Ammonia is the major end product of digestion of dietary protein and nonprotein nitrogen (urea and amino acids), as well as the major source of nitrogen for protein synthesis by ruminal bacteria (Cotta and Russell, 1997; Morrison and Mackie, 1997; Wallace et al., 1997). Indeed, ammonia is the preferred nitrogen source for growth of many bacteria and archaea. Results over a wide range of feed and N intakes demonstrate that 60-80% of bacterial N is derived from ammonia as a precursor (Mackie and White, 1990) with the balance coming from di and tri-peptides and amino acids. Rumen microbes approach 10% N in organic matter (Czerkawski, 1976; Fessenden et al., 2017) unless storage carbohydrate increases as a result of unbalanced (typically N-limited) growth (Hackmann and Firkins, 2015b). Therefore, bacterial protein synthesis and growth are greatly affected by the efficiency of ammonia assimilation. Despite its importance and central role as an intermediate in the degradation as well as assimilation of dietary nitrogen by intestinal bacteria, our understanding of the mechanisms and regulation of ammonia assimilation in ruminal bacteria remains superficial.
Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In bacteria and archaea, Amt's are used as environmental NH4+ scavengers for uptake and assimilation of nitrogen, and current dogma posits that bacteria are prepared to expend energy in the form of ATP to procure a critical nutrient under growth-limiting conditions. It is possible that under conditions of high ruminal ammonia concentrations that passive diffusion of ammonia can occur and satisfy N requirements of the cell. The protonated and positively charged ammonium can exist in a deprotonated and gaseous form, ammonia.The gaseous ammonia is able to diffuse through the membrane and become protonated ammonium. The pKa of ammonium is 8.95 at 35°C and at physiological pH (6.5-7.5) only 1 % of the total ammonium/ammonia exist in the gaseous ammonia (Martinelle and Haggstrom, 1997). Thus, microbial cells require an ammonium translocation mechanism and indeed ammonia transport systems are ubiquitous among bacteria isolated from a variety of habitats including the rumen and human gut systems.
Depending on the total ammonium/ammonia concentration, gaseous ammonia diffusion is potentially responsible for ammonia transport across the cytoplasmic membrane. The known transport protein, AmtB, is not expressed unless the ammonium concentration is very low(Soupene et al., 1998,2002; van Heeswijk et al., 1996; Winkler, 2006). Further evidence provided by organisms completely lacking ammonium transport facilitators, Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Salmonella enterica, and Saccharomyces cerevisiae are still able to grow optimally with high concentrations of ammonium/ammonia (Detsch and Stulke, 2003; Meier-Wagner et al., 2001; Soupene et al., 1998). In contrast, mixed ruminal microbes have an increased concentration gradient with 160 mg/L higher concentration in the cytoplasm compared to extracellular concentration, indicating active transport of ammonium (Russell and Strobel, 1987). When concentration of ammonium becomes too low, facilitated transport is required to move ammonium across the cytoplasmic membrane. Ammonium analogs, like methyl ammonium and ethyl ammonium, have been used to study the transport of ammonium across the bacterial membrane. Although useful, the results should be interpreted with caution for two primary reasons: transporters are very selective of their ligands especially for substrates as small as ammonium, and the diffusion of the three different molecules are not equal and can bias results (Kleiner, 1982; Kleiner and Castorph, 1982; Stevenson and Silver, 1977).
Once ammonium enters the cytoplasm, glutamate and glutamine are the key metabolic intermediates central to intracellular nitrogen cycling. Glutamate is the most abundant metabolite in the cell, 96 mM in E. coli, and directly links nitrogen metabolism with carbon metabolism via a-ketoglutarate, onto which ammonium is appended during ammonium assimilation (Bennett et al., 2009). Bacterial cells primarily incorporate ammonium into glutamate and glutamine, irrespective of the nitrogen conditions in the environment, which are then used as nitrogenous building blocks for a wide range of N-containing metabolites, including amino acids, purines, pyrimidines, and other metabolites.
