Presence Of Different Nitrogen Sources Research Articles

Function of a hydrophobin in growth and development, nitrogen regulation, and abiotic stress resistance of Ganoderma lucidum

Abstract Fungal hydrophobins have many important physiological functions, such as maintaining hydrophobicity and affecting virulence, growth, and development. In Ganoderma lucidum, the molecular regulation mechanisms of hydrophobins in mushroom are unclear. In this study, we investigated a hydrophobin protein 1 (Hyd1) in G. lucidum, which belongs to the fungal Class I hydrophobins. The hyd1 gene was highly expressed during the formation of primordia, and expression was the lowest in fruiting bodies. Through the construction of hyd1 silenced strains, we found that primordia formation was not initiated in these strains. This finding indicated that Hyd1 played an important role in the development of G. lucidum. Second, AreA, a key transcription factor in nitrogen metabolism, negatively regulated the expression of hyd1. In an areA-silenced strain, the expression of hyd1 increased by ∼14-fold compared with that of the wild-type (WT) strain. Electrophoretic mobility shift assays (EMSA) indicated binding of AreA to the promoter of hyd1. Additionally, expression of hyd1 was determined in the presence of different nitrogen sources. Compared with that in the ammonia nitrogen source, the expression of hyd1 in nitrate nitrogen source significantly increased. Finally, we found that hyd1 plays important roles not only in nitrogen regulation but also in the resistance to other abiotic stresses. After silencing of hyd1, the resistance to heat, cell wall, and salt stresses decreased. Our findings reveal the important roles of Hyd1 in the development and resistance to abiotic stresses in G. lucidum and provide insights into the nitrogen regulation mechanism of hydrophobins in higher basidiomycetes.

Effect of nitrogen source on mycelial growth, blastospore production and thermotolerance of Cordyceps fumosorosea (Hypocreales: Cordycipitaceae)

Among the entomogenous fungi, Cordyceps fumosorosea has great potential for the control of piercing-sucking insects in greenhouses. The blastospores of C. fumosorosea developed in liquid fermentation are more efficient for industrialization because they can kill insects faster and be produced faster than conidia from solid cultivation. Thermotolerance of blastospores plays a major role in their application in greenhouses. The added nutrient elements during its cultivation process have a significant impact on the thermotolerance of blastospores. Understanding the effects of nitrogen sources on the mycelial growth, sporulation and thermotolerance of C. fumosorosea is essential for its commercial application. Frequently used nitrogen sources were investigated in fermentation medium, including peptone, soya peptone, yeast extract, ammonium sulfate, urea and so on. Soya peptone provided the maximum blastospore yield and GT50, which were 5.17 × 108 blastospores mL−1 and 1.91 h, respectively. To explore the difference in intracellular substances involved in the thermotolerance of blastospores in the presence of different nitrogen sources, intracellular trehalose and mannitol were extracted and then qualified by ion chromatography. The results showed that blastospores with better thermotolerance generally had lower intracellular trehalose content, indicating that the intracellular trehalose content of blastospores could be a factor that the fungus responds to thermal stress and that its content in the blastospores could be adjusted by the supplemented nitrogen source.

Novel Glutamate-Putrescine Ligase Activity in Haloferax mediterranei: A New Function for glnA-2 Gene.

