Role of sodium in the growth of a ruminal selenomonad

The ruminal selenomonad strain H18 grew rapidly (mu = 0.50 h-1) in a defined medium containing glucose, ammonia, purified amino acids, and sodium (95 mM); little if any ammonia was utilized as a nitrogen source. When the sodium salts were replaced by potassium salts (0.13 mM sodium), there was a small reduction in growth rate (mu = 0.34 h-1), and under these conditions greater than 95% of the cell nitrogen was derived from ammonia. No growth was observed when the medium lacked sodium (less than 0.35 mM) and amino acids were the only nitrogen source. At least six amino acid transport systems (aspartate, glutamine, lysine, phenylalanine, serine, and valine) were sodium dependent, and these systems could be driven by an electrical potential (delta psi) or a chemical gradient of sodium. H18 utilized lactate as an energy source for growth, but only when sodium and aspartate were added to the medium. Malate or fumarate was able to replace aspartate, and when these acids were added, sodium was no longer required. Glucose-grown cells accumulated large amounts of polysaccharide (64% of dry weight), and when the exogenous glucose was depleted, this material was converted to acetate and propionate as long as sodium was present. When the cells were incubated in buffers lacking sodium, succinate accumulated and exogenous succinate could not be decarboxylated. Because sodium had little effect on the transmembrane pH gradient at pH 6.7 to 4.5, it did not appear that sodium was required for intracellular pH regulation.     

Molecular cloning of a coding sequence of Bordetella pertussis filamentous hemagglutinin gene

Abstract The halophilic phototrophic bacterium Rhodospirillum salexigens was tested for growth on a variety of organic and inorganic nitrogenous compounds as sole nitrogen sources. In media containing acetate as carbon source, the amino acids glutamate, proline, and aspartate supported good growth of R. salexigens ; several other amino acids or ammonia did not support growth. Attempts to grow R. salexigens on ammonia led to the discovery that this organism excretes a highly basic substance under certain nitrogen nutritional conditions which raises the pH above that supporting growth. Cultures of R. salexigens transferred to media containing both pyruvate and acetate as carbon sources grew on ammonia as sole nitrogen source and the culture pH did not rise. Dual substrate experiments showed that R. salexigens utilized glutamate in preference to ammonia when both were present at equimolar concentrations.     

EFFECT OF NITROGEN SOURCE ON PLATYMONAS (CHLOROPHYTA) CELL COMPOSITION AND AMINO ACID UPTAKE RATES1

The effect of nitrogen source (nitrate, ammonia and/or amino acids) on cell composition and amino acid uptake rates was examined. Substantial levels of free amino acids accumulated intracellularly with all nitrogen sources used. Ammonia accumulated only when provided in the medium. The presence of ammonia in the medium decreased the intracellular accumulation of free amino acids, especially arginine. Amino acid uptake rates were suppressed by the presence of excess nitrogen, especially ammonia. However, the suppression of uptake did not show any particular relation to the nitrogenous cell composition.     

Nutrient utilization during biomass and anthocyanin accumulation in suspension cultures of wild carrot cells

The medium used for the growth of anthocyanin-accumulating wild carrot (D. carota) suspension cultures contained ammonia as a sole nitrogen source and was buffered with succinate. Ammonia was the first nutrient to be completely utilized.The uptake of carbohydrate, phosphate and succinate continued after ammonia depletion. Biomass accumulation was faster and greater when sucrose was initially present in the medium than when glucose was present. When sucrose was provided in the medium it was rapidly hydrolysed to glucose and fructose and the fructose was used preferentially to glucose. Anthocyanin accumulation was rapid after ammonia fell below 3 mM and until the pH of the medium rose from 4.5 to 5.1 or 5.2.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Nitrogen Metabolism in Plant Cell Suspension Cultures: II. Role of Organic Acids during Growth on Ammonia

Tobacco cells (Nicotiana tabacum) are capable of growth on ammonia as a sole nitrogen source only when succinate, malate, fumarate, citrate, α-ketoglutarate, glutamate, or pyruvate is added to the growth medium. A ratio between the molar concentrations of ammonia to succinate (as a complementary organic acid) in the growth medium of 1.5 was optimal. Succinate had no effect on the rate of uptake of ammonia from the medium into the cells although it did affect the intracellular concentration of ammonia. However, the changes were not sufficient to explain inhibition of growth as being due to ammonia toxicity. The radioactivity from ¹⁴C-succinate was incorporated into malate, glutamate, and aspartate within 2 minutes.     

