Temperature has reciprocal effects on colanic acid and polysialic acid biosynthesis in <Emphasis Type="Italic">E. coli</Emphasis> K92

Escherichia coli K92 is an opportunistic pathogen bacterium able to produce polysialic acid (PA) capsules when grows at 37°C. PA polysaccharides are cell-associated homopolymers tailored from acid sialic monomers that function as virulence factors in different neuroinvasive diseases caused by certain Enterobacteriaceae. Conversely, when grows at 19°C (restrictive conditions), PA synthesis was negligible, whereas in such condition, a slimy substance started to be accumulated in the culture broths. Analysis by uronic acids colorimetric determinations, gas chromatography–mass spectrometry, and Fourier transform infrared spectroscopy allowed the isolation and identification of mucoid substance as colanic acid (CA). CA is a heteropolymer containing glucose, galactose, fucose, and glucuronic acid as monomers which seems to be involved in the protection of this bacterium against environment assaults. The study of physicochemical conditions required for CA synthesis revealed that in E. coli K92, nutrient (carbon and nitrogen sources) modulates CA production, reaching the maximal values when glucose and proline were as carbon and nitrogen sources, respectively. Furthermore, we have found that E. coli K92 is able to produce CA at all temperatures tested (from 42°C to 15°C), whereas PA synthesis only occurred when bacteria were cultured at temperatures higher than 25°C. Additionally, genetic engineering approaches revealed that the CA cluster including several genes required for synthesis was placed into a DNA fragment of 100 kb using polymerase chain reaction methodology.     

Improvement of Bacillus thuringiensis Bacteriocin Production Through Culture Conditions Optimization

Abstract

BUPM4 is a Bacillus thuringiensis subsp. kurstaki strain, isolated from Tunisian soil, producing an original bacteriocin named Bacthuricin F4. The optimization of the latter production conditions was carried out under several physicochemical conditions. It was found that the highest bacteriocin activity was reached at low aeration while bacteriocin synthesis yields were strongly reduced at higher ones. A balance between growth and bacteriocin synthesis, both highly dependent on aeration, was taken into account for the overproduction of bacteriocin. Both glucose and glycerol were shown to be necessary for Bacthuricin F4 maximal synthesis. In addition, the optimal carbon/nitrogen ratio for bacteriocin production is 9. In such optimal conditions, more than 4-fold greater bacteriocin production was obtained than when using TSB medium.     

Effect of external pH on inorganic carbon assimilation in unicellular marine green algae

Carbonic anhydrase (CA) induction has been studied in three marine green algae under acidic (pH 4.5) or alkaline (pH 8.0) conditions. An inhibition of the induction of the external CA in acidic conditions, similar to that observed in some freshwater green algae, could be observed in only Chlorella saccharophila. In the two other species, Chlorococcum littorale and Stichococcus bacillaris, no significant difference in CA induction was found under two pH conditions. The exact function of the external CA of C. saccharophila remains unclear, since cells grown under acidic conditions (under which this enzyme is repressed) possess the same abilities to use inorganic carbon (Ci) as alkaline‐grown cells. Internal pH values were not modified by the pH of the medium used to cultivate C. saccharophila. Regardless of the growth conditions, activities related to carbon fixation, that is, photosynthetic oxygen evolution, Ci uptake and assimilation were enhanced when the measurements were performed at acidic pH. This indicates that this marine alga is able to use CO₂ more efficiently than HCO₃^?. No evidence could be found for a specific Ci uptake and assimilation system in the acid‐grown cells.     

Internal Glutamine and Glutamate Pools in Klebsiella pneumoniae Grown Under Different Conditions of Nitrogen Availability

Internal pool sizes of glutamine and glutamate in Klebsiella pneumoniae grown under nitrogen limitation or nitrogen sufficiency were measured to study the signal transduction of external nitrogen limitation. K. pneumoniae cells were grown in an anaerobic, ammonium-limited chemostat culture. At a growth rate of 0.217 h⁻¹, the steady state ammonium concentration in the culture was 55 μm, correlating with repression of the nitrogen fixation (nif) genes. At growth rates below 0.138 h⁻¹, the ammonium concentration in the culture dropped below 0.5 μm and the nif genes became derepressed. During the transition from nitrogen sufficiency to nitrogen limitation, the internal glutamine pool in K. pneumoniae decreased by a factor of approximately 6. The glutamate pool, however, remained stable. Similarly, in anaerobic batch cultures with different limiting nitrogen sources, the glutamine pool generally decreased by a factor of 7 to 9 when nif gene derepression was achieved. All the limiting nitrogen sources used resulted in decreased growth rates compared with growth under nitrogen excess, suggesting an inverse relationship between glutamine pool size and doubling time. These studies indicate that K. pneumoniae perceives external nitrogen limitation as internal glutamine limitation. Received: 3 April 2000 / Accepted: 5 June 2000     

