Lysine Depletion Research Articles

Lysine Deprivation Suppresses Adipogenesis in 3T3-L1 Cells: A Transcriptome Analysis.

Growing evidence proves that amino acid restriction can reverse obesity by reducing adipose tissue mass. Amino acids are not only the building blocks of proteins but also serve as signaling molecules in multiple biological pathways. The study of adipocytes' response to amino acid level changes is crucial. It has been reported that a low concentration of lysine suppresses lipid accumulation and transcription of several adipogenic genes in 3T3-L1 preadipocytes. However, the detailed lysine-deprivation-induced cellular transcriptomic changes and the altered pathways have yet to be fully studied. Here, using 3T3-L1 cells, we performed RNA sequencing on undifferentiated and differentiated cells, and differentiated cells under a lysine-free environment, and the data were subjected to KEGG enrichment. We found that the differentiation process of 3T3-L1 cells to adipocytes required the large-scale upregulation of metabolic pathways, mainly on the mitochondrial TCA cycle, oxidative phosphorylation, and downregulation of the lysosomal pathway. Single amino acid lysine depletion suppressed differentiation dose dependently. It disrupted the metabolism of cellular amino acids, which could be partially reflected in the changes in amino acid levels in the culture medium. It inhibited the mitochondria respiratory chain and upregulated the lysosomal pathway, which are essential for adipocyte differentiation. We also noticed that cellular interleukin 6 (IL6) expression and medium IL6 level were dramatically increased, which was one of the targets for suppressing adipogenesis induced by lysine depletion. Moreover, we showed that the depletion of some essential amino acids such as methionine and cystine could induce similar phenomena. This suggests that individual amino acid deprivation may share some common pathways. This descriptive study dissects the pathways for adipogenesis and how the cellular transcriptome was altered under lysine depletion.

The Effect of Terpenoid Compounds on the Formation of Advanced Glycation Endproducts (AGEs) in Model Systems

Background: Terpenoid compounds, despite their established antioxidant ability, are neglected as potential glycation regulators. Methods: In-vitro model systems of lysine (0.1 M) with glucose (0.1 M and 1 M) were incubated at 80 °C and 100 °C for 3 h in the presence of aniseed oil, thymol and linalool (2–8 μΜ). Color development, absorbance at UV-Vis (280 nm, 360 nm, 420 nm), fluorescence intensity (λexc = 370 nm, λemm = 440 nm) and lysine depletion (HPLC-FL) were measured to monitor the progress of the Maillard reaction. Response Surface Methodology was used to analyze the impact of the five experimental conditions on the glycation indices. Results: All terpenoid compounds promoted color development and did not affect lysine depletion. The choice of terpenoid compound impacted glycation at 280 nm, 360 nm and 420 nm (p < 0.02). The effect was stronger at lower temperatures (p < 0.002) and 0.1 M glucose concentrations (p < 0.001). Terpenoid concentration was important only at 360 nm and 420 nm (p < 0.01). No impact was seen for fluorescence intensity from the choice of terpenoid compounds and their dose (p = 0.08 and p = 0.44 respectively). Conclusion: Terpenoid compounds show both anti- and proglycative activity based on the incubation conditions. Thymol showed the largest antiglycative capacity, followed by aniseed oil and linalool. Maximal antiglycative capacity was seen at 0.1 M glucose, 2 μΜ terpenoid concentration, 80 °C and 1 h incubation.

Lysine Depletion during Different Feeding Phases: Effects on Growth Performances and Meat Quality of Broiler Chickens.

Simple SummaryIn the past, many studies have been carried out to investigate the effect of lysine supplementation on broilers’ growth performances and feed efficiency. However, the knowledge concerning the reduction of the dietary content of this essential amino acid is limited and mainly restricted to the production performances of the birds. Within this context, the present study aimed at assessing the impact of lysine restriction during grower I (10–20 d) and grower I and II (10–20 and 21–34 d) feeding phases on live performances, breast meat quality traits and technological properties, as well as on the incidence and severity of abnormalities affecting the pectoral muscles (i.e., white striping, wooden breast and spaghetti meat). Lysine restriction during different feeding phases exerts negligible effects on the production performances of the broilers due to their compensatory growth. In addition, the increased anserine content following lysine depletion might have exerted a protective role against protein denaturation by buffering the acidic end-products generated during post-mortem rigor development.The present study aimed at assessing the impact of lysine restriction performed during different feeding phases on growth performances, meat quality traits and technological properties as well as on the incidence and severity of breast muscle abnormalities. For this purpose, a total of 945 one-day-old Ross 308 male chicks was randomly divided into three experimental groups: CONT, fed a four feeding phases commercial diet, GRW I, and GRW I + II fed CONT diet with the depletion of synthetic lysine during grower I and grower I and II feeding phases, respectively. Productive performances were recorded throughout the whole rearing cycle and the incidence of breast muscle growth-related abnormalities assessed at slaughter (49 d) on 280 breasts/group. Quality traits and technological properties of breast meat were measured on a total of 54 Pectoralis major muscles. Lysine restriction only marginally affected the productive performances and the quality parameters of breast meat. The increased (p < 0.05) solubility of the protein fraction along with the remarkably higher (p < 0.05) anserine content found in GRW I + II suggests an increased energy requirement in the pectoral muscles belonging to lysine-restricted birds and supports the hypothesis of a reduced protein synthesis taking place within these muscles.

