Amino Acid-supplemented Medium Research Articles

Putative Horizontally Acquired Genes, Highly Transcribed during Yersinia pestis Flea Infection, Are Induced by Hyperosmotic Stress and Function in Aromatic Amino Acid Metabolism.

While alternating between insects and mammals during its life cycle, Yersinia pestis, the flea-transmitted bacterium that causes plague, regulates its gene expression appropriately to adapt to these two physiologically disparate host environments. In fleas competent to transmit Y. pestis, low-GC-content genes y3555, y3551, and y3550 are highly transcribed, suggesting that these genes have a highly prioritized role in flea infection. Here, we demonstrate that y3555, y3551, and y3550 are transcribed as part of a single polycistronic mRNA comprising the y3555, y3554, y3553, y355x, y3551, and y3550 genes. Additionally, y355x-y3551-y3550 compose another operon, while y3550 can be also transcribed as a monocistronic mRNA. The expression of these genes is induced by hyperosmotic salinity stress, which serves as an explicit environmental stimulus that initiates transcriptional activity from the predicted y3550 promoter. Y3555 has homology to pyridoxal 5'-phosphate (PLP)-dependent aromatic aminotransferases, while Y3550 and Y3551 are homologous to the Rid protein superfamily (YjgF/YER057c/UK114) members that forestall damage caused by reactive intermediates formed during PLP-dependent enzymatic activity. We demonstrate that y3551 specifically encodes an archetypal RidA protein with 2-aminoacrylate deaminase activity but Y3550 lacks Rid deaminase function. Heterologous expression of y3555 generates a critical aspartate requirement in a Salmonella entericaaspC mutant, while its in vitro expression, and specifically its heterologous coexpression with y3550, enhances the growth rate of an Escherichia coli ΔaspC ΔtyrB mutant in a defined minimal amino acid-supplemented medium. Our data suggest that the y3555, y3551, and y3550 genes operate cooperatively to optimize aromatic amino acid metabolism and are induced under conditions of hyperosmotic salinity stress.IMPORTANCE Distinct gene repertoires are expressed during Y. pestis infection of its flea and mammalian hosts. The functions of many of these genes remain predicted or unknown, necessitating their characterization, as this may provide a better understanding of Y. pestis specialized biological adaptations to the discrete environments of its two hosts. This study provides functional context to adjacently clustered horizontally acquired genes predominantly expressed in the flea host by deciphering their fundamental processes with regard to (i) transcriptional organization, (ii) transcription activation signals, and (iii) biochemical function. Our data support a role for these genes in osmoadaptation and aromatic amino acid metabolism, highlighting these as preferential processes by which Y. pestis gene expression is modulated during flea infection.

An enhanced genome-scale metabolic reconstruction of Streptomyces clavuligerus identifies novel strain improvement strategies.

In this work, we expanded and updated a genome-scale metabolic model of Streptomyces clavuligerus. The model includes 1021 genes and 1494 biochemical reactions; genome-reaction information was curated and new features related to clavam metabolism and to the biomass synthesis equation were incorporated. The model was validated using experimental data from the literature and simulations were performed to predict cellular growth and clavulanic acid biosynthesis. Flux balance analysis (FBA) showed that limiting concentrations of phosphate and an excess of ammonia accumulation are unfavorable for growth and clavulanic acid biosynthesis. The evaluation of different objective functions for FBA showed that maximization of ATP yields the best predictions for cellular behavior in continuous cultures, while the maximization of growth rate provides better predictions for batch cultures. Through gene essentiality analysis, 130 essential genes were found using a limited in silico media, while 100 essential genes were identified in amino acid-supplemented media. Finally, a strain design was carried out to identify candidate genes to be overexpressed or knocked out so as to maximize antibiotic biosynthesis. Interestingly, potential metabolic engineering targets, identified in this study, have not been tested experimentally.

New Beauveriolides Produced by Amino Acid‐Supplemented Fermentation of Beauveria sp. FO‐6979

AbstractFor Abstract see ChemInform Abstract in Full Text.

Bew Beauveriolides Produced by Amino Acid-supplemented Fermentation of Beauveria sp. FO-6979.

Five new beauveriolides were isolated from the acetone extracts of Beauveria sp. FO-6979 mycelia fermented in amino acid-supplemented media. The structures were elucidated by spectroscopic studies including NMR experiments and chemical degradation. All the beauveriolides are cyclodepsipeptides consisting of one 3-hydroxy-4-methyl fatty acid, two L-amino acids and one D-amino acid in common. Beauveriolide VII with the structure of cyclo-[3-hydroxy-4-methyloctanoyl-L-phenylalanyl-L-alanyl-D-valyl] inhibited lipid droplet formation and cholesteryl ester synthesis in macrophages, but the other beauveriolides showed only slight or almost no effect on lipid droplet formation.