The genome of the halophilic archaea Haloferax mediterranei contains three ORFs that show homology with glutamine synthetase (GS) (glnA-1, glnA-2, and glnA-3). Previous studies have focused on the role of GlnA-1, suggesting that proteins GlnA-2 and GlnA-3 could play a different role to that of GS. Glutamine synthetase (EC 6.3.1.2) belongs to the class of ligases, including 20 subclasses of other different enzymes, such as aspartate–ammonia ligase (EC 6.3.1.1), glutamate–ethylamine ligase (EC 6.3.1.6), and glutamate–putrescine ligase (EC 6.3.1.11). The reaction catalyzed by glutamate–putrescine ligase is comparable to the reaction catalyzed by glutamine synthetase (GS). Both enzymes can bind a glutamate molecule to an amino group: ammonium (GS) or putrescine (glutamate–putrescine ligase). In addition, they present the characteristic catalytic domain of GS, showing significant similarities in their structure. Although these proteins are annotated as GS, the bioinformatics and experimental results obtained in this work indicate that the GlnA-2 protein (HFX_1688) is a glutamate–putrescine ligase, involved in polyamine catabolism. The most significant results are those related to glutamate–putrescine ligase’s activity and the analysis of the transcriptional and translational expression of the glnA-2 gene in the presence of different nitrogen sources. This work confirms a new metabolic pathway in the Archaea domain which extends the knowledge regarding the utilization of alternative nitrogen sources in this domain.

Rhizosphere microbiome dynamics in tropical seagrass under short-term inorganic nitrogen fertilization.

Rhizosphere microbes are crucial to seagrass meadows because they promote plant growth and heath. However, information concerning the response of rhizosphere microorganisms in seagrass sediment in the presence of different nitrogen sources is lacking. Here, by means of high-throughput sequencing, we investigated how addition of inorganic nitrogen affects the rhizosphere microbiome of the tropical seagrass Thalassia hemperichii. A seagrass culture system was set up to conduct a nitrogen addition (ammonium and nitrate) simulation experiment. We found that the relative abundance of Proteobacteria and Bacteroidetes was increased in inorganic nitrogen-enriched samples, whereas that of Acidobacteria decreased under ammonium enrichment, especially after 35days. High levels of inorganic nitrogen addition caused a significant decrease in the relative abundance of Desulfobacteraceae, Sulfurovaceae, and Spirochaetes, which are primarily involved in sulfur cycling. Additionally, the abundance of microbes in the seagrass rhizosphere reached the highest after the ammonium-enrichment treatment. Among the analyzed seagrass photosynthetic characteristics, seagrass leaves presented the highest light utility in treatments receiving nitrate, followed by the control groups and ammonium-enrichment groups. Moreover, 16S rRNA gene-predicted functional analysis suggested that some functions related to metabolism of amino acids and signal transduction were enriched in samples receiving high ammonium, whereas nitrate addition enriched predicted functions related to diseases. These findings provide new insights into the response of microbial communities to different types of nitrogen additions in seagrass ecosystems.

Description of a new marine planktonic cyanobacterial species Synechococcus moorigangaii (Order Chroococcales) from Sundarbans mangrove ecosystem

The genus Synechococcus is widespread across marine environments globally including in coastal habitats. In this study, culture of a new isolate (CMS01) of Synechococcus has been established and described based on polyphasic taxonomy from the world’s largest mangrove ecosystem, Sundarbans. This planktonic photoautrotroph has been proposed as a new species Synechococcus moorigangaii sp. nov. belonging to the order Chroococcales. The cells representing this proposed new species are solitary and can also form chain comprising of 4–6 cells. The shape of cell is oval to cylindrical and length ranges from 1.2–3 µm while the width ranges from 0.8–2 µm. The distribution pattern of photosynthetic filaments was found to be from the periphery of cell. Based on robust phylogeny of 16S rRNA, in addition to functional genes such as psbA, ureC, rbcL, and cbbA (multi gene phylogeny), the proposed new species differed from closest described species of Synechococcus under order Chroococcales. The fatty acid analysis indicated the presence of C12 and C14 chain fatty acids exclusive to isolate CMS01. The new isolate can grow across a range of salinity and in presence of different nitrogen sources. It has the ability to fix atmospheric di-nitrogen into ammonium ion. This new isolate of Synechococcus spp. is the first marine planktonic cyanobacterium described from a mangrove ecosystem and characterized using polyphasic approaches. Based on 16S rRNA phylogeny, this proposed new species clustered with Synechococcus strains PCC 7117, PCC 73109, PCC 7002, PCC 7003, PCC 7376 and NKNG15041c belonging to the order Chroococcales. The new species Synechococcus moorigangaii sp. nov. can serve as a model organism to understand ecophysiology and adaptation of planktonic cyanobacterial communities in mangrove ecosystems.

