen metabolism, including an enzyme involved in acetate metabolism, acetyl-CoA synthetase (acsA), and genes required for the development of competence, comK and the srfA operon (Serror and Sonenshein, 1996a; J. M. Zalieckas, L. V. Wray, Jr and S. H. Fisher, unpublished). CodY-dependent regulation of comK and the srfA operon explains, at least in part, previous observations that higher levels of competence occur in nitrogen-limited cells than in cells growing rapidly with amino acids (Dubnau and Roggiani, 1990; Kunst et al., 1994). Although purified CodY protein binds in vitro to the promoter regions of the dpp, srfA and comK, no CodY consensus binding sequence has been identified (Serror and Sonenshein, 1996a,b). Instead, CodY has been proposed to recognize and bind to a DNA structure formed by AT-rich DNA sequences.
While the signal regulating CodY activity has not been identified, CodY-dependent regulation is inversely correlated with growth rate (Fisher et al., 1996). Thus, CodY-dependent regulation reflects the total nutritional state of the cell, whereas GlnR and TnrA respond to nitrogen availability. CodY-dependent regulation was previously described as amino acid repression or nutritional repression, because the highest levels of regulation are seen in cells growing in a medium rich in amino acids. Only low levels of CodY-dependent repression occur in cells growing with excess carbon and nitrogen (but no amino acids). Carbon or nitrogen limitation of growth almost completely abolishes CodY-dependent regulation. As a result, several CodY-regulated genes, including dpp, hut, ureABC, srfA and comK, are induced when cells enter stationary phase resulting from exhaustion of the carbon or nitrogen source (Slack et al., 1995; Fisher et al., 1996; Serror and Sonenshein, 1996a; Wray et al., 1997). Regulation of the utilization of nitrogen (and carbon) sources in response to growth rate makes good sense, because it ensures that nutrients are available for adaptation to non-optimal growth conditions.
The σL regulon
The B. subtilis sigL gene encodes a homologue of the enteric RNA polymerase σ54 sigma factor (Débarbouilléet al., 1991a). Analysis of sigL mutant strains revealed that σL is required for the utilization of arginine, ornithine, isoleucine and valine as nitrogen sources and high-level expression of the levanase operon (lev ). Multiple σL transcriptional activators have been identified. LevR activates the expression of the lev operon (Débarbouilléet al., 1991b). RocR is required for the utilization of arginine and ornithine (Gardan et al., 1997). Three additional B. subtilis genes encoding members of the family of σ54 transcriptional enhancer proteins have been identified by polymerase chain reaction (PCR) amplification (Kaufman and Nixon, 1996) and analysis of the B. subtilis genome sequence. One of these proteins, BkdR, positively regulates the bkd operon, which encodes gene products involved in the utilization of isoleucine and valine as nitrogen sources (M. Débarbouillé, personal communication). Phosphorylation of the enteric σ54 transcriptional activator NtrC is regulated by the signal transduction protein PII (Magasanik, 1996). Although the B. subtilis NrgB protein is a PII homologue, NrgB does not seem to regulate the activity of any of the σL transcriptional activators (Wray and Fisher, 1994).
RocR is required for the expression of two operons (rocABC and rocDEF ) that encode gene products involved in arginine and ornithine utilization as well as the monocistronic rocG gene, which encodes a catabolic glutamate dehydrogenase (Gardan et al., 1997; Belitsky and Sonenshein, 1998; B. R. Belitsky and A. L. Sonenshein, personal communication). Ornithine induces the expression of the roc regulon and is proposed to regulate RocR activity by interaction with the RocR protein (Gardan et al., 1997). AhrC, a homologue of the enteric ArgR repressor, also positively regulates roc expression by a mechanism that remains to be clarified. AhrC has been shown to bind in vitro to the rocABC promoter region in the presence of arginine (Klingel et al., 1995). There is also evidence that AhrC may interact directly with the RocR protein (Gardan et al., 1997).
