Glutamate dehydrogenases GudB/RocG

Under growth conditions without an appropriate carbon source, glutamate can be converted via the oxidative deamination of L-glutamate into ammonium and α-ketoglutarate, feeding into the TCA cycle (Brunhuber and Blanchard, 1994).

The GDHs in B. subtilis use NAD⁺ as co-factor and

αKGαKGαKG αKG

Box II

E E

E E

E E E

E

Box I Box II Box III

gltA

A

B

C

Introduction Glutamate homeostasis in B. subtilis

have a very low affinity for ammonium (Gunka et al., 2010), which is typical for strictly catabolic GDHs. Other GDHs as for instance the E. coli GDH GdhA which is catabolically and anabolically active, uses NADP⁺ and has a higher affinity for ammonium (Brunhuber and Blanchard, 1994;

Reitzer, 2003; Sharkey and Engel, 2008).

B. subtilis harbors rocG and gudB two paralogous genes encoding for GDHs, which share 74 % amino acid sequence identity (Belitsky and Sonenshein, 1998). The rocG gene expression is strongly regulated by different nitrogen and carbon sources, whereas the promoter of the gudB gene is constitutively expressed (Fig. 1.6).

Fig. 1.6 Transcript levels of the gudB^CR and the rocG genes under different growth conditions.

The transcript level overview of the gudB^CR and the rocG gene in B. subtilis under different growth conditions is derived from SubtiWiki (Michna et al., 2016). The transcript level from the gudB^CR gene is constant whereas the transcript level from rocG gene depends on the different conditions (Nicolas et al., 2012):

A: high & low phosphate defined media containing arginine (Müller et al., 1997). B: Sporulation after 1 h in sporulation medium (Sterlini and Mandelstam, 1969). C: 0.3 h, 1 h, 1.3 h (maximum), 2 h, 2.3 h and 3 h after glucose exhaustion in modified M9 medium (Hardiman et al., 2007). D: Stationary growth in LB and sporulation after 0 h in sporulation medium (Sterlini and Mandelstam, 1969).

A B. subtilis strain deficient of the σ^L sigma factor is not able to use arginine or ornithine as sole nitrogen sources. The genes involved in arginine catabolism were shown to be under the control of the σ^L sigma factor and a corresponding transcriptional activator RocR encoded by the rocR gene (Calogero et al., 1994; Gardan et al., 1995). In contrast, the rocR gene is under the control of a σ^A sigma factor, not induced by arginine, and autoregulated (Gardan et al., 1995). However, the regulation of the rocG gene

and the rocABC operon is special, because the binding site of the RocR protein is located downstream of the rocG gene. It acts as downstream activating sequence (DAS) for the expression of the rocG gene and as upstream activating sequence (UAS) for the expression of the rocABC operon (Fig. 1.3) (Belitsky and Sonenshein, 1999). DNase I footprinting experiments defined the bidirectional enhancer element as doubled 8 bp inverted repeat separated by one base which leads to a curved DNA facilitating the interaction of RocR with the σ^L-RNAP (Ali et al., 2003).

As previously mentioned a GDH makes glutamate accessible as a carbon source. This is only necessary in the absence of a good carbon source. Therefore, the promoter is repressed in the presence of glucose by CcpA, the global regulator of CCR (Belitsky and Sonenshein, 1999;

Belitsky, 2004). Hence, under this conditions the RocG protein cannot inhibit the GOGAT activity (Commichau et al., 2007a) and GOGAT in turn can synthesize glutamate. In perfect agreement with this is the observation that a ∆ccpA strain deficient of CCR, grows poorly on medium with ammonium and glucose as sole nitrogen and carbon sources, respectively (Faires et al., 1999).

In this mutant strain CcpA does not repress rocG gene expression, but it is also not induced by RocR. Interestingly, it was shown that a readthrough effect of the upstream located sivA gene is responsible for a low level of rocG gene expression, which is normally shielded by CcpA

promoter region of the rocDEF genes (Choi and Saier, 2005). This indicates that the regulation of the σ^L sigma factor, the arginine catabolism genes and especially the rocG gene, all belonging to the nitrogen metabolism are strongly linked to global regulators of the carbon metabolism.

