**4. Regulation of bacterial GDH gene expression**

296 Dehydrogenases

high temperature for life.

sites (NAD+, ADP, NAD(P)H) [19]

hypothesized than the latter acts specifically in arginine catabolism by converting glutamate, a product of the ammonia-producing arginine succinyl transferase (AST) pathway, into 2-OG, since it is allosterically modulated by arginine (positively) and citrate (negatively) [25]. Similarly, the only active GDH from *Bacillus subtilis* (RocG, NAD+ dependent) appears to be involved in arginine and proline catabolism [46]. On the other hand, despite the catabolic function assigned to NAD+-GDHs, the existence of an NAD+ specific GDH with an unusual biosynthetic role has been reported in the oral bacterium *Capnocytophaga ochraea* [35]. In this case, it was found that only the NAD+-GDH ammonium assimilating activity could be detected in cell free extracts, probably due to the high concentration of ammonium and ammonium precursors that can be found in the gingival crevicular fluid. Interestingly, GDHs have been shown to play a substantial and even predominant role in nitrogen assimilation in conditions of N2 fixation in the Gram positive bacteria *Bacillus macerans* and *Bacillus polymyxa* [44, 45]. Nitrogen fixation only occurs under extreme nitrogen-limiting conditions, when nitrogen from other sources is very scarce. In these conditions nitrogen is always assimilated using the GS/GOGAT pathway, since the Km of GS for ammonium is much lower than that of GDH. This is not the case in *B. macerans* and *B. polymyxa*, as in these organisms the GOGAT activity is much lower than GDH activity in nitrogen-fixing cells. A NAD+-GDH involved in glutamate fermentation has also been described in the anaerobic Gram-positive bacteria *Peptostreptococcus asaccharolyticus* [54]. In this organism, GDH is the first enzyme of the glutamate fermentation via the hydroxyglutarate pathway, and can represent as much as 10% of total protein when grown on glutamate. Very high levels of GDH production has also been reported in some hyperthermophilic archaea like *Pyrococcus furiosus* or some *Thermococcus* strains [5, 6, 71]. These preferentially biosynthetic enzymes represent an exceptionally high percentage of total soluble protein of the cell, in some cases up to 20%, which suggests an important role of these enzymes in these organisms at an extremely

Determination of the GDH enzymatic structure has allowed the elucidation of the mechanisms for allosteric regulation and negative cooperativity. The activity of glutamate dehydrogenase in animals is allosterically regulated by purine nucleoside phosphates and other metabolic intermediates. In brief, GTP and ATP are allosteric inhibitors whereas GDP and ADP are allosteric activators. Hence, a lowering of the energy charge accelerates the oxidation of amino acids. Their *in vivo* regulation may be dependent on the metabolic status of the cell, or in the tissue they are located. The intracellular compartmentalization of the cofactors, and the GDH itself, may also drive the reaction in one or the other direction. In vertebrate cells, GDH appears to be localized primarily in the mitochondrial matrix [1]. The effects of nucleosides on mammalian GDH are complex. For the bovine liver GDH, four binding sites per subunit have been described, being the active site (site I), the adenine nucleotide regulatory site (site II), the guanine nucleotide regulatory site (site III) and the reduced coenzyme regulatory site (site IV). Some substrate and effectors bind just the active site (glutamate, oxoglutarate, ammonia, NADP+) while others are able to bind two different The diverse roles of bacterial GDH in different organisms provide for a variety of regulatory mechanisms. Here we show a few examples for selected bacteria in which transcriptional regulation of GDH genes has been characterized.

