Changes In Amino Acid Concentrations Research Articles

The effect of arsenic contamination on amino acids metabolism in Spinacia oleracea L.

Changes of amino acid concentrations (proline, glutamate, asparagine, aspartate, alanine) and glutamate kinase activity (GKA) in plants under arsenic chronic stress reported here reveal their role in plant arsenic stress adaptation. Results of the pot experiment confirmed the toxic effect of arsenic at tested levels (As1=25mgAskg−1 soil, As2=50mgAskg−1 soil, As3=75mgAskg−1 soil) for spinach. Growing available arsenic contents in soil were associated with the strong inhibition of above-ground biomass and with the enhancement of As plant content. The changes of glutamate, asparagine, aspartate and proline levels in the plants showed strong linear dependences on arsenic concentration in plants (R2=0.60–0.90). Compared to the untreated control, concentrations of free proline and aspartate of As3 treatment were enhanced up to 381% and 162%, respectively. The significant changes of glutamate were observed on As2 and As3 treatments (increased level up to 188, i.e. 617%). Arsenic in plants was shown to be an inhibitor of glutamase kinase activity (R2=0.91). Inhibition of GKA resulted in an increase in the content of glutamate that is used in synthesis of phytochelatins in plant cells. Concentration of alanine did not have a confirmed linear dependence on arsenic concentration in plant (R2=0.05). The changes of its concentrations could be affected by changes of pH in plant cell or induction of alanine aminotransferase by hypoxia.

Effect of processing on the amino acid content of New Zealand spinach (Tetragonia tetragonioides Pall. Kuntze)

SummaryThe aim of this work was to evaluate the effect of processing on the amino acid content and protein quality of New Zealand spinach (Tetragonia tetragonioides Pall. Kuntze). In this research, fresh and cooked New Zealand spinach as well as two frozen products prepared for consumption, one obtained using the traditional method (blanching–freezing–storage–cooking) and the other a convenience food product obtained using a modified process (cooking–freezing–storage–defrosting and heating in a microwave oven), were analysed. Glutamic acid was the dominant amino acid in fresh New Zealand spinach, and the limiting amino acids were cystine and methionine. Technological and culinary processing caused a significant increase in amino acid content in 100 g of edible portion, with the exception of methionine and cystine in frozen products prepared for eating. Changes in amino acid content expressed in g/16 g of N (which corresponded to 100 g tissue protein) were not significant, with the exception of the lower glutamic acid content in the frozen product obtained using traditional processing method.

Ex-vivo changes in amino acid concentrations from blood stored at room temperature or on ice: implications for arginine and taurine measurements

BackgroundDetermination of the plasma concentrations of arginine and other amino acids is important for understanding pathophysiology, immunopathology and nutritional supplementation in human disease. Delays in processing of blood samples cause a change in amino acid concentrations, but this has not been precisely quantified. We aimed to describe the concentration time profile of twenty-two amino acids in blood from healthy volunteers, stored at room temperature or on ice.MethodsVenous blood was taken from six healthy volunteers and stored at room temperature or in an ice slurry. Plasma was separated at six time points over 24 hours and amino acid levels were determined by high-performance liquid chromatography.ResultsMedian plasma arginine concentrations decreased rapidly at room temperature, with a 6% decrease at 30 minutes, 25% decrease at 2 hours and 43% decrease at 24 hours. Plasma ornithine increased exponentially over the same period. Plasma arginine was stable in blood stored on ice, with a < 10% change over 24 hours. Plasma taurine increased by 100% over 24 hours, and this change was not prevented by ice. Most other amino acids increased over time at room temperature but not on ice.ConclusionPlasma arginine concentrations in stored blood fall rapidly at room temperature, but remain stable on ice for at least 24 hours. Blood samples taken for the determination of plasma amino acid concentrations either should be placed immediately on ice or processed within 30 minutes of collection.