Studies on “Effect of Organic Nitrogen Source on Production of Antifungal Substance from Clostridium Species”

Objective: In this study, antifungal substances were isolated from different soil bacteria like Clostridium species in the presence of different nitrogen sources. The isolated antifungal substances were then tested against Aspergillus and Trichoderma species to analyse their potential activity. Methodology and Results: Antifungal substances were isolated from different soil bacterial species and it was observed that only Clostridium species was suitable to isolate active antifungal substances against Aspergillus and Trichoderma species in the presence of different nitrogen sources. Peptone and yeast extract were good nitrogen sources to isolate most active antifungal substances from Clostridium species. Agar well diffusion method was used to measure the zone of inhibitions and the maximum zone of inhibition was found up to 22 mm. Conclusion: Yeast extract and Peptone were found to be good nitrogen sources for the growth of Clostridium sp. YLC and antibiotic production.

Characterization of a new peptide transport system in Streptococcus thermophilus

In silico analysis of the genome of Streptococcus thermophilus LMD-9 revealed that this strain has a potential new peptide/nickel ABC transporter. We named this system OTS for Oligopeptide Transporter of S. thermophilus. It is composed of a peptide/nickel binding protein OtsA, two permeases OtsB and OtsC and a double ATPase OtsD. This system was presumably acquired by horizontal transfer from Actinobacteria or distant species like Lactococcus raffinolactis or Enterococcus asini may be via an intermediate like Lactococcus lactis or its ancestor. RT-PCR experiments proved that OTS gene cluster is transcribed and that at least the otsB, otsC, and otsD genes constitute an operon. A mutant LMD-9Δots, partially deleted for the otsA and otsB genes was constructed. Growth of LMD-9 and LMD-9Δots strains was monitored in the presence of different nitrogen sources and in the presence of urea and nickel. Results revealed that OTS is not implicated in nickel transport, but constitutes a new characterized transporter of peptides of small size, possibly di- and tripeptides in S. thermophilus.

Steady-state and dynamic gene expression programs in Saccharomyces cerevisiae in response to variation in environmental nitrogen.

Cell growth rate is regulated in response to the abundance and molecular form of essential nutrients. InSaccharomyces cerevisiae(budding yeast), the molecular form of environmental nitrogen is a major determinant of cell growth rate, supporting growth rates that vary at least threefold. Transcriptional control of nitrogen use is mediated in large part by nitrogen catabolite repression (NCR), which results in the repression of specific transcripts in the presence of a preferred nitrogen source that supports a fast growth rate, such as glutamine, that are otherwise expressed in the presence of a nonpreferred nitrogen source, such as proline, which supports a slower growth rate. Differential expression of the NCR regulon and additional nitrogen-responsive genes results in >500 transcripts that are differentially expressed in cells growing in the presence of different nitrogen sources in batch cultures. Here we find that in growth rate-controlled cultures using nitrogen-limited chemostats, gene expression programs are strikingly similar regardless of nitrogen source. NCR expression is derepressed in all nitrogen-limiting chemostat conditions regardless of nitrogen source, and in these conditions, only 34 transcripts exhibit nitrogen source-specific differential gene expression. Addition of either the preferred nitrogen source, glutamine, or the nonpreferred nitrogen source, proline, to cells growing in nitrogen-limited chemostats results in rapid, dose-dependent repression of the NCR regulon. Using a novel means of computational normalization to compare global gene expression programs in steady-state and dynamic conditions, we find evidence that the addition of nitrogen to nitrogen-limited cells results in the transient overproduction of transcripts required for protein translation. Simultaneously, we find that that accelerated mRNA degradation underlies the rapid clearing of a subset of transcripts, which is most pronounced for the highly expressed NCR-regulated permease genesGAP1,MEP2,DAL5,PUT4, andDIP5 Our results reveal novel aspects of nitrogen-regulated gene expression and highlight the need for a quantitative approach to study how the cell coordinates protein translation and nitrogen assimilation to optimize cell growth in different environments.