### **4.1. Regulation of GDH synthesis in the enterobacteria**

Transcriptional regulation of the *gdhA* gene, encoding NADP-GDH, was first described in the diazotrophic enterobacterium *Klebsiella pneumoniae*, and then in *Escherichia coli* [73]. Transcription of *gdhA* is repressed in both enteric bacteria under nitrogen limitation by the general nitrogen control system (Figure 3,A). This is consistent with the fact that low affinity for ammonium limits the use of GDH for glutamate synthesis at ammonium concentrations below 1 mM. The enterobacterial general nitrogen control (Ntr) system is a very well characterized signal transduction and regulatory network encompassing seven elements: the alternative σ factor σ54, encoded by *rpoN*, the uridylyl transferase-uridylyl removing enzyme GlnD, two PII signal transduction proteins, GlnB and GlnK, the two-component system NtrB-NtrC and the LysR-type transcriptional regulator Nac. Nitrogen status is signalled by the intracellular pools of glutamine (indicative of nitrogen sufficiency), and 2-OG (indicative of nitrogen limitation). These signals are perceived by the PII proteins by means of their reversible GlnD-dependent uridylylation in response to decreased glutamine levels and by allosteric modulation via 2-oxoglutarate binding. Interaction with PII proteins in either their uridylylated or deuridylylated states is responsible for posttranslational regulation of the activities of a variety of proteins involved in nitrogen metabolism, including glutamine synthetase, nitrogenase and the sensor kinase/phosphatase NtrB. Deuridylylated GlnB promotes NtrB-dependent dephosphorylation of NtrC under nitrogen excess [2, 74]. In nitrogen-limiting conditions, phosphorylated NtrC (NtrC-P) activates multiple σ54 dependent promoters, controlling the expression of over one hundred genes related in *E. coli*  [75]. NtrC-regulated genes encode multiple functions related to nitrogen metabolism, including transport and utilization pathways for diverse nitrogen sources, the nitrogen fixation sensor-regulator pair NifL-NifA (in the diazotrophic *Klebsiella pneumoniae*), and the nitrogen regulator Nac. Transcription of Nac is initiated from an NtrC-activated σ54 dependent promoter and autorepressed [76, 77]. Nac in turn activates and represses a set of genes whose products are mostly related to nitrogen metabolism. Nac-activated genes include those involved in the catabolism of histidine, proline, urea and alanine, among others [78-80]. Notably, Nac represses the genes coding for the two enzymes that synthesize glutamate, *gdhA,* encoding GDH, and *gltAB*, encoding GOGAT [81]. Unlike most LysR-type transcriptional regulators, the activity of Nac is not modulated by the presence of a small ligand, and the protein is synthesized in its active form [79]. Thus, the nitrogen limitation dependency of Nac regulation exclusively reflects the increase in concentration under nitrogen limitation due to its transcriptional regulation. Nac is not present in many bacteria containing the Ntr system, and is conspicuously absent in the closely related and wellcharacterized *S. enterica* [82]. As the need for two different regulators (NtrC and Nac) has been questioned, Nac has been proposed to act as an "adaptor" that integrates genes transcribed from σ70-dependent promoters (which cannot be directly regulated by the activator of σ54-dependent promoters NtrC) into the general nitrogen control network [83].

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 299

"weak" repression is in fact the result of the antagonist action of Nac on ArgP-mediated activation [82, 85]. The role of lysine as a signal can be rationalized if one considers that glutamate must serve two roles in the cell, as both an amino group donor for over 80% of the nitrogenous compounds in the cell, and as a counterion to K+ in osmotic pressure homeostasis. In order to sense the amount of glutamate being used for biosynthesis, the pools of one or more amino group acceptor may be used instead of glutamate itself. Lysine is likely one of those nitrogenous compounds that serve as surrogates for glutamate in signaling the extent of glutamate overflow from osmotic pressure homeostasis into the biosynthetic pathways for other nitrogenous compounds [85]. Consistently, *gdhA*p activity is decreased in rich media containing amino acids [81], and lysine levels may be one of several signals that mediate the feedback regulation of *gdhA*p. However, the identity of

**Figure 3.** Regulatory circuits for bacterial glutamate dehydrogenase genes. The cartoons represent the known regulatory circuits for *K. pneumoniae* (A), *P. putida* (B), *P. aeruginosa* (C), *B. subtilis* (D), *S. coelicolor*  (E) and *C. glutamicum* (E). Sigma 54- or SigL-dependent promoters are displayed as white arrows. Other promoter types are displayed as black arrows. Question marks represent aspects that are not well

characterized. CCR: carbon catabolite repression.

other compounds that may fulfill this role is as of yet unknown.