Another repression of the rocG gene and the rocABC operon is mediated by the transition state regulator AbrB, under conditions of good nutrient supply, when cells are in exponential growth phase (Chumsakul et al., 2011).

Furthermore, sensing the arginine pool in the cell, the transcriptional regulator of the arginine catabolism AhrC activates in the presence of arginine expression of the rocABC, the rocDEF, and the rocG genes and represses genes involved in arginine biosynthesis (Czaplewski et al., 1992;

Gardan et al., 1995; Klingel et al., 1995;

Commichau et al., 2007b).

To summarize, RocG is expressed in the presence of arginine or ornithine or to a lesser extent proline or citrulline in the absence of glucose (Belitsky and Sonenshein, 1998; Belitsky et al., 2004).

However, there is a second GDH in B. subtilis and growth experiments with ∆gudB and ∆rocG knock-out mutants of the B. subtilis NCIB 3610 wild type strain and biochemical analyses of the two proteins revealed that GudB is the major contributor for glutamate degradation (Noda‐

Garcia et al., 2017). In contrast to the less domesticated B. subtilis strains as the NCIB 3610, the major GDH GudB of the laboratory B. subtilis strains 160, 166, and 168 is not functional and very instable (Zeigler et al., 2008). This cryptic gudB^CR gene harbors a directly repeated sequence of 9 bp, termed tandem repeat (TR), within its coding region resulting in a duplication of three amino acids (VKA-VKA) in the positions 93-95 and 96-98 of the catalytically active center of the GudB protein. In strains deficient of the rocG gene, suppressor mutants (SM) emerge

rapidly on selective medium, that have precisely excised one part of the TR from the gudB^CR gene (Belitsky and Sonenshein, 1998). The resulting gudB⁺ gene encodes the functional GDH GudB⁺ that restores the glutamate homeostasis. The mutation rate of the gudB^CR gene is about 10^-4 and the highest reported so far (Gunka et al., 2012).

It is assumed that the gudB gene was inactivated during domestication of the laboratory wild type strain 168, because in contrast to the soil, B. subtilis’ natural environment, a lack of exogenous glutamate in laboratory culture media might have provided a selective growth advantage for mutants that have inactivated the gudB gene (Gunka et al., 2013). The acquisition of an inactive gudB^CR gene conferred a selective growth advantage. However, presence of a constitutively expressed gudB gene seems not to be disadvantageous, as recent studies revealed that the NCIB 3610 wild type strain shows no growth defect on medium with glucose and ammonium as carbon and nitrogen sources, respectively (Noda‐Garcia et al., 2017).

Contradictory, this medium does not provide glutamate for the cell, which consequently must be synthesized. Its constant degradation by the GDH GudB should lead to a futile cycle. However, in this study, it was shown that an exchange of the open reading frames of the gudB and the rocG gene leads to an impaired growth phenotype (Noda‐Garcia et al., 2017). This indicates, that high levels of GudB are not dangerous, but high levels of RocG are a serious problem for the cell. The RocG protein can form stable enzymatically active hexamers under a broader range of pH and with more varying concentrations of glutamate. Whereas the GudB protein is only present in its active hexameric form at distinct pH and high glutamate concentrations (Noda‐Garcia et al., 2017).

Furthermore, the authors observed that GudB

Introduction High frequency mutagenesis of the gudB

gene

and RocG are allosterically regulated by ATP and α-ketoglutarate even though the regulation is rather minor (Noda‐Garcia et al., 2017). Taken together, the rocG gene expression is tightly regulated but the resulting GDH RocG is stable and active under a broad range of conditions. In contrast, the gudB gene is constitutively expressed, but the resulting GDH GudB is only stable under defined environmental conditions.

However, the stability of the RocG and GudB complexes might be influenced further by their secondary function as so called moonlighting or trigger enzymes (Commichau and Stülke, 2008).

As it is the case for the GS (Ch.1.2.2), the trigger enzymes have besides their metabolic function a regulatory function. To prevent the emergence of a futile cycle of glutamate synthesis and degradation, GltC activity is inhibited by binding to the GDH RocG or GudB (Commichau et al., 2007a; Stannek et al., 2015b), which of course could also be important for RocG or GudB stability. However, two paralogous enzymes so differently regulated are likely to provide B. subtilis a selective growth advantage in adaptation to specific growth conditions.

1.4. High frequency mutagenesis of