Sensitivity of Nostoc commune UTEX 584 (Cyanobacteria) to water stress

Cells of Nostoc commune UTEX 584 from liquid cultures expressed an upshift in nitrogenase activity when immobilised on inert supports and exposed to matric water potentials between -1.10 and -99.5 MPa. Cells incubated at 0.10 MPa (a_w=c 1.0) maintained increased activity for at least 48 h following immobilization. At water potentials below -23.1 MPa (a_w=0.85), the upshift was transitory. Nitrogenase activity decreased rapidly when immobilised cells were incubated at lower values of _m.Desiccated cells stored at -99.5 MPa (a_w=0.50) underwent an upshift in nitrogenase activity, and in the size of the intracellular ATP pool, when rewetted with either distilled water or liquid MB_o medium (_o =-0.18 MPa). The upshift in nitrogenase activity was chloramphenicol-sensitive and was preceeded by a lag. The duration of the lag depended on the time taken to equilibrate cells to-99.5 MPa, the time desiccated, and the conditions of storage and rewetting. Cells that had no, or very low, nitrogenase activity when rewetted in air, showed a marked stimulation of nitrogenase activity in the presence of 5% v/v CO₂ under both aerobic and anerobic conditions.When rewetted in the presence of 1% w/v glucose (_o =-0.14 MPa), vegetative cells remained intact, but heterocysts underwent autolysis and nitrogenase activity was not detected, even in the presence of 5% v/v CO₂.Abbreviations TTC 2,3,5-triphenyl-2-tetrazolium chloride - _m matric water potential - _o osmotic water potential - a_w water activity     

Shifts in the intracellular ATP pools of immobilisedNostoc cells (Cyanobacteria) induced by water stress

Summary Immobilised, desiccated cells ofNostoc commune UTEX 584 have the capacity to increase the size of their extractable intracellular ATP pool upon rewetting. The time taken to recover the pool size depends on the conditions of storage at a particular water potential and the duration of storage. Under the conditions employed, the rewetting of cells induced an increase in ATP pool size at the expense of photophosphorylation or electron transport (oxidative) phosphorylation. The rise in the ATP pool size was instantaneous and was shown to be due to ATP synthesis. This increase did not occur when cells were rewetted in the presence of sodium azide (10 mmol/l), while a partial inhibition was observed with CCCP (carbonyl cyanidem-chlorophenylhydrazone; 2 mol/l). For cells dried at more extreme water potentials, the lag ofc 48 h observed before the ATP pool reached control values is of similar duration to that observed in the recovery of nitrogenase upon rewetting. Chloramphenicol (10 mol/l) stimulated significantly the upshift in the size of the ATP pool ofNostoc cells upon rewetting, yet inhibited completely the rise in nitrogenase activity.     

In vitro translation of mRNA from Nostoc commune (Cyanobacteria)

RNA pools were extracted from cells of Nostoc commune UTEX 584 in exponential growth (liquid cultures) and from cells which had been immobilized and dried rapidly at -99.5 MPa. Levels of incorporation of ³⁵S-methionine, five- to sixfold higher than the endogenous level, were obtained after in vitro translation of the RNA preparations in a heterologous S30 cell-free system purified from Escherichia coli Q13. The levels of incorporation, obtained with a homologous N. commune UTEX 584 S30 system, were much lower. The requirement for magnesium in the heterologous system was 15–21 mM, translation of N. commune UTEX 584 RNA was inhibited when the RNA concentration was greater than 0.3 mg ml^–1, and translation was stimulated significantly by the presence of ammonium chloride. Few qualitative differences were observed between the pattern of proteins (SDS-PAGE) obtained after translation of the RNA pools from cells in exponential growth, and from those cells subjected to immobilization and rapid drying. The data suggest that short-term desiccation of N. commune UTEX 584 does not have a marked selective effect on the composition of the mRNA pool. In contrast, preparations of RNA from field materials of Nostoc commune HUN (desiccated for 5 years) were unable to drive high rates of translation in any of the systems tested and optimized for use in this study.     

Polysome turnover in immobilized cells of Nostoc commune (cyanobacteria) exposed to water stress.

Water stress induced changes in the polysome content of immobilized cells of the desiccation-tolerant cyanobacterium Nostoc commune UTEX 584. Cells maintained an intact protein synthesis complex during 2 h of drying at -99.5 MPa. Polysomes were not recovered from cells subjected to extended periods of desiccation.     

Distribution and ecology of the edible cyanobacterium Ge-Xian-Mi (Nostoc) in rice fields of Hefeng County in China

Ge-Xian-Mi (an edible species of Nostoc) grows insome mountain paddy fields in China during winter and forms macroscopicallyvisible subspherical colonies. The geology and climate at one of its locations,Hefeng County, were investigated, and the present-day situation of Ge-Xian-Miwas assessed in order to raise awareness that it may be endangered. There wereformerly 796 ha of rice fields suited to its growth in HefengCounty and the maximum annual yield ever reached was 25 t. Theannual mean temperature is about 12.2 °C, and the annualrainfall is 1934 mm with mean relative humidity of 78–87%.The distribution of Ge-Xian-Mi was found to be associated with the source ofwater and the pH values of water suited to its growth were 6.2–6.3. Soilsin its habitats were enriched with phosphorus and contained more microbes thanthose without its distribution. With changing agricultural techniques most ofthe habitats are endangered or already extinct. The widespread use ofherbicides, pesticides and fertilizers containing chlorine had been suggestedtobe an important factor limiting its distribution. The taxonomic identity ofGe-Xian-Mi is discussed.     

