Glutamine synthesis is essential for growth of Streptococcus thermophilus in milk and is linked to urea catabolism

Growth of a glutamine synthetase-deficient mutant of Streptococcus thermophilus was compared to that of the parent strain in milk that was not supplemented or was supplemented with ammonium chloride, glutamine, or the urease inhibitor flurofamide. It was concluded that one of the functions of urease is to supply ammonia for the synthesis of glutamine.     

Glutamine Involvement in Nitrogen Control of Gibberellic Acid Production in Gibberella fujikuroi

When the fungus Gibberella fujikuroi ATCC 12616 was grown in fermentor cultures, both intracellular kaurene biosynthetic activities and extracellular GA₃ accumulation reached high levels when exogenous nitrogen was depleted in the culture. Similar patterns were exhibited by several nonrelated enzymatic activities, such as formamidase and urease, suggesting that all are subject to nitrogen regulation. The behavior of the enzymes involved in nitrogen assimilation (glutamine synthetase, glutamate dehydrogenase, and glutamate synthase) during fungal growth in different nitrogen sources suggests that glutamine is the final product of nitrogen assimilation in G. fujikuroi. When ammonium or glutamine was added to hormone-producing cultures, extracellular GA₃ did not accumulate. However, when the conversion of ammonium into glutamine was inhibited by L-methionine-DL-sulfoximine, only glutamine maintained this effect. These results suggest that glutamine may well be the metabolite effector in nitrogen repression of GA₃ synthesis, as well as in other nonrelated enzymatic activities in G. fujikuroi.     

Urease of Klebsiella aerogenes: control of its synthesis by glutamine synthetase.

Urease was purified 24-fold from extracts of Klebsiella aerogenes. The enzyme has a molecular weight of 230,000 as determined by gel filtration, is highly substrate specific, and has a Km for urea of 0.7 mM. A mutant strain lacking urease was isolated; it failed to grow with urea as the sole source of nitrogen but did grow on media containing other nitrogen sources such as ammonia, histidine, or arginine. Urease was present at a high level when the cells were starved for nitrogen; its synthesis was repressed when the external ammonia concentration was high. Formation of urease did not require induction by urea and was not subject to catabolite repression. Its synthesis was controlled by glutamine synthetase. Mutants lacking glutamine synthetase failed to produce urease, and mutants forming glutamine synthetase at a high constitutive level also formed urease constitutively. Thus, the formation of urease is regulated like that of other enzymes of K. aerogenes capable of supplying the cell with ammonia or glutamate.     

Nitrogen control in Pseudomonas aeruginosa: A role for glutamine in the regulation of the synthesis of NADP-dependent glutamate dehydrogenase,urease and histidase

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.     

Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium.

Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease.     

The regulation of urease activity in Aspergillus nidulans

Aspergillus nidulans can utilize urea as a sole source of nitrogen but not as a carbon source. Urea is degraded by a urease. Mutation at any one of three genes, ureB, ureC, and ureD, may result in deficient urease activity. The ureB gene is closely linked to ureA, the structural gene for the urea transport protein. The heat lability of a ureB ^– revertant strain, intragenic complementation tests, and the linkage of ureB to ureA suggest that ureB is the urease structural gene. The ureD gene is probably involved in the synthesis or incorporation of a nickel cofactor essential for urease activity. The function of the ureC gene is not known. Urease is not induced but is subject to nitrogen regulation. The urease activities of ammonium-derepressed mutants show that the effector of nitrogen regulation is more likely to be glutamine than ammonium. When glutamine is present in the medium, urease appears to be inactivated by some means which does not involve a newly synthesized protease or a direct interaction between glutamine and urease.     