Conditions governing haematochrome formation and loss in the alga haematococcus pluvialis flotow

Summary Culture experiments withHaematococcus pluvialis show that carotenogenesis may be independent of available nitrogen but not of the carbon supply, and that cell multiplication not only precludes the accumulation of carotenoids even under conditions which are otherwise favourable to it, but also effects their disappearance.Chodat's hypothesis concerning the importance of the carbon/nitrogen balance has only limited application and is replaced by one of a more general nature.     

Biosynthesis of citric and isocitric acids from ethanol by mutant Yarrowia lipolytica N 1 under continuous cultivation

The effect of ethanol, zinc, and iron (Fe²⁺ and Fe³⁺) concentration and of oxygen supply on cell growth and the production of citric acid (CA) and isocitric acid (ICA) from ethanol by mutant Yarrowia lipolytica N 1 was studied under continuous cultivation. The following peculiarities of Y. lipolytica metabolism were found: (1) intensive CA production occurred under yeast growth limitation by nitrogen; (2) inhibition of yeast growth by ethanol was accompanied by significant alterations in fatty acid composition of lipids; (3) the production of CA and ICA from ethanol required high concentrations of zinc and iron ions; (4) the intracellular iron concentration determined whether CA or ICA was predominantly formed; (5) the cell's requirement for oxygen depended on the intracellular iron concentration. The events taking place in the production of CA and ICA were evaluated through the activities of enzyme systems involved in the metabolism of ethanol and CA in this strain. Electronic Publication     

The ORF encoding a putative ferredoxin-like protein downstream of the vnfH gene in Azotobacter vinelandii is involved in the vanadium-dependent alternative pathway of nitrogen fixation

Summary An open reading frame (ORF) in the same operon as, but downstream of, vnfH in Azotobacter vinelandii can code for a ferredoxin-like protein. The role this ORF may play in the vnf (vanadium-dependent alternative) pathway of nitrogen fixation was investigated. Site-directed mutagenesis was used to alter one base in each of the codons specifying amino acids 18 and 19 generating a unique BglII site. A kanamycin resistance cartridge was cloned into the BglII site. This construct was mobilized into A. vinelandii CA12 ( nifHDK) strain by conjugation and the mutation was introduced into the genome by marker exchange. The resulting mutant was unable to fix nitrogen under conditions in which the vnf pathway of nitrogen fixation operates. This suggests that this ORF is functional and is essential for the vanadium-dependent alternative pathway of nitrogen fixation in A. vinelandii.     

Influence of feeding conditions on clavulanic acid production in fed-batch cultivation with medium containing glycerol

First, the effect of different levels of nitrogen source on clavulanic acid (CA) production was evaluated in batch cultivations utilizing complex culture medium containing glycerol and three different levels of soy protein isolate (SPI). Cellular growth, evaluated in terms of the rheological parameter K, was highest with a SPI concentration of 30 g.L⁻¹ (4.42 g.L⁻¹ N total). However, the highest production of CA (380 mg.L⁻¹) was obtained when an intermediate concentration of 20 g.L⁻¹ of SPI (2.95 g.L⁻¹ total N) was used. To address this, the influences of volumetric flow rate (F) and glycerol concentration in the complex feed medium (Cs_F) in fed-batch cultivations were investigated. The best experimental condition for CA production was F=0.01 L.h⁻¹ and Cs_F=120 g.L⁻¹, and under these conditions maximum CA production was practically twice that obtained in the batch cultivation. A single empirical equation was proposed to relate maximum CA production with F and Cs_F in fed-batch experiments.     