Delayed Deproteinization Causes Methodological Errors in Amino Acid Levels in Plasma Stored at Room Temperature or -20°C

Deproteinization has been recognized as a prerequisite for amino acid analysis of plasma samples. For plasma stored at room temperature, delaying deproteinization for 30, 60 or 120 minutes did not result in significant changes in the mean CV (coefficient of variation), which ranged from 4.4 to 5.6%. However the mean CV of aspartic acid, α-aminoadipic acid, alanine and lysine was about 10%. When the plasma was stored frozen at -20°C, the CV was increased at 0 and 120 minutes after thawing, to 12.4% (range, 4.1 to 35.3%) and 8.0% (2.5 to 30.7%), respectively. The concentrations in plasma during storage at room temperature of all the amino acids analyzed showed significant changes. In plasma stored for 30 minutes at room temperature, 17 amino acids increased in concentrations and two decreased. Extending this period to 60 or 120 minutes increased the instability as compare to the reference group. Storing plasma at -20°C for 2 weeks resulted in significantly greater changes in the amino acid concentrations than at room temperature. On extending the storage time at room temperature, after thawing, to 30, 60, and 120 minutes, 21, 20, and all 22 amino acids respectively changed significantly (p<0.01). The present study indicates that methodological errors occur in the concentrations determined for all amino acids when plasma is left at room temperature. The storage of frozen non-deproteinized plasma accompanied more significant changes in most amino acid concentrations and thus should be avoided. Deproteinization should be performed as soon as possible after plasma collection.

Local and systemic effects of two herbivores with different feeding mechanisms on primary metabolism of cotton leaves

Caterpillars and spider mites are herbivores with different feeding mechanisms. Spider mites feed on the cell content via stylets, while caterpillars, as chewing herbivores, remove larger amounts of photosynthetically active tissue. We investigated local and systemic effects of short-term caterpillar and spider mite herbivory on cotton in terms of primary metabolism and growth processes. After short-term caterpillar feeding, leaf growth and water content were decreased in damaged leaves. The glutamate/glutamine ratio increased and other free amino acids were also affected. In contrast, mild spider mite infestation did not affect leaf growth or amino acid composition, but led to an increase in total nitrogen and sucrose concentrations. Both herbivores induced locally increased dark respiration, suggesting an increased mobilization of storage compounds potentially available for synthesis of defensive substances, but did not affect assimilation and transpiration. Systemically induced leaves were not significantly affected by the treatments performed in this study. The results show that cotton plants do not compensate the loss of photosynthetic tissue with higher photosynthetic efficiency of the remaining tissue. However, early plant responses to different herbivores leave their signature in primary metabolism, affecting leaf growth. Changes in amino acid concentrations, total nitrogen and sucrose content may affect subsequent herbivore performance.

Control of mammalian gene expression by amino acids, especially glutamine

Molecular data rapidly accumulating on the regulation of gene expression by amino acids in mammalian cells highlight the large variety of mechanisms that are involved. Transcription factors, such as the basic-leucine zipper factors, activating transcription factors and CCAAT/enhancer-binding protein, as well as specific regulatory sequences, such as amino acid response element and nutrient-sensing response element, have been shown to mediate the inhibitory effect of some amino acids. Moreover, amino acids exert a wide range of effects via the activation of different signalling pathways and various transcription factors, and a number of cis elements distinct from amino acid response element/nutrient-sensing response element sequences were shown to respond to changes in amino acid concentration. Particular attention has been paid to the effects of glutamine, the most abundant amino acid, which at appropriate concentrations enhances a great number of cell functions via the activation of various transcription factors. The glutamine-responsive genes and the transcription factors involved correspond tightly to the specific effects of the amino acid in the inflammatory response, cell proliferation, differentiation and survival, and metabolic functions. Indeed, in addition to the major role played by nuclear factor-kappaB in the anti-inflammatory action of glutamine, the stimulatory role of activating protein-1 and the inhibitory role of C/EBP homology binding protein in growth-promotion, and the role of c-myc in cell survival, many other transcription factors are also involved in the action of glutamine to regulate apoptosis and intermediary metabolism in different cell types and tissues. The signalling pathways leading to the activation of transcription factors suggest that several kinases are involved, particularly mitogen-activated protein kinases. In most cases, however, the precise pathways from the entrance of the amino acid into the cell to the activation of gene transcription remain elusive.

