Matching the supply of bacterial nutrients to the nutritional demand of the animal host

Various animals derive nutrients from symbiotic microorganisms with much-reduced genomes, but it is unknown whether, and how, the supply of these nutrients is regulated. Here, we demonstrate that the production of essential amino acids (EAAs) by the bacterium Buchnera aphidicola in the pea aphid Acyrthosiphon pisum is elevated when aphids are reared on diets from which that EAA are omitted, demonstrating that Buchnera scale EAA production to host demand. Quantitative proteomics of bacteriocytes (host cells bearing Buchnera) revealed that these metabolic changes are not accompanied by significant change in Buchnera or host proteins, suggesting that EAA production is regulated post-translationally. Bacteriocytes in aphids reared on diet lacking the EAA methionine had elevated concentrations of both methionine and the precursor cystathionine, indicating that methionine production is promoted by precursor supply and is not subject to feedback inhibition by methionine. Furthermore, methionine production by isolated Buchnera increased with increasing cystathionine concentration. We propose that Buchnera metabolism is poised for EAA production at certain maximal rates, and the realized release rate is determined by precursor supply from the host. The incidence of host regulation of symbiont nutritional function via supply of key nutritional inputs in other symbioses remains to be investigated.     

Sources of variation in dietary requirements in an obligate nutritional symbiosis

The nutritional symbiosis between aphids and their obligate symbiont, Buchnera aphidicola, is often characterized as a highly functional partnership in which the symbiont provides the host with essential nutrients. Despite this, some aphid lineages exhibit dietary requirements for nutrients typically synthesized by Buchnera, suggesting that some aspect of the symbiosis is disrupted. To examine this phenomenon in the pea aphid, Acyrthosiphon pisum, populations were assayed using defined artificial diet to determine dietary requirements for essential amino acids (EAAs). Six clones exhibiting dependence on EAAs in their diet were investigated further. In one aphid clone, a mutation in a Buchnera amino acid biosynthesis gene could account for the clone''s requirement for dietary arginine. Analysis of aphid F₁ hybrids allowed separation of effects of the host and symbiont genomes, and revealed that both affect the requirement for dietary EAAs in the clones tested. Amino acid requirements were minimally affected by secondary symbiont infection. Our results indicate that variation among pea aphids in dependence on dietary amino acids can result from Buchnera mutation as well as variation in the host genotype.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Effect of an exogenous energy source and amino acids on DNA synthesis in regenerating rat liver

Rats on a protein-free diet synthesized less DNA after partial hepatectomy than rats on a normal diet. In regenerating livers of animals on the protein-free diet, induction of several enzymes involved in the DNA precursor synthetic pathway, and especially ribonucleotide reductase, were depressed. When young rats were maintained solely by parenteral nutrition after partial hepatectomy, exogenous amino acids were more important than the exogenous energy source for induction of enzymes involved in synthesis of DNA and its pyrimidine nucleotide precursors. In particular, induction of ribonucleotide reductase appeared to be controlled by exogenous amino acids. Tryptophan, methionine, phenylalanine, leucine, valine, isoleucine and threonine seemed to stimulate the induction of this enzyme most after partial hepatectomy.     

Classification of essential amino acids for the weanling pig

Feeding experiments with mixtures of purified amino acids show that arginine, leucine, phenylalanine, and valine are required for good growth of weanling pigs. The pig resembles the rat in its ability to synthesize part, but not all of the arginine required. It is now possible to tentatively classify the known amino acids with respect to major growth effects in weanling pigs. The amino acids which must be present in the diet for good growth are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. The remaining amino acids may be tentatively classed as dispensable.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

Nutrient media for plant parasitic nematodes. V. Amino acid nutrition of Aphelenchoides rutgersi

Nutrition of axenically cultured A. Rutgersi was investigated by deletion and addition of various levels of essential amino acids. Reproduction was lacking when isoleucine, leucine, methionine, phenylalanine, threonine, histidine, tryptophan, and lysine were individually deleted from M-10. Normal reproduction was observed over a range of concentrations but declined to nothing at still higher concentrations of tryptophan and histidine. On the basis of these tests and analyses of both the nematode and host tissue, M-12 was devised and tested. The amino acid group of M-12 contained 7 fewer amino acids and concentrations of another 15 amino acids were adjusted; but no significant differences in reproduction occurred.     

Amino acid synthesis from glucose-U-14C in Glossina morsitans

Amino acid synthesis from glucose-U-¹⁴C was investigated in 2 day post-emergent and pregnant females of Glossina morsitans. This insect can synthesize alanine, aspartic acid, cystine, glutamic acid, glycine, proline, and serine from glucose. Arginine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, threonine, and valine showed no radioactivity and hence may be classified as nutritionally indispensable amino acids. Although tyrosine and hydroxyproline were not synthesized from glucose, they are at least partially dispensable nutrients for this insect because their synthesis from phenylalanine has been demonstrated. After the labelled glucose injection the highest radioactivity was recovered in the proline fraction. This is probably related to its rôle as an important energy reserve for flight. The radioactive amino acids recovered from females and from their offspring following glucose-U-¹⁴C injection were similar to those recovered from younger females. Radioactivity was also detected in the expired CO₂ and the excreta. The amino acids alanine, arginine, cystine, glycine, histidine, leucine/isoleucine, lysine, methionine, proline, and valine were identified in the excreta, of which arginine and histidine were in the largest amounts. Only excreted alanine, glycine, and proline showed radioactivity.     

Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts

The metabolism of ¹⁴C-labeled aspartic acid, diaminopimelic acid, malic acid and threonine by isolated pea (Pisum sativum L.) chloroplasts was examined. Light enhanced the incorporation of [¹⁴C] aspartic acid into soluble homoserine, isoleucine, lysine, methionine and threonine and protein-bound aspartic acid plus asparagine, isoleucine, lysine, and threonine. Lysine (2 millimolar) inhibited its own formation as well as that of homoserine, isoleucine and threonine. Threonine (2 millimolar) inhibited its own synthesis and that of homoserine but had only a small effect on isoleucine and lysine formation. Lysine and threonine (2 millimolar each) in combination strongly inhibited their own synthesis as well as that of homoserine. Radioactive [1,7-¹⁴C]diaminopimelic acid was readily converted into [¹⁴C]threonine in the light and its labeling was reduced by exogenous isoleucine (2 millimolar) or a combination of leucine and valine (2 millimolar each). The strong light stimulation of amino acid formation illustrates the point that photosynthetic energy is used in situ for amino acid and protein biosynthesis, not solely for CO₂ fixation.     

Purification and characterization of neutrophil chemotactic factors of Streptococcus sanguis

Two neutrophil chemotactic factors were isolated from the culture filtrates of Streptococcus sanguis ATCC 10556 and were chemically characterized as N-terminal blocked peptides of low molecular weight. One of the factors consisted of proline, valine, methionine, isoleucine and leucine and the other of methionine, isoleucine, leucine and phenylalanine. In both factors, methionine was detected as the sole N-terminal amino acid, but the amino group was blocked. The removal of N-terminal methionine yielded several N-terminal amino acids, suggesting that S. sanguis produced several N-terminal blocked methionyl peptides, all of which could be chemotactically active.     

Transport of Biosynthetic Intermediates: Regulation of Homoserine and Threonine Uptake in Escherichia coli

Homoserine is transported by a single system that it shares with alanine, isoleucine, leucine, phenylalanine, threonine, valine and perhaps cysteine, methionine, serine, and tyrosine. We investigated the regulation of this transport system and found that alanine, isoleucine, leucine, methionine, and valine each repress the homoserine-transporting system. From the concentration resulting in 50% repression of this transport system and the maximal amount of repression, we ranked the amino acids according to their effectiveness in repressing homoserine transport (in decreasing order): leucine>methionine>alanine>valine>isoleucine. The exponential rate of decrease in transport capacity after leucine addition equals the exponential growth rate of the culture, and protein synthesis is necessary for the derepression seen when leucine is removed. Threonine, in addition to using the above system, is transported by a second system shared with serine. We present further evidence for this serine-threonine transport system and show that it is not regulated like the homoserine-transporting system.     

Differential effects of visible light on active transport in E. coli

Light of wavelengths above 400 nm inactivated several active transport systems in E. coli ML 308. Rates of inactivation for uptake of threonine, glycine, leucine and methionine were similar and differed from those for methyl thio-β-D-galactoside and phenylalanine. These differential effects indicate that inactivation of the threonine, glycine, leucine and methionine systems is linked to a common photochemical lesion differing from that involved in the inactivation of the methyl thio-β-D-galactoside and phenylalanine systems. These lesions may serve as labels to identify molecules involved in transport or energy coupling processes.     

Determination of nutrient requirements for growth and maintenance of growing pigs under tropical condition

Data from 27 feeding trials conducted on growing pigs from different research institutes across India were subjected to mixed model regression analysis to derive requirements of digestible energy (DE), crude protein (CP) and essential amino acids for maintenance and body weight gains. The ranges of maintenance requirements were determined to be: DE 516 to 702 kJ/kg M^0.75, CP 6.98 to 11.62, lysine 0.431 to 0.664, methionine 0.265 to 0.458, methionine + cystine 0.327 to 0.466, cystine 0.055 to 0.184, threonine 0.205 to 0.511, arginine 0.377 to 1.21, isoleucine 0.241 to 0.775, leucine 0.604 to 1.54, phenylalanine + tyrosine 0.496 to 1.33, tryptophan 0.078 to 0.213, and valine 0.330 to 0.892 g/kg M^0.75, respectively for different body weight ranges. The corresponding requirements for 1 g gain in body weight were: DE 28.6 to 38.6 kJ, CP 0.27 to 0.44 g, lysine 0.0071 to 0.0126 g, methionine 0.0047 to 0.0133 g, methionine + cystine 0.0151 to 0.0261 g, cystine 0.0043 to 0.0094 g, threonine 0.0052 to 0.0165 g, arginine 0.0045 to 0.0301 g, isoleucine 0.0023 to 0.0198 g, leucine 0.0150 to 0.0447 g, phenylalanine + tyrosine 0.0091 to 0.0382 g, tryptophan 0.0005 to 0.0044 g, and valine 0.0061 to 0.0222 g. Regression equations had high R² values (ranging from 0.50 to 0.99 for different estimates), low coefficients of variation, low variance of error estimates and the coefficients were highly significant (P < 0.001). Regressed values were used to develop feeding standards. As the new standards derived in the present study are based on a thorough analysis of a larger database than previous Indian standards, the new feeding standard seems to be more appropriate for India and other tropical countries.     