Although expression of the arginine degradative enzymes is inducible, their synthesis is not controlled by TnrA or GlnR. However, expression of these enzymes may be regulated in response to nutrient availability by unidentified mechanisms. Glutamine, but not ammonium, represses the induction of the arginine degradative enzymes in cells growing logarithmically in minimal medium (Baumberg and Harwood, 1979). Furthermore, expression of the arginine degradative enzymes is activated during late logarithmic growth in nutrient broth sporulation medium (Deutscher and Kornberg, 1968).
The roles of GlnR and TnrA in the B. subtilis life cycle
Three global regulatory proteins, CodY, GlnR and TnrA, control the expression of gene products involved in nitrogen metabolism in response to nutrient availability in B. subtilis. Each of these systems is active under different nutritional conditions. CodY represses in cells growing rapidly with amino acids; GlnR represses only in cells growing with excess nitrogen; and TnrA activates or represses transcription only during nitrogen-limited growth. All three proteins regulate ureABC expression (Wray et al., 1997). CodY and TnrA control gabP expression (Ferson et al., 1996), while both GlnR and TnrA regulate glnRA and tnrA (Wray et al., 1996; L. V. Wray, Jr, J. M. Zalieckas and S. H. Fisher, unpublished). The use of multiple systems to regulate gene products involved in nitrogen metabolism allows non-co-ordinate gene expression under diverse nutritional conditions.
While the regulation of gene products involved in nitrogen metabolism in B. subtilis has not been characterized exhaustively, certain general trends can be noted. GlnR and TnrA regulate gene products involved in ammonium transport or assimilation (nrgA, glnA, gltAB ) and the utilization of nitrogen-containing compounds whose expression is not subject to pathway-specific induction (ureABC, nas, gabP ). Expression of the ureABC, nas and gabP genes is most probably not inducible because urea, nitrate and γ-aminobutyric acid (which is found in root nodules) are all readily available in the soil, the natural habitat of B. subtilis. Although B. subtilis can initiate sporulation under nitrogen-limited growth conditions, neither GlnR nor TnrA are required for successful sporulation. Regulation of gene expression by GlnR and TnrA is not altered during the early stages of sporulation in nutrient broth sporulation medium (Wray et al., 1997; 1998). GS levels do not increase when sporulation is initiated by transfer to medium lacking a source of nitrogen (Pan and Coote, 1979). Moreover, TnrA may indirectly inhibit the initiation of sporulation during nitrogen-limited growth by activating the expression of KipI, an inhibitor of the KinA sporulation kinase (Wang et al., 1997). All these observations suggest that GlnR and TnrA most probably direct the cell towards adaptative vegetative growth, rather than towards sporulation, during nitrogen-limited growth conditions.
Why do TnrA and GlnR not regulate the expression of amino acid degradative enzymes?
In enteric bacteria, the Ntr system regulates the expression of gene products involved in the utilization of the amino acids arginine, tryptophan, asparagine, glutamine, histidine and proline (Reitzer, 1996a). The expression of the B. subtilis enzymes required for the degradation of aspartate, asparagine, alanine, arginine, histidine and proline is substrate inducible, but their expression is not activated during nitrogen limitation by GlnR or TnrA (Fisher, 1993). (Asparaginase is the one exception to this generalization; see below.) One possible explanation for this difference between B. subtilis and enteric bacteria is that amino acid catabolism plays an important role in the developmental life cycle of B. subtilis (see Setlow, 1981). During the initial stage of spore germination, the degradation of small acid-soluble spore proteins (which comprise 15–20% of the spore protein) generates copious amounts of free amino acids. In germinating spores of Bacillus megaterium, which is closely related to B. subtilis, up to 50% of the free amino acids generated by proteolysis are degraded, producing 10–20 times the energy obtained from the catabolism of 3-phosphoglycerate, the other major energy source in the dormant spore (Setlow, 1981). Although B. subtilis spores have not been examined for the presence of all known amino acid degradative enzymes, these spores contain significant levels of the catabolic enzymes for aspartate, asparagine, arginine, alanine, valine and isoleucine (Setlow, 1981).