ASN2 is a key enzyme in asparagine biosynthesis under ammonium sufficient conditions

The modification and enhancement of plant amino acid accumulation is potentially beneficial in terms of the production of suitable crop plants for food or feeds, and also with respect to possible environmental improvement via the effective use of nitrogen nutrition. It is also expected that modified plants will produce beneficial nitrogenous compounds, such as proteins, secondary metabolites, or nucleic acids. The size of amino acid pools in plants is strictly controlled by certain environmental factors such as light and nutrition. In order to gain an understanding of the mechanisms that control amino acid biosynthesis and metabolism, we investigated the diurnal changes in amino acid contents and also how the amino acid contents change with the progression of growth. We observed that almost all the amino acids in plant leaves undergo diurnal changes and that the pattern of these changes was different in young and old stages. We focused on correlation of the asparagine content and the expression of ASN2 asparagine synthetase, since under our experimental conditions their fluctuating patterns were found to be similar. The asparagine contents of ASN2-overexpressing and -underexpressing plants were increased and decreased, respectively, when they were grown under normal light and nutrient conditions. These changes in asparagine content were marked when the plants were grown under conditions where ammonium was the sole nitrogen source. It has been previously reported that ASN2 expression and ammonium metabolism are correlated. Our findings and these previous observations suggest that ASN2 functions as a regulator of asparagine biosynthesis and metabolism and that it mediates the effective use of nitrogen under ammonium-sufficient conditions.

Colonic luminal ammonia and portal blood l-glutamine and l-arginine concentrations: a possible link between colon mucosa and liver ureagenesis

The highest ammonia concentration in the body is found in the colon lumen and although there is evidence that this metabolite can be absorbed through the colonic epithelium, there is little information on the capacity of the colonic mucosa to transfer and metabolize this compound. In the present study, we used a model of conscious pig with a canula implanted into the proximal colon to inject endoluminally increasing amounts of ammonium chloride and to measure during 5 h the kinetics of ammonia and amino acid concentration changes in the portal and arterial blood. By injecting as a single dose from 1 to 5 g ammonia into the colonic lumen, a dose-related increase in ammonia concentration in the portal blood was recorded. Ammonia concentration remained unchanged in the arterial blood except for the highest dose tested, i.e. 5 g which thus apparently exceeds the hepatic ureagenesis capacity. By calculating the apparent net ammonia absorption, it was determined that the pig colonic epithelium has the capacity to absorb 4 g ammonia. Ammonia absorption through the colonic epithelium was concomitant with increase of L-glutamine and L-arginine concentrations in the portal blood. This coincided with the expression of both glutamate dehydrogenase and glutamine synthetase in isolated colonic epithelial cells. Since L-glutamine and L-arginine are known to represent activators for liver ureagenesis, we propose that increased portal concentrations of these amino acids following increased ammonia colonic luminal concentration represent a metabolic link between colon mucosa and liver urea biosynthesis.

Changes in amino acid concentration in plasma and type I and type II fibres during resistance exercise and recovery in human subjects

Eight male subjects performed leg press exercise, 4 x 10 repetitions at 80% of their maximum. Venous blood samples were taken before, during exercise and repeatedly during 2 h of recovery. From four subjects, biopsies were taken from the vastus lateralis muscle prior to, immediately after and following one and 2 h of recovery. Samples were freeze-dried, individual muscle fibres were dissected out and identified as type I or type II. Resistance exercise led to pronounced reductions in the glutamate concentration in both type I (32%) and type II fibres (70%). Alanine concentration was elevated 60-75% in both fibre types and 29% in plasma. Glutamine concentration remained unchanged after exercise; although 2 h later the concentrations in both types of fibres were reduced 30-35%. Two hours after exercise, the plasma levels of glutamate and six of the essential amino acids, including the branched-chain amino acids were reduced 5-30%. The data suggest that glutamate acts as an important intermediate in muscle energy metabolism during resistance exercise, especially in type II fibres.

Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles

The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40-50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and (13)C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. (13)C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20-24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down-regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.

Effects of cochlear ablation on amino acid concentrations in the chinchilla posteroventral cochlear nucleus, as compared to rat

Using a microchemical approach, we measured changes of amino acid concentrations in the chinchilla caudal posteroventral cochlear nucleus (PVCN) after cochlear ablation to determine to what extent slow decreases of glutamate and aspartate concentrations after carboplatin treatment resulted from slower effects of cochlear damage in chinchillas than in rats and guinea pigs, as opposed to effects of carboplatin treatment being slower than those of cochlear ablation. Our results indicate that both factors are involved: decreases of glutamate and aspartate concentrations after cochlear ablation are much slower in chinchillas than in rats and guinea pigs, but they are much faster than the decreases after carboplatin treatment. Further, aspartate and glutamate concentrations in the chinchilla caudal PVCN decreased by larger amounts after cochlear ablation than in rats or guinea pigs, and there was a transient increase of aspartate concentration at short survival times. Detailed mapping of amino acid concentrations in the PVCN of a chinchilla with 1 month survival after cochlear ablation and a rat with 7 days' survival indicated that the reductions of glutamate and aspartate occurred throughout the PVCN but were somewhat larger in ventral and caudal parts in chinchilla. Any decreases in the adjacent granular region were very small. There were also sustained bilateral decreases in concentrations of other amino acids, notably GABA and glycine, in the caudal PVCN of cochlea-ablated chinchillas but not rats. The effects of cochlear ablation on the concentrations of most of these other amino acids in chinchilla caudal PVCN differed from those of carboplatin treatment. Thus, although a major effect of auditory nerve damage on the cochlear nucleus—decreases of glutamate and aspartate concentrations—occurs across species and types of lesions, the details of timing and magnitude and the effects on other amino acids can vary greatly.