Edwardsiella ictaluri Encodes an Acid-Activated Urease That Is Required for Intracellular Replication in Channel Catfish (Ictalurus punctatus) Macrophages

Genomic analysis indicated that Edwardsiella ictaluri encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type E. ictaluri in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of wild-type E. ictaluri was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome.Identification of virulence factors is vitally important to an understanding of the pathogenesis of Edwardsiella ictaluri and to the development of methods for controlling the spread of disease. Although the pathogenesis of E. ictaluri was reviewed in 1993 (28, 31), recent reports demonstrated that E. ictaluri is a facultative intracellular pathogen (3) and that a type III secretion system is required for intracellular survival and replication within channel catfish head kidney-derived macrophages (HKDM) (30). Using signature-tagged mutagenesis (STM) in an immersion challenge model for E. ictaluri, Thune et al. (30) identified 50 transconjugants carrying transposon insertions in genes required for survival and replication in the channel catfish host. Two of those mutants had insertions in genes encoding homologs of UreG and UreF, proteins that are essential for the production of an active urease enzyme in other bacteria (6, 10, 14, 26). UreG is a GTP-binding accessory protein that functions in energy-dependent assembly of the urease holoenzyme (19), while UreF is a urease accessory protein that functions in the generation or delivery of carbon dioxide to the urease metallocenter assembly site (19). Both the ureG and ureF mutant strains were further characterized in a competitive challenge with the wild-type (WT) parental strain and were confirmed to be significantly attenuated (30). The identification of two mutants with insertions in urease-associated genes suggests an important role for urease activity in E. ictaluri pathogenesis, despite the fact that E. ictaluri is urease negative in standard biochemical tests. Consequently, the objectives of this study are to characterize the E. ictaluri urease pathogenicity island (PAI), to evaluate conditions for E. ictaluri urease activity, and to establish a possible role for urease in E. ictaluri pathogenesis.     

Regulation of amidase formation in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity

The formation of amidase was studied in mutants from Pseudomonas aeruginosa PAO lacking glutamine synthetase activity. It appeared that catabolite repression of amidase synthesis by succinate was partially relieved when cellular growth was limited by glutamine. Under these conditions, a correlation between amidase and urease formation was observed. The results suggest that amidase formation in strain PAO is subject to nitrogen control and that glutamine or some compound derived from it mediates the nitrogen repression of amidase.     

Aspartate biosynthesis is essential for the growth of Streptococcus thermophilus in milk, and aspartate availability modulates the level of urease activity

We investigated the carbon dioxide metabolism of Streptococcus thermophilus, evaluating the phenotype of a phosphoenolpyruvate carboxylase-negative mutant obtained by replacement of a functional ppc gene with a deleted and inactive version, Deltappc. The growth of the mutant was compared to that of the parent strain in a chemically defined medium and in milk, supplemented or not with L-aspartic acid, the final product of the metabolic pathway governed by phosphoenolpyruvate carboxylase. It was concluded that aspartate present in milk is not sufficient for the growth of S. thermophilus. As a consequence, phosphoenolpyruvate carboxylase activity was considered fundamental for the biosynthesis of L-aspartic acid in S. thermophilus metabolism. This enzymatic activity is therefore essential for growth of S. thermophilus in milk even if S. thermophilus was cultured in association with proteinase-positive Lactobacillus delbrueckii subsp. bulgaricus. It was furthermore observed that the supplementation of milk with aspartate significantly affected the level of urease activity. Further experiments, carried out with a p(ureI)-gusA recombinant strain, revealed that expression of the urease operon was sensitive to the aspartate concentration in milk and to the cell availability of glutamate, glutamine, and ammonium ions.     

Dietary l-glutamine supplementation increases Pasteurella multocida burden and the expression of its major virulence factors in mice

This study was conducted to determine the effects of graded doses of l-glutamine supplementation on the replication and distribution of Pasteurella multocida, and the expression of its major virulence factors in mouse model. Mice were randomly assigned to the basal diet supplemented with 0, 0.5, 1.0 or 2.0 % glutamine. Pasteurella multocida burden was detected in the heart, liver, spleen, lung and kidney after 12 h of P. multocida infection. The expression of major virulence factors, toll-like receptors (TLRs), proinflammatory cytokines (interleukin-1 beta, interleukin-6, and tumor necrosis factor alpha) and anti-oxidative factors (GPX1 and CuZnSOD) was analyzed in the lung and spleen. Dietary 0.5 % glutamine supplementation has little significant effect on these parameters, compared to those with basal diet. However, results showed that a high dose of glutamine supplementation increased the P. multocida burden (P < 0.001) and the expression of its major virulence factors (P < 0.05) as compared to those with a lower dose of supplementation. In the lung, high dose of glutamine supplementation inhibited the proinflammatory responses (P < 0.05) and TLRs signaling (P < 0.05). In the spleen, the effect of glutamine supplementation on different components in TLR signaling depends on glutamine concentration, and high dose of glutamine supplementation activated the proinflammatory response. In conclusion, glutamine supplementation increased P. multocida burden and the expression of its major virulence factors, while affecting the functions of the lung and spleen.     