Selective production of fungal beauveriolide I or III by fermentation in amino acid-supplemented media.

Beauveriolides I and III, cyclic depsipeptides composed of L-Phe, L-Ala, D-Leu and (3S,4S)-3-hydroxy-4-methyloctanoic acid, and L-Phe, L-Ala, L-allo-Ile and (3S,4S)-3-hydroxy-4-methyloctanoic acid, respectively, were previously isolated from the culture broth of fungal Beauveria sp. FO-6979 as inhibitors of macrophage foam cell formation. To improve the production of these compounds by fermentation, the culture conditions were studied. The production of both beauveriolides was increased five to ten folds by fermentation in the culture media containing tryptone. Further study revealed that addition of L-Leu/L-Ile, but not D-Leu/D-allo-Ile, to the culture medium yielded a high and selective production of beauveriolide I or III. As a result, regardless of their separation difficulty due to the similar physico-chemical properties, a large amount of beauveriolide I or III was prepared from the culture broth obtained from L-Leu- or L-Ile-supplemented fermentation, respectively, by one step purification using silica gel column chromatography.

Effect of amino acids, growth regulators and genotype on androgenesis in barley

The effects of an amino acid mixture and of plant growth regulators added to the FHG barley anther culture medium were examined using three barley cultivars (Cadette, Leger, and Igri) grown in two environments (growth cabinet and glasshouse). ‘Leger’ and ‘Igri’ were known as responsive, and ‘Cadette’ as recalcitrant to androgenesis. Our first experiment showed that the amino acid-supplemented medium was best for embryogenesis and regeneration of ‘Cadette’ and ‘Igri’ in both environments, and if ‘Leger’ in the growth cabinet. The addition of ABA and TDZ did not improve embryogenesis and plant regeneration, and PAA decreased them in the growth cabinet. The addition of the amino acid mixture in the FHG medium also reduced the percentage of albino plants in the growth cabinet, but growth regulators did not improve the percentage of albino plants, and in some cases increased it. In the growth cabinet, disregarding media, ‘Leger’ produced more embryos than ‘Cadette’ and ‘Igri’, and Leger' and ‘Igri’ produced more green plants than ‘Cadette‘. Percentages of albino plants were higher or ‘Cadette’ than for ‘Igri’ or ‘Leger’. In a second experiment, we compared seven hybrids with their parents for androgenic responsiveness. Hybrids had a higher ability to generate green plants than expected based upon the weighted average reflecting the contribution of each parent.

Characterization of Vacuolar Arginine Uptake and Amino Acid Efflux inNeurospora crassaUsing Cupric Ion to Permeabilize the Plasma Membrane

Treatment ofNeurospora crassamycelia with cupric ion has been shown to permeabilize the plasma and mitochondrial membranes. Permeabilized mycelia were shown to take up arginine into the vacuoles. Uptake was ATP-independent and appeared to be driven by an existing K+-gradient. The kinetic characteristics of the observed uptake were similar to those observed using vacuolar membrane vesicles: theKmfor arginine uptake was found to be 4.2–4.5 mM. Permeabilized mycelia were used to study the regulation of arginine uptake into vacuoles. The results suggest that uptake is relatively indifferent to the contents of the vacuoles and is not affected by growth of mycelia in amino acid-supplemented medium. Efflux of arginine, lysine, and ornithine from vacuoles was also measured using mycelia permeabilized with cupric ion. Arginine release was shown to be specifically enhanced by cytosolic ornithine and/or increases in the vacuolar pool of arginine or ornithine. Lysine efflux was shown be indifferent to the presence of other amino acids. These observations emphasize the importance of vacuolar compartmentation in controlling arginine and ornithine metabolism and suggest that vacuolar compartmentation may play an important role in nitrogen homeostasis of filamentous fungi.