Formation of l(-)malate by Saccharomyces cerevisiae during fermentation

Summary When grown in a synthetic medium most of the 51 strains of the genera Saccharomyces, Saccharomycodes, Zygosaccharomyces and Schizosaccharomyces investigated formed l-malate during fermentation. The quantity varied between 0.1 and 2.6 g malate per liter. Two strains of Saccharomyces cerevisiae synthesized malate at a rate of about 1.5 g/l. Malate was liberated during the growth phase and not metabolized during the stationary phase. Optimum malate formation was observed at a sugar concentration of about 20% (w/v), at pH 5 and at suboptimal nitrogen concentrations of less than 300 mg N/liter. Of the amino acids aspartate and glutamate were most favourable. If ammonium salts were used as the nitrogen source, significant amounts of malate were formed when the pH was kept constant by buffering. Trace metals had no or only little influence on malate synthesis. Biotin and pantothenate were essential for growth. Added ¹⁴CO₂ led to the formation of approximately equal quantities of labelled malate and succinate by S. cerevisiae strain 52, whereas about ten times more malate than succinate was formed by Saccharomyces uvarum. Avidin strongly inhibited the formation of malate while the inhibiton of succinate synthesis and of growth was comparatively much less. Malate is obviously formed by reduction of oxalacetate, the synthesis of which is catalysed by a biotin-dependent pyruvate carboxylase.     

Effect of nitrogen source on biosynthesis of rapamycin by Streptomyces hygroscopicus

Six non-amino acid nitrogen compounds were examined as nitrogen source for growth of Streptomyces hygroscopicus and biosynthesis of rapamycin. Of the nitrogen sources studied, ammonium sulfate was the best with respect to formation of rapamycin, and supported cell growth comparable to the organic nitrogen sources used in the control chemically defined medium, ie, aspartate, arginine plus histidine. In the new chemically defined medium, which is buffered with 200 mM 2-(N-morpholino)ethanesulfonic acid to prevent decline of pH during fermentation, an ammonium sulfate concentration of 40 mM was optimal for biosynthesis of rapamycin. Rapamycin production increased by more than 30% on both volumetric and specific bases as compared to the previous medium containing the three amino acids as nitrogen source. Received 08 November 1996/ Accepted in revised form 07 April 1997     

Metabolism of aspartate in Mycobacterium smegmatis

Mycobacterium smegmatis grows best on L-asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol.mg dry cells-1.h-1 from 1 mM L-asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen. Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso-diaminopimilate, had no discernable effect on either enzyme activity.     

Relative role of the glutaminase, glutamate dehydrogenase, and AMP-deaminase pathways in hepatic ureagenesis: studies with 15N.

