Studies on nitrate reductase from Cyanidium caldarium

Summary A nitrate reductase from the thermophilic acidophilic alga, Cyanidium caldarium, was studied. The enzyme utilises the reduced forms of benzyl viologen and flavins as well as both NADPH₂ and NADH₂ as electron donors to reduce nitrate.Heat treatment has an activating effect on the benzyl viologen (FMNH₂, FADH₂) nitrate reductase. At 50°C the activation of the enzyme is complete in about 20 min of exposure, whereas at higher temperatures (until 75°C) it is virtually an instantaneous phenomenon. The observed increase in activity is very low in extracts from potassium nitrate grown cells, whereas it is 5 or more fold in extracts from ammonium sulphate supplied cells. The benzyl viologen nitrate reductase is stable at 60°C and is destroyed at 75°C after 3 min; the NADPH₂ nitrate reductase is destroyed at 60°C. The pH optimum for both activities was found in the range 7.8–8.2.Ammonium nitrate grown cells possess a very low level of nitrate reductase: when they are transferred to a nitrate medium a rapid synthesis of enzyme occurs. By contrast, when cells with fully induced activity are supplied with ammonia, a rapid loss of NADPH₂ and benzyl viologen nitrate reductase occurs; however, activity measured with heated extracts shows that the true level of benzyl viologen nitrate reductase is as high as before ammonium addition. It is suggested that the presence of ammonia causes a rapid inactivation but no degradation of the enzyme.Cycloheximide inhibits the formation of the enzyme; the drug is without effect on the loss of nitrate reductase activity induced by ammonium. The nitrate reductase is reactivated in vivo by the removal of the ammonium, in the absence as well as in the presence of cycloheximide.     

Thermophilic methanogenesis from pectin by a mixed defined bacterial culture

Thermophilic degradation of pectin was studied in batch cultures at 55°C by different associations of anaerobic bacteria, includingClostridium thermocellum, Methanobacterium sp., andMethanosarcina sp.Clostridium thermocellum alone produced large amounts of methanol along with some isopropanol and H₂. The inoculation ofMethanobacterium sp. in the culture did not affect the metabolism ofC. thermocellum; this demonstrates the absence of interspecies hydrogen transfer. In the presence of the methylotrophicMethanosarcina sp., methanol was reduced to methane without effect on pectin hydrolysis; a small amount of the H₂ produced was also used to reduce methanol.     

Characteristics of nitrogen-fixingKlebsiella oxytoca isolated from wheat roots

Summary Of 45 fermentative gram negative bacterial isolates examined from wheat roots, three were capable of fixing atmospheric nitrogen as determined by the acetylene reduction technique and by protein contents of cells. A gram negative non-motile facultatively anaerobic bacterial strain capable of N₂ fixation was identified asKlebsiella oxytoca ZMK-2.Optimal growth and N₂ fixation occurred at pH 6.5. The optimum temperatures for growth under anaerobic conditions ranged between 30°–37°C. Acetylene reduction by intact cells was strikingly inhibited by 0.1 atm. or greater partial pressure of O₂. Furthermore, the accumulation of H₂ in the gas phase over cultures ofKlebsiella oxytoca ZMK-2 at partial pressures greater than 0.02 atm. resulted in a striking inhibition in the rate of C₂H₂ reduction. The addition of suspensions of eitherKlebsiella oxytoca ZMK-2 orAzotobacter vinelandii or a mixed culture of these two organisms to axenic cultures of wheat plants produced no significant increase in plant growth as measured by plant dry weight or nitrogen content of plants.     

Low-temperature limitations of photosynthesis in three tropical Vigna species: A chlorophyll fluorescence study

The temperature-dependence of photosynthesis and chlorophyll (Chl) fluorescence quenching components was studied between 0 and 45°C in three tropical, chilling-sensitive Vigna species and in chilling-tolerant pea. Photosynthesis of the Vigna spp. was approx. 20% more reduced by temperatures between 7 and 30°C than in pea. The latter revealed significant changes in Chl fluorescence parameters at much lower temperature than the Vigna spp. Below 15°C, the reduction state of Q_A increased quickly in pea, while in Vigna already below 30°C, an increase of reduced Q_A was obtained. The analysis of different components of non-photochemical Chl fluorescence quenching (q_N) revealed, that in pea photoinhibitory quenching (q_I) occurred below 13°C. Below ca. 7°C, a sudden breakdown of both q_P and the fast relaxing component of q_N was observed in pea.In Vigna, susceptibility of LHC II phosphorylation or limitation of electron flow by damage to PS I, the PS II reaction centre or the water-splitting system were not responsible for the chilling-sensitivity of photosynthesis between 5 and 30°C. Instead, photosynthesis was gradually limited by an inefficient use of reduction equivalents. This, in turn may increase susceptibilty to photoinhibition, which occurred below 20°C in Vigna. The combined study of q_P and of the different components of q_N allowed the demonstration of the subsequent occurrence of different limiting processes with decreasing temperature in the chilling-sensitive Vigna species.     