Refinement of the Peroxidase Peptide Reactivity Assay and Prediction Model for Assessing Skin Sensitization Potential.

A peptide reactivity assay with an activation component was developed for use in screening chemicals for skin sensitization potential. A horseradish peroxidase-hydrogen peroxide (HRP/P) oxidation system was incorporated into the assay for characterizing reactivity of hapten and pre-/prohapten sensitizers. The assay, named the Peroxidase Peptide Reactivity Assay (PPRA) had a predictive accuracy of 83% (relative to the local lymph node assay) with the original protocol and prediction model. However, apparent false positives attributed to cysteine depletion at relatively high chemical concentrations and, for some chemicals expected to react with the -NH2 group of lysine, little to no depletion of the lysine peptide were observed. To improve the PPRA, cysteine peptide reactions with and without HRP/P were modified by increasing the number of test concentrations and refining their range. In addition, removal of DL-dithiothreitol from the reaction without HRP/P increased cysteine depletion and improved detection of reactive aldehydes and thiazolines without compromising the assay's ability to detect prohaptens. Modification of the lysine reaction mixture by changing the buffer from 0.1 M ammonium acetate buffer (pH 10.2) to 0.1 M phosphate buffer (pH 7.4) and increasing the level of organic solvent from 1% to 25% resulted in increased lysine depletion for known lysine reactive chemicals. Refinement of the prediction model improved the sensitivity, specificity, and accuracy for hazard identification. These changes resulted in significant improvement of the PPRA making it is a reliable method for predicting the skin sensitization potential of all chemicals, including pre-/prohaptens and directly reactive haptens.

Lysine oxidase exposes a dependency on the thioredoxin antioxidant pathway in triple-negative breast cancer cells.

Transformed cells are vulnerable to depletion of certain amino acids. Lysine oxidase (LO) catalyzes the oxidative deamination of lysine, resulting in lysine depletion and hydrogen peroxide production. Although LO has broad antitumor activity in preclinical models, the cytotoxic mechanisms of LO are poorly understood. Triple (ER/PR/HER2)-negative breast cancer (TNBC) cells were treated with control media, lysine-free media or control media supplemented with LO and examined for cell viability, caspase activation, induction of reactive oxygen species (ROS) and antioxidant signaling. To determine the role of nuclear factor erythroid 2-related factor 2 (NRF2) and thioredoxin reductase-1 (TXNRD1) in LO-induced cell death, NRF2 and TXNRD1 were individually silenced by RNAi. Additionally, the pan-TXNRD inhibitor auranofin was used in combination with LO. LO activates caspase-independent cell death that is suppressed by necroptosis and ferroptosis inhibitors, which are inactive against lysine depletion, pointing to fundamental differences between LO and lysine depletion. LO rapidly induces ROS with a return to baseline levels within 24h that coincides temporally with induction of TXNRD activity, the rate-limiting enzyme in the thioredoxin antioxidant pathway. ROS induction is required for LO-mediated cell death and NRF2-dependent induction of TXNRD1. Silencing NRF2 or TXNRD1 enhances the cytotoxicity of LO. The pan-TXNRD inhibitor auranofin is synergistic with LO against transformed breast epithelial cells, but not untransformed cells, underscoring the tumor-selectivity of this strategy. LO exposes a redox vulnerability of TNBC cells to TXNRD inhibition by rendering tumor cells dependent on the thioredoxin antioxidant pathway for survival.

Inhibition Mechanism of Indoleamine 2, 3-Dioxygenase 1 (IDO1) by Amidoxime Derivatives and Its Revelation in Drug Design: Comparative Molecular Dynamics Simulations.

