The evolutionary aspect of GltC regulation via RocG and GudB

The divergence and convergence of functions are central tools for evolution. Accordingly, the different promoters, enzyme properties, and putative different interaction sites for GltC could result from the divergence of a common ancestor. However, in turn could the common ancestor also be lacking the moonlighting function and this property was developed later in RocG and GudB in a convergent manner, explaining the presence of putative different interaction sites. The question of the evolutionary origin of the RocG and GudB GDHs remains elusive. However, how important is the regulatory function in comparison to the metabolic function of the GDH?

4.3.3.1. The importance of glutamate synthesis regulation

It was observed that the E. coli the GDH GdhA is not able to trigger the repressor function of GltC at all (Fig. 3.27). In E. coli, the gltBD genes encoding for the GOGAT are regulated by many global and local TFs (van Heeswijk et al., 2013).

Some of these TF are also involved in the regulation of the genes encoding for the GS and the GDH in E. coli (van Heeswijk et al., 2013). A vicious cycle of simultaneous glutamate

biosynthesis and degradation is not observed until now. This might be because the GDH in E. coli is anabolically active. However, under conditions favoring the anabolic reaction of the GDH, still 85 % of ammonium assimilation and glutamate synthesis occurs via the energetically unfavorable GS-GOGAT cycle (Yuan et al., 2006;

van Heeswijk et al., 2013). However, there are first hints showing that GdhA is also catabolically active under high levels of glutamate (van Heeswijk et al., 2013). Maybe the GdhA lost its catabolic activity, because the prevention of the emergence of a vicious cycle is more important for the cell than the advantage of having a catabolically active GDH. Especially, because the GDH is not needed in E. coli for arginine degradation. Arginine catabolism is mediated by the astCADBE operon encoding for enzymes of the AST pathway that degrades arginine to succinate and glutamate and thereby generates ammonium serving as nitrogen source for the cell (Schneider et al., 1998).

In Rhizobium etli, which lives in a mutually beneficial relationship with legumes, the nitrogen assimilation is of such high importance that a GDH is entirely missing (Bravo and Mora, 1988). R. etli lives in nodules of legumes, fixes atmospheric nitrogen, and ensures a proper organic nitrogen supply for the plant, which in turn supplies the bacterium with nutrients. R. etli relies solely on the GS-GOGAT cycle for nitrogen fixation (Bravo and Mora, 1988; Castillo et al., 2000). The heterologous expression of the E. coli derived GdhA leads to a strong negative effect in symbiosis, as no or only ineffective nodules are formed (Mendoza et al., 1998). This is a very special situation, but shows nicely that GDH has a crucial impact on the nitrogen metabolism.

Moreover, in B. subtilis the regulatory function of the GDHs seems to be more important compared to their metabolic function. When the B. subtilis strain 168, that harbors an inactivated gudB^CR

gene, is additionally deficient of the rocG gene and is streaked on SP medium containing high levels of glutamate and arginine, rapidly gudB⁺ suppressor mutants emerged compensating for the loss of the rocG gene (Ch. 1.3.3 and 1.4).

However, this was not observed in a B. subtilis strain deficient of the rocG and the gltC gene. In a rocG deficient B. subtilis strain high levels of glutamate accumulate, which presumably lead to detrimental conditions for the cell. The additional loss of the gltC gene might partly compensate for the loss of the rocG gene, as no additional glutamate can be synthesized in the absence of the GOGAT. Interestingly, the SP medium does not contain glucose and the use of secondary carbon sources is important for the survival of the cell. Another GDH making glutamate accessible as carbon source would be beneficial for the cell, however gudB⁺ suppressor mutants do not emerge. This observation might be a first hint for the importance of the regulatory function of RocG & GudB in inhibiting GltC compared to their actual metabolic function. Corroborating this assumption, a study searching of monofunctional and super repressor variants of RocG, only one monofunctional exclusively metabolically active variant was found in contrast to ten GltC super repressor variants (Gunka et al., 2010).

In all examples presented here, the metabolic function either anabolic or catabolic is not as important as it is to precisely control the activity of the GOGAT. The GDH is in some cases a means to an end. It might be important under specific nutrient conditions, but it solely enables the use of secondary carbon sources.

4.3.3.2. Evolution of moonlighting proteins The metabolically active site consists in most cases only of a few relevant residues though many proteins have sizes between 30 and 50 kDa (Srere, 1984). So why are proteins so big? This

Discussion Two GDHs, one GltC, two evolutionary routes?

question was already discussed in a paper from 1984 and many ideas were presented. For instance, the size could serve as scaffold, for localization or to ensure proper protein-protein interactions (Srere, 1984). However, in large scale proteomics many proteins were found exhibiting post translational modifications (PTMs) for an unknown reason (Jeffery, 2016).

PTMs can switch the function of an enzyme and taken together, this could indicate an enormous number of proteins having moonlighting function yet undiscovered (Jeffery, 2016). Of course, many moonlighting enzymes were found to have homologs. However, the moonlighting function is not always maintained (Jeffery, 2016).

For instance, in duck eyes there are the delta 1 and delta 2 homologs of crystallins present.

Though they only differ in 27 aa, exclusively the delta 2 crystallin has an arginosuccinate lyase function (Chiou et al., 1991).