In Vitro Antioxidant and Antimicrobial Activities of Two Edible Mushroom Mycelia Obtained in the Presence of Different Nitrogen Sources

The antioxidant and antimicrobial properties of two edible mushrooms (Pleurotus ostreatus PBS281009 and Coprinus comatus M8102) were evaluated in this study. The mycelium ethanolic extract obtained in the presence of four different nitrogen sources was investigated with regard to their production of phenolic compounds, β-carotene, lycopene, and their antimicrobial and antioxidant properties. Significant differences in phenol and β-carotene concentrations in mycelia grown in different nitrogen sources were observed. Corn extract proved to be the most appropriate source for increasing bioactive properties, leading to the lowest EC₅₀ and minimum inhibitory concentration values in the P. ostreatus PBS281009 species, while peptone was the most suitable for the C. comatus M8102 species. The antimicrobial capacity was screened against Gram-positive and Gram-negative bacteria and against two species of Candida. These results indicate that the mycelia from these two edible mushrooms can be used as nutraceuticals and functional products.

Two aspartic proteinases secreted by the pathogenic yeast Candida parapsilosis differ in expression pattern and catalytic properties

Secreted aspartic proteinases (Sap) play a role in the virulence of pathogenic Candida spp. Candida parapsilosis possesses three genes encoding these enzymes: SAPP1, SAPP2, and SAPP3. We analyzed the expression of the SAPP1 and SAPP2 genes and the production of Sapp1p and Sapp2p proteinases in the presence of different nitrogen sources. While the SAPP2 transcript was present under all of the conditions tested, expression of SAPP1 was induced only by the presence of exogenous protein as the sole nitrogen source. The concentration of Sapp1 p in the medium upon induction was at least one order of magnitude higher than the concentration of Sapp2p in all media tested in this study. Enzymological characterization of purified Sapp1 p and Sapp2p demonstrated that Sapp2p has a more restricted substrate specificity and significantly lower catalytic activity than Sapp1p. Homology models of Sapp1p and Sapp2p revealed structural motifs that may be responsible for the differences between these two enzymes. Our results indicate that C. parapsilosis secretes a low level of Sapp2p proteinase with narrow substrate specificity and low proteolytic activity under most conditions, while expression and secretion of a higher amount of catalytically efficient Sapp1p enzymes is triggered in the presence of exogenous protein serving as a nitrogen source.

Bioactive properties of the medicinal mushroom Leucopaxillus giganteus mycelium obtained in the presence of different nitrogen sources

Leucopaxillus mushroom species are used in the chemical industry for extraction of clitocybin antibiotic and particularly the edible mushroom Leucopaxillus giganteus can be found in Northeast Portugal. Nevertheless, the production of its mycelium for pharmacological applications has not been explored. Herein, the mycelium obtained in the presence of four different nitrogen sources was investigated with regard to phenol production and antimicrobial and antioxidant properties. Phenol concentration increased along the growth time as a response to the oxidative stress and therefore free radical production. Although significant differences for mycelium growth between the nitrogen sources had not been observed, (NH 4) 2HPO 4 proved to be the most appropriate to increase bioactive properties, leading to the highest phenol content and lowest EC 50 and MIC values. The antimicrobial capacity was screened against Gram positive and Gram negative bacteria, and fungi. The samples selectively inhibited the growth of Gram positive bacteria, Staphylococcus aureus being the most susceptible one. Gram negative bacteria and fungi were resistant to the extracts. Antioxidant activity was calculated by the reducing power assay, the scavenging effect on DPPH radicals, and hemolysis and lipid peroxidation inhibition. Significantly negative linear regressions were established between phenols/flavonoids contents, which increased along the mycelia growth time and antioxidant activity.

