Ammonium assimilation in rumen bacteria

Three major discoveries from research on human colonic Bacteroides reveal a widely different nature of ammonium assimilation and regulation from the enteric model described above. This potential paradigm shift includes increased GDH activity under nitrogen limitation, increased GS transcription under ammonium excess, and an entirely new family of glutamine synthetases (GSIII) that are common in gut anaerobes (Kim et al., 2014, 2017). Features of regulation are critical to ascertain before a true enteric model can be applied to microbial ammonium assimilation within the human or animal gastrointestinal tract. In addition, the multiplicity of functional genes needs to be addressed to identify the roles for each putative glutamine synthetase and glutamine dehydrogenase encoded within gut Bacteroidales. Although investigations into ammonium assimilation within Bacteroidetes are limited, the breadth of the information primarily describes deviations from the classical enteric paradigm.

Recent genomic and transcriptomic research on two model rumen bacteria, Ruminococcus albus (a specialist plant cell wall degrading species with a Gram-positive cell wall type within the Firmicutes phylum; Kim et al.,

2014) and Prevotella ruminicola (a metabolically versatile, Gram-negative cell wall type of bacterial species within the Bacteroidetes phylum; Kim et al., 2017) has recently been published and is summarized here to provide a case study relevant to ammonia assimilation and N metabolism in two representative rumen bacteria.

Bioinformatic analysis of the Ruminococcus albus 8 draft genome sequence (3.8 Mb) revealed a number of genes encoding enzymes critical for nitrogen metabolism and ammonia assimilation (Fig. 3). We identified genes for an NADFI-dependent GDH (gdh), an NADPH-dependent GDH (gdhA), a high- affinity ammonium transporter (amtB), a regulatory protein (glnK), a putative urease, two different types of GS (type I GS [glnA] and type III GS [glnN]), and the two subunits of a typical bacterial GOGAT (large subunit [gltB] and small subunit [gltD]). R. albus 8 was grown with ammonia, urea, peptides, or amino acids as the nitrogen source, and transcript abundances and enzymatic activities of nitrogen metabolism proteins were analyzed to investigate nitrogen utilization and flux.

R. albus 8 utilized ammonia and urea and showed similar growth patterns on both substrates. R. albus 8 was also able to grow on peptides as a nitrogen source; although the growth yield was lower than that on preferred nitrogen sources (urea and ammonia), the maximum specific growth rate did not change. This is the first report of R. albus 8 using peptides as a nitrogen source. Bioinformatic analysis of the draft genome sequence revealed that R. albus 8 is also equipped with several peptide transporters and peptidases for uptake and utilization of peptides, including three peptide ABC transporters, two dipeptide ABC transporters, and 25 genes involved in peptidase metabolism. This finding is also consistent with patterns of nitrogen utilization in the rumen, where free amino acids are rapidly deaminated to produce ammonia and amino acids are transported into bacterial cells in peptide form, enabling energy conservation at the transport level.

Prevotella ruminicola strain 23 is non-cellulolytic, but can efficiently degrade hemicellulose and pectin and could potentially degrade the proteoglycan of the host. Regarding nitrogen metabolism, P. ruminicola 23 can efficiently utilize both ammonia and peptides (preferentially larger peptides, up to 2kDa) as a nitrogen source for growth. For breakdown of oligopeptides, P. ruminicola 23 harbors the greatest range and specific activity of dipeptidyl peptidases in comparison to species belonging to other abundant genera in the rumen, such as Butyrivibrio, Ruminococcus, and Fibrobacter. Whole genome transcriptional responses to environmental changes in the available nitrogen source and ammonium concentrations in P. ruminicola 23 were studied under defined medium and culture conditions, as well as proteome changes and the enzymatic activity of central enzymes in ammonium assimilation (Fig. 4).

