Solubilization of inorganic phosphate byRhizobium

A large number of strains ofRhizobium were able to solubilize the insoluble phosphate compound, hydroxy-apatite, in liquid culture. Solubilization of hydroxyapatite byRhizobium was not mediated by an enzyme but acidity developed in the cultures was involved in the process. An inverse relationship between the level of soluble phosphate and medium pH was evident. The ability to solubilize hydroxyapatite varied among the strains. In a medium without NH ₄ ⁺ , some of the strains showed better activity than when NH ₄ ⁺ was present, suggesting involvement of different mechanisms for phosphate solubilization.R. meliloti SU 47 produced 2-ketogluconic acid along with an unidentified acid in the medium containing NH ₄ ⁺ . 2-Ketogluconic acid was identified as the major factor in inorganic phosphate solubilization. Initial presence of soluble phosphate in the medium had no discernible influence on the extent of hydroxyapatite solubilization. Initial presence of calcium reduced solubilization of phosphate and addition of EDTA to stationary phase cultures caused an increase in the level of soluble phosphate.     

NH4 + and NO3 − requirement for wheat somatic embryogenesis

The influence of three nitrogen salts: NH₄NO₃, KNO₃ and NH₄Cl on wheat in vitro cultures was investigated. Both NO ₃ ⁻ and NH ₄ ⁺ ions were indispensable for proliferation of embryogenic calli and development of wheat somatic embryos. It is possible to obtain wheat somatic embryos when the medium is enriched with NH₄NO₃ only as a source of inorganic nitrogen. The results of the statistical analysis showed that the level of NH₄NO₃ and KNO₃ in the medium had a great influence on the efficiency of somatic embryogenesis. We observed tendency that calli on media containing 50 mM NH₄NO₃ and 0 to 20 mM KNO₃ turned out to be more embryogenic than on control MS medium. High concentrations of KNO₃- 100 mM inhibited somatic embryogenesis, while 100 mM NH₄NO₃ did not. The level of total N did not have significant influence on wheat somatic embryogenesis. Ratio NO ₃ ⁻ :NH ₄ ⁺ also turned out to be not substantial. We observed that mutual connection of concentration levels between NH₄NO₃ and KNO₃ and between NH₄Cl and KNO₃ was more important. The efficiency of somatic embriogenesis obtained in the experiment with NH₄Cl and KNO₃ was significantly lower than in experiment with NH₄NO₃ and KNO₃.     

Nitrate reductase activity of radish cotyledons as affected by phytohormones and different nitrogen sources

Radish (Raphanus sativus L.) seedlings pretreated with different hormones viz. kinetin, gibberellic acid and abscisic acid were subjected to different N-forms. The seedlings were treated with different concentrations of KNO₃, NH₄Cl and NH₄NO₃ and the changes in nitrate reductase activity were seen in light and dark conditions in the cotyledons. Nitrate reductase activity was affected differently by hormone application. Nitrate increased and ammonia decreased nitrate reductase activity; in both light and dark-grown seedlings KNO₃ induced more in vitro nitrate reductase activity. NH ₄ ⁺ when combined with NO ₃ ⁻ , however, could level up to some extent, with KNO₃ in light, except in kinetin. A transient response of induction of NR activity was evident with decreased levels after a certain specific ambient N-concentration, despite the presence of high N in the medium. However, the pattern of transition varied with the hormones applied. Further, hormones are found to affect induction of different isoforms of nitrate reductase by NH ₄ ⁺ and NO ₃ ⁻ . NH ₄ ⁺ induced isoform was prominently promoted by kinetin treatment in dark. The data documents a particular kind of interaction between controlling factors (light, N-source and phytohormones) which affect nitrate reductase levels.     

Characterization of the Mineral Phosphate-Solubilizing Activity of <Emphasis Type="Italic">Pantoea aglomerans</Emphasis> MMB051 Isolated from an Iron-Rich Soil in Southeastern Venezuela (Bolívar State)