The carbon monoxide- and oxygen-reacting haemoproteins of Streptomyces clavuligerus: cytochrome aa 3 is the predominant terminal oxidase of the respiratory chain

The nature of the carbon monoxide- and oxygen-reacting haemoproteins in the respiratory chain of the filamentous antibiotic-producing bacterium Streptomyces clavuligerus has been investigated. CO-difference (i.e. CO+ reduced minus reduced) spectra of intact cells showed the presence of cytochrome aa ₃, a CO binding b-type cytochrome, and a pigment resembling cytochrome d. In addition, cells that were approaching the end of the growth phase showed the presence of cytochrome P450: this pigment was undetectable in cells harvested early in the growth cycle. High speed centrifugation of cell-free extracts prepared from cells broken by sonication showed that cytochrome aa ₃ was tightly membrane-bound and that cytochrome P450 was soluble. Inhibition of oxygen uptake rates of cells by cyanide indicated that one component, which showed 50% inhibition at 2–4 mM CN^–, was acting as major terminal oxidase: this was observed in cells harvested from all stages of growth. Photodissociation (i. e. photolysed, CO reduced minus CO reduced) spectra at-118°C, in the absence of oxygen, showed cytochrome aa ₃ to be the sole photolysable CO-reacting haemoprotein. At higher temperature (-87°C), in the presence of oxygen, cytochrome aa ₃ formed a complex with oxygen that could not be photolysed by similar intensities of light. By raising the temperature to-43°C, the oxidation of c-type cytochromes was observed. It is concluded that cytochrome aa ₃ is the predominant terminal oxidase in S. clavuligerus and that the other CO reacting haemoproteins, of unknown function, are unlikely to be oxidases.     

Thermotolerant nalidixic acid-resistant mutants ofEscherichia coli

Nalidixic acid-resistant mutants ofEscherichia coli CGSC #6353 capable of growth at 48°C were obtained by mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine. Cotransductional analyses employing phage P1 indicated that the mutation resulting in the phenotype of growth at 48°C is an allele of thegyrA structural gene. Similar thermal inactivation kinetics were observed for ribosomes isolated from a thermotolerant (T/r) mutant grown at both 37°C and 48°C and from the parental strain grown at 37°C. Cell-free extracts prepared from the T/r mutant grown at 48°C exhibited a sharp increase in protein synthesis at 55°C, whereas this effect was not displayed by extracts from the mutant or parental strains grown at 37°C. In addition, preincubation at 55°C enhanced protein synthesis at 37°C up to 15-fold in an extract prepared from the T/r mutant grown at 48°C, whereas comparable values were 2.6- to 3.0-fold for extracts from the mutant and parental strains grown at 37°C.     

Metabolism ofl-proline toN-acetyl-d-glucosamine during germ tube formation ofCandida albicans

The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[¹⁴C]proline were taken up by 1×10⁶ cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [³H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[¹⁴C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.     

The role of the epidermis in the generation of the circadian rhythm of carbon dioxide fixation in leaves of Bryophyllum fedtschenkoi

The role of the epidermis in the generation of the endogenous circadian rhythm of CO₂ exchange in leaves of Bryophyllum fedtschenkoi has been examined. At 25° C the rhythm of CO₂ output exhibited by whole leaves kept in continuous darkness and an initially CO₂-free air stream also occurs in isolated pieces of mesophyll. The sensitivity to light of the rhythms in whole leaves and in isolated mesophyll appears to be identical. At 15° C, however, no rhythm is observed in isolated mesophyll tissue, despite there being a conspicuous rhythm in intact leaves. The rhythm of net CO₂ assimilation in whole leaves kept in continuous light and a stream of normal air at either 25° C or at 15° C is abolished by removal of the epidermis, although at 15° C and under the higher of the two light levels used, there is an indication that rhythmicity may begin to reappear after the third day of the experiment. Thus, only under certain environmental conditions is the rhythm of CO₂ exchange in Bryophyllum leaves independent of the epidermis. The results indicate that the rhythm of carbon dioxide fixation in continuous darkness and CO₂-free air is generated primarily in the mesophyll cells, whereas the rhythm in continuous light and normal air is generated in the stomatal guard cells or in an interaction of these cells with the mesophyll cells.Abbreviation PEPCase phosphoenolpyruvate carboxylase     