Effect of the respiratory activity on photoinhibition of the cyanobacterium Synechocystis sp.

The influence of respiratory activity on photosynthesis in Synechocystis cells that had been exposed to high light intensity was studied using distinct conditions of nitrogen supply. The photoinhibitory rate of N-sufficient cells was not influenced by the presence of different nitrogen sources. In contrast, when N-starved cells were resupplied with ammonium, they were protected from photoinhibition. Although N-starved cells presented a higher rate of dark O(2) uptake than N-sufficient ones, the photoinhibitory rate increased in both cases after addition of sodium azide or sodium azide plus salicylhydroxamic acid in the photoinhibitory treatment. In the absence of the D(1) protein repair mechanism, photodamage to Photosystem II was faster in N-sufficient cells than in N-starved ones. Mitigation of photodamage disappeared when the respiratory activity of N-starved cells was partially suppressed by the addition of sodium azide or sodium azide and salicylhydroxamic acid. Our results suggest that electron flow through cyanobacterial terminal oxidases can assist Photosystem I in removing electrons from the reduced plastoquinone pool, thus contributing to both reopening of Photosystem II reaction centers and avoiding photogeneration of reactive oxygen species under photoinhibitory conditions.

NifL of Klebsiella pneumoniae: redox characterization in relation to the nitrogen source

In Klebsiella pneumoniae, NifL modulates the activity of the transcriptional activator NifA in response to combined nitrogen or external molecular oxygen. We recently showed that K. pneumoniae NifL is a flavoprotein which apparently senses oxygen through a redox-sensitive, conformational change. In order to study whether the nitrogen signal might be transmitted to NifA through a stable modification of NifL we characterized the redox properties of NifL synthesized in Escherichia coli in the presence of different nitrogen sources. FAD analyses showed that purified NifL carried FAD as cofactor independent of nitrogen and oxygen availability. The redox potential of NifL synthesized in the presence of ammonium was −277±5 mV at pH 8.0 and 25°C, as determined by reduction with dithionite or with enzymatic reduction by xanthine oxidase in the presence of methyl viologen as redox mediator. When synthesized under nitrogen-limiting conditions, NifL showed a redox potential of −274±6 mV at pH 8.0 and 25°C. Fully reduced NifL fractions, synthesized under either condition listed above, reoxidized rapidly in the presence of molecular oxygen. These results indicate that for NifL synthesized in E. coli, the redox potential of the NifL-bound FAD is not influenced by the nitrogen source. The two NifL fractions differed, however, in that a non-flavin specific absorbance at 420 nm was found only in NifL synthesized in the presence of ammonium.

Regulation of nitrogen catabolic enzymes in Bacillus spp.

The levels of the inducible nitrogen catabolic enzymes arginase (L-arginine amidinohydrolase, EC 3.5.3.1) and alanine dehydrogenase (L-alanine:NAD+ oxidoreductase [deaminating], EC 1.4.1.1) from Bacillus licheniformis and histidase (L-histidine ammonia-lyase, EC 4.3.1.3) from Bacillus subtilis and the ammonia assimilatory enzymes from B. licheniformis were determined in cultures grown in the presence of different nitrogen sources. Although the levels of these enzymes were dependent upon the nitrogen source present, induction of the catabolic enzymes in response to the addition of inducer occurred even in the presence of preferred nitrogen sources. Intracellular pool sizes of ammonia, glutamate, glutamine, and alpha-ketoglutarate were measured in continuous cultures of b. licheniformis growing in the presence of different nitrogen sources. A comparison of the pool sizes of these metabolites with the ammonia assimilatory enzyme levels showed that the pools of the metabolites did not change in a manner consistent with their use as regulators of the synthesis of any of these enzymes.