The mineral phosphate-solubilizing (MPS) activity of a Pantoea agglomerans strain, namely MMB051, isolated from an iron-rich, acidic soil near Ciudad Piar (Bolívar State, Venezuela), was characterized on a chemically defined medium (NBRIP). Various insoluble inorganic phosphates, including tri-calcium phosphate [Ca₃(PO₄)₂], iron phosphate (FePO₄), aluminum phosphate (AlPO₄), and Rock Phosphate (RP) were tested as sole sources of P for bacterial growth. Solubilization of Ca₃(PO₄)₂ was very efficient and depended on acidification of the external milieu when MMB051 cells were grown in the presence of glucose. This was also the case when RP was used as the sole P source. On the other hand, the solubilization efficiency toward more insoluble mineral phosphates (FePO₄ and AlPO₄) was shown to be very low. Even though gluconic acid (GA) was detected on culture supernatants of strain MMB051, a consequence of the direct oxidation pathway of glucose, inorganic-P solubilization seemed also to be related to other processes dependent on active cell growth. Among these, proton release by ammonium (NH₄⁺) fixation appeared to be of paramount importance to explain inorganic-P solubilization mediated by strain MMB051. On the contrary, the presence of nitrate (NO₃^–) salts as the sole N source affected negatively the ability of MMB051 cells to solubilize inorganic P.     

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.     

The effect of ammonium on assimilatory nitrate reduction in the haloarchaeon Haloferax mediterranei

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO₃⁻, NO₂⁻ or NH₄⁺) for growth. Different approaches were considered to study the effect of NH₄⁺ on nitrogen assimilation in Hfx. mediterranei cells grown in KNO₃ medium. The NH₄⁺ addition to KNO₃ medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH₄⁺ media. Both activities increased when NH₄⁺ was removed from culture, showing that the negative effect of NH₄⁺ on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO₃⁻ and NH₄⁺, NH₄⁺ was preferentially consumed, but NO₃⁻ uptake was not completely inhibited by NH₄⁺ at prolonged time scale. The addition of MSX to NH₄⁺ or NO₃⁻ cultures results in an increase of Nas and NiR activities, suggesting that NH₄⁺ assimilation, rather than NH₄⁺ per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of cation transport pathways and glycolytic enzyme activity by alterations in red cell volume

In the presence of NH₄Cl and hypotonic solutions, Rana balcanica red cells respond by increasing their volume. The stimulation of cellular volume by hypotonicity is more rapid than that of NH₄Cl, while the maximum value is less than that observed in the presence of NH₄Cl. Depending on the cause of swelling, (net uptake of NH₄Cl or decrease in external osmolality) cells show specific responses. The NH₄Cl treatment causes a significant increase in intracellular Na⁺, from 5·14±0·78 to 29·84±0·47 mmoles l⁻¹ cell, while hypotonicity leads to a significant decrease of this cation, to 3·85±0·25 mmoles l⁻¹ cell in relation to the control, after 30 min of incubation of Rana balcanica erythrocytes. In addition, amiloride significantly reverses the NH₄Cl effect with respect to intracellular Na⁺. Both treatments cause a significant K⁺ loss in comparison with controls. Two glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and pyruvate kinase (PK) of Rana balcanica haemolysate were found to respond to the NH₄Cl effect by significantly decreasing their activity. Copyright © 1999 John Wiley & Sons, Ltd.     

Effect of Nitrogen Source on End Products of Naphthalene Degradation

Soil cultures, enrichment cultures, and pure culture isolates produced substantial quantities of salicylic acid from naphthalene in a mineral salts medium containing NH₄Cl as the nitrogen source. However, when KNO₃ was substituted for NH₄Cl, these same cultures failed to accumulate detectable quantities of salicylic acid but did turn the medium yellow. When an isolate identified as a Pseudomonas species was used, viable cell numbers were much greater in the medium containing KNO₃, but up to 94% of the naphthalene was utilized in both media. After 48 h of incubation in a 0.1% naphthalene-mineral salts medium, the cultures containing NH₄Cl showed irregular clumped cells, a pH of 4.7, 42 μg of salicylic acid per ml, and the production of 4.4 ml of CO₂. Under the same conditions, the cultures in the medium containing KNO₃ showed uniform cellular morphology, a pH of 7.3, no salicylic acid, the production of 29.7 ml of CO₂, and a distinct yellow coloration of the medium. The differences between nitrogen sources could not be accounted for by pH alone since results obtained using buffered media were similar. Growth with NH₄NO₃ displayed a pattern similar to that obtained when NH₄Cl was used. The yellow coloration in the medium containing KNO₃ was apparently due to more than one compound, none of which were 1,2-naphthoquinone or acidic in nature, as suggested by other investigators. Further attempts to identify the yellow compounds by high-pressure liquid chromatography, infrared analysis, and gas chromatography-mass spectrometry have been unsuccessful thus far.     