Changes in Nitrogen Metabolism of Vigna Radiata in Response to Elevated CO2

With the aim to determine the effects of CO₂ on nitrogen metabolism mungbean (Vigna radiata) plants were grown from seedling emergence to maturity inside open top chambers under ambient CO₂ (CA, 350 ± 25 µmol mol^–1) and elevated CO₂ (CE, 600 ± 50 µmol mol^–1) concentrations at the Indian Agricultural Research Institute, New Delhi. Leaflet blades of the same physiological age were sampled at 20, 35 and 50 d after germination. Total nitrogen concentration in dry mass was consistently lower under CE than in CA. Non-protein nitrogen and protein nitrogen were also decreased under CE Total soluble protein content also decreased up to 35 d after germination under CE. However, a 27 % increase in protein content at 50 d after germination due to CE was observed. A significant decrease in total free amino acid under CE at 20 d after germination was observed. CE also brought about a remarkable decrease in the activity of nitrate reductase in leaves at 20 d after germination but increase at 35 d and 50 d after germination. Nitrogenase activity increased at all growth stages due to CE. Although total harvested leaves of CE plants accumulated more nitrogen, the relative amount of nitrogen on a percentage basis was low, probably due to a comparatively greater accumulation of sugars in the leaves of CE plants.     

Carbon and Hydrogen Isotope Ratios in Bromeliads Growing under Different Light Environments in Natural Conditions

Wild Ananas species in northern South America occur in shady environments and appear to be relatively intolerant to droughts associated with growth under full sun exposure. This behaviour contrasts with the higher productivity of commercial varieties of Ananas comosus when grown under full sun exposure. Such differentiation within the genus offers an opportunity to study the process of adaptation of apparently high light avoiding species into true sun plants. As a first approximation, the analysis of nitrogen content and carbon and hydrogen isotope ratios of bromeliads growing under natural conditions was undertaken to test the following hypotheses: 1. Leaf nitrogen content of plants grown under partial shade is higher than that of the same species in the same habitat growing under full sun exposure, due to the higher availability to nitrogen in the under-canopy, but also to the lower proportion of structural carbohydrates in shade leaves; 2. δ¹³C values are usually more negative in CAM bromeliads growing under partial shade because of the lower contribution of CAM to total carbon gain, and the probable fixation of CO₂ originating from soil respiration; 3. δD values of CAM bromeliads are less negative than those of C3 bromeliads, but CAM bromeliads grown in shady habitats tend to have more negative δD values because of the lower relative accumulation of deuterium in leaf tissue water, and also because of their relatively lower CAM activity. The results show a clear differentiation between CAM and C3 bromeliads based on δ¹³C values, and in general δD values are less negative in CAM bromeliads. However, in several species overlapping δD values between C3 and CAM bromeliads were observed. The analysis of paired samples of the same species grown under contrasting light intensity usually conformed with the expectations. A number of deviations from the hypotheses were observed which appeared to be related to particular environmental conditions. The interpretation of δD values obtained from total organic matter is made difficult by the local variation of hydrogen/deuterium ratios in water available to the plant.     

Biomass production and nitrogen contents of the CAM plants Kalanchoe daigremontiana and K. tubiflora in cultures with different nitrogen and water supply

Summary Two CAM plant species (Kalanchoe daigremontiana and K. tubiflora) were cultivated in pure cultures with two different levels of nitrogen and water supply. A comparison of the plant dry weight showed that the productivity was severely reduced under low nitrogen or/and water conditions. Additionally the proportion of the different organs contributing to the total biomass, and thus the ratio of root/shoot dry weight shifted substantially. At the same time the production of leaf buds in ratio to leaf biomass was increased. Concentrations of organic nitrogen and nitrate in the shoots were drastically reduced under low nitrogen or/and water conditions, but organic nitrogen concentration in the roots remained relatively high. The leaf conductance over a day was investigated only for K. daigremontiana, and decreased with reduced water and nitrogen supply. The results indicate that CAM plants do not have the predicted high nitrogen use efficiency. Under environmental stress they change from biomass production towards provisions for life preservation.     

Biomass production and nitrate metabolism of Atriplex hortensis L. (C3 plant) and Amaranthus retroflexus L. (C4 plant) in cultures at different levels of nitrogen supply