Effects of Nitrate and Ammonium on Gene Expression of Phosphoenolpyruvate Carboxylase and Nitrogen Metabolism in Maize Leaf Tissue during Recovery from Nitrogen Stress

We previously showed that the selective accumulation of phosphoenolpyruvate carboxylase (PEPC) in photosynthetically maturing maize (Zea mays L.) leaf cells induced by nitrate supply to nitrogen-starved plants was primarily a consequence of the level of its mRNA (B Sugiharto, K Miyata, H Nakamoto, H Sasakawa, T Sugiyama [1990] Plant Physiol 92: 963-969). To determine the specificity of inorganic nitrogen sources for the regulation of PEPC gene expression, nitrate (16 millimolar) or ammonium (6 millimolar) was supplied to plants grown previously in low nitrate (0.8 millimolar), and changes in the level of PEPC and its mRNA were measured in the basal region of the youngest, fully developed leaves of plants during recovery from nitrogen stress. The exogenous supply of nitrogen selectively increased the levels of protein and mRNA for PEPC. This increase was more pronounced in plants supplemented with ammonium than with nitrate. The accumulation of PEPC during nitrogen recovery increased in parallel with the increase in the activity of glutamine synthetase and/or ferredoxin-dependent glutamate synthase. Among the major amino acids, glutamine was the most influenced during recovery, and its level increased in parallel with the steady-state level of PEPC mRNA for 7 hours after nitrogen supply. The administration of glutamine (12 millimolar) to nitrogen-starved plants increased the steady-state level of PEPC mRNA 7 hours after administration, whereas 12 millimolar glutamate decreased the level of PEPC mRNA. The results indicate that glutamine and/or its metabolite(s) can be a positive control on the nitrogen-dependent regulation of PEPC gene expression in maize leaf cells.     

Preventive oral supplementation with glutamine and arginine has beneficial effects on the intestinal mucosa and inflammatory cytokines in endotoxemic rats

The objective of this study was to evaluate the effect of oral supplementation with a combination of arginine and glutamine on the intestinal mucosa and inflammatory cytokines of lipopolysaccharide (LPS)-induced adult rats. Fifty Sprague-Dawley rats (average weight of 185 ± 15 g) were randomly divided into five groups: control group A (CA) and control group B (CB), both orally supplemented with 0.9% saline; group Arg, supplemented with 300 mg/kg day(-1) arginine; group Gln, supplemented with 300 mg/kg day(-1) glutamine; group AG, supplemented with 150 mg/kg day(-1) arginine and 150 mg/kg day(-1) glutamine. The experiment lasted for 2 weeks. Food intake and body weight were measured during the experiment. At 10.00 h of day 15, animals were injected with 4 mg/kg LPS (group CB, Arg, Gln, and AG) or sterile saline (group CA) after supplementation. Then at 14.00 h, all animals were killed and blood and tissue collected. The results showed that compared with group CB, arginine concentration tended to be increased (P > 0.05) in group Arg and AG, while there was no significant difference in glutamine concentration among the groups challenged with LPS. Oral supplementation with arginine or/and glutamine mitigated morphology impairment (lower villus height, P < 0.05) in the jejunum and ileum induced by LPS challenge. LPS administration resulted in a significant increase in TNF-α, IL-1β, IL-6 and IL-10 mRNA abundance. Arginine only significantly decreased TNF-α mRNA abundance in the ileum, while glutamine significantly decreased both TNF-α and IL-10 mRNA in the ileum. A combination of arginine and glutamine significantly decreased TNF-α and IL-1β mRNA abundance in both the jejunum and ileum, while they also significantly decreased anti-inflammatory IL-10 in the ileum. These results revealed that an oral supply of combined arginine and glutamine had more favorable effects on the intestinal mucosa and inflammatory cytokines than a supply of arginine or glutamine alone.     