As expected two redundant enzymes are evolutionary instable, but in B. subtilis they are stable for some reason. As recently reported, the GDHs underwent a promoter-enzyme co-evolution: The gudB gene is continuously expressed and the resulting GudB is regulated, whereas the rocG gene is highly regulated and the resulting RocG is stably active (Noda‐Garcia et al., 2017). In this study, the function of RocG and GudB as moonlighting enzymes are neglected. But as both GDHs can inhibit GltC there must be also a co-evolution of the interaction sites of RocG-GltC and GudB-GltC.

The co-evolution of regulatory functions is extensively studied for two-component systems and phosphorelay systems (Salazar and Laub, 2015). Two-component systems consist of a histidine kinase recognizing a signal and subsequently phosphorylating a response regulator that in turn regulates gene expression.

One essential aspect in the evolution of novel two-component systems is that the connection

between a histidine kinase and its response regulator must be stable and must not interfere with the novel system. To prevent interconnections between novel and existing two-component systems, duplicated histidine kinases and response regulators only arise under non-selective conditions and diverge fast to eliminate cross-talk between the systems (Capra et al., 2012). In two-component systems the recognition site consists only of four residues and it was shown that these residues could be exchanged with those of another two-component system resulting in accurate recognition in vitro (Podgornaia et al., 2013).

Investigating the interaction surfaces of RocG-GltC and GudB-RocG-GltC the binding sites might be also exchangeable. Another explanation for functional evolution of interaction surfaces was investigated in toxin-antitoxin systems where the toxin-antitoxin system was also shown to co-evolve without ever disrupting their interaction (Aakre et al., 2015), which would be detrimental for the cell. In the toxin-antitoxin system promiscuous enzymes with broadened substrate specificity served as mutational intermediates to allow specific mutations in the opposite gene and subsequent adaptive mutations that restrict substrate specificity (Aakre et al., 2015). This might also be the case for the evolution of the GDHs, as the loss of GltC regulation might be detrimental for the cell. The general problem in continuously maintaining the connection between two proteins is the reduction of possible mutations allowing the general evolution of the system.

One model describing the evolution of enzymes in general is the innovation-amplification-divergence (IAD) model (Näsvall et al., 2012). In the innovation state of the IAD model an enzyme acquires a weak secondary function and subsequently its copy number is increased via duplication and amplification, which is the major

source of novel proteins (Andersson and Hughes, 2009; Näsvall et al., 2012). One study in 1985 even suggested that B. subtilis will do gene amplification whenever it is possible (Jannière et al., 1985) and for Salmonella typhimurium it was estimated that 10 % of all cells growing in non-selective medium contain a gene duplication somewhere in their genome (Roth et al., 1996;

Andersson and Hughes, 2009). During the divergence state, beneficial mutations accumulate and finally copies of non-beneficial enzyme versions are lost during segregation. The final outcome is either a novel generalist having its original and the novel secondary function or two specialized enzymes (Näsvall et al., 2012).

In conclusion, there is a promoter-enzyme co-evolution for RocG and GudB and simultaneously a co-evolution of the interaction sites of RocG-GltC and GudB-RocG-GltC. Regarding, all these evolutionary aspects the successful co-evolution of enzymes, promoters, and interaction sites appears to be a miracle. But what if this evolution could only take place because these proteins are so highly interconnected?

4.3.3.3. The hypothetical evolution of GudB and RocG

Similar as the paradigm one gene, one protein, one function from Garrod, Beadle and Tatum is not state of the art anymore, it might be time to reevaluate Darwins paradigm of survival of the fittest (Darwin, 1859; Garrod, 1923; Beadle and Tatum, 1941). In a recent review, the evolution of moonlighting proteins was discussed (Fares, 2014). It is postulated that genetical robustness is the key for evolution. In harsh contrast to the general term that redundancy is genetically instable, redundancy might support evolution.

Moreover, genetical robustness can be achieved by the accumulation of different silent mutations within a bacterial community, because these mutations increase the genetic variability and

might be precursors for different novel phenotypes (Fares, 2014). As a result, a genetically diverse bacterial culture might faster react to challenging conditions, because the genetical repertoire and the concomitant possibilities for novel functions will increase the statistical chance of a beneficial mutation to occur. Evolution is not a single step method that either fails or wins. It is a gradual process of many small steps (Fares, 2014).

Hypothetically, the ancestor GDH of RocG and GudB acquired the ability to weakly inhibit GltC.

This leads to a growth advantage in the presence of glutamate. According to the IAD model the respective gene of the ancestor GDH duplicated or even amplified. Along with the growth advantage occurred the problem that the ancestor GDH was a very stable enzyme encoded by a constitutively expressed gene. The presence of several enzymes with overlapping functions enabled the fast and complex promoter-enzyme co-evolution, because once one enzyme weakened the connection to GltC the other GDH could buffer that loss. Silent mutations might contribute to gradual stepwise evolution of a highly regulated promoter or a protein that is only stable under certain conditions. Of course, this evolution does not exclude the emergence of better or different binding sites of RocG-GltC or GudB-GltC. For sure, the buffering effect and the acceptance of silent mutations contributed to a fast evolution as they allowed many more possibilities leading to improved binding sites for GltC and the respective GDH (Podgornaia and Laub, 2015).

This shows how restricted our view of nature is and that there are always possibilities we do not consider because of our self-made paradigms. A first step is to erase fixed paradigms from our memory, regard them as general rules and to broaden our point of view.