Shortage Of Amino Acids Research Articles

Serine Hydroxamate and the Transcriptome of High Cell Density Recombinant Escherichia coli MG1655

For more than 30 years, serine hydroxamate has been used to chemically stimulate a stringent response in Escherichia coli and other bacteria. These studies have elucidated numerous characteristics of the classical stringent response beyond the simple cellular response to an amino acid shortage, including phospholipid synthesis and protease upregulation. In this study, the effects of a serine hydroxamate addition on high-cell-density recombinant E. coli were examined and compared to the effects of recombinant protein production to determine overlaps, as recombinant protein production stress has often been attributed to amino acid shortages. Both the transcriptome and growth characteristics were evaluated and compared. The serine hydroxamate addition profoundly decreased the culture growth rate, whereas recombinant protein production did not. Conversely, the transcriptome profile of the recombinant E. coli cultures were relatively unaffected by the serine hydroxamate addition, yet recombinant protein production dramatically changed the transcriptome profile. A subset of the classical stringent response genes were effected by the serine hydroxamate addition, whereas recombinant protein production regulated numerous classical stringent response genes but not all. The genes that were regulated by the serine hydroxamate addition include numerous fatty acid synthesis genes, in agreement with altered phospholipids synthesis reports. These results indicate that recombinant protein production and the stringent response have many overlapping responses but are far from identical.

Streptomyces griseusEnhances Denitrification byRalstonia pickettiiK50, Which Is Possibly Mediated by Histidine Produced during Co-Culture

Ralstonia pickettii K50 (strain K50) is a denitrifying bacterium that produces low levels of N(2)O under aerobic conditions. In this study, we found that co-culturing of strain K50 with Streptomyces griseus significantly enhanced the denitrification activity of strain K50 in an artificial wastewater (AWW) system. Most factors that enhance denitrification activity were in the high molecular weight fraction of the cell-free broth of S. griseus, and were suggested to be extracellular proteases. Further investigation revealed that the cultivation of strain K50 in protease-treated AWW medium fully enhanced denitrification, and that a shortage of amino acids in the medium limited it. Among the 20 standard amino acids tested, only histidine had a significant effect in inducing denitrification by strain K50. Our results indicate that histidine is a novel inducer of bacterial denitrification.

Nutritional Regulation of GLUT Expression, Glucose Metabolism, and Intramuscular Fat Content in Porcine Muscle

We conducted a series of investigations in order to elucidate role of nutritional status in regulating GLUT expression and energy metabolism in porcine muscle. Firstly, the role of mild undernutrition in regulating muscle GLUT gene expression and function was studied in growing pigs (3 wk of age) on a high (H) or low (L) food intake (H = 2L) at 35°C or 26°C. Low food intake selectively upregulates GLUT1 and GLUT4 gene expression; mRNA levels were elevated in longissimus dorsi (L. dorsi) and rhomboideus muscles but not in diaphragm or cardiac muscles. Our next step was to determine whether dietary lysine, a major primary limiting amino acid in diets for pigs, affects muscle GLUT4 expression. Pigs of 6 wk of age were pair-fed a control or low lysine (LL) diet. The control diet contained optimal amounts of all essential amino acids, including 1.15% lysine. The LL diet was similar but contained only 0.70% lysine. GLUT4 mRNA expression was upregulated by the LL diet in L. dorsi and rhomboideus muscles, whereas that in cardiac muscle was unaffected. GLUT4 protein abundance was also higher in rhomboideus muscle of animals on the LL diet. We conducted another investigation in order to elucidate effects of the LL diet on post-GLUT4 glucose metabolism. Activity of hexokinase was unaffected by dietary lysine levels while that of citrate synthase was higher both in L. dorsi and rhomboideus muscles of pigs fed on the LL diet. Glucose 6-phosphate content was higher in L. dorsi msucle in the LL group. Glycogen content was higher both in L. dorsi and rhomboideus muscles in the LL group. Further, we determined the effects of dietary lysine levels on accumulation of intramuscular fat (IMF) in L. dorsi muscle of finishing pigs. A low lysine diet (lysine content was 0.40%) meeting approximately 70% of the requirement of lysine was given to finishing pigs for two months. IMF contents in L. dorsi of the pigs given the low lysine diet were twice higher than those of the pigs fed on a control diet (lysine content was 0.65%). Finally, we proved that a well known effect of breadcrumbs feeding to enhance IMF of finishing pigs could be attributed to shortage of amino acids in diets including breadcrumbs.