Transcription of the *gdhA* gene is repressed by Nac under nitrogen limitation in both *E. coli*  and *K. pneumoniae* (in contrast, *gdhA* expression is not nitrogen-regulated in *S. enterica*). Nac exhibits two modes of transcriptional repression of the *gdhA*p promoter. "Weak" repression involves Nac binding as a dimer to the promoter region in a single site located at -100 to -75 relative to the transcriptional start site [84]. "Strong" repression is observed when a Nac tetramer is simultaneously bound to the aforementioned site, centered at -89, and a downstream site centered at +47. The proposed regulatory mechanism for "strong" repression involves Nac bending DNA, looping out the intervening region, and forming a repressor loop reminiscent of those formed by LacI and GalR at the *lac* and *gal* promoters, respectively [79].

In addition to Nac-mediated repression, *gdhA* is subjected to positive regulation by a second LysR-type transcriptional regulator, ArgP. ArgP is an activator whose activity is antagonized by lysine. ArgP interacts with the *gdhA* promoter region at a single site between -100 and -50. Lysine inhibits interaction of ArgP with this site, thus preventing activation [85]. Activation is also prevented by Nac interaction with the overlapping binding site centered at -89, as both regulatory proteins bind in a mutually exclusive fashion. Thus "weak" repression is in fact the result of the antagonist action of Nac on ArgP-mediated activation [82, 85]. The role of lysine as a signal can be rationalized if one considers that glutamate must serve two roles in the cell, as both an amino group donor for over 80% of the nitrogenous compounds in the cell, and as a counterion to K+ in osmotic pressure homeostasis. In order to sense the amount of glutamate being used for biosynthesis, the pools of one or more amino group acceptor may be used instead of glutamate itself. Lysine is likely one of those nitrogenous compounds that serve as surrogates for glutamate in signaling the extent of glutamate overflow from osmotic pressure homeostasis into the biosynthetic pathways for other nitrogenous compounds [85]. Consistently, *gdhA*p activity is decreased in rich media containing amino acids [81], and lysine levels may be one of several signals that mediate the feedback regulation of *gdhA*p. However, the identity of other compounds that may fulfill this role is as of yet unknown.

298 Dehydrogenases

respectively [79].

reversible GlnD-dependent uridylylation in response to decreased glutamine levels and by allosteric modulation via 2-oxoglutarate binding. Interaction with PII proteins in either their uridylylated or deuridylylated states is responsible for posttranslational regulation of the activities of a variety of proteins involved in nitrogen metabolism, including glutamine synthetase, nitrogenase and the sensor kinase/phosphatase NtrB. Deuridylylated GlnB promotes NtrB-dependent dephosphorylation of NtrC under nitrogen excess [2, 74]. In nitrogen-limiting conditions, phosphorylated NtrC (NtrC-P) activates multiple σ54 dependent promoters, controlling the expression of over one hundred genes related in *E. coli*  [75]. NtrC-regulated genes encode multiple functions related to nitrogen metabolism, including transport and utilization pathways for diverse nitrogen sources, the nitrogen fixation sensor-regulator pair NifL-NifA (in the diazotrophic *Klebsiella pneumoniae*), and the nitrogen regulator Nac. Transcription of Nac is initiated from an NtrC-activated σ54 dependent promoter and autorepressed [76, 77]. Nac in turn activates and represses a set of genes whose products are mostly related to nitrogen metabolism. Nac-activated genes include those involved in the catabolism of histidine, proline, urea and alanine, among others [78-80]. Notably, Nac represses the genes coding for the two enzymes that synthesize glutamate, *gdhA,* encoding GDH, and *gltAB*, encoding GOGAT [81]. Unlike most LysR-type transcriptional regulators, the activity of Nac is not modulated by the presence of a small ligand, and the protein is synthesized in its active form [79]. Thus, the nitrogen limitation dependency of Nac regulation exclusively reflects the increase in concentration under nitrogen limitation due to its transcriptional regulation. Nac is not present in many bacteria containing the Ntr system, and is conspicuously absent in the closely related and wellcharacterized *S. enterica* [82]. As the need for two different regulators (NtrC and Nac) has been questioned, Nac has been proposed to act as an "adaptor" that integrates genes transcribed from σ70-dependent promoters (which cannot be directly regulated by the activator of σ54-dependent promoters NtrC) into the general nitrogen control network [83].

