Caesium uptake and toxicity in the cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis>

A NH₄⁺ transport-defective mutant and a K⁺ transport-defective mutant of the cyanobacterium Nostoc muscorum were analysed with regard to percentage survival as a function of CsCl toxicity and Cs⁺ uptake activity. Neither survival nor Cs⁺ uptake was affected in either of the two mutants when compared with the wild type. The results indicate that the toxicity of Cs⁺ is determined at more than one cellular site in this organism.     

Nutrient uptake and alkaline phosphatase (ec 3:1:3:1) activity of emiliania huxleyi (PRYMNESIOPHYCEAE) during growth under n and p limitation in continuous cultures

Emiliania huxleyi (strain L) expressed an exceptional P assimilation capability. Under P limitation, the minimum cell P content was 2.6 fmol P·cell^?1, and cell N remained constant at all growth rates at 100 fmol N·cell^?1. Both, calcification of cells and the induction of the phosphate uptake system were inversely correlated with growth rate. The highest (cellular P based) maximum phosphate uptake rate (V_max^P) was 1400 times (i.e. 8.9 h^?1) higher than the actual uptake rate. The affinity of the P‐uptake system (dV/dS) was 19.8 L·μmol^?1·h^?1 at μ = 0.14 d^?1. This is the highest value ever reported for a phytoplankton species. V_max and dV/dS for phosphate uptake were 48% and 15% lower in the dark than in the light at the lowest growth rates. The half‐saturation constant for growth was 1.1 nM. The coefficient for luxury phosphate uptake (Q_maxt/Q_min) was 31. Under P limitation, E. huxleyi expressed two different types of alkaline phosphatase (APase) enzyme kinetics. One type was synthesized constitutively and possessed a V_max and half‐saturation constant of 43 fmol MFP·cell^?1·h^?1 and 1.9 μM, respectively. The other, inducible type of APase expressed its highest activity at the lowest growth rates, with a V_max and half‐saturation constant of 190 fmol MFP·cell^?1·h^?1 and 12.2 μM, respectively. Both APase systems were located in a lipid membrane close to the cell wall. Under N‐limiting growth conditions, the minimum N quotum was 43 fmol N·cell^?1. The highest value for the cell N‐specific maximum nitrate uptake rate (V_max^N) was 0.075 h^?1; for the affinity of nitrate uptake, 0.37 L·μmol^?1·h^?1. The uptake rate of nitrate in the dark was 70% lower than in the light. N‐limited cells were smaller than P‐limited cells and contained 50% less organic and inorganic carbon. In comparison with other algae, E. huxleyi is a poor competitor for nitrate under N limitation. As a consequence of its high affinity for inorganic phosphate, and the presence of two different types of APase in terms of kinetics, E. huxleyi is expected to perform well in P‐controlled ecosystems.     

Genetically modified cyanobacteriumNostoc muscorum overproducing proline in response to salinity and osmotic stresses

In the parentNostoc muscorum an active proline oxidase enzyme is required to assimilate exogenous proline as a fixed nitrogen source. Cyanobacterial mutants, resistant to growth inhibitory action of proline analogue L-azetidine-2-carboxylate (Ac-R), were deficient in proline oxidase activity, and were over-accumulators of proline. Proline over-accumulation, resulting either from mutational acquisition of the Ac-R phenotype, or from salinity-induced uptake of exogenous proline, confirmed enhanced salinity/osmotic tolerance in the mutant strain. The nitrogenase activity and photosynthetic O₂ evolution of the parent were sensitive to both salinity as well as osmotic stresses than of Ac-R mutant strain. In addition, the mutation to Ac-resistant phenotype showed no alteration in salinity inducible potassium transport system in the cyanobacterium.     

Physiological and biochemical analysis of the glutamine synthetase-impaired mutants of the nitrogen-fixing cyanobacteriumNostoc muscorum

