Nitrogen-sufficient Conditions Research Articles

A novel gene whose expression in Medicago truncatula roots is suppressed in response to colonization by vesicular-arbuscular mycorrhizal (VAM) fungi and to phosphate nutrition.

A cDNA clone (Mt4) was isolated as a result of a differential screen to identify genes showing altered expression during the interaction between Medicago truncatula and the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus versiforme. Mt4 represents a M. truncatula mRNA that contains numerous short open reading frames, the two longest of which are predicted to encode polypeptides of 51 amino acids each. One of these open reading frames shares a short region of identity with a phosphate starvation-inducible gene from tomato. Mt4 gene expression is regulated in response to colonization by mycorrhizal fungi: transcripts were detected in non-colonized roots and levels decreased in both M. truncatula and M. sativa (alfalfa) roots after colonization by G. versiforme. Transcript levels also decreased during the incomplete interaction between G. versiforme and a M. sativa mycorrhizal minus (myc-) line, indicating that the down-regulation of this gene occurs early during the interaction between the fungus and its host plant. Phosphate levels in the nutrient media also affected the expression of the Mt4 gene: transcripts were present in the roots of plants grown under phosphate-deficient conditions, but were undetectable in the roots of plants grown under phosphate sufficient conditions. Furthermore, expression was only observed when plants were grown under nitrogen-sufficient conditions. Northern blot analyses indicate that Mt4 transcripts are present primarily in roots and barely detectable in stems or leaves. Thus, Mt4 represents a M. truncatula gene whose expression is regulated in response to both colonization by mycorrhizal fungi and to the phosphate status of the plant.

Activities of ligninolytic enzymes of the white rot fungus, phanerochaete chrysosporium and its recalcitrant substance degradability

The factors influencing the activities of extracellular ligninolytic enzymes synthesized by white rot fungus,Phanerochaete chrysosporium , were investigated by batch culture experiments. The LiP activity was maximal under the nitrogen-sufficient condition, compared with the activities under both conditions of the nitrogen-limited and nitrogen-excess. The manganese-dependent peroxidase (MnP) activity was highest under nitrogen-limited condition. Veratryl alcohol was found to be the most important substrate enhancing lignin peroxidase (LiP) activity in carbon-limited medium. The decolorization of azo dye (Reactive Red 22) by P. chrysosporium in the presence of both LiP and MnP under carbon-limited condition was greater than that in the presence of MnP under nitrogen-limited condition. Two chlorinated substances (2,6-DCP and MCPA) were degraded under conditions of nitrogen-limitation and notrogen-sufficiency. The azo dyes were also decolorized by the crude extracellular ligninolytic enzymes from P. chrysosporium.

Hydrogen Peroxide Production as a Limiting Factor in Xenobiotic Compound Oxidation by Nitrogen-Sufficient Cultures of Bjerkandera sp. Strain BOS55 Overproducing Peroxidases

The overproduction of ligninolytic peroxidase by the N-deregulated white rot fungus Bjerkandera sp. strain BOS55 under nitrogen-sufficient conditions had no noteworthy effect on the oxidation of anthracene or the decolorization of the polymeric aromatic dye Poly R-478 in 6-day-old cultures. Only when the endogenous production of H(inf2)O(inf2) was increased by the addition of extra oxygen and glucose could a 2.5-fold increase in the anthracene oxidation rate and a 6-fold increase in the Poly R-478 decolorization rate be observed in high-N cultures with 10- to 35-fold higher peroxidase activities than N-limited cultures. Further increase of the H(inf2)O(inf2) generation rate in high-N cultures with glucose oxidase led to an additional 3.5-fold increase in the anthracene oxidation rate (350 mg liter(sup-1) day(sup-1)) and a 10-fold increase in the Poly R-478 decolorization rate. These results indicate that xenobiotic compound oxidation by white rot fungi cannot be improved by overproducing peroxidases without increasing the endogenous production of H(inf2)O(inf2). The absence of Mn, which decreased the manganese peroxidase titers and increased the lignin peroxidase titers, was associated with up to 95% improvements in the anthracene oxidation rate. The simultaneous presence of Mn and veratryl alcohol was observed to have a synergistic negative effect on the oxidation of anthracene and the decolorization of Poly R-478.