Temperature control of nitrogen fixation in Klebsiella pneumoniae

At growth temperatures above 37°C, Klebsiella pneumoniae does not grow in a medium containing N₂ or NO ₃ ^- as nitrogen sources. However, both the growth in the presence of other nitrogen sources as well as the in vitro nitrogenase activity are not affected at this temperature. The inability to fix N₂ at high temperature is due to the failure of the cells to synthesize nitrogenase and other nitrogen fixation (nif) gene encoded proteins. When cells grown under nitrogen fixing conditions at 30°C were shifted to 39°C, there was a rapid decrease of the rate of de novo biosynthesis of nitrogenase (component 1), nitrogenase reductase (component 2), and the nifJ gene product. There was no degradation of nitrogenase at the elevated temperature since preformed enzyme remained stable over a period of at least 3 h at 39°C. Thus, temperature seems to represent a third control system, besides NH ₄ ⁺ and O₂, governing the expression of nif genes of K. pneumoniae.     

The polyamine analogue N1,N11‐diethylnorspermine can induce chondrocyte apoptosis independently of its ability to alter metabolism and levels of natural polyamines

We have been investigating the effects of natural polyamines and polyamine analogues on the survival and apoptosis of chondrocytes, which are cells critical for cartilage integrity. Treatment of human C‐28/I2 chondrocytes with N¹,N¹¹‐diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, rapidly induced spermidine/spermine N¹‐acetyltransferase (SSAT) and spermine oxidase (SMO), key enzymes of polyamine catabolism and down‐regulated ornithine decarboxylase, the first enzyme of polyamine biosynthesis, thus depleting all main polyamines within 24 h. The treatment with DENSPM did not provoke cell death and caspase activation when given alone for 24 h, but caused a caspase‐3 and ‐9 dependent apoptosis in chondrocytes further exposed to cycloheximide (CHX). In other cellular models, enhanced polyamine catabolism or polyamine depletion has been implicated as mechanisms involved in DENSPM‐related apoptosis. However, the simultaneous addition of DENSPM and CHX rapidly increased caspase activity in C‐28/I2 cells in the absence of SSAT and SMO induction or significant reduction of polyamine levels. Moreover, caspase activation induced by DENSPM plus CHX was not prevented by a N¹‐acetylpolyamine oxidase (PAO)/SMO inhibitor, and depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not reproduce DENSPM effects in the presence of CHX. DENSPM/CHX‐induced apoptosis was associated with changes in the amount or activation of signalling kinases, Akt and MAPKs, and increased uptake of DENSPM. In conclusion, the results suggest that DENSPM can favour apoptosis in chondrocytes independently of its effects on polyamine metabolism and levels. J. Cell. Physiol. 219: 109–116, 2009. © 2008 Wiley‐Liss, Inc.     

Improvement of<Emphasis Type="Italic"> Citrus clementina</Emphasis> Hort. ex Tan. microspore-derived embryoid induction and regeneration

An improvement of the protocol for haploid induction through anther culture of Citrus clementina Hort. ex Tan. cv. Nules was achieved following the evaluation of a number of the factors affecting androgenesis. The influence of thidiazuron (TDZ) and three temperature pre-treatments (4°C, 25°C, 32°C) on the floral buds with respect to anther culture of C. clementina Hort. ex Tan., cv. Nules was investigated. An increased embryoid production was induced in the medium supplemented with TDZ. Pre-treatment temperatures of 4°C and 25°C were more favorable for embryo production than 32°C. Regeneration of androgenic haploid plantlets from cv. SRA 63 of C. clementina is reported here for the first time.Abbreviations 6-BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - KI Kinetin - NAA -Naphthaleneacetic acid - TDZ Thidiazuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea) - ZEA Zeatin Communicated by L. PeñaBoth authors have contributed equally to this article.     