Suppression of C-Hordein Synthesis in Barley by Antisense Constructs Results in a More Balanced Amino Acid Composition

Barley has for feeding purposes a shortage of essential amino acids, especially lysine, threonine, and methionine, and an excess of proline and glutamine. In the present study, we have introduced into barley an antisense construct against C-hordeins, the storage protein with the lowest nutritional quality. SDS-PAGE and reverse phase HPLC revealed a relative reduction in the amounts of C-hordeins and relative increases in the content of the other storage proteins. The five different lines analyzed had lower amounts of proline, glutamic acid/glutamine, and phenylalanine (up to 12%, 6%, and 9% reductions), while the lysine, threonine, and methionine content was increased with up to 16%, 13% and 11%. It is concluded that antisense mediated suppression of C-hordein synthesis may be a promising approach for improving the nutritional value of barley as a feed crop while at the same time reducing the environmental nitrogen load.

Troubleshooting coupled in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins

Cell-free coupled transcription–translation systems with bacterial lysates are widely used to synthesize recombinant proteins in amounts of several mg per ml. By using reporter green fluorescence protein (GFP) we demonstrate that proteins are synthesized with an unsatisfyingly low-active fraction of (50 ± 20)%. One reason is probably the T7 polymerase used, being up to eight times faster than the intrinsic transcriptase and thus breaking the coupling between transcription and translation in bacterial systems. The active fraction of the synthesized protein was improved by using either a slower T7 transcriptase mutant or lowering the incubation temperature to 20°C. A drop of protein synthesis observed after 7 h incubation time was not due to a shortage of nucleotide triphosphates, but rather to a shortage of amino acids. Accordingly, a second addition of amino acids after 10 h during an incubation at 20°C led to synthesis of up to 4 mg/ml of GFP with virtually 100% activity.

Structured model to predict intracellular amino acid shortages during recombinant protein overexpression in E. coli

Recombinant protein overexpression and the classical stringent response have been shown to induce the same proteases. Since the stringent response was the result of an intracellular amino acid shortage, it was hypothesized that the overexpression of the recombinant protein also caused an intracellular amino acid shortage. A structured non-segregated kinetic mathematical model of recombinant Escherichia coli was developed to predict intracellular amino acid shortages during recombinant protein overexpression, and thus the induction of the stringent response. Two model recombinant proteins were examined, chloramphenicol acetyl-transferase (CAT) and an ‘average protein’. The model predicted an aromatic amino acid shortage during CAT overexpression, as predicted based on the CAT's amino acid content. The model also predicted a shortage of the intracellular alanine family amino acid pool during CAT overexpression. This was unexpected due to the relatively low content of alanine family amino acids in CAT compared to the average E. coli protein. The model predicted alanine, glutarate, and aspartate family amino acid shortages during recombinant ‘average protein’ overexpression. Additionally, the model predicted a decrease in the ribosome pool at induction for both recombinant proteins, which agrees with published experimental results. Thus, the structured kinetic model was able to predict amino acid shortages, that could potentially cause a stringent response and elevated protease activity.

Life in protein-rich environments: the relA-independent response of Streptococcus pyogenes to amino acid starvation.

Considering that group A streptococci are multiple auxotrophs that may encounter shortage of amino acids during specific stages of the infectious process, we studied their adaptive response to amino acid deprivation. We found that, in addition to the (p)ppGpp-mediated stringent response characterized previously, Streptococcus pyogenes exhibits a relA-independent response comprising transcriptional modulation of a specific subset of genes involved in pathogenesis. Genes/operons transcriptionally upregulated during starvation of both wild type and relA mutants included the two-component signal transduction system covRS, the positive regulator (ropB) of the pyrogenic exotoxin B gene, speB, the oligopeptide (opp) and dipeptide (dpp) permease systems and the pepB gene putatively involved in the intracellular processing of oligopeptides. Upregulation of covRS was accompanied by downregulation of ska, one of the target genes of the negative CovR regulator, and the net effect of amino acid starvation also favoured repression of speB. A significant feature of upregulated opp expression was stimulated readthrough transcription of the operon-internal oppA terminator, leading to increased mRNA levels for synthesis of the translocator complex relative to the substrate-binding protein. Based on these and previous results, a stimulus-response network is proposed that counteracts the stringent response and may enable the pathogen to mount a dynamic response to the protein-rich environment provided by its human host.