Selective hyperproduction of manganese peroxidases byPhanerochaete chrysosporium l-1512 immobilized on nylon net in a bubble-column reactor

Manganese peroxidases were overproduced byPhanerochaete chrysosporium I-1512 immobilized on nylon net in a bubble-column reactor. This study investigates a new design of bioreactor, a compromise between a pneumatic reactor and an immobilized biofilm reactor. The carrier, a sheet of nylon net, was maintained by a cylindrical stainless-steel frame installed vertically. It was characterized by its hydrophilic nature, its surface morphology and its surface roughness.P. chrysosporium adhesion was highly efficient; mycelial hyphae invaded the tridimensional structure and strengthened the bonding to the network, as shown by electron scanning microscopy. High levels of Mn peroxidases were produced by strain I-1512 under conditions of glycerol and nitrogen sufficiency when the medium was supplemented with phospholipid and veratryl alcohol. Yields of 3600 U/l Mn peroxidase were produced after 95 h of incubation, indicating significant productivity for industrial purposes (900 U day−1 l−1).

Activities of ligninolytic enzymes of the white rot fungus, Phanerochaete chrysosporium and its recalcitrant substance degradability

The factors influencing the activities of extracellular ligninolytic enzymes synthesized by white rot fungus, Phanerochaete chrysosporium, were investigated by batch culture experiments. The LiP activity was maximal under the nitrogen-sufficient condition, compared with the activities under both conditions of the nitrogen-limited and nitrogen-excess. The manganese-dependent peroxidase (MnP) activity was highest under nitrogen-limited condition. Veratryl alcohol was found to be the most important substrate enhancing lignin peroxidase (LiP) activity in carbon-limited medium. The decolorization of azo dye (Reactive Red 22) by P. chrysosporium in the presence of both LiP and MnP under carbon-limited condition was greater than that in the presence of MnP under nitrogen-limited condition. Two chlorinated substances (2,6-DCP and MCPA) were degraded under conditions of nitrogen-limitation and notrogen-sufficiency, The azo dyes were also decolorized by the crude extracellular ligninolytic enzymes from P. chrysosporium.

Regulation of poly-β-hydroxybutyrate biosynthesis by nicotinamide nucleotide in Alcaligenes eutrophus

Poly-β-hydroxybutyrate biosynthesis was studied in Alcaligenes eutrophus under various nutrient-limiting conditions. When the cells were cultivated in nitrogen-limited media, both the levels of NAD(P)H and the ratios of NAD(P)H/NAD(P) were higher than those under nitrogen-sufficient conditions. The specific poly-β-hydroxybutyrate production rate was found to increase with the values of both NADH/NAD and NADPH/NADP, indicating that poly-β-hydroxybutyrate synthesis is directly regulated by the ratios of nicotinamide nucleotides. The effects of nicotinamide nucleotides on poly-β-hydroxybutyrate biosynthesis was investigated with regard to enzyme kinetics. Citrate synthase activity was significantly inhibited by NADH and NADPH, indicating that poly-β-hydroxybutyrate accumulation could be enhanced by facilitating the metabolic flux of acetyl-CoA to poly-β-hydroxybutyrate synthetic pathway. It was also found that cellular NADPH was a limiting substrate for NADPH-linked reductase, controlling the overall biosynthetic activity of poly-/3-hydroxybutyrate in this strain.

Regulation of poly-β-hydroxybutyrate biosynthesis by nicotinamide nucleotide in Alcaligenes eutrophus

Poly-β-hydroxybutyrate biosynthesis was studied in Alcaligenes eutrophus under various nutrient-limiting conditions. When the cells were cultivated in nitrogen-limited media, both the levels of NAD(P)H and the ratios of NAD(P)H/NAD(P) were higher than those under nitrogen-sufficient conditions. The specific poly-β-hydroxybutyrate production rate was found to increase with the values of both NADH/NAD and NADPH/NADP, indicating that poly-β-hydroxybutyrate synthesis is directly regulated by the ratios of nicotinamide nucleotides. The effects of nicotinamide nucleotides on poly-β-hydroxybutyrate biosynthesis was investigated with regard to enzyme kinetics. Citrate synthase activity was significantly inhibited by NADH and NADPH, indicating that poly-β-hydroxybutyrate accumulation could be enhanced by facilitating the metabolic flux of acetyl-CoA to poly-β-hydroxybutyrate synthetic pathway. It was also found that cellular NADPH was a limiting substrate for NADPH-linked reductase, controlling the overall biosynthetic activity of poly-/3-hydroxybutyrate in this strain.