Increasing the stability of immobilizedLactococcus lactis cultures stored at 4 °C

Summary Immobilized cell technology was used to prepare concentrated cultures ofLactococcus lactis that lost only 22% of viability over a 30-day storage period at 4°C. Concentrated cultures ofL lactis CRA-1 were immobilized in calcium alginate beads and added to glycerol, NaCl or sucrose-NaCl solutions in order to obtain a_w readings ranging from 0.91 to 0.97. The suspensions were subsequently placed at 4°C and viability (CFU g^–1 of bead) was followed during storage. Viability losses were high at a_w readings of 0.95 and 0.97 and pH dropped significantly (up to one unit) in the unbuffered solutions. Addition of 1% soytone or glycerophosphate helphed stabilize pH, and a beneficial effect on viability during storage was observed in the glycerol-soytone mix when the beads were added to the conservation solutions immediately following immobilization. When beads were added to the conservation solution immediately following immobilization, a 70% drop in cell counts occurred during the first 5 days of incubation. Dipping theL lactis-carrying beads in milk for 2h before mixing with the glycerolsoytone 0.93 a_w solution reduced this initial 5-day viability loss. Cultures grown in the alginate beads also had good stability in the 0.93 a_w glycerol-soytone solution, where 78% of the population was viable after 30 days at 4°C. The process could be used to store immobilized cells at a processing plant, or by suppliers of lactic starters who wish to ship cultures without freezing or drying.     

Control of the activity of intracellular nucleases in Neurospora crassa.

Summary At least four species of nucleases (nuclease N₁, N₂, N₃ and N₄) and one ribonuclease (ribonuclease N₃) were detected in extract of wild type mycelia grown in high phosphate media by gel filtration of 0–65% ammonium sulfate precipitate through Sephadex G-100. Nuclease N₄ eluted the first is a latent nuclease, the activity of which is not detectable within a week after preparation of the extract but a significant increase in nuclease activity was observed during additional one or two weeks by standing the fraction at 4°C. Nuclease N₁ eluted the second is very labile and nuclease N₂ eluted the third is stable at the temperature. Nuclease N₃ eluted the last was activated within two or three weeks at 4°C. Although all the four nucleases were detected independent of the concentration of orthophosphate in culture media, significantly large amounts of latent ribonuclease (ribonuclease N₃) and a number of nucleases including at least one latent nuclease were observed in wild type mycelia grown in low phosphate media. Ribonuclease N₃ was determined to be a repressible enzyme. The activities of these constitutive latent nucleases, ribonuclease N₃ and a number of nucleases specifically present in wild type mycelia grown in low phosphate media were not observed or significantly reduced in both nuc-1 and nuc-2 mutants, which were deficient to derepress at least eight orthophosphate repressible enzymes relating to phosphate metabolism. A revertant from nuc-2 restored the ability to show activation of at least one of the constitutive latent nucleases.     

Fructose-induced dark germination ofAnabaena akinetes

Standard sporulation medium was used to produce akinetes from vegetative cells ofAnabaena variabilis and culturedAnabaena azollae isolated fromAzolla mexicana. Singlecelled, axenic akinetes were incubated on BG-11 medium supplemented with 1% Bacto-agar. Three different sources of combined nitrogen–KNO₃ (5 mM), NH₄Cl(5 mM), and glutamine (5 mM)–were added singly or in combination with fructose (50 mM) to the BG-11 plates. The akinetes were incubated under continuous light (5500 lux) or in the dark at 25±2°C. Akinetes of both species germinated in the dark when BG-11 was supplemented with fructose. Akinetes incubated in the dark on plates containing NH₄Cl and fructose germinated less than those germinated in the presence of fructose alone. Akinetes of both species germinated in the light.     

A temperature-sensitive osmophilic mutant of Zygosaccharomyces rouxii

Summary A temperature-sensitive osmophilic mutant of Zygosaccharomyces rouxii, OS15, was isolated, which required high salt or sugar concentration for growth above 30°C. Cell viability at 35°C in the presence of NaCl was higher than in the absence of NaCl, and a survival ratio of the mutant cells after incubation at 55°C was also higher in the presence of NaCl than NaCl-free condition. Furthermore, resistance to UV light, hygromycin B and geneticin was improved in the presence of NaCl. There was no difference between the parent and the mutant in fatty acid saturation and microscopic cell shape under NaCl condition.     