For cancer treatment, in addition to the three standard therapies of surgery, chemotherapy, and radiotherapy, immunotherapy has become the fourth internationally-recognized alternative treatment. Indoleamine 2, 3-dioxygenase 1 (IDO1) catalyzes the conversion of tryptophan to kynurenine causing lysine depletion, which is an important target in the research and development of anticancer drugs. Epacadostat (INCB024360) is currently one of the most potent IDO1 inhibitors, nevertheless its inhibition mechanism still remains elusive. In this work, comparative molecular dynamics simulations were performed to reveal that the high inhibitory activity of INCB024360 mainly comes from two aspects: disturbing the ligand delivery tunnel and then preventing small molecules such as oxygen and water molecules from accessing the active site, as well as hindering the shuttle of substrate tryptophan with product kynurenine through the heme binding pocket. The scanning of key residues showed that L234 and R231 residues both were crucial to the catalytic activity of IDO1. With the association with INCB024360, L234 forms a stable hydrogen bond with G262, which significantly affects the spatial position of G262-A264 loop and then greatly disturbs the orderliness of ligand delivery tunnel. In addition, the cleavage of hydrogen bond between G380 and R231 increases the mobility of the GTGG conserved region, leading to the closure of the substrate tryptophan channel. This work provides new ideas for understanding action mechanism of amidoxime derivatives, improving its inhibitor activity and developing novel inhibitors of IDO1.

Fatty acid starvation activates RelA by depleting lysine precursor pyruvate.

Bacteria undergoing nutrient starvation induce the ubiquitous stringent response, resulting in gross physiological changes that reprograms cell metabolism from fast to slow growth. The stringent response is mediated by the secondary messengers pppGpp and ppGpp collectively referred to as (p)ppGpp or 'alarmone'. In Escherichia coli, two paralogs, RelA and SpoT, synthesize (p)ppGpp. RelA is activated by amino acid starvation, whereas SpoT, which can also degrade (p)ppGpp, responds to fatty acid (FA), carbon and phosphate starvation. Here, we discover that FA starvation leads to rapid activation of RelA and reveal the underlying mechanism. We show that FA starvation leads to depletion of lysine that, in turn, leads to the accumulation of uncharged tRNALys and activation of RelA. SpoT was also activated by FA starvation but to a lower level and with a delayed kinetics. Next, we discovered that pyruvate, a precursor of lysine, is depleted by FA starvation. We also propose a mechanism that explains how FA starvation leads to pyruvate depletion. Together our results raise the possibility that RelA may be a major player under many starvation conditions previously thought to depend principally on SpoT. Interestingly, FA starvation provoked a ~100-fold increase in relA dependent ampicillin tolerance.

In chemico assessment of potential sensitizers: Stability and direct peptide reactivity of 24 fragrance ingredients.

Twenty-four pure fragrance ingredients of concern as potential skin sensitizers were previously subjected to degradation studies and evaluated using the high throughput with dansyl cysteamine (HTS-DCYA) method. The experimental results showed that two-thirds of the 24 fragrance ingredients underwent chemical degradation. In some cases, such degradation was accompanied by an increase in thio-reactivity. These results prompted us to investigate the reactivity of the same ingredients using the direct peptide reactivity assay (DPRA). In the present work, the 24 chemicals were subjected to forced degradation for 150days, and evaluated with both DPRA and HTS-DCYA methods. At the end of the study, four and eight compounds remained non-reactive in the DPRA and DCYA assay, respectively. Coumarin, benzyl salicylate, benzyl cinnamate and hexyl cinnamal were found unreactive in both assays, while cinnamal, cinnamyl alcohol, hydroxycitronellal and lilial were found negative in the DCYA but positive in the DPRA method. The incongruity in reactivity of these four compounds was attributed to a possible role of pro-oxidants formed upon degradation, resulting in depletion of peptide without formation of apparent covalent adducts with the test chemical. To validate this hypothesis, the effect of hydrogen peroxide as model pro-oxidant on both lysine- and cysteine-heptapeptide depletion in the DPRA method was thus investigated. The obtained results showed little effect of oxidative conditions on lysine depletion, while cysteine depletion was significantly affected by concentrations above 1.1mg/L of hydrogen peroxide. Overall, both in chemico methods confirmed chemical instability should be considered when assessing the skin sensitization potential of (un)known chemicals with alternative methods.