As the expression of very few amino acid catabolic enzymes has been examined carefully during sporulation, the mechanism(s) ensuring their presence in the sporulating cells (and dormant spores) are poorly understood. During growth and sporulation in rich medium, the expression of many B. subtilis amino acid degradative enzymes is induced before the initiation of sporulation (Fisher, 1993). The synthesis of these enzymes can also be induced (or their synthesis maintained) by free amino acids generated from increased protein turnover during sporulation. Sporulation-specific regulatory mechanisms may also exist, because the ansAB operon (which encodes asparaginase and aspartase) is preferentially expressed in the developing forespore, but not in the mother cell, during sporulation (Sun and Setlow, 1991). Thus, the regulation of amino acid degradative enzymes in B. subtilis appears to have evolved so that these enzymes are present at high levels in sporulating cells and spores, rather than preferentially expressed during nitrogen-limited growth.
Although the synthesis of amino acid degradative enzymes is generally not nitrogen regulated in B. subtilis, a hierarchy for amino acid utilization is present in cells growing with mixtures of amino acids. Examination of the extracellular concentrations of amino acids during growth of a B. subtilis culture in medium containing ammonium and casamino acids as nitrogen sources revealed that glutamate, aspartate, serine and alanine were depleted from the growth medium at similar rates throughout exponential growth (Liebs et al., 1988). The majority of the extracellular arginine, glycine and proline was taken up only during late exponential growth, while histidine, isoleucine, threonine and valine were not used significantly until the onset of the stationary phase (Liebs et al., 1988). The mechanism(s) preventing the utilization of certain amino acids in cells growing rapidly with amino acid mixtures has only been determined for histidine, isoleucine and valine. In amino acid-grown cells, CodY represses transcription of the hut and bkd operons (Fisher et al., 1996; M. Débarbouillé, personal communication). In addition, reduced rates of histidine uptake in amino acid-grown cells prevent hut expression by interfering with histidine induction (Wray et al., 1994).
Regulation of asparaginase expression
Asparaginase is the only amino acid degradative enzyme whose synthesis is known to be activated by TnrA during nitrogen-limited growth (Atkinson and Fisher, 1991; S. H. Fisher, unpublished). The ansAB operon encodes asparaginase (ansA) and aspartase (ansB ) (Sun and Setlow, 1991). Expression of ansAB is negatively regulated by the product of the ansR gene (Sun and Setlow, 1993), and aspartate appears to be the inducer (Sun and Setlow, 1991). As the transcription of ansAB has not been examined under nitrogen-limited growth conditions, it is not known whether ansA encodes the nitrogen-regulated asparaginase. In fact, the B. subtilis genome sequence contains another open reading frame, yccC, that encodes a protein with an amino acid sequence that is highly similar to the asparaginase from Erwinia chrysanthemi (Kumano et al., 1997). A sequence with similarity to the GlnR/TnrA consensus sequence is located upstream of the yccC gene. Thus, B. subtilis may encode two differently regulated asparaginase genes, ansA and yccC. In this scenario, the expression of ansA would be induced by aspartate, while yccC expression would be activated by TnrA during nitrogen-limited growth.