Enzymes of ammonium assimilation inStreptomyces avermitilis

Glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine dehydrogenase (ADH) and alanine aminotransferase (GPT) were detected in the cell-free homogenate ofStreptomyces avermitilis grown in a defined medium containing ammonium sulfate as the only nitrogen source. At an initial NH₄ ⁺ concentration of 7.5 mmol/L, high activities of GS, GOGAT and GDH were found while that of ADH was low. The ADH activity was markedly increased at initially millimolar NH₄ ⁺ concentrations. In some characteristics of its NH₄ ⁺-assimilating system (e.g. control of some enzyme activities, the NADPH specificity of GOGAT, the presence of alanine aminotransferase),S. avermitilis differs from other known streptomycetes.     

K+-independent effects of valinomycin in photosynthetic systems

With chromatophores ofRhodospirillum rubrum, valinomycin inhibited electron transport in the presence or absence of K⁺. NH₄Cl had no effect on photophosphorylation but uncoupled with valinomycin present. ATPase activity was stimulated by NH₄Cl plus valinomycin but not by either alone. K⁺ partially reversed the inhibition of phosphorylation and the stimulation of ATPase by valinomycin plus NH₄Cl.With chloroplasts, valinomycin inhibited coupled but not basal electron transport. The inhibition was only partially reversed by uncouplers. Valinomycin stimulated the light-activated Mg²⁺-dependent ATPase similar to several uncouplers such as quinacrine, methylamine, and S-13. In addition, valinomycin inhibited delayed light emission and stimulated the H⁺/e^– ratio. These contrasting activities in chloroplasts are not easily explained.Contribution number 389 of the Charles F. Kettering Research Laboratory.     

POTASSIUM ALLEVIATES THE INHIBITORY ACTION OF AMMONIUM UPON THE PHOTOSYNTHETIC RECOVERY OF NOSTOC FLAGELLIFORME (CYANOPHYCEAE) DURING REHYDRATION1

Effects of ammonium on the photosynthetic recovery of Nostoc flagelliforme Berk. et M. A. Curtis were assayed when being rehydrated in low‐K⁺ or high‐K⁺ medium. Its photosynthetic recovery was K⁺ limited after 3 years of dry storage. The potassium absorption of N. flagelliforme reached the maximum after 3 h rehydration in low‐K⁺ medium but at 5 min in high‐K⁺ medium. The K⁺ content of N. flagelliforme rehydrated in high‐K⁺ medium was much higher than that in low‐K⁺ medium. The maximal PSII quantum yield (F_v/F_m) value of N. flagelliforme decreased significantly when samples were rehydrated in low‐K⁺ medium treated with 5 mM NH₄Cl. However, the treatment of 20 mM NH₄Cl had little effect on its F_v/F_m value in high‐K⁺ medium. The relative F_v/F_m 24 h EC₅₀ (concentration at which 50% inhibition occurred) value of NH₄⁺ in high‐K⁺ medium (64.35 mM) was much higher than that in low‐K⁺ medium (22.17 mM). This finding indicated that high K⁺ could alleviate the inhibitory action of NH₄⁺ upon the photosynthetic recovery of N. flagelliforme during rehydration. In the presence of 10 mM tetraethylammonium chloride (TEACl), the relative F_v/F_m 24 h EC₅₀ value of NH₄⁺ was increased to 46.34 and 70.78 mM, respectively, in low‐K⁺ and high‐K⁺ media. This observation suggested that NH₄⁺ entered into N. flagelliforme cells via the K⁺ channel. Furthermore, NH₄⁺ could decrease K⁺ absorption in high‐K⁺ medium.     

Use of controlled-release polymer to feed ammonium to Streptomyces clavuligerus cephalosporin fermentations in shake flasks

Summary Controlled-release polymers containing NH₄Cl were used to feed NH ₄ ⁺ to Streptomyces clavuligerus cultures making cephalosporins in shake flasks. Production was improved in comparison to free NH₄Cl included in the medium at various concentrations; it approached the performance of l-asparagine, the best single nitrogen source for production.     

Control of histidase formation and improvement of urocanic acid production inSerratia marcescens

Summary In the wild-type strain ofSerratia mar-cescens Sr41, histidase was subjected to both cata-bolite repression (CR) by glucose and nitrogen re-pression (NtR) by excess NH⁺ ₄. CR-or NtR-re-sistant mutants for histidase were isolated from urocanase-deficient strain Ud-8, which was more sensitive to CR and NtR than wild-type strain. CR-resistant strain Uc1016 showed a high forma-tion of histidase as well as of D-serine deaminase in the presence of glucose. However, the addition of 2% (NH₄)₂SO₄ to the minimal medium de-creased the histidase formation to a large extent. NtR-resistant strain Gcl98 showed a high forma-tion of histidase as well as of adenylylated glu-tamine synthetase even in the presence of glucose and excess NH⁺ ₄. NtR-resistant strain Gc2031, isolated from urocanic acid-producing strain UdT122, produced urocanic acid more rapidly than strain UdT122 and in proportion to the rate of growth, although the maximum amounts of urocanic acid produced were the same. During cultivation, histidine accumulation was less and histidase activity was four times higher in strain Gc2031 than in the strain UdT122.     

