Metabolic networks for nitrogen utilization in Prevotella ruminicola 23

Abstract

Nitrogen metabolism in gut systems remains poorly studied in spite of its importance for microbial growth and its implications for the metabolism of the host. Prevotella spp. are the most predominant bacteria detected in the rumen, but their presence has also been related to health and disease states in the human gut and oral cavity. To explore the metabolic networks for nitrogen assimilation in this bacterium, changes in gene expression profiles in response to variations in the available nitrogen source and to different concentrations of ammonium were analyzed by microarray and reverse transcription quantitative PCR, and linked with function by further proteomic analysis. The observed patterns of transcript abundances for genes involved in ammonium assimilation differed from the classical "enteric paradigm" for nitrogen utilization. Expression of genes encoding high substrate affinity nitrogen assimilation enzymes (GS-GOGAT system) was similar in growth-limiting and non-limiting nitrogen concentrations in P. ruminicola 23, whereas E. coli and Salmonella spp. responses to excess nitrogen involve only low substrate affinity enzymes. This versatile behavior might be a key feature for ecological success in habitats such as the rumen and human colon where nitrogen is rarely limiting for growth, and might be linked to previously reported Prevotella spp. population imbalances relative to other bacterial species in gut systems.

Figure 1

(A) Prevotella ruminicola 23 can utilize ammonium sulfate supplemented with 3 mM methionine (green line), peptides (tryptone, orange), and a mixture of both (light blue) for growth. The bacterium displayed no growth using amino acids as the sole nitrogen source (dark blue). Growth was measured by determining average optical densities at 600 nm (OD₆₀₀) on triplicate tubes. Error bars represent standard deviations. (B) Combined substrate depletion and growth curves indicated that growth on ammonium sulfate + Met corresponded to a decrease in the concentration of ammonia nitrogen (1); growth on tryptone + ammonium sulfate + Met showed a decrease in ammonium nitrogen and amino acids, the latter indicating depletion of peptides (2); growth on tryptone was supported by a slight decrease in ammonia nitrogen and amino acids (3); and lack of growth on casamino acids was linked with insignificant changes in their concentration during growth (4).

Figure 2

Growth of P. ruminicola 23 in continuous culture shifting from excess ammonium (10 mM) to limiting conditions (0.7 mM), and residual ammonium concentration in culture medium. Higher ammonium concentrations yielded higher ODs in comparison with growth–limiting conditions. Growth was measured by determining average optical densities at 600 nm (OD₆₀₀) in triplicate. Error bars represent standard deviations.

Figure 3

Gene clusters (1 to 6) for nitrogen uptake and assimilation in the genome of Prevotella ruminicola 23 (A), and genome map with corresponding numbered locations and gene coordinates (B). Color code represents functional categories for genes relevant to nitrogen metabolism in Prevotella ruminicola 23.

Figure 4

(A) Log transformation of the fold change in transcript abundances obtained through microarray and qRT-PCR indicates consistency in the results obtained through both techniques (B) Heat map displaying normalized changes in transcript abundances for a subset of genes implicated in nitrogen metabolism. Highest obtained values under non-limiting nitrogen conditions corresponded to genes related to ammonium transport (amtB, ammonium transporter gene; PRU_1977, hypothetical transporter), ammonium assimilation and its regulation (PRU_2048, NAD-dependent glutamate dehydrogenase; PRU_2071, gdhA, NADP-dependent glutamate dehydrogenase; glnA, glutamine synthetase type I; glnN-1 glutamate synthetase type III-1; glnN-2 glutamate synthetase type III-2; gltB, glutamate synthase, large subunit; gltD, glutamate synthase, small subunit; glnK, nitrogen regulatory protein P_II), amino acid and protein biosynthesis (dapF, diaminopimelate epimerase; asnB, asparagine synthase; PRU_1974, aminotransferase, homolog; PRU_1973, glutamine amidotransferase). Highest values under growth on peptides corresponded to genes involved in protein biosynthesis (PRU_2971, O-acetylhomoserine aminocarboxypropyltransferase; cysK, cysteine synthase; PRU_2042, diaminopimelate dehydrogenase), or had unclear roles in nitrogen metabolism (PRU_2827, outer membrane receptor RagA) (H, growth on excess ammonium; L, growth on limiting ammonium concentrations; A, growth on ammonium; P, growth on peptides). (C) Comparison of log transformed fold changes in transcript abundances obtained by qRT-PCR in the assayed growth conditions (AP, Ammonium vs Peptides, HL, excess (H) vs growth-limiting (L) ammonium concentrations). Gene symbols and observed trends as in (B). (D) Principal component analysis integrating fold change in transcript abundances obtained through microarray and qRT-PCR on P. ruminicola 23 grown in different nitrogen sources (ammonium or peptides) or ammonium concentrations (excess [high] or growth-limiting [low]). Arrows point in the direction of the maximum correlation. Gene symbols as in (B) and (D).

Figure 5

Metabolic networks for nitrogen utilization in Prevotella ruminicola 23. The bacterium can utilize both ammonium and peptides for growth through activation of different biochemical pathways. In contrast to E. 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.