Growth studies showed that P. ruminicola 23 can efficiently utilize peptides and ammonium as nitrogen sources for growth, but not amino acids (Kim et al., 2017). Shifts in its overall transcriptional profiles were shown when growth occurred on ammonium or peptides; specifically, ammonium assimilation pathways were not induced when the bacterium was grown on peptides. These results suggest that the bacterium utilizes peptides directly for protein synthesis after uptake and intracellular hydrolysis and entry into the intracellular amino acid pool. Interestingly, growth on ammonium sulfate was not observed in the absence of supplementation with methionine. Methionine is stimulatory and might be essential for growth of P. ruminicola 23, although this is likely not as a N source but as a methyl or methanethiol (CH3S) donor in other biosynthetic reactions.

These results collectively show that P. ruminicola 23 responds to changes in environmental nitrogen differently from enteric species of Proteobacteria, such as £. coli or Salmonella spp (Kim et al., 2017). These responses reflect differential transcriptional regulation of genes involved in nitrogen metabolism and variations in related enzyme activities (Fig. 3). Previous studies had shown that P. ruminicola possesses both NADP- (anabolic) and NAD-dependent (catabolic) GDP! activities. Correspondingly, higher transcript abundances of gdhA and elevated NADP-GDH activity were detected under excess concentrations of ammonium. The GS-GOGAT pathway constitutes a major ammonium assimilation pathway for enteric bacteria grown under ammonium- limiting conditions. Nevertheless, transcription of GS-GOGAT genes was highly induced in non-limiting concentrations of ammonium in Prevotella

Ruminal protein breakdown and ammonia assimilation


Metabolic networks for nitrogen utilization in Prevotella ruminicola 23

Figure 4 Metabolic networks for nitrogen utilization in Prevotella ruminicola 23 (Kim et al„ 2017). The bacterium can utilize both ammonium and peptides for growth through activation of different biochemical pathways. In contrast to £ coli and Salmonella spp., growth on non-limiting ammonium conditions is maximized by both GDH and GS/GOGAT-dependent ammonium assimilation. Growth on peptides might rely on extracellular hydrolysis and transport of resulting amino acids, or intracellular deamination of imported oligopeptides. High induction of genes involved in cysteine synthesis could indicate generation of labile amino acids in the cell. Chemical/biochemical species are represented by grey circles. Nitrogen-containing species are represented in green, and sulfur-containing species are depicted in blue. EX and IN stand for extracellular and intracellular cell locations, respectively. Dashed arrows are used for clarity in order to represent that many steps are implicated in the generation of the displayed species. Red arrows represent pathways induced on ammonium and yellow arrows represent pathways induced on peptides.

ruminicola 23. In contrast to the enteric paradigm, our results demonstrate that P. ruminicola 23 utilizes the high substrate affinity GS-GOGAT enzymatic system to grow in non-limiting ammonium conditions, a pattern that has also been observed in Ruminococcus albus 8. To our knowledge, this is the first description of an organism that uses both GDH and GS-GOGAT pathways for ammonium assimilation when grown under non-limiting concentrations of ammonium. Even though the GS-GOGAT system requires ATP, and its down-regulation would prevent energy waste when ammonium assimilation relies on GDH, the observed behavior could reflect simultaneous utilization of GDH and GS-GOGAT pathways to maintain the glutamate pool for the biosynthesis of amino acids. This may represent an evolutionary adaptation of strain 23 to rumen conditions, where nitrogen concentrations are rarely growth limiting (ammonium concentration typically ranges from 4 to 70 mM). This strategy would enable the organism to maintain an active growth in the natural environment, outcompeting other microbes in the utilization of ammonium as a nitrogen source, consistently agreeing with the observation that Prevotella constitutes the most abundant reported bacterial genus in the rumen.