Transcription of the *gdhA* gene is repressed by Nac under nitrogen limitation in both *E. coli*  and *K. pneumoniae* (in contrast, *gdhA* expression is not nitrogen-regulated in *S. enterica*). Nac exhibits two modes of transcriptional repression of the *gdhA*p promoter. "Weak" repression involves Nac binding as a dimer to the promoter region in a single site located at -100 to -75 relative to the transcriptional start site [84]. "Strong" repression is observed when a Nac tetramer is simultaneously bound to the aforementioned site, centered at -89, and a downstream site centered at +47. The proposed regulatory mechanism for "strong" repression involves Nac bending DNA, looping out the intervening region, and forming a repressor loop reminiscent of those formed by LacI and GalR at the *lac* and *gal* promoters,

In addition to Nac-mediated repression, *gdhA* is subjected to positive regulation by a second LysR-type transcriptional regulator, ArgP. ArgP is an activator whose activity is antagonized by lysine. ArgP interacts with the *gdhA* promoter region at a single site between -100 and -50. Lysine inhibits interaction of ArgP with this site, thus preventing activation [85]. Activation is also prevented by Nac interaction with the overlapping binding site centered at -89, as both regulatory proteins bind in a mutually exclusive fashion. Thus

**Figure 3.** Regulatory circuits for bacterial glutamate dehydrogenase genes. The cartoons represent the known regulatory circuits for *K. pneumoniae* (A), *P. putida* (B), *P. aeruginosa* (C), *B. subtilis* (D), *S. coelicolor*  (E) and *C. glutamicum* (E). Sigma 54- or SigL-dependent promoters are displayed as white arrows. Other promoter types are displayed as black arrows. Question marks represent aspects that are not well characterized. CCR: carbon catabolite repression.

### **4.2. Regulation of GDH synthesis in pseudomonads**

Little is known about the transcriptional regulation of GDH synthesis in most Gramnegative bacteria. However, the regulatory mechanisms behind *gdhA* regulation were recently revealed in the soil bacterium *Pseudomonas putida* [86]. Similarly to its enteric counterparts, *P. putida* represses *gdhA* expression during nitrogen-limited growth in a Ntr system-dependent fashion (Figure 3,B). *P. putida* harbors a simplified general nitrogen control system that lacks the major PII protein, GlnB, and the transcriptional regulator Nac. In this scheme, the only PII protein, GlnK, controls the phosphorylation/dephosphorylation balance of NtrC in response to nitrogen availability using mechanisms likely similar to those found in the enterics [87]. In the absence of Nac, NtrC has been shown to directly regulate some of the functions that are controlled by Nac in the enterics, including activation of *codB*  and *ureD*, encoding cytosine deaminase and urease respectively [88], and repression of the GDH gene *gdhA* [86]. NtrC binds the σ70-dependent *gdhA*p promoter region cooperatively at four different sites, centered at positions -118, -95, -21 and +12 relative to the transcriptional start. While simultaneous occupancy of all four sites yields the maximal levels of repression, only the promoter-proximal I and II sites were found to be absolutely required for repression. A mechanism based on a repressor loop involving four NtrC dimers has been proposed for negative regulation of the *gdhA*p promoter in *P. putida*. Although repression is partially sensitive to the phosphorylation state of NtrC *in vitro*, *in vivo* evidence indicated that phosphorylation of NtrC is not required for efficient repression of the *gdhAp* promoter. Since NtrC synthesis is itself nitrogen regulated *via* the *glnA*p promoter, the increase in repressor concentration under nitrogen limitation appears to be the major determinant of nitrogen-regulated expression of *gdhA* in *P. putida* [86].

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 301

The *Bacillus subtilis* genome contains two genes encoding GDHs, *rocG* and *gudB*. While RocG is an enzymatically active GDH, GudB is inactive, due to a duplication of three amino acid residues at its active center. Decryptification of *gudB* in a *rocG* background is achieved by high-frequency acquisition of a suppressor mutation consisting of the precise deletion of part of the 9-bp direct repeat that prevents activity [46, 47]. Both RocG and decryptified GudB are primarily catabolic dehydrogenases, and *de novo* glutamate synthesis in *B. subtilis* 