Synthesis of the translational apparatus is regulated at the translational level.

The synthesis of many mammalian proteins associated with the translational apparatus is selectively regulated by mitogenic and nutritional stimuli, at the translational level. The apparent advantages of the regulation of gene expression at the translational level are the speed and the readily reversible nature of the response to altering physiological conditions. These two features enable cells to rapidly repress the biosynthesis of the translational machinery upon shortage of amino acids or growth arrest, thus rapidly blocking unnecessary energy wastage. Likewise, when amino acids are replenished or mitogenic stimulation is applied, then cells can rapidly respond in resuming the costly biosynthesis of the translational apparatus. A structural hallmark, common to mRNAs encoding many components of the translational machinery, is the presence of a 5' terminal oligopyrimidine tract (5'TOP), referred to as TOP mRNAs. This structural motif comprises the core of the translational cis-regulatory element of these mRNAs. The present review focuses on the mechanism underlying the translational control of TOP mRNAs upon growth and nutritional stimuli. A special emphasis is put on the pivotal role played by ribosomal protein S6 kinase (S6K) in this mode of regulation, and the upstream regulatory pathways, which might be engaged in transducing external signals into activation of S6K. Finally, the possible involvement of pyrimidine-binding proteins in the translational control of TOP mRNAs is discussed.

Effects of the sulfonylurea herbicide metsulfuron methyl on growth and macromolecular synthesis in the green alga Selenastrum capricornutum

The sulfonylureas (SU), are highly phytotoxic due to the inhibition of the enzyme acetolactate synthase (ALS), the key enzyme in the synthesis of branched-chain amino acids. In order to find a short-term algal test to be used as an indicator of the environmental toxicity of SU, the effects of the sulfonylurea metsulfuron methyl (MSM) in the green alga Selenastrum capricornutum were studied. As a comparison of long-term and short-term effects of MSM in Selenastrum, effects on growth were compared to short-term inhibition of protein synthesis and the synthesis of nucleic acids. The EC:50 for inhibition of growth of S. capricornutum by MSM was determined to be 4.1 μmol/l and the No Effect Concentration (NEC) to be between 0.1 and 0.2 μmol/l. The inhibition of growth could be substantially reduced by the addition of the branched chain amino acids and valine was especially important to restore growth. Protein synthesis measured as incorporation of [ 3H]methionine was, in spite of the shortage of amino acids, not inhibited by MSM in a short-term test. In contrast, the incorporation of [ 3H]adenine into cold-TCA-insoluble macromolecules, was inhibited by MSM, although maximal inhibition was restricted to about 50%. The No Effect Concentration for inhibition of adenine incorporation was 0.3 μmol/l, similar to the NEC value for growth inhibition. If alkaline hydrolysis was used to specifically hydrolyse RNA prior to the precipitation with cold TCA, a greater portion of the adenine incorporation could be inhibited by MSM. Adenine incorporation into nucleic acids seems to be appropriate for a short-term test of effects of SU on natural microbial communities, since adenine incorporation is quickly inhibited at concentrations of SU known to inhibit growth.

High level production of a cellulase-free xylanase in glucose-limited fed batch cultures of a thermophilic Bacillus strain

Bacillus sp. strain XE and its mutant derivative strain D3 produce a thermostable xylanase which is suitable for enzymatic bleaching of kraft pulp. Xylanase synthesis was shown to be induced by the soluble products of xylan hydrolysis (xylooligosaccharide) and equally, catabolically-repressed when these oligosaccharides accumulate in the medium. An optimal balance between these two antagonistic effects was obtained in a carbon-limited, fed-batch culture with continuous xylooligosaccharide feeding. To reduce substrate cost, the xylooligosaccharide could be 90% substituted by glucose without reduction of the xylanase production rate. Xylanase production ceased when the activity reached approximately 380 U ml −1 due to an amino acid shortage. A continuous supply of exogenous amino acids allowed the production to continue to more than 1000 U ml −1.

