A study of the nitrate and nitrite discharge from the mutant cells of Neurospora crassa lacking nitrate and nitrite reductase activities

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.     

Evaluation of <Emphasis Type="Italic">Galleria mellonella</Emphasis> larvae as an in vivo model for assessing the relative toxicity of food preservative agents

Larvae of Galleria mellonella are widely used for evaluating the virulence of microbial pathogens and for measuring the efficacy of anti-microbial agents and produce results comparable to those that can be obtained using mammals. In this work, the suitability of using G. mellonella larvae to measure the relative toxicity of a variety of food preservatives was evaluated. The response of larvae to eight commonly used food preservatives (potassium nitrate, potassium nitrite, potassium sorbate, sodium benzoate, sodium nitrate, sodium chloride, sodium nitrite and sodium acetate) administered by feeding or by intra-haemocoel injection was measured. A significant correlation between the LD₅₀ (R ²?=?0.8766, p?=?0.0006) and LD₈₀ (R ²?=?0.7629, p?=?0.0046) values obtained due to oral or intra-haemocoel administration of compounds was established. The response of HEp-2 cells to the food preservatives was determined, and a significant correlation (R ²?=?0.7217, p?=?0.0076) between the LD₅₀ values of the compounds administered by feeding in larvae with the IC₅₀ values of the compounds in HEp-2 cells was established. A strong correlation between the LD₅₀ values of the eight food preservatives in G. mellonella larvae and rats (R ²?=?0.6506, p?=?0.0156) was demonstrated. The results presented here indicate that G. mellonella larvae may be used as a model to evaluate the relative toxicity of food preservatives, and the results show a strong positive correlation to those obtained using established cell culture and mammalian models.     

On the control mechanisms of the nitrite level in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells: the mathematical model

Background

Due to a high toxicity of nitrite and its metabolites, it is of high interest to study mechanisms underlying the low NO₂ level maintenance in the cell. During anaerobic growth of Escherichia coli the main nitrite-reducing enzymes are NrfA and NirB nitrite reductases. NrfA reductase is localized in the cell periplasm and uses NO₂ as an electron acceptor to create a proton gradient; NirB reductase is restricted to the cytoplasm and metabolizes excessive nitrite inside the cell, the uptake of which is mediated by the transporter protein NirC. While it is known that these three systems, periplasmic, cytoplasmic and transport, determine nitrite uptake and assimilation in the cell as well as its excretion, little is known about their co-ordination.

Results

Using a mathematical model describing the nitrite utilization in E. coli cells cultured in a flow chemostat, the role of enzymes involved in nitrite metabolism and transport in controlling nitrite intracellular levels was investigated. It was demonstrated that the model adapted to the experimental data on expression of nrfA and nirB genes encoding NrfA and NirB nitrite reductases, can describe nitrite accumulation kinetics in the chemostat in the millimolar range of added substrate concentrations without any additional assumptions. According to the model, in this range, low intracellular nitrite level, weakly dependent on its concentration in the growth media, is maintained (mcM). It is not sufficient to consider molecular-genetic mechanisms of NrfA reductase activity regulation to describe the nitrite accumulation dynamics in the chemostat in the micromolar range (≤1 mM) of added nitrite concentrations. Analysis of different hypotheses has shown that the mechanism of local enzyme concentration change due to membrane potential-induced diffusion from the cytoplasm to the periplasm at low nitrite levels is sufficient to explain the nitrite accumulation dynamics in the chemostat.

Conclusions

At nitrite concentrations in the media more than 2 mM, the model adapted to the experimental data on nitrite utilization dynamics in E. coli cells cultured in the flow chemostat demonstrates the largest contribution of genetic mechanisms involved in nrf and nir operons activity regulation to the control of nitrite intracellular levels. The model predicts a significant contribution of the membrane potential to the periplasmic NrfA nitrite reductase activity regulation and nitrite utilization dynamics at substrate concentrations ≤1 mM.