Enzymatic pathways of ammonium assimilation
Current knowledge of enteric ammonium assimilation and regulation is largely based on research on the model Proteobacteria (£. coli, Klebsiella, Salmonella), and Bacillus (as a model for the Gram-positive type system), which does not necessarily reflect dominant gut microbes from Bacteroidetes and Firmicutes (Reitzer, 2003; Van Heeswijk et al., 2013). Research on Bacillus subtilis provides evidence for the divergence in regulation and mechanisms of the ammonium assimilation genes (Gunka and Commichau, 2012).The enteric paradigm based on the model Proteobacteria is structured around two competing pathways that are inversely regulated depending on the ammonium concentration and nitrogen status of the cell (Fig. 2). These two pathways are described as the high-affinity pathway and the low-affinity pathway. The first pathway is employed under limiting concentrations of ammonium and contains three functional enzymes including an ATP-dependent glutamine synthetase (GS), glutamate synthase (GOGAT), and an ammonium transporter (AmtB). In contrast, the low-affinity pathway is utilized under excess (non-limiting) concentrations of ammonium. This pathway consists of a NAD(P)H-oxidizing glutamate dehydrogenase (GDH). In B. subtilis, the GS/GOGAT system is solely responsible for the assimilation of ammonium, while the GDH enzymes run the reverse reaction for catabolism of glutamate.
In addition to the differences in energy expenditure and catalytic mechanisms, the regulation of these pathways has been characterized in detail. In £. coli, an elegant balance of transcriptional regulation and post- translational modification orchestrates the total contributions of both pathways.
Ruminal protein breakdown and ammonia assimilation
Figure 2 Enteric paradigm for ammonium assimilation based on E. coli. Enzymatic pathways include low-affinity and high-affinity pathways with the functional proteins glutamate dehydrogenase (GDH, gdhA), glutamine synthetase (GS, glnA), glutamate synthase (GOGAT, gltB), and the ammonium transporter (AmtB). The regulatory network includes UTase, ATase, and GlnB that modify the activity of functional proteins, and transcriptional regulators, NRI, NRII, and Nac, which regulate transcription of functional genes. Proteins encapsulated in red are not identified in Bacteroidetes by sequence homology. (PhD thesis, M. lakiviak 2018).
The non-competitive binding of metabolic intermediates further modulates the differential regulation and function of the catalytic enzymes and regulatory proteins. Although helpful in understanding how an organism modulates enzymatic activity through transcriptional and posttranslational means, this paradigm fails to explain gut nitrogen utilization. The complicated machinery of the high-affinity pathway is more energy demanding than the low-affinity pathway. As such, a strong regulatory network is required to minimize the energy expenditure of the cell to optimize growth. Significant efforts have uncovered the intricate network of regulation that Proteobacteria have evolved to regulate the flux of metabolites through both pathways (Van Heeswijk et al., 2013). The model has been extended to include all gut organisms and is termed the enteric paradigm.
Mechanisms of regulation
An elegant model of regulation has been proposed through studies of model Gram-negative and Gram-positive bacteria (Van Heeswijk et al., 2013). Global gene expression is also modulated primarily via NRI/NRII, a two-component system, and Nac, which is itself transcriptionally regulated by NRI/NRII. Rapid repression or activation of enzymatic occurs through the enzymatic modification of GS and GDH. The modulation of activity occurs through the regulatory proteins GlnB (also known as P-ll), ATase (adenylyltransferase/ adenylyl-removing enzyme), and UTase (uridylyltransferase/uridylyl-removing enzyme). In B. subtilis, the transcriptional regulators include TnrA and GlnR, which modulate transcription through protein-protein interaction with the functional enzymes (Fisher, 1999). Through very carefully fine-tuned enzymatic and regulatory pathways, organisms incorporate extracellular ammonium into intracellular a-ketoglutarate and glutamate to produce glutamate and glutamine, respectively.