Effects of Fis on Ribosome Synthesis and Activity and on rRNA Promoter Activities in Escherichia coli

The DNA-bending protein Fis of Escherichia coliis required for efficient initiation of chromosome replication and for activation of the P1 promoters of stable RNA (rRNA and tRNA) genes. Using fis +and fis −bacterial strains, we have determined ribosome synthesis and activity, rRNA gene dosage, and transcript initiation rates at the rrnBP1 and P2 promoters. Different growth media were used to achieve growth rates between 0.7 and 2.7 doublings/hour for the fis +strain, and between 0.7 and 2.2 doublings/hour for the fis −strain. In minimal media, the Fis deficiency only reduced the DNA and rrngene concentration (DNA, oriCcopies and rrngenes per protein); in amino acid-supplemented media, it also reduced rRNA synthesis rates per protein. Under all conditions, the ribosome activity (protein synthesis rate/ribosome) remained unchanged by the absence of Fis. In the presence of Fis, the absolute activities of the isolated rrnBP1 and P2 promoters increased from 2 to 88 and 10 to 50 initiations/minute, respectively, with increasing growth-rate. In the absence of Fis, these activities increased from 3 to 70 and 10 to 80 initiations/minute, respectively. Relative to the isolated rrnBP2 promoter, the strength of the rrnBP1 promoter was found to increase with increasing growth rate tenfold (from 0.17 to 1.7) in the presence, but only fivefold (from 0.17 to 0.85) in the absence of Fis. An evaluation of the data leads us to propose estimates of k catand relative K Mvalues for the two rRNA promoters, and relative values for free RNA polymerase concentrations during growth in different media. The analysis suggests that the reduced strength of P1 promoters of stable RNA genes in the absence of Fis, together with the reduced rrngene concentration, increases the concentration of free RNA polymerase. In addition, the lower rrnP1 promoter activity in the absence of Fis reduces the probability that the downstream P2 promoter is blocked (“occluded”) by a transcription elongation complex originating at the P1 promoter. The increased polymerase concentration and reduced P2 promoter occlusion both help to compensate for the Fis-deficiency, but during growth in rich media when the demand for ribosomes is high, this compensation is insufficient for fis −bacteria to achieve the wild-type level of ribosome synthesis and growth.

Suppression of oxidative envelope damage by pseudoreversion of a superoxide dismutase-deficient mutant of Escherichia coli

Mutants of Escherichia coli that are devoid of superoxide dismutase (SOD) fail to grow in aerobic minimal medium. This is largely because of the O2- sensitivities of several amino acid biosynthetic pathways, since amino acid supplements can restore growth, albeit at a slow rate. We now report that growth in amino acid-supplemented medium can be further stimulated by the presence of extracellular osmolytes. Osmolytes also partially suppress the amino acid requirements of the SOD mutant. These data suggest that the combination of oxidative injury and turgor pressure permeabilizes the cell envelope and that critical metabolites, including the limiting products of damaged biosynthetic pathways, escape from the cell. External osmolytes may offer protection by countervailing the usual turgor pressure and thus stabilizing the damaged envelope. This model is consistent with the previous observation that deficiency of cell wall components is lethal to SOD mutants. A pseudorevertant that can grow at a moderate rate in normosmotic medium without amino acid supplementation has been obtained (J. A. Imlay and I. Fridovich, Mol. Gen. Genet. 228:410-416, 1991). Analysis suggests that the suppressor mutation allows the envelope either to resist or to tolerate oxidative lesions. Study of the pseudorevertant may illuminate the molecular basis of this oxidative envelope injury.

The role of amino acids in the regulation of protein synthesis in perfused rat liver. II. Effects of amino acid deficiency on peptide chain initiation, polysomal aggregation, and distribution of albumin mRNA.

Decreased rates of protein synthesis which occurred in rat livers perfused with amino acid-deficient medium were accompanied by a loss of polysomes and a doubling of concentrations of ribosomal subunits and monomers as compared to unperfused liver or livers perfused with amino acid-supplemented medium. The loss of polysomes was not the result of mRNA degradation because this effect could be reversed by addition of amino acids to the perfusion medium. Instead, loss of polysomes indicated an impairment in peptide chain initiation. To determine whether the block of initiation could be the result of sequestration of mRNA in untranslatable pools, the content of albumin mRNA in membrane-bound and free polysomes and in the non-polysomal fraction isolated on sucrose density gradients was determined by hybridization to an albumin cDNA. In livers perfused with an amino acid-supplemented medium, 90% of the albumin mRNA was found in bound polysomes, and with amino acid-deficient medium, this value was decreased to 80%. Thus, there was no indication of a significant pool of mRNA which was not being translated. The block of initiation was, however, accompanied by a marked change in density and methionine-binding characteristics of the 40 S ribosomal subunit in livers perfused with deficient medium as compared to livers perfused with supplemented medium or to unperfused livers. There was a relative loss of 40 S subunits in a low density form, 1.41 g/cm3, an increase in the proportion in a high density form, 1.48 g/cm3, and a decrease in binding of [35S]methionine to 40 S ribosomal subunits. These changes provide evidence of reduced rates of formation of the 40 S initiation complex.