Three types of glutamine synthetase (GS)-impaired mutants (gln) ofNostoc muscorum were isolated as ethylenediamine (EDA)-resistant phenotypes and characterized with respect to heterocyst development, nitrogen fixation, ammonium metabolism, photosynthetic characteristics, and glutamine synthetase activity. The criterion for categorizing the mutants was the extent of loss of GS activity (both in transferase and biosynthetic assays) compared with wild type; it was 70% in EDA-1, 30% in EDA-2, and more than 90% in EDA-3 strains. The level of nitrogenase activity in mutant strains was proportionate to heterocyst frequency and was found refractory to ammonium and EDA repression. In EDA-resistant strains, development of heterocysts and their spacing pattern remained unaffected and did not respond to treatment of amino acid analogues, drugs, and ammoniacal compounds which otherwise either stimulated or suppressed the number and altered the spacing pattern in wild type. A biphasic pattern of ammonium uptake indicating two transport systems was observed in all the strains except that the Km values for both high- and low-affinity systems were altered in mutant strains. In vivo treatment with MSX or EDA significantly inhibited the GS activity in wild type, whereas mutant strains did not respond to these treatments and were able to liberate NH ₄ ⁺ continuously into the medium without MSX treatment. During NH ₄ ⁺ uptake, percentage inhibition of O₂ evolution and changes in increase of fluorescence intensity were low in EDA strains compared with wild type. Assessment of GS protein with antibodies against GS and quantitative polyacrylamide gel electrophoresis (PAGE) suggested that loss in specific activity of GS per milligram of extractable protein in EDA mutants was owing to low production of GS-specific protein. SDS-PAGE of purified GS enzyme from all the strains revealed only one polypeptide band of molecular weight of about 51.28 kDa.     

Differential response of NaCl-Resistant mutants of the cyanobacterium Nostoc muscorum to salinity and osmotic stresses

Summary In the parent strain of Nostoc muscorum, the percentage survival, nitrogenase activity and oxygenic photosynthesis were severely impaired by NaCl (ionic) and sucrose (non-ionic) stresses. Spontaneously occurring NaCl-Resistant mutant clones of the cyanobacterium N. muscorum were found to exhibit differential responses under ionic and non-ionic stresses. One of the mutants (NaCl-R) was found to show resistance in terms of percentage survival, nitrogenase activity and oxygenic photosynthesis under saline (ionic) as well as osmotic (non-ionic) stresses and showing compatible solute strategy for such adaptation. Another mutant (Na⁺-R) was found to show resistance only to salinity stress and showed an enhanced Na⁺-efflux system driven by H⁺. The Na⁺-R mutant differed from the NaCl-R mutant strain in the sense that it was sucrose sensitive.     

Evaluating the potential of <Emphasis Type="Italic">N. calcicola</Emphasis> and its bicarbonate resistant mutant as bioameleorating agents for ‘usar’ soil

The potential of Nostoc calcicola and its bicarbonate resistant mutant as bioameleorating agent was investigated, using laboratory simulation experiments, in terms of their growth potential, glutamine synthetase (GS) activity, heterocyst frequency and effect on pH of soil. Nostoc calcicola, exhibited a tendency to lower the pH of ‘usar’ soil significantly and showed better growth and pigment content at 20% soil extract as compared to basal medium. The bicarbonate resistant mutant (HCO₃ ^−R) exhibited a better ability to grow at higher percentage of soil extract (60%), besides bringing about a more significant change in soil pH as compared to wild type. The heterocyst frequency was much higher in the mutant strain, which was not significantly affected by growth in various concentrations of soil extract. The mutant strain holds promise as a potential bioameliorant for ‘usar’ soil after further evaluation of its reclamative properties at field level.     

Membrane Functions and Tolerance to Aromatic Hydrocarbon Fungitoxicants in Conidia of Fusarium solani

With the use of ³²P-labeled phosphate and ⁴²K₂CO₃ the effect of diphenyl on permeability and uptake properties of the cytoplasmic membrane in wild type and diphenyl-tolerant mutant conidia of Fusarium solani f. cucurbitae was studied. No general damage to the membrane with unspecific leakage of cell constituents was demonstrated under conditions in which diphenyl prevents germination of wild type conidia. The fresh conidia do not require exogenous supply of energy for the uptake of phosphate or of potassium. In the wild type the entry of ³²P is inhibited but that of ⁴²K strikingly stimulated by diphenyl. Independently of the tolerant mutant gene present, the mutant conidia are significantly less sensitive to the phosphate uptake inhibition and not affected at all by diphenyl with respect to the uptake of potassium. The latter difference from the wild type seems to indicate genetic control of some property of the potassium transport system in this fungus.     