Collective genomic and proteomic results provide strong evidence that GSIII-2 is the main enzyme implicated in ammonium assimilation when the nitrogen is non-limiting. Transcript abundances for the GOGAT genes were also higher than 22 fold on ammonium. Therefore, the GSIII-2-GOGAT coupling of enzymes is likely to play a major role in ammonium assimilation and ammonium recycling in P. ruminicola 23. An increased transcript abundance of the ammonium transporter coding gene (amtB) was shown during growth on non-limiting concentrations of ammonium, which suggests the ability of P. ruminicola 23 to detect and respond to fluctuations in the environmental ammonium concentration. However, when concentrations of ammonium became limiting, the bacterium induced peptide and polyamine ABC transporters, suggesting its capability to scavenge more complex and/ or alternative available nitrogen sources. In addition, transporting peptides at the level of a di- or tri-peptide (1/2 or 1/3 ATP per amino acid transported) would enable the cell to conserve energy compared to the transport of a single or free amino acid (1 ATP per amino acid transported. Transcript abundances for the nitrogen regulatory protein PM gene, glnK, were also higher on nonlimiting ammonium. The most recent mechanistic insight into the signaling role of the P protein suggests that a post-translational change driven by changes in its observed ATPase activity under fluctuating nitrogen levels sensed by intracellular a-KG would facilitate or inhibit ammonium uptake through the ammonium transporter channel. The glnK gene can be located upstream or downstream of amtB. This genetic linkage is highly conserved, and both resulting proteins are functionally related.

In summary, P. ruminicola 23 utilizes both NADP-GDH and the GSIII-2- GOGAT pathways for ammonium assimilation when nitrogen is non-limiting for growth. This may reflect an adaptation of P. ruminicola 23 to a more stable concentration of ammonium in the rumen relative to other environments (e.g. human gut) by enhancing glutamate production and maintaining the intracellular glutamate/glutamine pool for amino acid, purine/pyrimidine, and cell wall biosynthesis. In contrast, under limiting concentrations of ammonium,

P. ruminicola 23 may utilize basal levels of GDH or GS-GOGAT pathways to assimilate ammonium and synthesize amino acids.

Archael nitrogen metabolism

Much less is known about nitrogen metabolism in archaea than in bacteria. Methanogens in general use nitrogen in the biosynthesis of amino acids, purines, and pyrimidines and by employing most of the same reactions as Bacteria. In addition, ammonia assimilation in methanogens is similar to that seen in bacteria (DeMoll, 1993).

The availability of the complete genome sequences of several members of the euryarchaea has enabled new approaches to the understanding of methanogen physiology and biochemistry, including metabolic reactions involving nitrogen compounds in methanogens. Methanobrevibacter smithii is the dominant human-gut-associated hydrogenotrophic methanogen and uses ammonia as the preferred nitrogen source. The M. smithii proteome contains a transporter for ammonium (AmtB; MSM0234) plus two routes for its assimilation: (i) the ATP-dependent glutamine synthetase-glutamate synthase pathway, which has a high affinity for ammonium and thus is advantageous under nitrogen-limited conditions, and (ii) the ATP-independent glutamate dehydrogenase pathway, which has a lower affinity for ammonium (Samuel et al., 2007). Hansen et al. (2011) used RNA-Seq to perform expression profiles on five M. smithii isolates when grown on medium containing ammonia as a nitrogen source. Both pathways were expressed in all strains, with 0.4-1.21% of reads mapping to enzymes involved in assimilation of ammonia. The energy-dependent GlnA pathway was generally expressed at a much higher level than the low-affinity GDH pathway, although strain-specific differences in levels of expression were noted.

Methanococcus maripaludis and Methanosarcina mazei are bothmesophilic methanogenic members of the Euryarchaeota, and studies have enriched and extended our knowledge of nitrogen regulation (Leigh and Dodsworth, 2007). Both species assimilate ammonia apparently by the GS/GOGAT pathway, and both can fix nitrogen. Both species contain a nif operon composed of homologs to bacterial dinitrogenase and dinitrogenase reductase genes and other nif genes involved in nitrogenase cofactor synthesis. GlnA and nif genes are regulated as expected by nitrogen conditions, and GS and nitrogenase activities are also regulated. Not surprisingly, studies in the Archaea have revealed new aspects of nitrogen regulation. Highlights from studies of M. maripaludis and M. mazei include the novel transcriptional repressor NrpR, direct regulation of GS by GlnK, and direct regulation of nitrogenase by Nifl (Leigh and Dodsworth, 2007). Much further research is required on ruminal methanogens into the present research capabilities and promise.