Biofilm Lysine Decarboxylase, a New Therapeutic Target for Periodontal Inflammation

Lysine, a nutritionally essential amino acid, enters the oral cavity in gingival crevicular fluid (GCF). During oral hygiene restriction (OHR), lysine decarboxylase (LDC) in dento-gingival biofilms converts lysine to cadaverine. Lysine depletion impairs the dental epithelial barrier to bacterial proinflammatory products. Antibodies to LDC from Eikenella corrodens (Ecor-LDC) inhibit LDC activity and retard gingival inflammation in beagle dogs. Whether E. corrodens is the major source of LDC in dental biofilms and whether the lysine analog tranexamic acid (TA) inhibits LDC activity, biofilm accumulation, and GCF exudation in a human gingivitis model were examined. Antibodies raised in goats to LDC-rich extracts from E. corrodens cell surfaces were used to inhibit Ecor-LDC and detect it in biofilm extracts using Western blots. Ecor-LDC activity was measured at pH 4.0 to 11.0 and its TA dissociation constant (Ki) at pH 7.0. Young adults used a 5% or 10% TA mouthwash three times daily during OHR for 1 week. Ecor-LDC antibodies and TA inhibited biofilm LDC. Ki of TA for Ecor-LDC was 940 μM. TA reduced plaque index (PI) by downshifting the PI correlation with biofilm lysine content after OHR without TA. GCF was correspondingly suppressed. However, greater TA retention in saliva partially relieved GCF suppression but not biofilm lysine depletion. TA slightly inhibits LDC but strongly reduces biofilm by inhibiting bacterial lysine uptake. Unfortunately, TA may impair dental epithelial attachments by also inhibiting lysine transporter uptake. Ecor-LDC inhibitors other than lysine analogs may maintain sufficient lysine levels and attachment integrity to prevent periodontal inflammation.

Bacterial Lysine Decarboxylase Influences Human Dental Biofilm Lysine Content, Biofilm Accumulation, and Subclinical Gingival Inflammation

Dental biofilms contain a protein that inhibits mammalian cell growth, possibly lysine decarboxylase from Eikenella corrodens. This enzyme decarboxylates lysine, an essential amino acid for dentally attached cell turnover in gingival sulci. Lysine depletion may stop this turnover, impairing the barrier to bacterial compounds. The aims of this study are to determine biofilm lysine and cadaverine contents before oral hygiene restriction (OHR) and their association with plaque index (PI) and gingival crevicular fluid (GCF) after OHR for 1 week. Laser-induced fluorescence after capillary electrophoresis was used to determine lysine and cadaverine contents in dental biofilm, tongue biofilm, and saliva before OHR and in dental biofilm after OHR. Before OHR, lysine and cadaverine contents of dental biofilm were similar and 10-fold greater than in saliva or tongue biofilm. After 1 week of OHR, the biofilm content of cadaverine increased and that of lysine decreased, consistent with greater biofilm lysine decarboxylase activity. Regression indicated that PI and GCF exudation were positively related to biofilm lysine after OHR, unless biofilm lysine exceeded the minimal blood plasma content, in which case PI was further increased but GCF exudation was reduced. After OHR, lysine decarboxylase activity seems to determine biofilm lysine content and biofilm accumulation. When biofilm lysine exceeds minimal blood plasma content after OHR, less GCF appeared despite more biofilm. Lysine appears important for biofilm accumulation and the epithelial barrier to bacterial proinflammatory agents. Inhibiting lysine decarboxylase may retard the increased GCF exudation required for microbial development and gingivitis.

Reversible brain response to an intragastric load of l-lysine under l-lysine depletion in conscious rats

L-Lysine (Lys) is an essential amino acid and plays an important role in anxiogenic behaviour in both human subjects and rodents. Previous studies have shown the existence of neural plasticity between the Lys-deficient state and the normal state. Lys deficiency causes an increase in noradrenaline release from the hypothalamus and serotonin release from the amygdala in rats. However, no studies have used functional MRI (fMRI) to compare the brain response to ingested Lys in normal, Lys-deficient and Lys-recovered states. Therefore, in the present study, using acclimation training, we performed fMRI on conscious rats to investigate the brain response to an intragastric load of Lys. The brain responses to intragastric administration of Lys (3 mmol/kg body weight) were investigated in six rats intermittently in three states: normal, Lys-deficient and recovered state. First, in the normal state, an intragastric load of Lys activated several brain regions, including the raphe pallidus nucleus, prelimbic cortex and the ventral/lateral orbital cortex. Then, after 6 d of Lys deprivation from the normal state, an intragastric load of Lys activated the ventral tegmental area, raphe pallidus nucleus and hippocampus, as well as several hypothalamic areas. After recovering from the Lys-deficient state, brain activation was similar to that in the normal state. These results indicate that neural plasticity in the prefrontal cortex, hypothalamic area and limbic system is related to the internal Lys state and that this plasticity could have important roles in the control of Lys intake.