In E. coli, the functional proteins are transcriptionally regulated by the aforementioned two-component system NRI/NRII, as well as several other regulators including Nac, CRP-cAMP, IHF, Lrp, and ArgR (Van Heeswijk et al., 2013). The sensing protein, NRII, binds to ammonium and undergoes autophosphorylation, subsequently transferring the phosphate to the response regulator NRl.The phosphorylated NRI goes on to increase transcription of gin A, glnK, amtB, nac, and other genes (Magasanik, 1989). Interestingly, Nac represses gdhA without a coeffector molecule or covalent modification. The bacterium responds to amino acid deficit through Lrp by increasing transcription of gltBD, and to energy (ATP) deficit through Crp-cAMP inhibiting gltBD expression and modulating a basal level of expression of glnA (Van Heeswijk et al., 2013).
In the model Firmicute, B. subtilis, three transcriptional regulators have been identified, GlnR, TnrA, and GltC (Fisher, 1999; Fisher and Wray, 2002; Schumacher et al., 2015; Wray et al., 2001). The transcriptional activity of GlnR is mediated by the binding of GS, stabilizing DNA interaction when bound, and is affected by pH. In contrast,TnrA is inactive when bound to glutamine synthetase. Additionally, TnrA can be titrated away from DNA by association with the membrane by interactions with GlnK and AmtB. Finally, GltC is responsible for the activation of transcription of glutamate synthase under increased glutamate demand during higher growth rates (Gunka and Commichau, 2012). The P-ll proteins are central to regulation of protein activity as they incorporate signal from the intracellular metabolite pool and modulate enzymatic activity as well as transcription. A P-ll (GlnB) and a P-ll like protein (GlnK) are encoded by glnB and glnK, and glnK is commonly found adjacent to amtB (Arcondeguy et al., 2001; Blauwkamp and Ninfa, 2002; Detsch and Stulke, 2003; Forchhammer, 2008; Van Heeswijk et al., 1996). P-ll proteins are homotrimers and possess binding sites for a-ketoglutarate and ATP, as well as uridylylation sites by which UTase acts as an efficient glutamine sensor. In addition, P-ll proteins can undergo adenylylation in mycobacteria, phosphorylation in cyanobacteria, or remain unmodified (Forchhammer, 2008; Gunka and Commichau, 2012; Williams et al„ 2013). Several proteins directly interact with P-ll including AmtB, ATase, NRII, and UTase in proteobacteria, as well asTnrA in B. subtilis. The transport of ammonium across AmtB is regulated by direct insertion of a P-ll loop into the transport channel of the trimeric AmtB, preventing ammonium transport. This interaction is inhibited by UTase uridylylation of P-ll at the loop (Reitzer, 2003).
Glutamine synthetase type 1 (GS-1) activity is regulated via covalent modification by the ATase in Proteobacteria. The ATase adenylylates a subunit of the homododecameric GS-1 and inactivates that subunit. Since a range of adenylylation states can exist (between 0-12), GS-1 can exist in a range of activities. ATase is also capable of activating GS-1 by the deadenylylation activity present within the same polypeptide. The regulation of adenylylation/ deadenylylation is mediated by P-ll interaction with the ATase. The regulatory activity of P-ll toward ATase is dependent on its uridylylation state via the UTase's ability to uridylylate or deuridylylate P-ll. The UTase uridylylation/ deuridylylation activity is affected by glutamine and other small molecules (Fig. 3). Finally, transcription of ammonium assimilatory genes is also affected by P-ll through its interaction with NRII. Interaction between P-ll and NRII is affected by the metabolites ATP and a-ketoglutarate, resulting in decreased autophosphorylation under energy and nitrogen abundance. Extension of the enteric paradigm to representatives of the Bacteroidetes (Prevotella) and Firmicutes (Ru mi nococcus) phyla that are abundant in the rumen cannot be direct, as both genera lack homologs to the ammonium assimilation regulators, namely the two-component system (NRI/NRII), covalent modifiers (ATase/ UTase), and the transcriptional regulators. In addition, conflicting reports exist concerning the dominant enzymatic activity that Bacteroides sp. exhibit under varying nitrogen availability.
Figure 3 Nitrogen metabolism pathways identified in Ruminococcus albus 8 through bioinformatic analyses (Kim et al., 2014).