We have studied the relative roles of the glutaminase versus glutamate dehydrogenase (GLDH) and purine nucleotide cycle (PNC) pathways in furnishing ammonia for urea synthesis. Isolated rat hepatocytes were incubated at pH 7.4 and 37 degrees C in Krebs buffer supplemented with 0.1 mM L-ornithine and 1 mM [2-15N]glutamine, [5-15N]glutamine, [15N]aspartate, or [15N]glutamate as the sole labeled nitrogen source in the presence and absence of 1 mM amino-oxyacetate (AOA). A separate series of incubations was carried out in a medium containing either 15N-labeled precursor together with an additional 19 unlabeled amino acids at concentrations similar to those of rat plasma. GC-MS was utilized to determine the precursor product relationship and the flux of 15N-labeled substrate toward 15NH3, the 6-amino group of adenine nucleotides ([6-15NH2]adenine), 15N-amino acids, and [15N]urea. Following 40 min incubation with [15N]aspartate the isotopic enrichment of singly and doubly labeled urea was 70 and 20 atom % excess, respectively; with [15N]glutamate these values were approximately 65 and approximately 30 atom % excess for singly and doubly labeled urea, respectively. In experiments with [15N]aspartate as a sole substrate 15NH3 enrichment exceeded that in [6-NH2]adenine, indicating that [6-15NH2]adenine could not be a major precursor to 15NH3. Addition of AOA inhibited the formation of [15N]glutamate, 15NH3 and doubly labeled urea from [15N]aspartate. However, AOA had little effect on [6-15NH2]adenine production. In experiments with [15N]glutamate, AOA inhibited the formation of [15N]aspartate and doubly labeled urea, whereas 15NH3 formation was increased. In the presence of a physiologic amino acid mixture, [15N]glutamate contributed less than 5% to urea-N. In contrast, the amide and the amino nitrogen of glutamine contributed approximately 65% of total urea-N regardless of the incubation medium. The current data indicate that when glutamate is a sole substrate the flux through GLDH is more prominent in furnishing NH3 for urea synthesis than the flux through the PNC. However, in experiments with medium containing a mixture of amino acids utilized by the rat liver in vivo, the fraction of NH3 derived via GLDH or PNC was negligible compared with the amount of ammonia derived via the glutaminase pathway. Therefore, the current data suggest that ammonia derived from 5-N of glutamine via glutaminase is the major source of nitrogen for hepatic urea-genesis.     

Factors affecting lactate and malate utilization by Selenomonas ruminantium.

Lactate utilization by Selenomonas ruminantium is stimulated in the presence of malate. Because little information is available describing lactate-plus-malate utilization by this organism, the objective of this study was to evaluate factors affecting utilization of these two organic acids by two strains of S. ruminantium. When S. ruminantium HD4 and H18 were grown in batch culture on DL-lactate and DL-malate, both strains coutilized both organic acids for the initial 20 to 24 h of incubation and acetate, propionate, and succinate accumulated. However, when malate and succinate concentrations reached 7 mM, malate utilization ceased, and with strain H18, there was a complete cessation of DL-lactate utilization. Malate utilization by both strains was also inhibited in the presence of glucose. S. ruminantium HD4 was unable to grow on 6 mM DL-lactate at extracellular pH 5.5 in continuous culture (dilution rate, 0.05 h-1) and washed out of the culture vessel. Addition of 8 mM DL-malate to the medium prevented washout on 6 mM DL-lactate at pH 5.5 and resulted in succinate accumulation. Addition of malate also increased bacterial protein, acetate, and propionate concentrations in continuous culture. These results suggest that 8 mM DL-malate enhances the ability of strain HD4 to grow on 6 mM DL-lactate at extracellular pH 5.5.     

Growth of Trichophyton mentagrophytes on individual amino acids

Trichophyton mentagrophytes was tested for its ability to utilize individual amino acids as a source of carbon and nitrogen in basal medium containing 0.4 mM magnesium sulphate (0.1 g/l) in 0.05 M potassium phosphate buffer at pH6.5. Growth was quantitated by measurement of both dry weight and fungal protein. Seventeen naturally occurring amino acids supported growth, serving as a source of both carbon and nitrogen. Seven amino acids failed to support growth under these conditions; however three of these could be metabolized for nitrogen, but not for carbon.     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

Partial purification and characterization of succinyl-CoA synthetase from Saccharomyces cerevisiae

Succinyl-CoA synthetase from Saccharomyces cerevisiae was partially purified (20-fold) with a yield of 44%. The Michaelis-Menten constants were determined: Km (succinate) = 17 mM; Km (ATP) = 0.13 mM; Km (CoA) = 0.03 mM. The succinyl-CoA synthetase has a molecular weight of about 80000 dalton (as determined by polyacrylamide gradient gel electrophoresis). The pH optimum is at 6.0. During fermentation the activity of succinyl-CoA synthetase is lower than in aerobically grown yeast cells. The presence of succinyl-CoA synthetase in fermenting yeasts may be regarded as an indication for the oxidative formation of succinate. In fermenting yeast cells succinyl-CoA synthetase is repressed by glucose if ammonium sulphate serves as nitrogen source. This catabolite repression is not observed with disaccharides or when amino acids are used as nitrogen source.     