Changes in the Pi uptake and polyP accumulation in Saccharomyces cerevisiae strains deficient in the synthesis of trehalose and/or glycerol

The intracellular level of free inorganic orthophosphate (P_i) in yeast cells generally depends on the P_i uptake capacity, energy state of the cells in respect to the activity of the membrane-associated ATPases and on the activity of metabolic pathways involved in the production of glycerol and trehalose. Batch fermentation was performed to investigate the carbon substrate consumption, the P_i uptake capacity and product formation by four Saccharomyces cerevisiae strains differing in their ability to produce glycerol and/or trehalose. The consumption of P_i in mutant strains with a lack of the synthesis of the trehalose and/or glycerol exceeded the level for a wild type strain about two times. Maximum intracellular polyP content (29.9 mg/g DW) was shown for tps1Δ gpd1Δ mutant. In this study we showed that the P_i uptake and polyP accumulation level were closely connected with the changes in the synthesis of trehalose and glycerol.     

pho3: a phosphorus-deficient mutant of Arabidopsis thaliana (L.) Heynh

A novel P-deficient mutant of Arabidopsis thaliana, pho3, was isolated by screening for root acid phosphatase (APase) activity in plants grown under low-P conditions. pho3 had 30% less APase activity in roots than the wild type and, in contrast to wild-type plants, root APase activity did not increase in response to growth in low P. However, shoot APase activity was higher in pho3 than in the wild-type plants. In addition, the pho3 mutant had a P-deficient phenotype, even when grown in P-sufficient conditions. The total P content of 11-d-old pho3 plants, grown in agar media with a plentiful supply of P, was about 25% lower than the wild-type level in the shoot, and about 65% lower in the roots. In the rosette leaves of mature soil-grown pho3 plants the total P content was again reduced, to about 50% of wild-type levels. pho3 exhibited a number of characteristics normally associated with low-P stress, including severely reduced growth, increased anthocyanin content (at least 100-fold greater than the wild type in soil-grown plants) and starch accumulation. The results suggest that the mutant is unable to respond to low internal P levels, and may lack a transporter or a signalling component involved in regulating P nutrition. Received: 21 March 2000 / Accepted: 15 August 2000     

Response of Alkaline Phosphatases in the Cyanobacterium Anabaena sp. FACHB 709 to Inorganic Phosphate Starvation

Alkaline phosphatases (APases) play a crucial role in phosphorus (P) metabolism and regulation, but their physiological functions largely remain unclear in cyanobacteria. Here, we identified four putative APase genes, designated as phoA-709, phoD1-709, phoD2-709, and phoS-709, in the cyanobacterium Anabaena sp. FACHB 709, and investigated their response to inorganic phosphate (P_i) starvation. With the exception of phoD2-709, three other APase genes were expressed at a constant and relative low level in P_i-replete medium, whereas the expression of all four APase genes was elevated in response to P_i starvation but phoA-709 significantly. However, disruption of phoA-709 did not affect the total APase activity but caused the expressional up-regulation of phoD1-709 and phoS-709 under P_i-sufficient and P_i-limiting conditions. These suggest that, the four APases of Anabaena sp. FACHB 709 are involved in P metabolism and regulation, and PhoA-709 is the main, yet dispensable, APase.     

Functional characterization of the Aspergillus fumigatusPHO80 homologue

Phosphate is an ion that is essential for fungal growth. The systems for inorganic phosphate (P_i) acquisition in eukaryotic cells (PHO) have been characterized as a low-affinity (that assures a supply of P_i at normal or high external P_i concentrations) and a high-affinity (activated in response to P_i starvation). Here, as an initial step to understand the PHO pathway in Aspergillus fumigatus, we characterized the PHO80 homologue, PhoB^PHO80. We show that the ΔphoB^PHO80 mutant has a polar growth defect (i.e., a delayed germ tube emergence) and, by phenotypic and phosphate uptake analyses, establish a link between PhoB^PHO80, calcineurin and calcium metabolism. Microarray hybridizations carried out with RNA obtained from wild-type and ΔphoB^PHO80 mutant cells identify Afu4g03610 (phoD^PHO84), Afu7g06350 (phoE^PHO89), Afu4g06020 (phoC^PHO81), and Afu2g09040 (vacuolar transporter Vtc4) as more expressed both in the ΔphoB^PHO80 mutant background and under phosphate-limiting conditions of 0.1 mM P_i. Epifluorescence microscopy revealed accumulation of poly-phosphate in ΔphoB^PHO80 vacuoles, which was independent of extracellular phosphate concentration. Surprisingly, a phoD^PHO84 deletion mutant is indistinguishable phenotypically from the corresponding wild-type strain. mRNA analyses suggest that protein kinase A absence supports the expression of PHO genes in A. fumigatus. Furthermore, ΔphoB^PHO80 and ΔphoD^PHO84 mutant are fully virulent in a murine low dose model for invasive aspergillosis.     