     

Peroxidase activity of octaheme nitrite reductases from bacteria of the <Emphasis Type="Italic">Thioalkalivibrio</Emphasis> genus

Closely related penta- and octaheme nitrite reductases catalyze the reduction of nitrite, nitric oxide, and hydroxylamine to ammonium and of sulfite to sulfide. NrfA pentaheme nitrite reductase plays the key role in anaerobic nitrate respiration and the protection of bacterial cells from stresses caused by nitrogen oxides and hydrogen peroxide. Octaheme nitrite reductases from bacteria of the Thioalkalivibrio genus are less studied, and their function in the cell is unknown. In order to estimate the possible role of octaheme nitrite reductases in the cell resistance to oxidative stress, the peroxidase activity of the enzyme from T. nitratireducens (TvNiR) has been studied in detail. Comparative analysis of the active site structure of TvNiR and cytochrome c peroxidases has shown some common features, such as a five-coordinated catalytic heme and identical catalytic residues in active sites. A model of the possible productive binding of peroxide at the active site of TvNiR has been proposed. The peroxidase activity has been measured for TvNiR hexamers and trimers under different conditions (pH, buffers, the addition of CaCl₂ and EDTA). The maximum peroxidase activity of TvNiR with ABTS as a substrate (k _cat = 17 s^–1; k _cat/K _m = 855 mM^–1 s^–1) has been 100–300 times lower than the activity of natural peroxidases. The different activities of TvNiR trimers and hexamers indicate that the rate-limiting stage of the reaction is not the catalytic event at the active site but the electron transfer along the heme c electron-transport chain.     

Nitrogen transformation under different dissolved oxygen levels by the anoxygenic phototrophic bacterium <Emphasis Type="Italic">Marichromatium gracile</Emphasis>

Marichromatium gracile: YL28 (M. gracile YL28) is an anoxygenic phototrophic bacterial strain that utilizes ammonia, nitrate, or nitrite as its sole nitrogen source during growth. In this study, we investigated the removal and transformation of ammonium, nitrate, and nitrite by M. gracile YL28 grown in a combinatorial culture system of sodium acetate-ammonium, sodium acetate-nitrate and sodium acetate-nitrite in response to different initial dissolved oxygen (DO) levels. In the sodium acetate-ammonium system under aerobic conditions (initial DO?=?7.20–7.25 mg/L), we detected a continuous accumulation of nitrate and nitrite. However, under semi-anaerobic conditions (initial DO?=?4.08–4.26 mg/L), we observed a temporary accumulation of nitrate and nitrite. Interestingly, under anaerobic conditions (initial DO?=?0.36–0.67 mg/L), there was little accumulation of nitrate and nitrite, but an increase in nitrous oxide production. In the sodium acetate-nitrite system, nitrite levels declined slightly under aerobic conditions, and nitrite was completely removed under semi-anaerobic and anaerobic conditions. In addition, M. gracile YL28 was able to grow using nitrite as the sole nitrogen source in situations when nitrogen gas produced by denitrification was eliminated. Taken together, the data indicate that M. gracile YL28 performs simultaneous heterotrophic nitrification and denitrification at low-DO levels and uses nitrite as the sole nitrogen source for growth. Our study is the first to demonstrate that anoxygenic phototrophic bacteria perform heterotrophic ammonia-oxidization and denitrification under anaerobic conditions.     

Exogenously applied nitrate improves the photosynthetic performance and nitrogen metabolism in tomato (<Emphasis Type="Italic">Solanum</Emphasis><Emphasis Type="Italic">lycopersicum</Emphasis> L. cv Pusa Rohini) under arsenic (V) toxicity