CAMBIOS POSTCOSECHA DEL HONGO COMESTIBLE HUITLACOCHE (Ustilago maydis (D.C.) Corda)

Huitlacoche (Ustilago maydis ( D.C.) Corda) has been an important food in the diet of the inhabitants of the Central-Southern region of Mexico since pre-Columbian times. Recently, its acceptance has increased all around the world. Although it is a highly perishable mushroom, there are no postharvest studies. For these reasons the objective of this work was to identify changes in physical and physiological postharvest parameters of huitlacoche on the corn ear and of cut huitlacoche galls stored at room temperature, 10 °C, and 3 °C for 11 d. The measured variables were changes in appearance, color, weight loss, respiration, production of ethylene and amino acids. The average respiration rate of huitlacoche on the ear at room temperature, 10 and 3 °C was 320.8, 120.8 and 71.5 mLCO2·kg -1 ·h -1 , respectively, and 372.8, 346.7 and 164.1 mLCO2·kg -1 ·h -1 in cut galls, respectively. Weight loss was above 80, 60 and 22 % at room temperature, 10 and 3 °C at 7 days, respectively. Huitlacoche contains 18 amino acids, of these, essential amino acids accounted for 37.8 % of the total. Lysine was 27.2 % of the essential amino acids. After the storage period, there was no major change in amino acid content. The huitlacoche kept its appearance, color and weight better when it was stored at 3 oC on the ear. Ethylene production by huitlacoche was not detected.

Polymorphisms in large neutral amino acids transporter, system L, in association with CNS disorders

Maintenance of the amino acids (AAs) levels within the central nervous system (CNS) is of importance for the formation of neurotransmitters. Alterations of the l-tryptophan and l-tyrosine (precursors of serotonin and dopamine, respectively) in brain parenchyma may result in serious CNS disorders such as depression. Malfunction of the system L (in particular LAT1/4F2hc) transporter can result in inevitable fluctuation of the large neutral amino acids (NAAs). From our preliminary mutation detection analyses, we hypothesize that the light chain LAT1 (SLC7A5) polymorphisms may change functionality of the system L resulting in fluctuation of key large NAAs levels in CNS. Also, mutations in 4F2hc (SLC3A2), the heavy chain of various AAs transporters, may alter the functions of some key transporters and cause changes in AAs concentrations of the brain. If proven, this hypothesis would grant new insights in molecular biology of the large neutral amino acid transporters in relevance to CNS disorders.

Alteration of Brain Levels of Neurotransmitters and Amino Acids in Male F344 Rats Induced by Three-week Repeated Inhalation Exposure to 1-Bromopropane

The present study investigated the effects of 1-bromopropane (1BP) on brain neuroactive substances of rats to determine the extent of its toxicity to the central nervous system (CNS). We measured the changes in neurotransmitters (acetylcholine, catecholamine, serotonin and amino acids) and their metabolites or precursors in eight brain regions after inhalation exposure to 1BP at 50 to 1,000 ppm for 8 h per day for 7 d per week for 3 wk. Rats were sacrificed at 2 h (Case 1), or at 19 h (Case 2) after the end of exposure. In Case 1, the level of 5-hydroxyindoleacetic acid (5HIAA) was lowered in some brain regions by 1BP exposure. The decrease of 5HIAA in the frontal cortex was statistically significant at 50 ppm 1BP exposure. In Case 2, gamma-amino butyric acid (GABA) and taurine were decreased in many brain regions of exposed rats, and a significant decrease of taurine in the midbrain occurred at 50 ppm 1BP exposure. In both cases of 2-h and 19-h intervals from the end of exposure to sacrifice, aspartate and glutamine levels were elevated in many brain regions, but the acetylcholine level did not change in any brain region. Three-week repeated exposure to 1BP produced significantly changes in amino acid contents of rat brains, particularly at 1,000 ppm.