Primary culture and characteristic morphologies of medulla terminalis neurons in the eyestalks of Chinese shrimp, Fenneropenaeus chinensis

A method for culturing medulla terminalis (MT) neurons in the eyestalk of Chinese shrimp, Fenneropenaeus chinensis, was first established. The neurons showed immediate outgrowth in the culture medium supplemented with glutamine, glucose and antibiotics. The cells grew for about 2-7 days and then sustained for a week or more. At least six types of neurons were distinguished on the basis of size and form of soma and outgrowth pattern of cells.     

A soybean seed urease-null produces urease in cell culture

Itachi, a soybean (Glycine max [L.] Merr.) variety with 0.2% normal seed urease activity, was recovered from a screen of 6,000 entries in the United States Department of Agriculture soybean germplasm collection. No urease antigen in Itachi seed extracts was detected by double diffusion or by rocket immunoelectrophoresis. Native gels stained for protein or ureolytic activity revealed no detectable urease holoenzyme. An anti-urease antibody affinity column was used to remove all detectable urease activity and antigen from `wild type' (cv. Prize) seed extracts. Affinity column effluent and nonchromatographed Itachi extracts both lack a species which comigrates with purified urease subunits in sodium dodecylsulfate polyacrylamide gels. Inability to detect urease antigen or urease protein suggests that during development of Itachi seeds there is no synthesis of urease protein or that, at most, its synthesis is 0.2% of wild type (Prize).     

In Vitro Regeneration of a High Oil-yielding Variety of Safflower (Carthamus tinctorius var HUS-305)

A protocol for regular in vitro regeneration of Carthamus tinctorius var HUS-305 is described. The morphogenic response of seed explants and explants from seedlings of different ages were studied on Murashige and Skoog’s medium (MS) supplemented with different growth regulators. The protocol finally standardized involves culture of cotyledonary explants from 2 cm long, 2- to 6-day-old seedlings on MS supplemented with 1.0 mg l^?1 BAP and 0.1 mg l^?1 kinetin. Various other adjuvants viz., adenine sulphate, glutamine and casein hydrolysate were also tested. None of these promoted the caulogenic response significantly. The microshoots could be rooted on medium supplemented with different auxins. The highest rhizogenic response was on 1/2 MS supplemented with 0.2 mg l^?1 NAA.     

Co does not replace Ni with respect to urease activity in zucchini (Cucurbita pepo convar. giromontiina) and soybean (Glycine max)

In order to evaluate whether Co can functionally replace Ni in urease, as suggested by Watanabe et al. (Soil Sci. Plant Nutr. (1994) 40, 545–548), zucchini and soybean plants were grown in purified nutrient solutions containing nitrate and ammonium as sole N source supplemented either with 0.05 mmol m-3 Ni or Co or with no supplementation (control). In addition, isolated soybean cotyledons were incubated axenically with either 1 mmol m-3 Ni or Co supplements. Plant growth was not affected by the Ni and Co additions, but urease activity was only detected in Ni-supplemented tissue, and in no case did Co raise the urease activity level. Only Ni-deprived plants (–Ni – Co and –Ni + Co) accumulated appreciable amounts of urea in the leaves. These results strongly suggest, that Co does not replace Ni with respect to functional urease.     