Tomato (Solanum lycopersicum L.) being a widespread and most commonly consumed vegetable all over the world has an important economic value for its producers and related food industries. It is a serious matter of concern as its production is affected by arsenic present in soil. So, the present study, investigated the toxicity of As(V) on photosynthetic performance along with nitrogen metabolism and its alleviation by exogenous application of nitrate. Plants were grown under natural conditions using soil spiked with 25 mg and 20 mM, As(V) and nitrate, respectively. Our results revealed that plant growth indices, photosynthetic pigments, and other major photosynthetic parameters like net photosynthetic rate and maximum quantum efficiency (F _v /F _m ) of photosystem II (PSII) were significantly (P ≤ 0.05) reduced under As(V) stress. However, nitrate application significantly (P ≤ 0.05) alleviated As(V) toxicity by improving the aforesaid plant responses and also restored the abnormal shape of guard cells. Nitrogen metabolism was assessed by studying the key nitrogen-metabolic enzymes. Exogenous nitrate revamped nitrogen metabolism through a major impact on activities of NR, NiR, GS and GOGAT enzymes and also enhanced the total nitrogen and NO content while malondialdehyde content, and membrane electrolytic leakage were remarkably reduced. Our study suggested that exogenous nitrate application could be considered as a cost effective approach in ameliorating As(V) toxicity.     

Biotic factors in induced defence revisited: cell aggregate formation in the toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC 7806 is triggered by spent <Emphasis Type="Italic">Daphnia</Emphasis> medium and disrupted cells

Bioassays with the toxic cyanobacterium Microcystis aeruginosa PCC 7806, its non-toxic mutant ΔmcyB, and Daphnia magna as grazer were used to evaluate biotic factors in induced defence, in particular cyanobacterial and grazer-released info-chemicals. Three main questions were addressed in this study: Does Daphnia grazing lead to a loss of cyanobaterial biomass? Is the survival time of Daphnia shorter in a culture of the toxic cyanobacterium? Does direct grazing or the presence of spent Daphnia medium or a high number of disrupted toxic Microcystis cells in the assays lead to an increase in the cellular microcystin content in the remaining intact cells? The biovolume (growth) as well as size and abundance of Microcystis aggregates were determined by particle analysis, while the survival time of Daphnia individuals was recorded by daily observation and counting, with the relative concentration of cell-bound microcystin-LR, was measured by HPLC analysis. Compared to some recent studies in the field of induced defence, in this study, evidence was found for a direct grazing effect, i.e. the loss of biovolume in the toxic culture. In addition, Daphnia magna ingested more non-toxic than toxic cells, and survived longer with non-toxic cells. In terms of increased cell-bound toxin concentration as a means of defence reported in some studies, a higher cell-bound microcystin-LR content was not measured in this study in any of the treatments (P > 0.05). Under low light conditions with impaired growth of Microcystis, and the presence of a high number of particles with less than 1-μm diameter (possibly heterotrophic bacteria), Daphnia medium was associated with a strong reduction in cell-bound toxin concentration (P < 0.05). This study showed no increased cell aggregation under direct grazing (P > 0.05), but increased aggregation with spent Daphnia medium under high light conditions (P < 0.05). Further, the addition of cell-free extract from disrupted toxic Microcystis cells strongly increased the aggregation of the intact cells under low light (P < 0.05). These findings are discussed with the possible role of microcystin and other infochemicals in the expression of proteins and morphology changes in Microcystis.     

Effects of solid-phase denitrification on the nitrate removal and bacterial community structure in recirculating aquaculture system

A solid-phase denitrification (SPD) reactor packed with poly (3-hydroxybutyrate-co-3-hydroxyvalerate) as a carbon source was incorporated into a recirculating aquaculture system (RAS) to remove accumulated nitrate. Bacterial community structures in different parts of the RAS, including biofilter unit, SPD reactor, and culture water, were analyzed using Illumina MiSeq sequencing technology. The data showed that nitrate levels decreased remarkably in the RAS connected with SPD reactor (RAS-DR). In contrast, nitrate levels increased continuously in the conventional RAS without SPD reactor (RAS-CK). Biofilter unit and culture water in RAS-DR developed lower species richness and higher bacterial community diversity than that in RAS-CK. The bacterial community structure of RAS was significantly affected by the SPD process and the changes included an increase in the proportion of Proteobacteria and Firmicutes and a decrease in Nitrospira abundance in RAS-DR. Firmicutes was the most abundant phylum (56.9 %) and mainly consisted of Clostridium sensu stricto (48.3 %) in SPD reactor.     