Visualization of Arginine Influx into Plant Cells Using a Specific FRET-sensor

Amino acids are not only the building blocks of proteins, but are the metabolic precursors of a variety of primary and secondary metabolites. In order to detect and visualize how plants transport, sense, and store amino acids with sub-cellular specificity, chimeric fluorescent proteins that respond to changes in amino acid concentrations were constructed. The reporter element of these sensors consists of a periplasmic bacterial protein that undergoes large, non-enzymatic conformational changes upon binding of its substrate. The receptor protein was attached to ECFP and an environmentally insensitive YFP derivative at opposite ends. Fluorescence resonance energy transfer changes were specifically observed after addition of arginine and to a lesser extent ornithine. The recombinant sensor showed a concentration-dependent increase in the fluorescence ratio with an apparent in vitro affinity for arginine of approximately 2 mM. A mutation in the binding pocket lowered the affinity and decreased the specificity. When expressed in E. coli, an increase in the fluorescence ratio was specifically detected after exposure to arginine and ornithine. Transient expression of the sensor in plant cell protoplasts and stable expression in Arabidopsis roots revealed specific fluorescence changes upon addition of arginine. The analysis suggests that fluorescent amino acid sensors may be versatile tools for studying the in vivo dynamics of metabolism and compartmentalization in plants.

Acute Alterations of Glutamate, Glutamine, GABA, and Other Amino Acids After Spinal Cord Contusion in Rats

Spinal cord injury (SCI) leads to an alteration of energetic metabolism. As a consequence, glutamate, glutamine, aspartate and other important amino acids are altered after damage, leading to important disregulation of the neurochemical pathways. In the present study, we characterized the acute-phase changes in tissue concentration of amino acids involved in neurotransmitter and non-neurotransmitter actions after SCI by contusion in rats. Animals were submitted to either laminectomy or SCI by contusion and sacrificed at 2, 4, 8, and 12 h after lesion, for the analysis of tissue amino acids by HPLC. Results showed that both aspartate and glutamate contents diminished after SCI, while glutamine concentrations raised, however, the sum of molar concentrations of glutamate plus glutamine remained unchanged at all time points. GABA concentrations increased versus control group, while glycine remained unchanged. Finally, citrulline levels increased by effect of SCI, while taurine-increased only 4 h after lesion. Results indicate complex acute-phase changes in amino acids concentrations after SCI, reflecting the different damaging processes unchained after lesion.

Changes of amino acid concentrations in the rat vestibular nuclei after inferior cerebellar peduncle transection

Although there is a close relationship between the vestibular nuclear complex (VNC) and the cerebellum, little is known about the contribution of cerebellar inputs to amino acid neurotransmission in the VNC. Microdissection of freeze-dried brain sections and high-performance liquid chromatography (HPLC) were combined to measure changes of amino acid concentrations within the VNC of rats following transection of the cerebellovestibular connections in the inferior cerebellar peduncle. Distributions of 12 amino acids within the VNC at 2, 4, 7, and 30 days after surgery were compared with those for control and sham-lesioned rats. Concentrations of gamma-aminobutyric acid (GABA) decreased by 2 days after unilateral peduncle transection in nearly all VNC regions on the lesioned side and to lesser extents on the unlesioned side and showed partial recovery up to 30 days postsurgery. Asymmetries between the two sides of the VNC were maintained through 30 days. Glutamate concentrations were reduced bilaterally in virtually all regions of the VNC by 2 days and showed complete recovery in most VNC regions by 30 days. Glutamine concentrations increased, starting 2 days after surgery, especially on the lesioned side, so that there was asymmetry generally opposite that of glutamate. Concentrations of taurine, aspartate, and glycine also underwent partially reversible changes after peduncle transection. The results suggest that GABA and glutamate are prominent neurotransmitters in bilateral projections from the cerebellum to the VNC and that amino acid metabolism in the VNC is strongly influenced by its cerebellar connections.