Summary Pure and mixed cultures of the dicotyledons Atriplex hortensis L. (C₃ plant) and Amaranthus retroflexus L. (C₄ plant) were maintained under open air conditions in standard soil at low and high nitrogen supply levels.A comparison of shoot dry weight and shoot length in the various series shows that the growth of the aboveground parts of both species was severely reduced under low N conditions. In both pure and mixed cultures the differences resulting from low N vs. high N conditions was less pronounced with Atriplex (C₃ plant) than with Amaranthus (C₄ plant). The root dry weight of the two species was not reduced so much under low N conditions as was the shoot dry weight. The low N plants were found to contain a larger proportion of their biomass in the roots than did the high N plants. In general the root proportion of Atriplex was greater than that of Amaranthus. The contents of organic nitrogen and nitrate and the nitrate reductase activity (NRA) per g dry weight of both species decreased continually throughout the experiments. With the exception of young plants, the low N plants always had tower contents of organic nitrogen and nitrate and nitrate reductase activities than did the high N plants. The highest values of NRA were measured in the leaf laminae. The eaves also exhibited the highest concentrations of organic nitrogen. The highest nitrate concentrations, however, were observed in the shoot axis, and in most cases the lowest nitrate values were found in the laminae. At the end of ne growing season this pattern was found to have been reversed with Atriplex, but not with Amaranthus. Thus Atriplex was able to maintain a higher NRA in the laminae than Amaranthus under low N conditions.The transpiration per leaf area of the C₄ plant Amaranthus during the course of a day was substantially lower than that of the C₃ plant Atriplex. There were no significant differences in transpiration between the low N and high N series of Amaranthus. The low N plants of Atriplex, however, clearly showed in most cases higher transpiration rates than the corresponding high N plants. These different transpiration rates of the high N and the low N Atriplex plants were also reflected in a distinct ¹³C discrimination.The sum of these results points to the conclusion that the C₃ plant Atriplex hortensis can maintain a better internal inorganic nitrogen supply than the C₄ plant Amaranthus retroflexus under low N conditions and an ample water supply, due to the larger root proportion and the more pronounced and flexible transpiration of the C₃ plant.Dedicated to Prof. Dr. Karl Mägdefrau, Deisenhofen, on the ocasion of his 80th birthday     

Phospholipase Dε enhances Braasca napus growth and seed production in response to nitrogen availability

Phospholipase D (PLD), which hydrolyses phospholipids to produce phosphatidic acid, has been implicated in plant response to macronutrient availability in Arabidopsis. This study investigated the effect of increased PLDε expression on nitrogen utilization in Brassica napus to explore the application of PLDε manipulation to crop improvement. In addition, changes in membrane lipid species in response to nitrogen availability were determined in the oil seed crop. Multiple PLDε over expression (PLDε‐OE) lines displayed enhanced biomass accumulation under nitrogen‐deficient and nitrogen‐replete conditions. PLDε‐OE plants in the field produced more seeds than wild‐type plants but have no impact on seed oil content. Compared with wild‐type plants, PLDε‐OE plants were enhanced in nitrate transporter expression, uptake and reduction, whereas the activity of nitrite reductase was higher under nitrogen‐depleted, but not at nitrogen‐replete conditions. The level of nitrogen altered membrane glycerolipid metabolism, with greater impacts on young than mature leaves. The data indicate increased expression of PLDε has the potential to improve crop plant growth and production under nitrogen‐depleted and nitrogen‐replete conditions.     

Response patterns of Vaccinium myrtillus and V. vitis-idaea along nutrient gradients in boreal forest

Abstract. The relationships between biomass of dwarf shrub species and nutrient gradients of forest soils was studied under field conditions in boreal forests. The biomass-response curves of Vaccinium myrtillus and V. vitis-idaea were fitted against soil nutrient gradients using Generalized Linear Models (GLM). Ecological niches of Vaccinium myrtillus and V. vitis-idaea were evaluated, and effects of nitrogen addition (manipulation of the nutrient gradient) on response function were tested. The Vaccinium species showed statistically significant Gaussian responses along soil nitrogen, phosphorus and calcium gradients, but not along other gradients (K and Mg). Furthermore, manipulation of the nitrogen gradient seemed to have a minor effect on response functions, i.e. addition of nitrogen did not change ecological niches of these species. Ecological optima of V. myrtillus and V. vitis-idaea on the nutrient gradients were about the same. This study suggests that differences in dominance between Vaccinium myrtillus and V. vitis-idaea in boreal forest is not determined by nutrient gradients, but may rather be explained by light conditions and/or moisture availability.     