A reliable and efficient protocol for inducing hairy roots in Papaver bracteatum

An efficient and reliable transformation system for a very important medicinal plant Papaver bracteatum was developed through optimization of several factors that affect the rate of effective A. rhizogenes-mediated transformation and growth rate of hairy root. Five bacterial strains, A4, ATCC15834, LBA9402, MSU440 and A13, and three explants types, hypocotyls, leaves and excised shoots were examined. The highest frequency of transformation was achieved using LBA9402 strain in the excised shoots. Several inoculation and co-cultivation media and different concentration of arginine were evaluated using LBA9402 strain and the excised shoots as explant. Interestingly, a drastic increase in the frequency of transformation (47.3 %) was observed when Murashige and Skoog medium containing 1 mM arginine and lacking NH₄NO₃ KH₂PO₄, KNO₃ and CaCl₂ was used. The effect of sucrose concentration and the ratio of NH₄ ⁺: NO₃ ^? on hairy root biomass was examined. Maximum biomass was obtained in 30 g/l sucrose and 20:10 mM ratio of NH₄ ⁺ to NO₃ ^? on MS medium. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR) and Southern hybridization.     

Decoloration of azo dyes by three whiterot fungi: influence of carbon source

Two strains of Phanerochaete chrysosporium and a local isolate of white-rot fungus, if pre-cultured in a high nitrogen medium with glucose, could decolorize two azo dyes (Amaranth and Orange G) and a heterocyclic dye (Azure B). When starch was used in the pre-cultivation medium, decoloration of Orange G occurred if the medium also contained 12mM NH₄Cl, whether or not veratric acid was present. In medium containing 1.2mM NH₄Cl and veratric acid, greater decoloration occurred with one strain of P. chrysosporium and the local isolate. In preculture medium with cellulose and 1.2mM NH₄Cl, decoloration by the local isolate was enhanced but not that by the other strains.The authors are with the Department of Microbiology, Soochow University, Shih Lin, Taipei, Taiwan     

Simultaneous phosphate solubilization potential and antifungal activity of new fluorescent pseudomonad strains, Pseudomonasaeruginosa, P. plecoglossicida and P. mosselii

Of 80 fluorescent pseudomonad strains screened for phosphate solubilization, three strains (BFPB9, FP12 and FP13) showed the ability to solubilize tri-calcium phosphate (Ca₃(PO₄)₂). During mineral phosphate solubilization, decrease of pH in the culture medium due to the production of organic acids by the strains was observed. These phosphate solubilizing strains produced indole-3-acetic acid (IAA) and protease as well as exhibited a broad-spectrum antifungal activity against phytopathogenic fungi. When tested in PCR using the gene-specific primers, strain BFPB9 showed the presence of hcnBC genes that encode hydrogen cyanide. On the basis of phenotypic traits, 16S rRNA sequence homology and subsequent phylogenetic analysis, strains BFPB9, FP12 and FP13 were designated as Pseudomonas aeruginosa, P. plecoglossicida and P. mosselii, respectively. Present investigation reports the phosphate solubilization potential and biocontrol ability of new strains that belong to P. plecoglossicida and P. mosselii. Because of the innate potential of phosphate solubilization, production of siderophore, IAA, protease, cellulase and HCN strains reported in this study can be used as biofertilizers as well as biocontrol agents.     

The Amount of Phosphate Solubilization Depends on the Strain,C-Source,Organic Acids and Type of Phosphate