Liver histology of reindeer calves during the winter season.

Seventeen reindeer calves were divided into three groups and liver biopsies were taken during the winter. Group 1 consisted of 6 animals allowed to graze freely under natural conditions, those in groups 2 (n = 5) and 3 (n = 6) were kept in pens and fed with lichens, the diet being designed to achieve a slight (group 2) or moderate (group 3) weight loss. Weight losses from November to April averaged 14.9%, 13.2% and 24.8% in the three groups respectively. The relative volumes of the different structures in the liver were determined by light microscopic morphometry. The mean size of the hepatocytes was slightly greater in March than in January in groups 1 and 2, and slightly smaller in group 3 (no statistical differences) and then decreased markedly. These changes could also be seen in the sinusoidal lumina. The decrease in the size of the hepatocytes may reflect a shortage of amino acids in the cells or it may be hormone-mediated. Unlike the situation in cows, there was no fatty accumulation in the hepatocytes. This may be a species adaptation to an annual period of starvation.

Effect of L-Methionine Sulphoximine on the Products of Photosynthesis in Wheat (Triticum aestivum) Leaves

Methionine sulphoximine, an inhibitor of glutamine synthetase, caused ammonia accumulation in detached wheat leaves. The rate was increased by increased oxygen in the atmosphere and by simultaneously supplying glycine or giving extra nitrate; it was decreased by isonicotinyl hydrazide. Ammonia production was light-dependent and continued at a constant rate in air for at least 2 h. Photosynthesis was progressively inhibited after the first hour; this inhibition was not because of increased stomatal resistance. Leaves supplied with 30 mol m−3 ammonium chloride, without methionine sulphoximine, accumulated more ammonia than leaves treated with the inhibitor but showed less inhibition of photosynthesis. The inhibitor decreased synthesis of [14C] amino acids from 14CO2 in the light but increased the synthesis of [14C] malate and, relatively, the incorporation of 14C into sugar phosphates. In the absence of inhibitor, nitrate increased and ammonium ion decreased synthesis of malate. Methionine sulphoximine, by causing a shortage of amino acids, probably inhibited photosynthesis in part by decreasing the recycling of carbon from the photorespiratory cycle back to the Calvin cycle.

Requirement for an upstream element for optimal transcription of a bacterial tRNA gene.

Bacterial promoters are the sites at the 5' end of each gene that bind RNA polymerase and direct the initiation of transcription. The functional elements of Escherichia coli promoters are two highly conserved sequences, each about six nucleotides long, usually centred at sites -10 and -35, +1 being the initiating nucleotide. We have been interested in the structure of promoters of genes that are subject to stringent control, that is whose expression is reduced in conditions of amino acid shortage, such as rRNA and tRNA genes. We have therefore mapped the sequences involved in promoting in vivo transcription of a bacterial tRNATyr (tyrT) gene by fusing the tyrT promoter region to a galactokinase (galK) gene, and using in vivo expression of galactokinase activity to measure promoter strength. We show here that efficient expression from the tyrT promoter requires specific sequences upstream of the canonical promoter elements, and we suggest that these sequences constitute an extended promoter structure.

Adaptive regulation of amino acid transport in cultured human fibroblasts. Sites and mechanism of action.

The regulation of the transport of neutral amino acids across the cell membrane by adaptive mechanisms has been studied in cultured human fibroblasts. Among the three transport systems (A, ASC, and L) individually discriminated, only the Na+-dependent System A was subject to adaptive regulation, showing enhancement of its activity when the cells were incubated under conditions of amino acid shortage (derepression phase) and decrease of its activity when the cells were exposed to a medium supplied with Site A-reactive amino acids (repression phase). Starvation-induced derepression of transport activity and its reversal by amino acid refeeding required active RNA and protein synthesis. Derepression involved an early mRNA synthesis which started within 30 min from the abrupt change in extracellular amino acid concentration and apparently lasted 90 min. The transcribed mRNA was rather stable and translatable for a few hours (presumably into transport proteins) in cells maintained in the absence of amino acids. Repression by amino acid refeeding also involved an early mRNA synthesis, the product of its translation being presumably a protein capable of causing degradation or inactivation of transport proteins. The rate of decay in transport activity of previously derepressed cells was somewhat faster in the presence of added Site A-reactive amino acids than in their absence. A model is proposed in which the concentration of Site A-reactive amino acids affects transport activity of System A by modulating transcription of mRNA species coding for transport proteins and their putative inactivators and by regulating the efficiency of transport protein inactivation at the cell membrane.