Reductions catalyzed by a quinone and peroxidases from Phanerochaete chrysosporium

A quinone produced from veratryl alcohol by lignin peroxidase from the white rot fungus Phanerochaete chrysosporium was tested for its ability to mediate reduction. The quinone (2-hydroxymethyl-5-methoxy-1,4-benzoquinone), reduced chemically or by cellobiose:quinone reductase isolated from cultures of the fungus, mediated the reduction of cytochrome c in reactions containing either Mn(III), a manganese-dependent peroxidase, Mn(II) and H 2O 2, or lignin peroxidase and H 2O 2. Formation of the semiquinone, the species responsible for reducing cytochrome c, was observed by electron spin resonance spectroscopy in these reactions. The production of the quinone was observed in the extracellular fraction of cultures grown under nutrient nitrogen-deficient conditions (2.4 m m ammonium tartrate) for over 10 days, starting on Day 2, but not under nutrient nitrogen-sufficient conditions. These results suggest that a quinone produced by lignin peroxidase can serve as a physiological mediator of reductive reactions catalyzed by the fungal peroxidases.

Enhanced Epiphytic Coexistence of Near-Isogenic Salicylate-Catabolizing and Non-Salicylate-Catabolizing Pseudomonas putida Strains after Exogenous Salicylate Application

The hypothesis that epiphytic bacterial populations can coexist through nutritional resource partitioning was tested with the near-isogenic bacterial strain pair Pseudomonas putida R20 and R20(pNAH7). The plasmid pNAH7 conferred upon R20 the ability to catabolize salicylate as a sole carbon source in vitro. P. putida R20(pNAH7) also catabolized exogenously applied salicylate in planta and reached a significantly larger epiphytic population size than the near-isogenic parental strain R20 under the same conditions. This supports previous observations that epiphytic populations on plants grown under nitrogen-sufficient conditions are limited by carbon availability. In the absence of exogenous salicylate, R20 and R20(pNAH7) competed for and partitioned endogenous carbon according to their inoculum proportion in replacement series experiments, exhibiting a low level of coexistence. In the presence of exogenous salicylate, however, R20(pNAH7) was solely able to catabolize the additional carbon and achieved a higher level of coexistence with R20 than was possible in the absence of exogenous carbon.

Toxicity of Pentachlorophenol to Six Species of White Rot Fungi as a Function of Chemical Dose

The growth of six species of white rot fungi was a function of pentachlorophenol (PCP) dose, expressed as mass of PCP per mass of mycelia, at PCP doses </=35 mug mg of mycelium, and not concentration. At higher doses, Inonotus dryophilus, Perenniporia medulla-panis, and Ganoderma oregonense removed less PCP than three other species of white rot fungi. Phanerochaete chrysosporium grown under nitrogen-deficient conditions was inactivated at PCP doses that under nitrogen-sufficient conditions resulted in only 2-day lag periods in growth. Trametes versicolor was the fastest-growing species that remained viable at higher PCP doses. Both Trametes versicolor and Phellinus badius were able to degrade PCP at higher PCP doses.

Ligninase production by immobilized cultures of Phanerochaete chrysosporium grown under nitrogen-sufficient conditions

The production of lignin peroxidase (ligninase) by free and immobilized cultures of P. chrysosporium was studied in the range of N-concentration from 2.2 to 22 m m. As expected, free fungal pellets produced significant levels of ligninase activity (200 U l −1) only under N-limitation (2.2 m m). In contrast, in cultures of P. chrysosporium immobilized on pieces of polyurethane sponge or foam, the level of lignin peroxidase increases with the N-concentration and up to 190 and 340 U l −1 of ligninase activity were found under conditions of N-sufficiency (22 m m), respectively.

Heat-denaturation kinetics of lignin peroxidases from Phanerochaete chrysosporium

Many practical applications have been proposed for the lignin peroxidases from Phanerochaete chrysosporium. To design and develop these technologies, more information regarding the stability of these enzymes is required. Heat-denaturation studies were conducted on crude and partially purified lignin peroxidases obtained from P. chrysosporium under nitrogen-limiting and nitrogen-sufficient conditions. At temperatures of 37°C, or below, no significant loss of activity was observed over a 5-d period. At temperatures between 50 and 60°C, lignin peroxidase activity rapidly decreased. The heat-denaturation kinetics under these conditions was not first order. A four-parameter model that describes the heat-denaturation kinetics of crude and partially purified lignin peroxidases was developed. Based on the successful application of this model, it is suggested that P. chrysosporium produces lignin peroxidases that can be separated into at least two groups with similar decay constants within a group and different decay constants between groups.

