The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20C and pH 69 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20 degrees C and pH 6.9 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.     

Characterization of a Ustilago maydis gene specifically induced during the biotrophic phase: evidence for negative as well as positive regulation

The phytopathogenic basidiomycete Ustilago maydis requires its host plant, maize, for completion of its sexual cycle. To investigate the molecular events during infection, we used differential display to identify plant-induced U. maydis genes. We describe the U. maydis gene mig1 (for "maize-induced gene"), which is not expressed during yeast-like growth of the fungus, is weakly expressed during filamentous growth in axenic culture, but is extensively upregulated during plant infection. mig1 encodes a small, highly charged protein of unknown function which contains a functional N-terminal secretion sequence and is not essential for pathogenic development. Adjacent to mig1 is a second gene (mdu1) related to mig1, which appears to result from a gene duplication. mig1 gene expression during the infection cycle was assessed by fusing the promoter to eGFP. Expression of mig1 was absent in hyphae growing on the leaf surface but was detected after penetration and remained high during subsequent proliferation of the fungus until teliospore formation. Successive deletions as well as certain internal deletions in the mig1 promoter conferred elevated levels of reporter gene expression during growth in axenic culture, indicative of negative regulation. During fungal growth in planta, sequence elements between positions -148 and -519 in the mig1 promoter were specifically required for high levels of induction, illustrating additional positive control. We discuss the potential applications of mig1 for the identification of inducing compounds and the respective regulatory genes.     

Increases in enzyme levels during the formation of phenolic acids in carrot cell cultures

The levels of glutamic acid dehydrogenase (GDH), phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CAH) and O-methyltransferase (OMT) were measured during the formation of phenolic acids in carrot cells in suspension culture. Caffeic, ferulic and p-hydroxybenzoic acids were always present as the culture proceded. Total content of these acids increased at the early logarithmic and linear phases. GDH showed high activity at the early logarithmic and stationary phases. PAL activity was much enhanced at the linear and stationary phases. CAH activity was found in actively growing cells, especially at the early and late logarithmic phases OMT behaved similarly to PAL. The increases in GDH and CAH might be responsible for the rapid synthesis of phenolic acid at the early logarithmic phase. The increase in phenolic acid at the linear phase would certainly be due to enhancements of both PAL and OMT. On the other hand, the accumulation of vanillic acid was observed in cells which were transferred and cultured on an agar medium, but not in cells in suspension culture. This accumulation is related to increases in OMT levels and also to changes in the degree of β-oxidation.     

Study of stationary phase metabolism via isotopomer analysis of amino acids from an isolated protein

Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully ¹³C‐labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Proliferation related acidic leucine-rich protein PAL31 functions as a caspase-3 inhibitor

Proliferation related acidic leucine-rich protein PAL31 (PAL31) is expressed in proliferating cells and consists of 272 amino acids with a tandem structure of leucine-rich repeats in the N-terminus and a highly acidic region with a putative nuclear localization signal in the C-terminus. We previously reported that PAL31 is required for cell cycle progression. In the present study, we found that the antisense oligonucleotide of PAL31 induced apoptosis to the transfected Nb2 cells. Stable transfectants, in which PAL31 was regulated by an inducible promoter, were generated to gain further insight into the signaling role of PAL31 in the regulation of apoptosis. Expression of PAL31 resulted in the marked rescue of Rat1 cells from etoposide and UV radiation-induced apoptosis and the cytoprotection was correlated with the levels of PAL31 protein. Thus, cytoprotection from apoptosis is a physiological function of PAL31. PAL31 can suppress caspase-3 activity but not cytochrome c release in vitro, indicating that PAL31 is a direct caspase-3 inhibitor. In conclusion, PAL31 is a multifunctional protein working as a cell cycle progression factor as well as a cell survival factor.     

Effects of Glyphosate on Metabolism of Phenolic Compounds: V. l-alpha-AMINOOXY-beta-PHENYLPROPIONIC ACID AND GLYPHOSATE EFFECTS ON PHENYLALANINE AMMONIA-LYASE IN SOYBEAN SEEDLINGS

The phenylalanine ammonia-lyase (PAL) inhibitor l-alpha-aminooxy-beta-phenylpropionic acid (AOPP) was root-fed to light-exposed soybean seedlings alone or with glyphosate [N-(phosphonomethyl)glycine] to test further the hypothesis that PAL activity is involved in the mode of action of glyphosate. Extractable PAL activity was increased by 0.01 and 0.1 millimolar AOPP. AOPP reduced total soluble hydroxyphenolic compound levels and increased phenylalanine and tyrosine levels, indicating that in vivo PAL activity was inhibited by AOPP. The increase in extractable PAL caused by AOPP may be a result of decreased feedback inhibition of PAL synthesis by cinnamic acid and/or its derivatives. AOPP alone had no effect on growth (fresh weight and elongation) at either concentration, but at 0.1 millimolar it slightly alleviated growth (fresh weight) inhibition caused by 0.5 millimolar glyphosate after 4 days. Reduction of the free pool of phenylalanine by glyphosate was reversed by AOPP. These results indicate that glyphosate exerts some of its effects through reduction of aromatic amino acid pools through increases in PAL activity and that not all growth effects of glyphosate are due to reductions of aromatic amino acids.     