Changes in vitreous amino acid concentrations in a rabbit model of cataract surgery

We previously reported marked elevation of glutamate, gamma-aminobutyric acid (GABA) and alanine in the vitreous after combined cataract surgery and vitrectomy for macular disorder. Photo-stimulation by the operating microscope and increased intraocular pressure (IOP) are possible causes of postoperative increases in amino acids. An animal model was used to verify this hypothesis. Forty-five coloured rabbits were studied; stress was applied to simulate cataract surgery. Simultaneous photic and pressure stress, photic stress alone, or pressure stress alone were exerted for 20 mins. The vitreous was extracted at the end of the stress period, or 10 mins or 30 mins later. Levels of six amino acids (asparate, glutamate, glycine, taurine, alanine and GABA) in the vitreous were analysed quantitatively. Significant increases in vitreous glutamate concentrations in treated eyes were observed following simultaneous photic and pressure stress, as well as after pressure stress alone, whereas no difference was found after photic stress alone. The mean glutamate concentrations in treated eyes and fellow eyes, respectively, were 5.59 +/- 2.03 microM and 4.36 +/- 2.09 microM (p < or = 0.05) 10 mins after simultaneous photic and pressure stress, and 4.32 +/- 0.97 microM and 2.29 +/- 0.51 microM (p < 0.05) 10 mins after pressure stress alone. Taurine concentration was elevated by pressure stress alone at 0 min and at 10 mins post-stress. Alanine concentration was reduced by photic stress alone at 10 mins post-stress. In all protocols, no significant differences between treated and fellow eyes were observed at 30 mins after stress. Time-dependent changes were observed not only in the treated eyes, but also in untreated fellow eyes. Elevations of vitreous glutamate in cataract surgery may be caused by pressure stress due to irrigation. Photic stress caused by the operating microscope has little influence. Sympathetic response may be involved in amino acid changes due to stress in cataract surgery.

Screening of Enterobacterial Contamination During Gelatin Production and its Effect on Pharmaceutical Grade Gelatin

Gelatin, an animal protein derived from collagen has many industrial applications; mainly in food and pharmaceutical products. In this study, enterobacterial contamination of gelatin during different stages of its manufacturing was examined. Since gelatin is extracted in the form of liquor by hot water treatment of ossein and undergoes a complex series of processing stages before being finally blended and packaged off as dry gelatin product, contamination at any stage affects the quality of the final product. In total, 142 samples of gelatin were analysed for the presence of enteric bacteria, which were obtained from different stages of gelatin processing. The Enterobacteriaceae-positive samples were processed and these enterobacterial species were isolated and identified as Proteus mirabilis, Serratia marcescens, E. coli, Salmonella typhi, Klebsiella oxytoca, Klebsiella pneumoniae, Shigella flexneri and Serratia liquefaciens. These were tested for gelatinase activity. The eight species producing maximum gelatinolysis were selected and protease zymography was performed using 1% (w/v) gelatin as a substrate in 7.5% (w/v) acrylamide gel and their molecular weights were determined. The effect of these bacteria on the quality of gelatin was assessed chromatographically in terms of change in amino acid content of gelatin broth inoculated by these species, which showed a marked change with length of incubation. Since these enteric bacteria possess pathogenic properties, some of them are gelatinolytic and also have a significant effect on the quality and amino acid content of gelatin, they are of great concern both for the manufacturers as well as for the consumers.

Regulation of Transcription in a Reduced Bacterial Genome: Nutrient-Provisioning Genes of the Obligate SymbiontBuchnera aphidicola

Buchnera aphidicola, the obligate symbiont of aphids, has an extremely reduced genome, of which about 10% is devoted to the biosynthesis of essential amino acids needed by its hosts. Most regulatory genes for these pathways are absent, raising the question of whether and how transcription of these genes responds to the major shifts in dietary amino acid content encountered by aphids. Using full-genome microarrays for B. aphidicola of the host Schizaphis graminum, we examined transcriptome responses to changes in dietary amino acid content and then verified behavior of individual transcripts using quantitative reverse transcriptase PCR. The only gene showing a consistent and substantial (>twofold) response was metE, which underlies methionine biosynthesis and which is the only amino acid biosynthetic gene retaining its ancestral regulator (metR). In another aphid host, Acyrthosiphon pisum, B. aphidicola has no functional metR and shows no response in metE transcript levels to changes in amino acid concentrations. Thus, the only substantial transcriptional response involves the one gene for which an ancestral regulator is retained. This result parallels that from a previous study on heat stress, in which only the few genes retaining the global heat shock promoter showed responses in transcript abundance. The irreversible losses of transcriptional regulators constrain ability to alter gene expression in the context of environmental fluctuations affecting the symbiotic partners.