The effect of essential amino acid deprivation on poly(A) metabolism in Neurospora crassa

Poly(A) messenger RNA was isolated from a methionine auxotroph of Neurospora crassa grown to exponential and early stationary phases, and from mycelia deprived of the essential amino acid. The polyadenylate regions were isolated from the mRNA preparations by enzymatic digestion and affinity chromatography, and their lengths determined by electrophoresis on 10% acrylamide 90% formamide gels. There was no significant difference in the lengths of the poly(A) regions between exponential and early stationary phase cultures. However, the poly(A) regions of mRNA isolated from cells undergoing amino acid deprivation were larger and more heterogeneous than the poly(A) regions isolated from cells grown in amino acid-supplemented medium. Whole cell extracts prepared from both amino acid-supplemented and amino acid-deprived cultures were assayed for their ability to synthesize and degrade poly(A). No significant difference between the two extracts was detected in either poly(A) polymerase or poly(A)-degrading activities. It was concluded that continuing translational activity is required for the shortening of poly(A) tracts in N. crassa.

Studies on vitamin B6 metabolism in microorganisms. Part XX. Vitamin B6 biosynthesis of a Pdx mutant,Escherichia coli B WG3,in amino acid-supplemented medium.

Alanine, cystine, cysteine, β-hydroxypyruvate, an amino acid mixture and glycolaldehyde, which were found to satisfy the vitamin B6 (B6) requirement of Escherichia coli B WG3, a pdxB mutant, by paper disc assay method, were examined about the effect on growth of the mutant in liquid cultivation. Alanine and β-hydroxypyruvate did not significantly affected growth under the culture conditions, whereas cysteine and cystine supported growth to some extent. The amount of B6 synthesized by the mutant in the amino acid-supplemented medium was from 0.05 to 0.12 nmol per ml. The amount of B6 synthesized in the medium, which was supplemented with an amino acid mixture composed of l-cysteine, l-threonine and l-methionine, was about 0.15 nmol per ml. This amount of B6 was lower than the amount (0.4 nmol per ml) biosynthesized in glycolaldehyde-supplemented medium. The data suggested that growth of the mutant supplemented with amino acids could not be supported by only the B6 biosynthesized from them, because such amo...

Chloramphenicol-Induced Changes in the Synthesis of Ribosomal, Transfer, and Messenger Ribonucleic Acids in Escherichia coli B/r

The synthesis of ribosomal ribonucleic acid (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA) was measured in Escherichia coli B/r after the addition of 100 mug of chloramphenicol (CAM) per ml to cultures growing either in one of three minimal media (succinate, glycerol, or glucose) or in one of the same three media supplemented with 20 amino acids. (i) During CAM treatment, rRNA and tRNA were synthesized in the same relative proportions (85:15) as during exponential growth. The faster accumulation of tRNA relative to rRNA in CAM was due to a decreased stability of rRNA that is synthesized in the presence of or immediately before the addition of CAM. (ii) CAM stimulated the synthesis of rRNA and tRNA two- to eightfold. The results fell into two groups; one group was from studies done in minimal media and the other was from amino acid-supplemented media. In each group the stimulation decreased with increasing growth rate of the culture during exponential growth before the addition of CAM; however, the stimulation in minimal media was lower than that in amino acid-supplemented media. (iii) CAM caused an increase in the proportion of rRNA and tRNA synthesis and a corresponding decrease in the proportion of mRNA synthesis. In minimal media, the residual proportion of mRNA synthesis after CAM treatment was 10 to 15% of total RNA synthesis; in amino acid-supplemented media this proportion was 0 to 10%. In either case, the residual proportion of mRNA synthesis was independent of the proportions observed during exponential growth in these media. (iv) The absolute rate of mRNA synthesis decreased severalfold with the addition of CAM; i.e., the rate of synthesis of rRNA and tRNA was increased at the expense of mRNA synthesis. (v) During exponential growth, the fraction of the instantaneous rate of total RNA synthesis that corresponds to mRNA is a function of both the growth rate and the presence or absence of amino acids in the growth medium: in the absence of amino acids, this fraction decreased with increasing growth rate; in the presence of amino acids, the fraction increased slightly with growth rate. These results are consistent with a regulation of rRNA and tRNA synthesis at the transcriptional level, e.g., with a CAM-induced increase in the affinity of RNA polymerase for the rRNA and tRNA promoters. The results also suggest the occurrence of a regulation of RNA polymerase enzyme activity, i.e., of an activation of RNA polymerase that is inactive during exponential growth. A distinction between these alternatives requires measurements of the rRNA chain growth rates during CAM treatment.