Host and symbiont genetic determinants of nutritional phenotype in a natural population of the pea aphid

A defining feature of the nutritional ecology of plant sap‐feeding insects is that the dietary deficit of essential amino acids (EAAs) in plant sap is supplemented by EAA‐provisioning microbial symbionts in the insect. Here, we demonstrated substantial variation in the nutritional phenotype of 208 genotypes of the pea aphid Acyrthosiphon pisum collected from a natural population. Specifically, the genotypes varied in performance (larval growth rates) on four test diets lacking the EAAs arginine, histidine and methionine or aromatic EAAs (phenylalanine and tryptophan), relative to the diet containing all EAAs. These data indicate that EAA supply from the symbiotic bacteria Buchnera can meet total aphid nutritional demand for only a subset of the EAA/aphid genotype combinations. We then correlated single nucleotide polymorphisms (SNPs) identified in the aphid and Buchnera genomes by reduced genome sequencing against aphid performance for each EAA deletion diet. This yielded significant associations between performance on the histidine‐free diet and Buchnera SNPs, including metabolism genes predicted to influence histidine biosynthesis. Aphid genetic correlates of performance were obtained for all four deletion diets, with associations on the arginine‐free diet and aromatic‐free diets dominated by genes functioning in the regulation of metabolic and cellular processes. The specific aphid genes associated with performance on different EAA deletion diets are largely nonoverlapping, indicating some independence in the regulatory circuits determining aphid phenotype for the different EAAs. This study demonstrates how variation in the phenotype of associations collected from natural populations can be applied to elucidate the genetic basis of ecologically important traits in systems intractable to traditional forward/reverse genetic techniques.     

Variations circadiennes des acides aminés libres du muscle de Penaeus kerathurus

Free amino acids of the abdominal muscle of Penaeus kerathurus show circadian variations which are proportionally more important in the case of essential amino acids (valine, leucine, isoleucine, threonine, lysine, histidine, phenylalanine, methionine) than in the case of non-essential amino acids. Total concentration of essential amino acids shows two diurnal peaks, at times which are dependent on environmental factors. In winter conditions, these peaks occur earlier (06 and 18 h) than in summer conditions (09 and 21 h). These 12-12 rhythms may be related to the circadian variations of digestive enzyme activities.     

Neoaplectana glaseri: essential amino acids

Required for a nematode's reproduction in a chemically defined medium are the nine mammalian essential amino acids (sensu strictu). Needed in addition to lysine, tryptophane, histidine, phenylalanine, leucine, isoleucine, threonine, methionine, and valine is arginine which is marginally essential for mammals. Tyrosine, nonessential for mammals, is not absolutely required by the nematode but is beneficial in terms of onset time and quantity of reproduction. Aspartic acid, nonessential for the nematode and mammals, is toxic for adult nematodes at and above 4.8 mg/ml medium. The nematode, Neoaplectana glaseri, is parasitic in insect grubs but the strain used has been cultivated in species isolation on kidney slices continuously since (1944).     

Lysosomal pool of free-amino acids

Free (non-protein) amino acids were measured in whole rat liver and in unmodified lysosomes which were prepared from rat liver by the technique of free-flow electrophoresis. Significant intralysosomal pools of threonine, serine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and arginine were found. No efflux occurred from rat liver lysosomes in isotonic buffered sucrose at 0°C, but all amino acids showed various degrees of efflux at 20⁰ and 37⁰.     

Characterization of soybean tissue culture cell lines resistant to methionine analogs

Several hundred soybean [Glycine max (L.) Merr.] cell lines resistant to ethionine were isolated either with or without chemical mutagenesis. of these, 26 were found to contain 2 to 22 times higher than normal levels of uncombined methionine. These 26 cell lines also contained higher than normal levels of S-adenosylmethionine and S-methylmethionine, but the levels of free lysine, threonine, cysteine, valine, tyrosine and phenylalanine were not elevated. Isoleucine levels were only slightly elevated. These results suggest that the regulation of methionine synthesis in vivo is more likely to be later in the pathway (after homoserine phosphate) than early in the pathway.Abbreviations AdoMet S-adenosylmethionine - SMM S-methylmethionine - trp tryptophan - met methionine - thr threonine - val valine - phe phenylalanine - lys lysine - ile isoleucine - cys cysteine