Chlamydia pneumoniae Seropositivity and Hyperhomocysteinemia Are Linked in Patients with Atherosclerosis

Atherosclerosis, with its different clinical manifestations, is the major cause of morbidity and mortality in the developed countries. Chlamydia pneumoniae infection and the ensuing chronic inflammation have been claimed to contribute to the atherosclerotic process (1). Concurrently, epidemiologic evidence suggests that hyperhomocysteinemia is associated with an increased atherosclerotic risk (2). Both C. pneumoniae infection and hyperhomocysteinemia have been assumed to increase the atherosclerotic risk independently of each other and independently of the classic risk factors. In vitro, the growth of C. pneumoniae is enhanced in serum-free media and particularly by the depletion of lysine or methionine (3). In human metabolism, homocysteine is produced by demethylation of methionine, and defects in the recycling pathway (in which homocysteine is remethylated to methionine) are one cause of hyperhomocysteinemia (4). This constellation prompted us to investigate whether C. pneumoniae seropositivity and plasma homocysteine concentrations are related in patients with established atherosclerosis. Approval for this project was obtained from the ethics committee of the University Hospital of Zurich. All patients gave informed consent to participate. The study subjects consulted our ward for follow-up visits and were recruited consecutively. Inclusion criteria were that subjects had to be male and have had an established atherosclerosis defined as the following: ( a ) history of myocardial infarction or …

Identification of lysine decarboxylase as a mammalian cell growth inhibitor in Eikenella corrodens: possible role in periodontal disease

The pathogenesis of inflammatory periodontal disease was studied by examining the mechanism of HeLa and HL60 cell growth inhibition by cell-free saline-soluble extracts of Eikenella corrodens and bacterial plaque. Previous studies identified a protein (p80) as causing growth inhibition by E. corrodens extracts. After purification by two-dimensional SDS-PAGE, p80 was digested with protease lysC. Amino acid sequences were obtained and backtranslated for use as PCR primers. A 5840 nucleotide sequence containing a lysine decarboxylase gene was obtained from a Sau3 A1 genomic library ofE. corrodens DNA. Lysine decarboxylase activity was present at physiologic pH in the E. corrodens extracts containing p80, and also in bacterial plaque. Both extracts caused growth inhibition by depleting lysine from cell culture media through conversion to cadaverine. Adding lysine, or immune goat IgG to a peptide derived from the active site sequence of E. corrodens lysine decarboxylase, retarded lysine depletion and growth inhibition. -Amino caproic acid specifically enhanced lysine decarboxylase activity at the low lysine concentration in HL60 cell culture media, and also increased the growth inhibition. Thus, lysine decarboxylases such as p80 inhibit growth by removing lysine from mammalian cell culture media. A new role for lysine decarboxylase activity in the microbial aetiology of periodontal disease is discussed.

Feeding a Lysine-free Diet Decreases Food Intake without Changes in Hypothalamic Monoamine Concentrations in Growing Chicks

The objective of this study was to determine the relationship between the early reduction in intake of a lysine-free diet and the changes in hypothalamic monoamine concentration in growing chicks. Food intake was hourly measured after presentation of the control and lysine-free diets for 7 hours. At first, third and seventh hours, chicks were decapitated and brains were removed. Monoamines were determined in the hypothalamus using a HPLC-ECD system. Food intakes from 4 to 5 and 5 to 6 hours were significantly less in chicks on the lysine-free diet than in the control chicks. Initial concentrations of monoamines (mean values with SD, ng per mg wet tissue) in the hypothalamic area including the three nuclei were as follows; serotonin (5-hydroxytryptamine, 5-HT) 9.0±1.8, norepinephrine 2.0±0.5, epinephrine 1.51±0.38 and dopamine O.405±0.100. In general, the concentration of hypothalamic monoamines did not significantly vary with the dietary lysine depletion. These results indicated that the early reduction of food intake by feeding the lysine-free diet occurred without the change in the overall concentration of monoamines in the hypothalamus.

