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

300 Dehydrogenases

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

nitrogen-regulated expression of *gdhA* in *P. putida* [86].

similar to that observed in *P. putida* [86], may also occur in this organism.

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

*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

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*  is performed exclusively by GOGAT.

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 precludes growth on glutamate as the sole carbon source.

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 contacts [94].

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 designated "trigger enzymes", which have catalytic as well as regulatory functions. RocG interacts with the LysR-type transcriptional regulator GltC, which is the cognate activator of the *gltAB* operon. RocG-GltC interaction results in inactivation of the latter, which in turn prevents activation of the assimilatory glutamate synthase in conditions in which glutamate is already being synthesized from arginine-related amino acids or proline. This mechanism allows tight control of glutamate metabolism by the availability of carbon and nitrogen sources [95-97].

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance 303

In addition to the different physiological roles of GDHs from different bacteria in nitrogen assimilation, amino acid catabolism, osmotic balance or tolerance to high temperatures, GDH catalysis is crucial for a number of biotechnological processes. These include industrial production of glutamate by *C. glutamicum* and related species, which involves catalysis in the aminating direction, and production of aromas by lactic acid bacteria during cheeses

*C. glutamicum* is a facultatively anaerobic, nonpathogenic, non-motile, biotin-auxotrophic, Gram-positive soil bacterium that was isolated more than 50 years ago in a screen for bacteria that excrete glutamate. Since then, derivative strains of this bacterium and related species have been isolated as glutamate producers, and industrial processes for biological glutamate production have been developed. Annual L-glutamate production is estimated to be over 2 million tons, with a continuously increasing demand (3% per year), above all in

In addition to its importance in industrial biotechnology, *C. glutamicum* has gained interest as a model organism for the *Corynebacterineae*, an industrially relevant suborder of the actinomycetes. Because of the academic and industrial interest, its physiology and metabolism have been deeply characterised, three research groups have independently determined the genomic sequence of *C. glutamicum* strains [103-105], and global analysis techniques such as proteomics, transcriptomics, metabolomics and metabolic flux analysis

*C. glutamicum* exponentially growing cells do not accumulate glutamate. However, glutamate production and excretion can be easily induced by an astonishing variety of treatments, which allow accumulation of glutamate in the culture medium to a concentration as high as 80 g L-1. These include biotin limitation [106], which was the first identified condition for glutamate overproduction, addition of fatty acid ester surfactants such as Tween 40 (polyoxyethylene sorbitan monopalmitate) or Tween 60 (polyoxyethylene sorbitan monoestearate) [107], addition of certain β-lactam antibiotics such as penicillin [108], use of glycerol-auxotrophic or fatty acid-auxotrophic strains [109], use of temperature-sensitive strains cultured at higher temperature [110], or addition of ethambutol, an inhibitor of cell wall arabinogalactan synthesis [111]. In spite of the apparent diversity of treatments to induce glutamate production and secretion, all of them have features in common and there is a unified view that glutamate production is triggered by environmental conditions that produce damage of the cell surface structures

Since *C. glutamicum* is a biotin auxotroph, supplementation of a defined medium with a limiting concentration of biotin in batch cultures results in reduced total biomass and concomitant glutamate production. Biotin is a co-factor of acetyl-CoA carboxylase, the first

**5. Involvement of GDH in biotechnological processes** 

ripening, which involves catalysis in the opposite direction.

**5.1. Production of L-Glutamate by** *Corynebacterium glutamicum*

developing countries. L-glutamate is mainly used in food as a flavour enhancer.

have been used to obtain a holistic view of the glutamate production process.

[112-114].