Isolation and characterization of a marine Anabaena sp. capable of rapid growth on molecular nitrogen

A marine filamentous cyanobacterium capable of rapid growth under N₂-fixing conditions has been isolated from the Texas Gulf Coast. This organism appears to be an Anabaena sp. and has been given the strain designation CA. Cultures grown on mineral salts medium bubbled with 1% CO₂-enriched air at 42°C show a growth rate of 5.6±0.1 generations per day with molecular nitrogen as the sole nitrogen source. This growth rate is higher than any other reported in the literature to date for heterocystous cyanobacteria growing on N₂. Under similar growth conditions, 7.5 mM NH₄Cl yields a growth rate of 6.6±0.1 generations per day while 7.5 mM KNO₃ allows for a growth rate of 5.8±0.4 generations-day. Nitrogen-fixation rates, as measured by acetylene reduction, show maximum activity values in the range of 50–100 nmoles ethylene produced/minxmg protein. These values compare favorably with those obtained from heterotrophic bacteria and are much higher than values reported for other cyanobacteria. Growth experiments indicate that the organism requires relatively high levels of sodium and grows maximally at 42°C. Because of its high growth rate on N₂, this newly isolated organism appears ideal for studying nitrogen metabolism and heterocyst development among the cyanobacteria.     

Studies on the Culture Conditions of Higher Plant Cells in Suspension Culture

To study the influence of cultural conditions on higher plant cells in suspension culture, the effects of nutritional conditions on the growth of suspended cells were investigated. Calluses were induced from 39 species of Nicotiana plants and 6 species of Populus plants on agar slant media, then these were transferred to suspension cultures. Concentrations of 2,4-D and kinetin suitable for incubation of callus from each plant were investigated and species having high growth rates in the appropriate medium were selected.

The effects of concentrations of auxins and kinetin, a variety of carbon and nitrogen sources, thiamin and myo-inositol on growth of the selected calluses were also studied. Of these calluses studied, N. glutinosa, N. tabacum var. Xanthi ova and P. hybrids were selected as calluses having high growth rates. Myo-inositol had no effect on any callus growth, and thiamin gave a distinct effect on Populus callus only. Nitrate as a nitrogen and sucrose as a carbon sources, and 2,4-D as an auxin were most effective in all calluses studied. Kinetin was essential for N. glutinosa among the calluses studied. Although high sugar concentrations tended to lengthen the lag period in the growth curve, there was no difference in the growth rates of the logarithmic phase among the concentrations.     

Nitrogen nutrition in the estuarine zone: the case of Suaeda maritima var. macrocarpa

Suaeda maritima L. var. macrocarpa is a halophytic species distributed in the lower parts of salt marshes of the French coasts. The influence of salinity on nitrogen nutrition and on levels of the key enzymes involved in nitrogen assimilation is analyzed by growing Suaeda under experimental conditions. Use of ¹⁵N-labelled NO₃ ^- and NH₄ ⁺ shows that both ions are effective sources of inorganic nitrogen for Suaeda. The plant is found to use NH₄ ⁺ ions with a good yield, chiefly at high salinities (up to 130 mM). Nitrate reduction and ammonium assimilation by the glutamine synthetase/glutamate synthase pathway occurs mainly in leaves when Suaeda is grown at optimal saline conditions (130 mM NaCl). Absence of NaCl creates less favourable conditions and lowers the activity of nitrate reductase and glutamine synthetase but leads to an important activity of glutamate dehydrogenase in roots. This enzyme could play a major role under suboptimal environmental conditions (i.e., absence of NaCl for Suaeda maritima).Part of this paper is taken from a thesis that was submitted by J. P. Billard in fulfillment of the Doctorat d'Etat degree at the University of Caen, France.     

Chelate-assisted phytoextraction of lead using Fagopyrum esculentum: laboratory vs. field experiments

Abstract

The development of more sustainable remediation techniques has been receiving greater attention, as an alternative to soil excavation plan in urban gardens. An in situ phytoextraction experiment with buckwheat (Fagopyrum esculentum) was performed with a 5?mmol kg^?1 citric acid (CA) application. Joint experiments under laboratory conditions were conducted using various cultivars of F. esculentum in two soils with a Pb contamination of either geogenic or anthropogenic origin and various chelate concentrations. Results show that a minimum dose of 50?mmol kg^?1 of CA is required to lower soil pH and raise the concentration of mobile Pb–CaCl₂ for both soils. Consequently, Pb shoot uptake is increased from 6.3 to 8.9 times depending on soil type. Phytoextraction efficiency is found to be 1.3 to 2.0 times higher in the anthropogenic contaminated soil than in the soil with geogenic Pb. A scale effect has also been identified since Pb root accumulation under laboratory conditions was 2.4 times higher than in the field experiment. Despite an increase in the Pb extraction rate with CA, buckwheat appears to lack the efficiency needed to remove Pb in moderately contaminated soils. The calculated remediation period would last 166?years to remove the mobile Pb fraction.