Inactivation, sequence, and lacZ fusion analysis of a regulatory locus required for repression of nitrogen fixation genes in Rhodobacter capsulatus

Transcription of the genes that code for proteins involved in nitrogen fixation in free-living diazotrophs is typically repressed by high internal oxygen concentrations or exogenous fixed nitrogen. The DNA sequence of a regulatory locus required for repression of Rhodobacter capsulatus nitrogen fixation genes was determined. It was shown that this locus, defined by Tn5 insertions and by ethyl methanesulfonate-derived mutations, is homologous to the glnB gene of other organisms. The R. capsulatus glnB gene was upstream of glnA, the gene for glutamine synthetase, in a glnBA operon. beta-Galactosidase expression from an R. capsulatus glnBA-lacZ translational fusion was increased twofold in cells induced by nitrogen limitation relative to that in cells under nitrogen-sufficient conditions. R. capsulatus nifR1, a gene that was previously shown to be homologous to ntrC and that is required for transcription of nitrogen fixation genes, was responsible for approximately 50% of the transcriptional activation of this glnBA fusion in cells induced under nitrogen-limiting conditions. R. capsulatus GLNB, NIFR1, and NIFR2 (a protein homologous to NTRB) were proposed to transduce the nitrogen status in the cell into repression or activation of other R. capsulatus nif genes. Repression of nif genes in response to oxygen was still present in R. capsulatus glnB mutants and must have occurred at a different level of control in the regulatory circuit.

Lignin peroxidase production by strains of Phanerochaete chrysosporium grown on glycerol

Lignin peroxidase production by several strains of Phanerochaete chrysosporium was determined during growth on glycerol under conditions of nitrogen sufficiency. Fungal strains which grew poorest on glycerol produced the highest titres of lignin peroxidase whereas enzyme levels were much lower when marginally greater biomass values were recorded. In the case of P. chrysosporium strain INA-12, the nature of the nitrogen source had a pronounced effect on both growth and enzyme production. Highest biomass values were obtained when l-glutamate or l-glutamine served as the major nitrogen source but enzyme synthesis was normally repressed completely. Lignin peroxidase activity in this strain was maximal when the initial pH of the culture medium was adjusted to pH 5.0.

CHEMICAL PROFILE OF SELECTED SPECIES OF MICROALGAE WITH EMPHASIS ON LIPIDS1

ABSTRACTThe lipid profile of seven species of unicellular eukaryotic microalgae grown under controlled conditions was studied with emphasis on the hydrocarbons and the fatty acids as part of a search for oil‐producing algae. Green, slow‐growing colonies of Botryococcus braunii Kutz contained the highest lipid content of 45% based on the organic weight, with an increase to 55% under nitrogen deficiency and with no effect of sodium chloride stress. Ankistrodesmus sp. Thomas, Dunaliella spp., Isochrysis sp., Nannochloris sp. Thomas, and Nitzschia sp. Chapman contained an average of 25% lipids under nitrogen sufficient conditions. Nitrogen deficiency resulted in significant increase in the lipid content in all species but Dunaliella spp., which produced a higher content of carbohydrates. Significant low amounts of acyclic hydrocarbons were detected only in Botryococcus braunii Kutz and not in the other algae. The major hydrocarbon fractions in nitrogen deficient Botryococcus braunii Kutz, Dunaliella salina Thomas, Isochrysis spp. and Nannochloris sp. Thomas were cyclic and branched polyunsaturated components which were identified as various isoprenoid derivatives. The polar lipid composition of glycolipids and phospholipids of all species investigated was fairly typical of photosynthetic eukaryotic algae. Fatty acid composition was species specific, with changes occurring in the relative amounts of individual acids of cells cultivated under different conditions and growth phases. All species synthesized C14:0, C16.0, C18:1, C18:2 and C18:3 fatty acids; C 16:4 in Ankistrodesmus sp. Thomas; C18:4 and C 22.6 in Isochrysis sp.; C16:2, C16:3 and C20:5 in Nannochloris sp. Thomas; C16:2, C16:3 and C20:5 in Nitzschia sp. Chapman. Nitrogen deficiency and salt stress induced accumulation of C18:1 in all treated species and to a lesser extent in Botryococcus braunii Kutz. The low production of hydrocarbons under optimal growth conditions and the high production of hydrocarbons under limited growth conditions cannot support the notion that microalgae can be utilized as biosolar energy converters for the production of liquid fuel, but point to the availability of a variety of neutral and polar lipid products.

Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sufficiency

Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sufficiency

Yields, photosynthetic efficiencies and proximate composition of dense marine microalgal cultures. II. Dunaliella primolecta and Tetraselmis suecica experiments

Yields and efficiencies of light utilization were determined in two species of green marine microalgae, Dunaliella primolecta and Tetraselmis suecica. Under nitrogen-sufficient conditions, the maximum yield of D. primolecta was 12.09 g dry weight m −2 day −1 and the corresponding photosynthetic efficiency was 4.3% (based on PAR supplied; 400–700 nm). When cells became N-deficient, the yield and efficiency decreased to 10.2 g m −2 day −1 and 2.8%, respectively. Under N-sufficient conditions the maximum yield of T. suecica was 19.1 g m −2 day −1 with an efficiency of 7.3%. Deficiency reduced these values to 12.7 g m −2 day −1 and 3.6%, respectively. Cellular protein levels in both species were very much reduced by N deficiency and photosynthetically fixed carbon was shunted into the production of carbohydrate, which increased to up to 65% of the dry weight. Carbohydrate yields increased about five-fold in deficient cells. Deficiency may be useful in manipulating cells in outdoor cultures to form carbohydrate, which in turn could be converted to useful biomass energy resources — methane or ethanol.

Yields, photosynthetic efficiencies and proximate composition of dense marine microalgal cultures. III. Isochrysis sp. and Monallantus salina experiments and comparative conclusions

Yields and efficiencies of light utilization were determined for the Chrysophyte alga, Isochrysis (Tahitian strain), and the Xanthophyte, Monallantus salina. Under conditions of nitrogen sufficiency the maximum yield of the former alga was 47·9 g m −2 day −1 with an efficiency of light utilization based on PAR supplied (400–700 nm) of 11·6%. Nitrogen deficiency decreased these values to 7·6 g m −2 day −1 and 1·9% respectively. For Monallantus (N-sufficient) the maximum values were 13·9 g m −2 day −1 and 6·4% respectively; N deficiency reduced these values to 7·5 g m −2 day −1 and 3·7%, respectively. Deficiency reduced protein levels in both species and in Isochrysis metabolism was shunted towards the formation of carbohydrate. Carbohydrate yield was doubled by deficiency. In Monallantus, lipid content was not increased by deficiency; this is in contrast to previous reports. Rather an appreciable amount of some fraction ‘other’ than protein, carbohydrate, lipid or ash was formed. The results of all five marine microalgae studied in this series are compared. Phaedactylum gave the highest yield and efficiency, but similar values were obtained by Tetraselmis and Isochrysis. To produce the maximum amount of a high-energy component such as lipid, one would choose Isochrysis, and for high protein yield, Phaeodactylum or Tetraselmis would be chosen. The highest carbohydrate yield would be attained by Dunaliella, Tetraselmis, or Isochrysis under N-deficient conditions. These comparative results may be useful in choosing species and conditions for maximizing various biomass products in outdoor cultures.

The role fo glutamine synthetase and glutamine metabolism in nitrogen metabolite repression, a regulatory phenomenon in the lower eukaryote Neurospora crassa.

Growth of Neurospora crassa on media containing NH4+ leads to the repression of a variety of permeases and alternative pathways which would generate NH4+, so called "ammonium repression." The mutant am2 which lacks NADP-GDH is not subject to ammonium repression of nitrate reductase or urea permease, but like the wild type has repressed levels of these systems when grown in the presence of proline, glutamate or glutamine. The glutamine synthetase (GS) mutant gln-1a has derepressed levels of the aforementioned systems unless grown with glutamine. The oligomeric state of GS depends upon the nitrogen sufficiency of the cell, a tetrameric form predominates under conditions of nitrogen limitation and an octameric form under conditions of nitrogen sufficiency. We have found that the tetrameric form GS predominates in the mutants am2 and gln-1a when they are ammonium derepressed. Th mechanism of NH4+ repression in N. crassa is thought to entail a cessation of positive gene action by the product of the nit-2 regulatory gene. We propose that under conditions of NH4+ sufficiency, and hence glutamine sufficiency, the octameric form of GS represses nit-2 gene expression and thereby achieves ammonium repression.