Proton- and sodium-coupled phosphate transport systems and energy status of Yarrowia lipolytica cells grown in acidic and alkaline conditions

In this study we have used a newly isolated Yarrowia lipolytica yeast strain with a unique capacity to grow over a wide pH range (3.5–10.5), which makes it an excellent model system for studying H⁺- and Na⁺-coupled phosphate transport systems. Even at extreme growth conditions (low concentrations of extracellular phosphate, alkaline pH values) Y. lipolytica preserved tightly-coupled mitochondria with the fully competent respiratory chain containing three points of energy conservation. This was demonstrated for the first time for cells grown at pH 9.5–10.0. In cells grown at pH 4.5, inorganic phosphate (P_i) was accumulated by two kinetically discrete H⁺/P_i-cotransport systems. The low-affinity system is most likely constitutively expressed and operates at high P_i concentrations. The high-affinity system, subjected to regulation by both extracellular P_i availability and intracellular polyphosphate stores, is mobilized during P_i-starvation. In cells grown at pH 9.5–10, P_i uptake is mediated by several kinetically discrete Na⁺-dependent systems that are specifically activated by Na⁺ ions and insensitive to the protonophore CCCP. One of these, a low-affinity transporter operative at high P_i concentrations is kinetically characterized here for the first time. The other two, high-affinity, high-capacity systems, are derepressible and functional during P_i-starvation and appear to be controlled by extracellular P_i. They represent the first examples of high-capacity, Na⁺-driven P_i transport systems in an organism belonging to neither the animal nor bacterial kingdoms. The contribution of the H⁺- and Na⁺-coupled P_i transport systems in Y. lipolytica cells grown at different pH values was quantified. In cells grown at pH values of 4.5 and 6.0, the H⁺-coupled P_i transport systems are predominant. The contribution of the Na⁺/P_i cotransport systems to the total cellular P_i uptake activity is progressively increased with increasing pH, reaching its maximum at pH 9 and higher. Received: 15 December 2000/Revised: 14 May 2001     

Response of two strains of Nostoc muscorum to metal stress and salinity

Two strains of a cyanobacterium Nostoc muscorum, wild-type N. muscorum (Cd^s) and an isolate having resistance to the heavy metal cadmium (Cd^r), were selected for characterisation of their growth potential and physiological assays in certain defined stress environments. The chosen determinants were copper (heavy metal) and NaCl (salt stress). The observations on growth, heterocyst frequency, chlorophyll and nucleic acid contents, photosynthetic O₂ evolution, ¹⁴C incorporation and acetylene reduction suggested that the strain Cd^r was also resistant to copper. This strain, however, was found to be more sensitive to NaCl in comparison to its wild-type counterpart. NaCl was found to enhance sugar accumulation in Cd^s and was more inhibitory to acetylene reduction rates than to the photosynthetic activities. The interaction between Cu and NaCl appeared to be antagonistic as the depression of growth and physiological activities by a mixture of the two was lesser than that caused by either of these. These observations form the first report on the response of a metal resistant strain of cyanobacterium to salinity.     

Isolation and characterisation of nitrate reductase mutants and regulation of nitrate reductase and nitrogenase in the cyanobacterium Nostoc muscorum

Summary Chlorate resistant mutants of the cyanobacterium Nostoc muscorum isolated after N-methyl-N-nitro-N-nitrosoguanidine (MNNG) mutagenesis were found to be defective/blocked in nitrate reductase (NR).The parent strain possessed active NR in the presence of nitrogen as nitrate and only basal levels of activity in ammonia and N-free grown cultures. Addition of ammonia suppressed the NR activity in the parent strain whereas addition of L-methionine DL-sulphoximine (MSX) restored NR activity. A similar repression by ammonia, glutamine and derepression with MSX were also observed for nitrogenase synthesis.One class of mutants lacked NR activity (nar ^-) whereas the specific activity of NR was low in another class of mutants (nar ^def). Unlike the parent, the mutants synthesized nitrogenase and differentiated heterocysts in the presence of nitrate nitrogen. Uptake studies of nitrite and ammonia in mutants revealed that they possessed both nitrite reductase and glutamine synthetases (GS) at low levels, and the same level respectively in comparison with the parent.     