Laue crystal structure of <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis> nitrite reductase from a high-yield expression system

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein “small tetraheme c” replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5–1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-Å-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein).     

Cloning,purification and characterization of novel Cu-containing nitrite reductase from the <Emphasis Type="Italic">Bacillus firmus</Emphasis> GY-49

Nitrite is generated from the nitrogen cycle and its accumulation is harmful to environment and it can be reduced to nitric oxid by nitrite reductase. A novel gene from Bacillus firmus GY-49 is identified as a nirK gene encoding Cu-containing nitrite reductase by genome sequence. The full-length protein included a putative signal peptide of 26 amino acids and shown 72.73% similarity with other Cu-containing nitrite reductase whose function was verified. The 993-bp fragment encoding the mature peptide of NirK was cloned into pET-28a (+) vector and overexpressed as an active protein of 36.41 kDa in the E.coli system. The purified enzyme was green in the oxidized state and displayed double gentle peaks at 456 and 608 nm. The specific activity of purified enzyme was 98.4 U/mg toward sodium nitrite around pH 6.5 and 35 °C. The K _m and K _cat of NirK on sodium nitrite were 0.27 mM and 0.36?×?10³ s^?1, respectively. Finally, homology model analysis of NirK indicated that the enzyme was a homotrimer structure and well conserved in Cu-binding sites for enzymatic functions. This is a first report for nitrite reductase from Bacillus firmus, which augment the acquaintance of nitrite reductase.     

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].     

Cryopreservation of <Emphasis Type="Italic">Citrus</Emphasis> anthers in the National Crop Genebank of China

Genebank conservation of pollen is valuable because it makes genetic resources immediately available for use in breeding programs. In the case of Citrus species, conserved anthers or pollen can be easily transported and used to develop new varieties with pathogen resistance and desirable quality and yield traits. The aim of this study was to develop and improve air-desiccation cryopreservation protocols for Citrus cavaleriei and Citrus maxima anthers in genebanks. In the current study, warming, rehydration, and in vitro germination conditions were optimized to achieve high levels of in vitro germination in Citrus pollen for ten cultivars after liquid nitrogen (LN) exposure. The optimal warming, rehydration, and in vitro germination medium formulations affected the germination levels after pollen cryopreservation, with species- and cultivar-dependent effects. The Citrus anthers were dehydrated to the moisture content of 5–14% before LN exposure and warmed at 25 (cryopreserved Citrus anthers with a moisture content of lower than 10%) or 37°C (a moisture content of 10% or higher), then rehydrated, and cultured on medium with 150-g L^?1 sucrose, 0.1-g L^?1 boric acid, 1.0-g L^?1 calcium nitrate, 0.1-g L^?1 potassium nitrate, 0.3-g L^?1 magnesium sulfate, and 10-g L^?1 agar. After 2 yr of storage, in vitro germination levels of Citrus pollen after cryopreservation were significantly higher (> 22% for all ten cultivars) than those of samples that were stored at 4°C (0%). In vitro germination levels of pollen from six of ten cultivars after cryopreservation remained relatively high after 2 yr of storage (38–93%). The highest viability of 93% was obtained for C. cavaleriei ‘2–3’. The methods identified in the current study could be used to cryopreserve C. cavaleriei and C. maxima anthers.     

Activity of nitrate reductase in <Emphasis Type="Italic">Desulfovibrio vulgaris</Emphasis> VKM 1388

Evidence was obtained of the inhibitory effect of nitrate on the metabolism of Desulfovibrio vulgaris 1388. Nitrate is reduced only at low concentrations and in the presence of sulfate in the medium. Genetic data suggest that the genome of D. vulgaris 1388 contains the information about the γ subunit and possibly the NarG catalytic subunit of the membrane-bound nitrate reductase.     