Some Effects of Protein Deficiency in Young Growing Pigs. III. Ferrokinetic Investigations

Ferrokinetic investigations have been carried out in pigs which had undergone protein starvation and rehabilitation. After long term protein deprivation haematocrit levels, blood haemoglobin levels, serum iron concentrations, serum total iron binding capacity and blood volume per kg body weight were all reduced in the deficient animals. The values for the rehabilitated animals were in the normal range. The erythrocytes of the protein starved pigs showed an increased rate of uptake of radioactive iron compared with the control pig. However, the turnover of iron as a whole was reduced greatly in the protein deprived group compared to the rehabilitated and control pigs. Accumulation of iron occurred in the livers, spleens and kidneys of the protein starved animals. The rehabilitated pigs had lower liver stores of iron than the control pig. On the basis of the data presented it has been concluded that these effects are mainly due to a shortage of amino acids for protein synthesis.

Mode of action of glucocorticoids: II. Early effects of triamcinolone on amino acid incorporation into various tissues in vivo

1. 1. Acute effects of the glucocorticoid triamcinolone (350 μg per 100 g body weight, 2 to 4 h) on labeled amino acid incorporation into protein of subcellar components in various tissues have been investigated in fed rats. 2. 2. The incorporation of uniformely labeled L-[ 14C]valine and DL-[3- 14C]phenylalanine into proteins of nuclei, mitochondria, microsomes and supernatant fractions of hepatic cells was not modified by the administration of triamcinolone, whereas the incorporation of these amino acids into proteins of each of these fractions of the thymus was reduced by triamcinolone given either 2 or 4 h previously. 3. 3. The administration of triamcinolone increased liver glycogen, but did not significantly affect the concentration of blood sugar and muscle glycogen in fed animals. The incorporation of 14C from uniformely labeled L-[ 14C]valine as well as DL-[3 14]phenylalanine into liver glycogen and blood glucose were s by triamcinolone administration, indicating increased gluconeogenesis from the carbon of phenylalanine as well as from that of valine. 4. 4. The size of the pool of free amino acids and the specific activities of free amino nitrogen in the blood and thymus, were not significantly modified by triamcinolone, but the size of the pool and the radioactivities of free amino acids in hepatic cells were increased. 5. 5. These findings suggest that triamcinolone inhibits protein synthesis in the thymus even in very short-term experiments and that this inhibiting effect is due neither to the effect of hormone on cell membrane transport of amino acids nor to the catabolism of amino acids in the liver for gluconeogenesis with resultant shortage of amino acids for protein synthesis in peripheral tissue.

THE LIPOTROPIC ACTIVITY OF PROTEIN

A distinct lipotropic effect of casein has been observed in rats under conditions that appear to preclude any explanation involving the methionine or choline content of the ration. Diets low in protein produce a type of fatty liver that is not prevented by choline and is histologically different from that of choline deficiency. Choline appears less effective in such diets than in ones more adequate in protein. Presumably choline has only a limited effect on abnormal fat in periportal regions because the biochemical basis of this lesion seems to involve faulty formation of lipoprotein due to shortage of essential amino acids.

THE LIPOTROPIC ACTIVITY OF PROTEIN

A distinct lipotropic effect of casein has been observed in rats under conditions that appear to preclude any explanation involving the methionine or choline content of the ration. Diets low in protein produce a type of fatty liver that is not prevented by choline and is histologically different from that of choline deficiency. Choline appears less effective in such diets than in ones more adequate in protein. Presumably choline has only a limited effect on abnormal fat in periportal regions because the biochemical basis of this lesion seems to involve faulty formation of lipoprotein due to shortage of essential amino acids.