General approach to bacterial nutrition: growth factor requirements of Moraxella nonliquefaciens.

A general procedure was devised for the determination of growth factor requirements of heterotrophic bacteria based upon identification of individual nutrients as they are successively depleted from a limited quantity of complex medium. By using this approach, it was possible to develop a defined medium for growth of Moraxella nonliquefaciens that contained nine amino acids and three vitamins. Three of the amino acids, proline, serine, and cysteine, were required in unusually high concentrations to obtain optimal growth. Methionine had a sparing action on the requirements for serine and cysteine. Glycine could substitute for serine. Although a required nutrient, cysteine was inhibitory for growth, but this inhibitory action was antagonized by valine or leucine. The requirement for cysteine was satisfied by cystine, glutathione, or sodium sulfide. M. nonliquefaciens could not use ammonia as a nitrogen source but could use glutamate or aspartate for this purpose. With the exception of 1 auxotrophic strain, the growth factor requirements of 23 independently isolated strains of M. nonliquefaciens were essentially the same.     

Helicobacter pylori utilises urea for amino acid synthesis

Abstract Helicobacter pylori has one of the highest urease activities of all known bacteria. Its enzymatic production of ammonia protects the organism from acid damage by gastric juice. The possibility that the urease activity allows the bacterium to utilise urea as a nitrogen source for the synthesis of amino acids was investigated. H. pylori (NCTC 11638) was incubated with 50 mM urea, enriched to 5 atom% excess ¹⁵N, that is the excess enrichment of ¹⁵N above the normal background, in the presence of either NaCl pH 6.0, or 0.2M citrate pH 6.0. E. coli (NCTC 9001) was used as a urease-negative control. ¹⁵N enrichment was detected by isotope ratio mass spectrometry. H. pylori showed intracellular incorporation of ¹⁵N in the presence of citrate buffer pH 6.0 but there was no significant incorporation of ¹⁵N in unbuffered saline or by E. coli in either pH 6.0 citrate buffer or unbuffered saline. The intracellular fate of the urea-nitrogen was determined by means of gas chromatography/mass spectrometry following incubation with ¹⁵N enriched 5 mM urea in the presence of either 0.2 M citrate buffer pH 6.0 or 0.2 M acetate buffer pH 6.0. After 5 min incubation in either buffer the ¹⁵n label appeared in glutamate, glutamine, phenylalanine, aspartate and alanine. It appears, therefore, that at pH and urea concentrations typical of the gastric mucosal surface, H. pylori utilises exogenous urea as a nitrogen source for amino acid synthesis. The ammonia produced by H. pylori urease activity thus facilitates the organism's nitrogen metabolism at neutral pH as well as protecting it from acid damage at low pH.     

Amino acid uptake systems in Bacteroides ruminicola

Uptake of amino acids by Bacteroides ruminicola was observed in cells grown in a complete defined medium, containing ammonia as the nitrogen source. A high rate of uptake occurred only in fresh medium, as an inhibitory substance, possibly acetate, apparently accumulated during growth. All amino acids except proline were taken up and incorporated into cold trichloroacetic acid precipitable material. Different patterns of incorporation and different responses to 2,4-dinitrophenol and potassium ferricyanide indicated multiple uptake systems were involved. Kinetic inhibition patterns suggested six distinct systems were present for amino acid uptake, with specificities related to the chemical structures of the amino acids. Thus, the failure of free amino acids to act as sole nitrogen sources for growth of B. ruminicola is not due to the absence of transport systems for these compounds.     