The B. subtilis TnrA/GlnR regulatory system has greater similarity to the GATA family of transcriptional factors that mediate nitrogen catabolite repression in Saccharomyces cerevisiae than to the enteric Ntr nitrogen-regulatory system. The major unanswered question regarding the B. subtilis nitrogen regulatory circuitry is the identity of the signal(s) regulating the activity of the GlnR and TnrA proteins. The only factor known to affect the in vivo activity of these proteins (other than growth conditions) is GS. This is an ironic and controversial observation. While the B. subtilis TnrA/GlnR nitrogen-regulatory system is quite different from the Ntr system, one of the original Ntr models proposed that GS played a regulatory role in enteric nitrogen metabolism. It was subsequently shown that NtrB and NtrC, whose genes are located within the glnA–ntrBC operon, were responsible for the altered regulation seen in glnA mutants of enteric bacteria. Nonetheless, biochemical and genetic studies have failed to reveal any alternative explanations for the defects in nitrogen regulation seen in B. subtilis glnA mutants. Thus, the mechanism by which GS participates in the nitrogen signalling pathway in B. subtilis remains an unsolved mystery.
Although the frequency at which sporulation occurs increases in nitrogen-limited cultures, it is also not known what acts as the nitrogen signal for sporulation. The initiation of sporulation in B. subtilis is controlled by a phosphorelay system that contains at least two histidine sensor kinases, KinA and KinB, and two response regulators (Hoch, 1993; Perego, 1998). The multicomponent structure of the phosphorelay provides multiple points for the entry and integration of nutritional, environmental and cell cycle signals that influence the initiation of sporulation. As glnA mutants are not deficient in sporulation, GS does not transduce a nitrogen signal to the KinA and KinB kinases (Dean et al., 1977; Fisher and Sonenshein, 1977). The observation that the transcription of the KipI, an inhibitor of KinA, is nitrogen regulated raises the possibility that KipI may be involved in transmitting nitrogen availability to the phosphorelay (Wang et al., 1997). The activity of KipI is negated by KipA, which is encoded in the same operon as kipI (Wang et al., 1997). Because the effector molecule(s) regulating the interaction between KipI and KipA have not been identified, it is not known whether these proteins respond directly to the nitrogen state of the cell.
Regulatory systems similar to the classical enteric Ntr system have been shown to control the expression of gene products required for ammonium assimilation, utilization of nitrogen-containing compounds and nitrogen fixation in many other bacteria (Merrick and Edwards, 1995). It is unclear to what extent nitrogen-regulatory systems analogous to the B. subtilis GlnR, TnrA and CodY systems are present in other bacteria. While the genetic organization and GlnR-dependent regulation of the glnRA operon is preserved in Bacillus cereus and Staphylococcus aureus (Nakano and Kimura, 1991; Gustafson et al., 1994), little is known about the regulation of other gene products involved in nitrogen metabolism in these two bacteria. Interestingly, unique regulatory mechanisms control GS expression in Streptomyces coelicolor, Clostridium acetobutylicum and cyanobacteria. In C. acetobutylicum, glnA expression is regulated by the production of antisense RNA from a downstream nitrogen-regulated promoter (Fierro-Monti et al., 1992). Transcription of the two genes encoding GS in S. coelicolor requires a response regulatory protein belonging to the VirG/OmpR family of transcriptional factors (Fisher, 1992). The global regulatory protein NtcA, a member of the Crp family of transcriptional activators, is required for regulation of GS and nitrate utilization enzymes in response to nitrogen availability in cyanobacteria (Luque et al., 1994). Thus, examination of the regulation of GS expression has identified four diverse regulatory mechanisms, all of which are distinct from the enteric Ntr system. This indicates that, while the Ntr system is an interesting and important paradigm for gene regulation, any investigation of microbial nitrogen metabolism should remain open to the possibility that novel mechanisms may be involved in this regulation.
I would like to thank B. Belitsky, M. Débarbouillé, M. Nakano, B. Révet, H. Schreier and A. L. Sonenshein for permission to cite results before publication. I am grateful to L. Wray, A. L. Sonenshein, L. Reitzer and S. Kustu for their insightful comments on the manuscript and apologize in advance to colleagues whose work was not included because of space limitations. Research in my laboratory has been supported by the National Science Foundation and the National Institute of General Medical Sciences.