Ammonium Metabolism Enzymes Aid Helicobacter pylori Acid Resistance

The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH₃ that is immediately protonated to form NH₄⁺. This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) the exit of protonated ammonium outward via the UreI permease, which was shown to facilitate diffusion of both urea and ammonium, and/or (ii) the assimilation of this ammonium, which is supported by evidence that H. pylori assimilates urea nitrogen into its amino acid pools. We investigated the second hypothesis by constructing strains with altered expression of the ammonium-assimilating enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and the ammonium-evolving periplasmic enzymes glutaminase (Ggt) and asparaginase (AsnB). H. pylori strains expressing elevated levels of either GS or GDH are more acid tolerant than the wild type, exhibit enhanced ammonium production, and are able to alkalize the medium faster than the wild type. Strains lacking the genes for either Ggt or AsnB are acid sensitive, have 8-fold-lower urea-dependent ammonium production, and are more acid sensitive than the parent. Additionally, we found that purified H. pylori GS produces glutamine in the presence of Mg²⁺ at a rate similar to that of unadenylated Escherichia coli GS. These data reveal that all four enzymes contribute to whole-cell acid resistance in H. pylori and are likely important for assimilation and/or efflux of urea-derived ammonium.     

Genetic tests of the roles of the embryonic ureases of soybean

We assayed the in vivo activity of the ureases of soybean (Glycine max) embryos by genetically eliminating the abundant embryo-specific urease, the ubiquitous urease, or a background urease. Mutant embryos accumulated urea (250-fold over progenitor) only when lacking all three ureases and only when developed on plants lacking the ubiquitous urease. Thus, embryo urea is generated in maternal tissue where its accumulation is not mitigated by the background urease. However, the background urease can hydrolyze virtually all urea delivered to the developing embryo. Radicles of 2-day-old germinants accumulated urea in the presence or absence of the embryo-specific urease (2 micromoles per gram dry weight radicle). However, mutants lacking the ubiquitous urease exhibited increased accumulation of urea (to 4-5 micromoles urea per gram dry weight radicle). Thus, the ubiquitous and not the embryo-specific urease hydrolyzes urea generated during germination. In the absence of both of these ureases, the background urease activity (4% of ubiquitous urease) may hydrolyze most of the urea generated. A pleiotropic mutant lacking all urease accumulated 34 micromoles urea per gram dry weight radicle (increasing 2.5-fold at 3 days after germination). Urea (20 millimolar) was toxic to in vitro-cultured cotyledons which contained active embryo-specific urease. Cotyledons lacking the embryo-specific urease accumulated more protein when grown with urea than with no nitrogen source. Among cotyledons lacking the embryo-specific urease, fresh weight increases were virtually unchanged whether grown on urea or on no nitrogen and whether in the presence or absence of the ubiquitous urease. However, elimination of the ubiquitous urease reduced protein deposition on urea-N, and elimination of both the ubiquitous and background ureases further reduced urea-derived protein. The evidence is consistent with the lack of a role in urea hydrolysis for the embryo-specific urease in developing embryos or germinating seeds. Because the embryo-specific urease is deleterious to cotyledons cultured in vitro on urea-N, its role may be to hydrolyze urea in wounded or infected embryos, creating a hostile environment for pest or pathogen. While the ubiquitous urease is operative in leaves and in seedlings, all or most of its function can be assumed by the background urease in embryos and in seedlings.     

Streamlined Regulation and Gene Loss as Adaptive Mechanisms in Prochlorococcus for Optimized Nitrogen Utilization in Oligotrophic Environments

Prochlorococcus is one of the dominant cyanobacteria and a key primary producer in oligotrophic intertropical oceans. Here we present an overview of the pathways of nitrogen assimilation in Prochlorococcus, which have been significantly modified in these microorganisms for adaptation to the natural limitations of their habitats, leading to the appearance of different ecotypes lacking key enzymes, such as nitrate reductase, nitrite reductase, or urease, and to the simplification of the metabolic regulation systems. The only nitrogen source utilizable by all studied isolates is ammonia, which is incorporated into glutamate by glutamine synthetase. However, this enzyme shows unusual regulatory features, although its structural and kinetic features are unchanged. Similarly, urease activities remain fairly constant under different conditions. The signal transduction protein P_II is apparently not phosphorylated in Prochlorococcus, despite its conserved amino acid sequence. The genes amt1 and ntcA (coding for an ammonium transporter and a global nitrogen regulator, respectively) show noncorrelated expression in Prochlorococcus under nitrogen stress; furthermore, high rates of organic nitrogen uptake have been observed. All of these unusual features could provide a physiological basis for the predominance of Prochlorococcus over Synechococcus in oligotrophic oceans.