Physiological,biochemical, and cytological characteristics of a halotolerant and alkalitolerant methanotroph grown on methanol

The halotolerant alkaliphilic methanotroph Methylomicrobium buryatense 5B is capable of growth at high methanol concentrations (up to 1.75 M). At optimal values of pH and salinity (pH 9.5 and 0.75% NaCl), the maximum growth rate on 0.25 M methanol (0.2 h-1) was twice as high as on methane (0.1 h-1). The maximum growth rate increased with increasing medium salinity and was lower at neutral than at alkaline pH. The growth of the bacterium on methanol was accompanied by a reduction in the degree of development of intracytoplasmic membranes, the appearance of glycogen granules in cells, and the accumulation of formaldehyde, formate, and an extracellular glycoprotein at concentrations of 1.2 mM, 8 mM, and 2.63 g/l, respectively. The glycoprotein was found to contain 23% protein and 77% carbohydrates, the latter being dominated by glucose, mannose, and aminosugars. The major amino acids were glutamate, aspartate, glycine, valine, and isoleucine. The glycoprotein content rose to 5 g/l when the concentration of potassium nitrate in the medium was augmented tenfold. The activities of sucrose-6-phosphate synthase, glycogen synthase, and NADH dehydrogenase in methanol-grown cells were higher than in methane-grown cells. The data obtained suggest that the high methanol tolerance of M. buryatense 5B is due to the utilization of formaldehyde for the synthesis of sucrose, glycogen, and the glycoprotein and to the oxidation of excess reducing equivalents through the respiratory chain.     

The culture of plant cells with ammonium salts as the sole nitrogen source

Soybean cell suspension cultures grew on defined media with ammonium as the sole nitrogen source if Krebs cycle acids were added. Satisfactory growth was obtained with ammonium salts of citrate, malate, fumarate, or succinate, when compared with the regular medium containing nitrate and ammonium. Little or no growth occurred when ammonium salts of shikimate, tartrate, acetate, carbonate, or sulfate were used. The cells also grew well with l-glutamine as nitrogen source. The specific activities of glutamine synthetase and isocitrate dehydrogenase (nicotinamide adenine dinucleotide phosphate) were lower than in cells grown on a nitrate medium, but ammonium enhanced the activity of glutamate dehydrogenase. Cells of soybean, wheat, and flax have been cultured for an extended period on the ammonium citrate medium.     

Metabolism of Aspartate by Propionibacterium freudenreichii subsp. shermanii: Effect on Lactate Fermentation

More than 90% of the aspartate in a defined medium was metabolized after lactate exhaustion such that 3 mol of aspartate and 1 mol of propionate were converted to 3 mol of succinate, 3 mol of ammonia, 1 mol of acetate, and 1 mol of CO₂. This pathway was also evident when propionate and aspartate were the substrates in complex medium in the absence of lactate. In complex medium with lactate present, about 70% of the aspartate was metabolized to succinate and ammonia during lactate fermentation, and as a consequence of aspartate metabolism, more lactate was fermented to acetate and CO₂ than was fermented to propionate. The conversion of aspartate to fumarate and ammonia by the enzyme aspartase and subsequent reduction of fumarate to succinate occurred in the five strains of Propionibacterium freudenreichii subsp. shermanii studied. The ability to metabolize aspartate in the presence of lactate appeared to be related to aspartase activity. The specific activity of aspartase increased during and after lactate utilization, and the levels of this enzyme were lower in cells grown in defined medium than levels in those cells grown in complex medium. Under the conditions used, no other amino acids were readily metabolized in the presence of lactate. The possibility that aspartate metabolism by propionibacteria in Swiss cheese has an influence on CO₂ production is discussed.     

Studies on Brevibacterium linens metabolism in fermentor

Summary Growth and metabolism of Brevibacterium linens were studied in a fermentor regulated for fixed levels of pH (7.5 to 8.5), temperature (20–30° C) and dissolved oxygen (40%–60% of air saturated medium). The curves of disappearance of l-lactate and amino acids were invariable, indicating that phenylalanine, tyrosine, arginine, proline, glutamic acid and histidine are growth-limiting nutrients. Ornithine appeared at the beginning of cultures when oxygen consumption was low. Ammonia was produced, but large quantities were observed only when amino acid concentrations were higher than that of the carbon source. When the latter was low, the ammonia produced was consumed before a number of amino acids as an easily assimilable nitrogen source. Whether alkali or acid was consumed to maintain constant pH depended on the pH of the medium and on maximal growth rates.