Similarly to *P. aeruginosa* GdhB, RocG is not nitrogen-regulated and is linked to arginine and proline catabolism, as its expression is induced by arginine, ornithine and, to a lesser extent, proline. Transcription from the *rocG* promoter depends on the product of the *Bacillus subtilis sigL* gene, an ortholog of the alternative σ factor σ54 [91], and is activated by the UAS binding protein RocR in response to the presence of arginine, ornithine, or proline with the assistance of the arginine-dependent activator AhrC (Figure 3,D). SigL, RocR and AhrC also control transcription of the two operons, *rocABC* and *rocDEF*, involved in arginine conversion into glutamate. In that regard, RocG-dependent deamination of glutamate to 2 oxoglutarate can be viewed as the final step in the use of arginine, ornithine, and proline as carbon or nitrogen, providing rapidly metabolizable carbon- or nitrogen-containing compounds for biosynthesis [92]. Interestingly, inducibility of RocG synthesis by arginine

The most salient feature of the *rocG*p SigL-dependent promoter is the absence of an upstream activator sequence (UAS) for RocR. Instead, the UAS present at the *rocA*p promoter, located immediately downstream from the *rocG* coding sequence, is the *cis-*acting element essential for RocR-dependent activation of *rocG*p. This sequence has been shown to be active when placed upstream or downstream and as far as 15 kb away from the target promoter [93]. According to the general model of σ54-dependent promoter activation, RocR bound to its target sequence activates transcription by interacting with promoter-bound σ54- RNA polymerase by a mechanism that involves looping out of the intervening DNA sequences. The AhrC protein is also required for activation of *rocG* (as well as *rocABC* and *rocDEF*), and it apparently modulates the activity of RocR by means of protein-protein

Unlike the arginine utilization operons *rocABC* and *rocDEF*, *rocG* transcription is subjected to carbon catabolite repression in the presence of glucose. Such repression is mediated by the regulatory protein CcpA. A *cre* (catabolite responsive element) site is located at positions +39 and +51, and is required for CcpA binding and *rocG*p repression. CcpA binding at this location prevents transcription from the *rocG*p promoter, but also acts as a roadblock for low-level readthrough transcription from an upstream promoter, which is relevant in the absence of both glucose and the cognate inducers [92]. As in other CcpA-regulated genes, CcpA-mediated repression requires the assistance of the accessory proteins HPr and Crh.

A final feature of the *B. subtilis* GDH RocG worth mentioning is its role in the regulation of the assimilatory glutamate synthase operon, *gltAB*. RocG belongs to a group of proteins

**4.3. Regulation of GDH synthesis in** *Bacillus subtilis*

precludes growth on glutamate as the sole carbon source.

is performed exclusively by GOGAT.

contacts [94].

*Pseudomonas aeruginosa* encodes two GDH enzymes, a NAD-dependent GDH encoded by *gdhB*, primarily involved in glutamate deamination to 2-oxoglutarate, and a NADP-dependent GDH, encoded by *gdhA*, primarily involved in ammonium assimilation. The catabolic enzyme is linked to the AST pathway for aerobic utilization of arginine as a carbon source. Glutamate is the end product of the AST pathway, and NAD-GDH serves the purpose of connecting arginine catabolism to the central metabolism *via* 2-oxoglutarate. Transcription of *gdhB* is activated by the arginine regulatory protein ArgR in response to the presence of arginine, which is also an allosteric modulator of the enzyme activity (Figure 3,C). An ArgR binding site is found immediately upstream from the -35 box of the *gdhB* promoter. Induction of the expression of *gdhB* and activation of the encoded dehydrogenase by arginine serve to direct the flow of glutamate into the TCA cycle [25]. Conversely, transcription of *gdhA* is repressed by ArgR in the presence of arginine. Such repression could serve to minimize the operation of an energy-consuming futile cycle involving the simultaneous function of *gdhA* and *gdhB* when *P. aeruginosa* uses arginine as a carbon source. Repression of *gdhA* expression is exerted from a single ArgR binding site centered at position -41, and a simple steric hindrance mechanism has been proposed at this promoter [89]. Downregulation of *gdhA* expression by the Ntr system under nitrogen limitation has also been reported [90], but the factors and mechanisms involved are uncharacterized. Since *P. aeruginosa* also lacks Nac, direct repression by NtrC similar to that observed in *P. putida* [86], may also occur in this organism.