Glycolysis in Ustilago maydis

The kinetic parameters of the 10 glycolytic enzymes and glycolytic fluxes were determined for the first time in Ustilago maydis. Enzyme activities in yeast grown in minimal medium and harvested in the stationary stage were twofold higher than those from yeast grown in rich medium. In contrast, in yeast harvested in the exponential stage, the enzyme activities were higher in cells grown in rich medium. Phosphofructokinase activity was the lowest in the four culture conditions analyzed, suggesting that this enzyme is a flux-controlling step in U. maydis glycolysis. The V(max) and K(m) values of hexokinase and pyruvate kinase were similar under all conditions. The results revealed that U. maydis aldolase belongs to the class II type of metalo-aldolases. 3-Phosphoglycerate mutase (PGAM) activity was 2,3-bisphosphoglycerate cofactor independent, which contrasted with the cofactor dependency predicted by the amino acid sequence alignment analysis. Pyruvate was secreted by U. maydis yeast in the presence and absence of external glucose. The glycolytic enzyme activities in the U. maydis mycelial form were similar to those found in yeast, except for one order of magnitude higher phosphofructokinase and PGAM activities, thus suggesting differences in the glycolysis regulatory mechanisms between the two cellular forms.     

Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated.     

Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides.

The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.     

Indole-3-acetic acid (IAA) biosynthesis in the smut fungus Ustilago maydis and its relevance for increased IAA levels in infected tissue and host tumour formation

Infection of maize (Zea mays) plants with the smut fungus Ustilago maydis is characterized by excessive host tumour formation. U. maydis is able to produce indole-3-acetic acid (IAA) efficiently from tryptophan. To assess a possible connection to the induction of host tumours, we investigated the pathways leading to fungal IAA biosynthesis. Besides the previously identified iad1 gene, we identified a second indole-3-acetaldehyde dehydrogenase gene, iad2. Deltaiad1Deltaiad2 mutants were blocked in the conversion of both indole-3-acetaldehyde and tryptamine to IAA, although the reduction in IAA formation from tryptophan was not significantly different from Deltaiad1 mutants. To assess an influence of indole-3-pyruvic acid on IAA formation, we deleted the aromatic amino acid aminotransferase genes tam1 and tam2 in Deltaiad1Deltaiad2 mutants. This revealed a further reduction in IAA levels by five- and tenfold in mutant strains harbouring theDeltatam1 andDeltatam1Deltatam2 deletions, respectively. This illustrates that indole-3-pyruvic acid serves as an efficient precursor for IAA formation in U. maydis. Interestingly, the rise in host IAA levels upon U. maydis infection was significantly reduced in tissue infected with Deltaiad1Deltaiad2Deltatam1 orDeltaiad1Deltaiad2Deltatam1Deltatam2 mutants, whereas induction of tumours was not compromised. Together, these results indicate that fungal IAA production critically contributes to IAA levels in infected tissue, but this is apparently not important for triggering host tumour formation.     

Peculiarities of amino acid transport inSchizosaccharomyces pombe: Effects of growth medium

Transport systems for amino acids in the wild-type strain ofSchizosaccharomyces pombe are not constitutive. During growth on different media no transport of acidic, neutral and basic amino acids is detectable. To acquire the ability to transport amino acids, cells must be preincubated with a metabolic source of energy, such as glucose. The appearance of transport activity is associated with protein synthesis (suppression by cycloheximide) at all phases of culture growth. After such preincubation the initial rate of amino acid uptake depends on the phase of growth of the culture and on the amount of glucose in the growth medium but not on the nitrogen source used.l-Proline and 2-aminoisobutyric acid are practically not transported under any of the conditions tested.     

L-proline is an essential amino acid for hepatocyte growth in culture

For improvement of the culture conditions of adult rat hepatocytes in primary culture in collagen coated dishes, effects of various commercial culture media on the induction of replicative DNA synthesis of the cells stimulated by insulin plus epidermal growth factor were studied. Proline-deficient media, such as Leibovitz's L-15, Eagle's minimal essential medium and Dulbecco's modified minimal essential medium, did not induce DNA synthesis in hepatocytes, whereas proline-rich media, such as Williams medium E, McCoy's 5A and Ham's F-12, induced markedly hepatocyte proliferation. Moreover, when the proline-deficient media were supplemented with L-proline, the cells synthesized DNA in response to the two hormones. Cis-4-hydroxyl-L-proline strongly inhibited the induction of DNA synthesis, without affecting protein synthesis of the cells or showing any cytotoxicity. This inhibition was recovered completely by adding excess proline to the medium. Addition of other amino acids not present in the medium did not promote DNA synthesis. These findings indicate that L-proline is essential for induction of hepatocyte proliferation in culture, through its affect on synthesis of intracellular collagen.     

Induction of phenolic compounds in wheat (Triticum aestivum L.) tissue cultures by streptomycin

The tissue cultures of wheat (Triticum aestivum L.) were induced from the mature embryos (explants) of the dry grains and grown on MS medium containing kinetin (0.1 mg/1) and 2,4 D (1.0 mg/l). The cultures were incubated for two weeks at (25+/-2) degrees C under a light/dark regime (16 h light daily). The formed calli were subcultured at the beginning of the stationary growth phase (15 days) with fresh MS medium containing 0, 5, 10, 25, 50, 100, 150 mg/l streptomycin elicitor and maintained for two weeks for three subcultures. A significant increase in phenylalanine ammonia lyase (PAL) activity coincided with the increase of the total phenolic compounds after elicitation with streptomycin. Maximum induction was recorded during the first two weeks, then gradually declined during the rest of the experimental period, but the values attained were still markedly higher than that of the control. The endogenous cinnamic acid content was also increased significantly with the increase in PAL activity making about 2-18% of the total phenolic acids. The growth and accumulation of phenolic compounds were inversely related. However, accumulation of phenolic compounds became limited for growth of wheat tissue culture especially during the long term cultivation.