Mutational Engineering of the Cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis> for Resistance to Growth-Inhibitory Action of LiCl and NaCl

The effect of NaCl on two vital processes of cyanobacterial metabolism, viz. N₂ fixation and oxygenic photosynthesis, was studied in the cyanobacterium Nostoc muscorum grown diazotrophically. An increase in NaCl concentration suppressed the formation of heterocyst and adversely affected the nitrogenase activity in the parent, whereas in Li⁺-R and Na⁺-R mutants NaCl stress did not cause any adverse effect. The rate of photosynthetic O₂-evolution was also adversely affected by the NaCl stress, but the magnitude was less than that of nitrogenase activity. L-Proline, the well-known osmoprotectant, provided protection to the cyanobacterium against NaCl stress. The parent strain utilized L-proline as a nitrogen source and suppressed heterocyst formation and nitrogenase activity, while mutants showed normal heterocyst frequency and nitrogenase activity. Therefore, it may be that the proline metabolism is altered as a result of mutation. The intracellular levels of proline in the parent were enhanced about threefold in the medium containing 1 mol m⁻³ proline, while in mutants there was no significant increase in the intracellular level of proline. In the medium containing both NaCl and proline, the intracellular level of proline was enhanced in the parent as well as in both mutant strains. This suggests that the parent strain possessed both normal proline uptake and salt-induced proline uptake systems, whereas the mutant strains were defective in normal proline uptake and had only salt-induced proline uptake. The over-accumulation of proline in the presence of NaCl stress is due either to the loss of proline oxidase activity or to the accumulation of exogenous proline. Received: 10 July 2002 / Accepted: 13 August 2002     

31P NUCLEAR MAGNETIC RESONANCE STUDY OF INTRACELLULAR PHOSPHATE POOLS AND POLYPHOSPHATE METABOLISM IN HETEROSIGMA AKASHIWO (HADA) HADA (RAPHIDOPHYCEAE)1

The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by ³¹P-NMR spectroscope of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) Hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol·cell⁻¹ and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (P_i). After addition of P_i, rapid uptake of P_i was observed and the P cell quota increased to 108 fmol·cell⁻¹ in 2 h, 134 fmol·cell⁻¹ in 5 h and 222 fmol·cell⁻¹ in 1 day after addition of phosphate. The ³¹P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of P_i.     

The effect of weak auxins on the growth of blue-green algae

Summary 5-Hydroxy indole-3-acetic acid promoted the growth (increase in dry weight) of Anacystis nidulans, Chlorogloea fritschii, Phormidium foveolarum, Nostoc muscorum and Tolypothrix tenuis. 5-Hydroxy tryptamine stimulated the growth of Chlorogloea fritschii and Nostoc muscorum. Phenyl-acetic acid promoted the growth of Nostoc muscorum and Tolypothrix tenuis. Tryptophol stimulated the growth of Chlorogloea fritschii, it failed to stimulate the growth of Nostoc muscorum. Isatin promoted the growth of Anacystis nidulans and Chlorogloea fritschii 2, 3, 5-triidobenzoic acid inhibited the growth of Anacystis nidulans, Chlorogloea fritschii, Phormidium foveolarum, Nostoc muscorum and Tolypothrix tenuis.     

Evidence for a sodium-dependent proline and glycine-betaine uptake in the cyanobacterium <Emphasis Type="Italic">Nostoc muscorum</Emphasis>

The cyanobacterium Nostoc muscorum is able to utilized proline and glycine-betaine as a nitrogen source under unstressed growth conditions. This cyanobacterium when grow in modified Chu No. 10 medium (without Na⁺) unable to utilized proline and glycine-betaine as a nitrogen source. Spontaneously occurring mutant clones defective in Na⁺ transport (Na⁺-R) was isolated and analyzed for proline and glycine-betaine utilization. The mutant phenotype showed normal heterocyst frequency and nitrogenase activity even in the medium containing 1 mM proline or 1 mM glycine-betaine, indicates the role of Na⁺ for proline/glycine-betaine uptake. The Na⁺-R mutant showed 100% survival at pH 11 and was simultaneously able to uptake and utilize proline/glycine-betaine at higher alkaline pH. This indicates that proline and glycinebetaine uptake systems are more efficient at higher alkaline pH. Since, the hypersaline environments are rich in Na⁺ contents and have alkaline pH, therefore it is suggested that the origin and evolution of specific compatible solutes may not depend only on the osmoregulatory role they play, but also on the other ecological factors operating simultaneously in the organism’s niche.