Gene <Emphasis Type="Italic">yddG</Emphasis> of <Emphasis Type="Italic">Escherichia coli</Emphasis> encoding the putative exporter of aromatic amino acids: Constitutive transcription and dependence of the expression on the cell growth rate

Gene yddG of Escherichia coli encodes a protein of the inner membrane. Data obtained earlier demonstrated that under conditions of aromatic amino acids overproduction YddG promotes their export from E. coli cells. In this work, a method of primer extension was used to localize the P _yddG promoter, which corresponds to E. coli promoters recognized by RNA polymerase in complex with σ⁷⁰ or σ^S subunits. By constructing a gene of the hybrid protein YddG’-LacZ at the intrinsic site of gene yddG location in the E. coli chromosome and analyzing the activity of β-galactosidase in cells growing on laboratory media LB and M9, the constitutive type of yddG expression at a low level was demonstrated (the activity was about 3 to 4% of the LacZ level under induction of the lac operon in E. coli wild-type cells). The expression of yddG had a twofold increase under conditions of retarded cell growth upon the stress caused by the high NaCl content (0.6 M) or by the presence of phenylalanine excess quantities (>1 mM) in the culture medium.     

Growth and uptake of caesium,rubidium, and potassium by ectomycorrhizal and saprotrophic fungi grown on either ammonium or nitrate as the N source

After the accident at the Fukushima Dai-ichi Nuclear Power Plant in 2011, high activities of radiocaesium have been reported in wild mushrooms in Japan. Fungi play an important role in the dynamics of radiocaesium in forest ecosystems. We examined the contents of caesium (Cs), rubidium (Rb), and potassium (K) in the mycelium of 15 isolates of ectomycorrhizal (EM) fungi and nine isolates of saprotrophic (SA) fungi in a synthetic medium with either ammonium chloride (NH₄Cl) or sodium nitrate (NaNO₃), supplemented with 1 ppm caesium chloride and rubidium chloride. The mycelia were harvested after 8 weeks of incubation, and the contents of Cs, Rb, and K were measured by inductively coupled plasma mass spectrometry. The dry weight of the mycelium in the medium with NH₄ was significantly higher than that with NO₃, although some EM species, Hebeloma, Astraeus, Scleroderma, and Pisolithus, grew well in the medium with NO₃. Among SA species, Crucibulum and Cyathus grew in the medium with NO₃. The uptakes of Cs, Rb, and K by Suillus, Pisolithus, and Rhizopogon were higher than that in other EM and SA species when they grew on the medium with NH₄, while the uptakes of these elements by Astraeus and Scleroderma were higher than those by other species grown on the medium with NO₃. The content of Rb was positively correlated with Cs (r = 0.85, p < 0.001) and K (r = 0.51, p < 0.001). The accumulation of Cs, Rb, and K was differently affected by the N source and fungal species.     

A novel methanotroph in the genus <Emphasis Type="Italic">Methylomonas</Emphasis> that contains a distinct clade of soluble methane monooxygenase

Aerobic methane oxidation is a key process in the global carbon cycle that acts as a major sink of methane. In this study, we describe a novel methanotroph designated EMGL16-1 that was isolated from a freshwater lake using the floating filter culture technique. Based on a phylogenetic analysis of 16S rRNA gene sequences, the isolate was found to be closely related to the genus Methylomonas in the family Methylococcaceae of the class Gammaproteobacteria with 94.2–97.4% 16S rRNA gene similarity to Methylomonas type strains. Comparison of chemotaxonomic and physiological properties further suggested that strain EMGL16-1 was taxonomically distinct from other species in the genus Methylomonas. The isolate was versatile in utilizing nitrogen sources such as molecular nitrogen, nitrate, nitrite, urea, and ammonium. The genes coding for subunit of the particulate form methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), and methanol dehydrogenase (mxaF) were detected in strain EMGL16-1. Phylogenetic analysis of mmoX indicated that mmoX of strain EMGL16-1 is distinct from those of other strains in the genus Methylomonas. This isolate probably represents a novel species in the genus. Our study provides new insights into the diversity of species in the genus Methylomonas and their environmental adaptations.