The autolytic ('suicidase') system of Enterococcus hirae: From lysine depletion autolysis to biochemical and molecular studies of the two muramidases of Enterococcus hirae ATCC 9790

The autolytic ('suicidase') system of Enterococcus hirae: From lysine depletion autolysis to biochemical and molecular studies of the two muramidases of Enterococcus hirae ATCC 9790

The autolytic ('suicidase') system of Enterococcus hirae: From lysine depletion autolysis to biochemical and molecular studies of the two muramidases of Enterococcus hirae ATCC 9790

The autolytic ('suicidase') system of Enterococcus hirae: From lysine depletion autolysis to biochemical and molecular studies of the two muramidases of Enterococcus hirae ATCC 9790

The effect of lysine deprivation on leukemic blood

Studies have shown that specific amino acids are required for optimal growth of leukemic versus normal cells, and it is believed that the depletion of selected amino acids can abrogate tumor growth. We have developed a technique for studying the effect of amino acid deprivation on leukemic cell proliferation. The technique is based on the controlled enzymatic removal of the amino acid from leukemic blood and the subsequent measurement of cell proliferative capacity. The specific system being studied is the removal of lysine from blood using immobilized L-lysineα-oxidase.A reactor has been designed that consists of L-lysineα-oxidase and catalase co-immobilized within the void space of the porous region of asymmetric hollow fiber (ultrafiltration) membranes. Blood from leukemic sheep is currently being treatedin vitro with this reactor. By varying treatment time, the amount of enzyme immobilized, and the blood flow rate, the amount of lysine removed from the blood can be varied and controlled. Preliminary data indicate that 80% depletion of lysine from leukemic blood is enough to cause a significant (25%) decrease in total white cell count as well as a decrease in the proliferative capacity of the leukemic cells.

Characteristics of two lysine-independent strains of Streptococcus faecalis.

SUMMARY The ease of isolation of two mutants of Streptococcus faecalis 8043 appeared to be the result of the presence in the parent strain of a system for slow synthesis of lysine. The mutation to lysine independence occurred when the population became large enough for a significant number of spontaneous mutational events to occur. The slow growth and lysine depletion lysis of the parent strain selected for the new strain. The hydroxylysine-resistant mutant can arise from the parent strain or from the lysine-independent strain and is selected for by growth in a medium containing hydroxylysine. The three strains appeared to be identical by all tests employed except in their responses to lysine and hydroxylysine.

Control of lysine biosynthesis in yeast by a feedback mechanism.

Homocitric acid (beta-hydroxy-beta-carboxyadipic acid; HC) is accumulated by a lysine-requiring yeast mutant when grown in a chemically defined medium, supplemented with limited amounts of lysine. A study of the formation of HC in relation to the depletion of lysine from the growth medium indicates that HC accumulated only when the concentration of lysine was low. The enzymatic formation of HC from alpha-ketoglutarate plus acetyl-coenzyme A in cell-free extracts of the same organism was also inhibited by lysine. The inhibitory effect of lysine on the formation of HC in both whole cells and cell-free extracts is indicative of the functional existence of a feedback control mechanism in the pathway for lysine biosynthesis in yeast.

The behaviour of aspartate semialdehyde as a precursor of the intra- and extracellular amino acids in Escherichia coli 26-26

1. 1. In an attempt to gain some data on the precursor behaviour of aspartate semialdehyde in the biosynthesis of intra- and extracellular amino acids by the lysine-requiring mutant Escherichia coli 26-26, [ 14C]aspartate semialdehyde has been used. 2. 2. The amino acids examined included the free and bound amino acids from the culture medium and the amino acids present in the cell-wall and cytoplasmic proteins. The amino acid composition and isotopic incorporation into amino acids of the cell walls, cytoplasmic proteins and extracellular lipopolysaccharide-protein are given. 3. 3. The aspartate semialdehyde causes a delay in the normal rate of biosynthesis of the extracellular diaminopimelic acid until depletion of lysine has occurred. The relative isotopic incorporation into the several forms of diaminopimelic acid and lysine allows one to suggest the existence of different pathways for the biosynthesis of lysine. 4. 4. The greatest isotopic incorporation into intra- and extracellularly bound amino acids is exhibited by glycine, threonine, valine and tyrosine. Diaminopimelic acid, alanine, valine and isoleucine are the free amino acids present in the culture medium, the biosynthetic levels of which are given. The radioactive distribution in the carbon atoms of valine is discussed in terms of the fate of aspartate semialdehyde. 5. 5. The paper concludes with a discussion of the results in terms of the general biochemical properties of the mutant.