Anaerobic rumen fungi

The nutritional requirements of ruminal fungi are relatively simple. Lowe et al. (1985) reported that they grow in medium lacking amino acids, implying that ammonia can be used to synthesize all amino acids. Orpin and Greenwood (1986) also demonstrated that Neocallimastix patriciarum grew in a defined medium, and that growth was stimulated by amino acids, particularly glutamate, serine, and methionine. However, little is known concerning amino acid metabolism in ruminal fungi, or the extent to which fungi may incorporate amino acids (Theodorou et al., 1994). In order to evaluate the extent of de novo synthesis of individual amino acids in Piromyces communis and Neocallimastix frontalis, isotope enrichment in amino acids was determined during growth on 15NH4CI in different media (Atasoglu and Wallace, 2002). Most amino acid N and hence cell N for P. communis and N. frontalis continued to be formed de novo from ammonia when 1 g I"1 trypticase was added to the medium; this concentration approximates the peak concentration of peptides in the rumen after feeding. Higher peptide/amino acid concentrations decreased de novo synthesis. Lysine was exceptional, in that its synthesis decreased much more than other amino acids when Trypticase or amino acids were added to the medium, suggesting that lysine synthesis might limit fungal growth in the rumen. These results need further study with more modern approaches to resolve which nitrogen sources are being utilized during rumen fermentation and metabolism.

Ciliate protozoa

The largest and most obvious protozoa in the rumen are the ciliates, which are divided into two main groups, the so-called isotrichid (Order: Vestibuliferida)and the entodiniomorphid (Order: Entodiniomorphida) protozoa that differ not only in morphology but also in their metabolism. The entodiniomorphid protozoa, although able to take up soluble compounds, feed principally by the engulfment of particulate matter. All entodiniomorphid protozoa, whether grown in vivo or in vitro, have bacteria in digestive vesicles in the cytoplasm and there is evidence that the species present reflect those in the surrounding medium and include methanogens (Williams and Coleman, 1997). Bacteria probably provide the most important source of nitrogenous compoundsfor protozoal growth, although plant protein and free amino acids also represent a valuable source with some species. After engulfment, which may be selective although this is not always consistent, bacteria are completely digested in a digestive vacuole. On incubation of 14C labeled amino acids in £. coli with a suspension of entodiniomorphid protozoa some of the labeled amino acids are incorporated into protozoal protein, some may be incorporated as a related amino acid, and the remainder is released into the cell amino acid pool or the medium. Constituents of bacterial nucleic acid are incorporated into protozoal nucleic acid, with the transfer taking place at the nucleotide level (Williams and Coleman, 1997).

The isotrichid ciliate protozoa occurring in the rumen are mostly the genera Isotricha and Dasytricha, and are easily observed under the microscope because of their size and motility compared to rumen bacteria. They are ellipsoid-shaped organisms with cilia covering the complete external surface of the cell. Both Isotricha and Dasytricha will ingest rumen bacteria and nonrumen bacteria, although some selectivity of bacteria being ingested has been observed. Following engulfment, the bacterial cells rapidly lose viability and are extensively degraded. Unchanged bacterial amino acids are directly incorporated into protozoal protein. The Isotrich protozoa obtain some of their nitrogen requirements from ingestion and digestion of bacteria, but they are also able to take up amino acids from the medium. These amino acids are assimilated directly and they are also capable of excretion of nitrogenous material (Williams and Coleman, 1997).

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