A fine-structural study of the freeze-preservation of plant tissue cultures

Summary Suspension culture cells of sycamore (Acer pseudoplatanus L.) and carrot (Daucus carota L.) were frozen to ultralow temperatures under rapid ( 100 °C s^–1) and slow, controlled (1 or 2 °C min^–1) rates, in the presence and absence of cryoprotective compounds. After storage at –196 °C, cells were recovered by thawing either slowly, in air at room temperature (ca. 20 °C min^–1) or rapidly, in a water bath at 40 °C (ca. 100 °C min^–1). The ultrastructure of the thawed cells was examined by thin-sectioning and compared with unfrozen controls and cells examined in the frozen state. Cells frozen rapidly, in the presence of cryoprotectants, or frozen slowly in their absence, suffered serious ultrastructural damage and a total loss of viability. Carrot cells frozen at a rate of 2 °C min^–1 in the presence of cryoprotectants and thawed at either rate, yielded up to 70% of viable cells. The recovered aggregates of carrot cells comprised some centrally located, seriously damaged cells and, at the periphery, groups of cells with a high electron opacity neighbouring well preserved cells, showing little ultrastructural modification compared with unfrozen controls. The highest rate of survival of sycamore cells (ca. 30%) was observed when they were frozen at a rate of 1 °C min^–1 and thawed rapidly. In all recoverd cells of sycamore some ultrastructural modifications were evident. These included: dilation of mitochondria, plastids, golgi and ER cisternae and the nuclear envelope, decrease in polysomes, increase in nuclear and cytoplasmic microfilaments and changes in nuclear and nucleolar granularity. The probable causes and timing of the ultrastructural changes and their effects on the potential for regrowth of the recovered cells are discussed.     

Chlorophyll a Fluorescence Analysis in Response to Excitation Irradiance in Bean Plants (Phaseolus vulgaris L. and Vigna unguiculata L. Walp) Submitted to High Temperature Stress

Bean plants Phaseolus vulgaris L. (cv. Carioca and Negro Huasteco) and Vigna unguiculata L. Walp (cv. Epace-10) were grown in a growth chamber with a photosynthetic photon flux density of 200 mol m^–2 s^–1 at leaf level and air temperature of 25+1 °C. Fully expanded, first pair leaves of 12-d-old plants were submitted for 90 min to high temperature (25, 30, 35, 40, 45, and 48 °C). Chlorophyll a fluorescence parameters (ETR, q_P, q_N, and F₀) were investigated using a modulated fluorimeter at 25 °C during recovery considered here as 48 h after stress induction period. An accentuated decrease in q_P and an increase in q_N at 48 °C in Carioca and Negro Huasteco was not observed in Epace-10. In response to excitation irradiance a great potential for ETR was found in Negro Huasteco at 25 °C, also demonstrated by net photosynthetic rate. At 48 °C ETR was high for Epace-10 while it was equal to zero for Carioca and Negro Huasteco. Tolerance to high temperature observed in Epace-10 provided important information about the adaptative characteristics of Vigna cultivars to warm climates.     

Catabolism of arginine byCarnobacterium spp. isolated from vacuum-packed sugar-salted fish

The ability ofCarnobacterium spp. originally isolated from vacuum-packed, sugar-salted fish to catabolize arginine was examined. All strains were able to produce citrulline, ornithine, and NH₃ from arginine, presumably by the arginine deiminase pathway. The metabolism of arginine was concurrent with acid production from glucose for one strain ofCarnobacterium sp. but delayed for one strain ofCarnobacterium piscicola. The arginine catabolism was not inhibited in the presence of 2% glucose for three strains of carnobacteria during growth in test broth and/or shrimp extract. Growth as well as arginine catabolism was delayed for two strains of carnobacteria by lowering the temperature from 9°C to 4°C. A similar result was obtained by incubating one strain ofC. piscicola in CO₂. None of the compoundsl-citrulline,l-ornithine hydrochloride, and (NH₄)₂SO₄ had any effect on growth or arginine catabolism of this strain. Neither did pH of the medium affect the time for initiation of arginine catabolism.     

Klebsiella sp.NIAB-I: A new diazotroph,associated with roots of kallar grass from saline sodic soils

A nitrogen fixing organism containing a plasmid has been isolated from the rhizosphere fraction ofLeptochloa fusca (L) Kunth (kallar grass) growing on saline soils in the Punjab area. This bacterium can grow aerobically in a medium containing 1M NaCl and can fix nitrogen efficiently under microaerobic conditions on semi-solid medium with glucose or sucrose as a carbon source. Maximum N₂-fixation in batch cultures occurred with 100 mM NaCl at pH 8.0 and 35°C. DNA hybridization and analysis of the protein pattern were carried out to establish its taxonomic position. On the basis of protein electrophoretic pattern, physiological characteristics, DNA relatedness, and better growth in the presence of high NaCl concentration, we regard this strain as a new species ofKlebsiella.