Although production of organic acids (OAs) is usually mentioned as the main mechanism of phosphate solubilization, the relationship between carbon sources (C-sources) and OAs produced during phosphate-solubilization by microorganisms is still poorly understood. We evaluated the influence of different C-sources on FePO₄·2H₂O and Ca₃(PO₄)₂ solubilization by bacteria and on the identity/quantity of the OAs produced. Our results showed that the amount of phosphate solubilization depends on the strain, C-source, OAs, and type of phosphate. Among the five strains under study isolated from cowpea nodules (Rhizobium tropici strain UFLA 03-08, Acinetobacter sp. strain UFLA 03-09, Paenibacillus kribbensis strain UFLA 03-10, P. kribbensis strain UFLA 03-106, and Paenibacillus sp. strain UFLA 03-116), three of them solubilized Ca₃(PO₄)₂ in all C-sources. The influence of C-sources on Ca₃(PO₄)₂-solubilization increased in the following order: cellulose?<?lactose?<?mannitol?<?glucose. A significant positive correlation between the amount of phosphorus solubilized from Ca₃(PO₄)₂ and the concentration of total OAs in the presence of glucose and mannitol was observed for these three strains. In the presence of glucose, the highest solubilization rates are associated with high concentrations of tartaric acid, and in the presence of mannitol, are associated with maleic acid. Only one strain produced OAs in the medium with lactose and Ca₃(PO₄)₂, but there was no OAs in the medium containing cellulose. Despite the production of OAs, albeit in small concentrations, in all the C-sources investigated, FePO₄·2H₂O-solubilization was not observed. Thus, a relationship among C-sources, OAs, and phosphate solubilization was not always verified.     

NH4 + transport system of a psychrophilic marine bacterium, Vibrio sp. strain ABE-1

NH₄ ⁺ transport system of a psychrophilic marine bacterium Vibrio sp. strain ABE-1 (Vibrio ABE-1) was examined by measuring the uptake of [¹⁴C]methylammonium ion (¹⁴CH₃NH^{3 +}) into the intact cells. ¹⁴CH₃NH₃ ⁺ uptake was detected in cells grown in medium containing glutamate as the sole nitrogen source, but not in those grown in medium containing NH₄Cl instead of glutamate. Vibrio ABE-1 did not utilize CH₃NH₃ ⁺ as a carbon or nitrogen source. NH₄Cl and nonradiolabeled CH₃NH₃ ⁺ completely inhibited ¹⁴CH₃NH₃ ⁺ uptake. These results indicate that ¹⁴CH₃NH₃ ⁺ uptake in this bacterium is mediated via an NH₄ ⁺ transport system and not by a specific carrier for CH₃NH₃ ⁺. The respiratory substrate succinate was required to drive ¹⁴CH₃NH₃ ⁺ uptake and the uptake was completely inhibited by KCN, indicating that the uptake was energy dependent. The electrochemical potentials of H⁺ and/or Na⁺ across membranes were suggested to be the driving forces for the transport system because the ionophores carbonylcyanide m-chlorophenylhydrazone and monensin strongly inhibited uptake activities at pH 6.5 and 8.5, respectively. Furthermore, KCl activated ¹⁴CH₃NH₃ ⁺ uptake. The ¹⁴CH₃NH₃ ⁺ uptake activity of Vibrio ABE-1 was markedly high at temperatures between 0° and 15°C, and the apparent K _m value for CH₃NH₃ ⁺ of the uptake did not change significantly over the temperature range from 0° to 25°C. Thus, the NH₄ ⁺ transport system of this bacterium was highly active at low temperatures. Received: August 1, 1998 / Accepted: October 8, 1998     

Adaptation to phosphate deprivation in osteoblast-like cells

The rat osteosarcoma cell line UMR-106–01 has an osteoblast-like phenotype. When grown in monolyer culture these cells transport inroganic phosphate and L-alanine via Na⁺-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60–70 per cent increase in Na⁺-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na⁺-dependent alanine uptake and Na⁺-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased V_max and was blocked completely by pretreatment with cycloheximide (70 μM). In these cells recovery of intracellular pH after acidification with NH₄Cl is due primarily to the Na⁺/H⁺ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na⁺/H⁺ exchange was inhibited during phosphate deprivation.     

Interactions between C and N metabolism in Dunaliella salina cells cultured at elevated CO2 and high N concentrations

The green algaDunaliella salina UTEX 200 was cultured at high (5 percnt;) [CO₂] in a medium containing 10 mmol/L of either NO₃⁻ or NH₄⁺ as the sole N source. Specific growth rate was 50 percnt; higher for NH₄⁺-grown cells than for their counterparts cultured in the presence of NO₃⁻. Cell size, protein content, Rubisco protein, phosphoenolpyruvate carboxylase (PEPC) activity, and light independent carbon fixation were enhanced by growth in the presence of NH₄⁺. On the other hand, maximal photosynthetic rate and cell glycerol concentration were lower when N was supplied as NH₄⁺. The activity of glutamine synthetase was affected very little by the N-source.D. salina UTEX 200 showed some peculiarities in its mechanism of adaptation to high [N] in comparison to other strains previously used for similar studies. This allowed dissection of the underlying mechanism of the growth response to high [N], highlighting the potential role of PEPC, the main anaplerotic enzyme, as a pivotal player in the adaptation of cells to these conditions.