Ultrastructural analysis of the rehydration of desiccatedNostoc commune HUN (cyanobacteria) with particular reference to the immunolabelling of NifH

Summary Vegetative cells and heterocysts of the filamentous desiccation-tolerant cyanobacteriumNostoc commune HUN retain their ultrastructural organisation and the integrity of their intra- and extracellular membranes after two years of desiccation and subsequent rehydration. Immunogold-labelling of thin sections demonstrated the presence of NifH (Fe protein of nitrogenase) in vegetative cells and heterocysts within five minutes of the rehydration of dried colonies. Immunogold label accumulated in discrete areas vegetative cells within 5 minutes of the rewetting of cells, and after 30 minutes a conspicuous association of NifH protein with heterocyst ribosomes was detected. After longer periods of rehydration, the deposition of gold particles became more random within both cell types but occurred with a greater frequency in heterocysts. Up to 24 hours after the rewetting of cells, two morphologically-distinct forms of heterocyst could be discerned. NifH protein was detected through Western blotting (subunit M_r=33,800) in protein extracts from samples ofNostoc commune, collected in different parts of the world and including some which had been desiccated for periods of up to 10 years. The results are discussed in relation to the sequential recovery of metabolic functions, particularly nitrogen fixation, which occurs upon the rehydration of cells after their prolonged storage in the air-dry state.     

The production of cyclopiazonic acid by Penicillium commune and cyclopiazonic acid and aflatoxins by Aspergillus flavus as affected by water activity and temperature on maize grains

The combined effects of water activity (a_w) and temperature on mycotoxin production by Penicilium commune (cyclopiazonic acid — CPA) and Aspergillus flavus (CPA and aflatoxins — AF) were studied on maize over a 14-day period using a statistical experimental design. Analysis of variance showed a highly significant interaction (P 0.001) between these factors and mycotoxin production. The minimum a_w/temperature for CPA production (2264 ng g^–1 P. commune, 709 ng g^–1 A. flavus) was 0.90 a_w/30 °C while greatest production (7678 ng g^–1 P. commune, 1876 ng g^–1 A. flavus) was produced at 0.98 a_w/20 °C. Least AF (411 ng g^–1) was produced at 0.90 a_w/20 °C and most (3096 ng g^–1) at 0.98 a_w/30 °C.     

Structural and functional characterization of galactooligosaccharides in Nostoc commune: beta-D-galactofuranosyl-(1-->6)-[beta-D-galactofuranosyl-(1-->6)]2-beta-D-1,4-anhydrogalactitol and beta-(1-->6)-galactofuranosylated homologues

A new class of galactooligosaccharides has been identified from the terrestrial cyanobacterium Nostoc commune by MS and NMR techniques. These consist of beta-D-galactofuranosyl-(1-->6)-[beta-D-galactofuranosyl-(1-->6)]n-beta-d-1,4-anhydrogalactitols with n ranging from 2 to 8, corresponding to compounds designated 1 through 7. In total these saccharides amounted to approximately 0.35% of the dry thallus of N. commune, while in several other cyanobacteria they were not detected. Possibly they play some role in protection from damage by heat and desiccation as suggested by experiments with heterologous systems. For example, phosphoglucomutase (EC 2.7.5.1) from rabbit muscle was protected against heat inactivation by these oligosaccharides, and alpha-amylase (EC 3.2.1.1) from porcine pancreas by the oligosaccharides 6 and 7. The homologues of lower molecular mass, however, enhanced heat sensitivity of alpha-amylase. The viability of Escherichia coli was completely abolished by desiccation, whereas in the presence of 4 survival rates were approximately 50% of controls not subjected to desiccation. The newly identified saccharides are compared with known galactofuranose-based oligo- and polysaccharides and possible biological functions of them are discussed.     

Growth of Agrostis palustris in subsurface black-layered sand induced by cyanobacteria and sulfate-reducing bacteria

The growth of Agrostis palustris was evaluated in sand columns in response to black-layer formed by the interaction of cyanobacteria in the genera Oscillatoria (isolates OS-1 and OS-2) and Nostoc (NS-1) with the sulfate-reducing bacterium Desulfovibrio desulfuricans. All plants of A. palustris transplanted into black-layered sand columns survived, and the black-layer cleared adjacent to roots as they grew down in the column. Black-layer remained in the columns below the advancing root tips. After 10 weeks of growth, numerous roots showed discontinuous reddish-brown discoloration on their surfaces. Shoot growth of A. palustris was reduced in response to all cyanobacteria and D. desulfuricans isolates alone or in combination. Root growth was unaffected by the microorganisms with the exception of stimulation by OS-1 and inhibition by D. desulfuricans. Interaction of the microbes and the formation of black-layer is discussed relative to the growth of A. palustris. Journal Paper J-14483 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011. Project 2616 Journal Paper J-14483 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011. Project 2616     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Production of palatinose using<Emphasis Type="Italic"> Serratia plymuthica</Emphasis> cells immobilized in chitosan

In recent decades, the production of palatinose has aroused great interest since this structural isomer of sucrose has interesting potential. We describe a simple and effective method of immobilizing Serratia plymuthica cells in chitosan. The sucrose isomerase activity of immobilized preparations was enhanced many times by activation with fresh nutrient medium and subsequent drying. The preparations obtained were physically very stable with high enzyme activity and excellent operational stability. The effect of temperature, pH and substrate concentration on enzyme activity of the immobilized cells was investigated. Using immobilized cells, a complete conversion of sucrose (40% solution) into palatinose was achieved in 4 h in a "batch"-type enzyme reactor. The use of free or immobilized cells had no effect on the composition of the solution, in particular the sugar content. The palatinose content was 80% and that of trehalulose 7%.     

pag gene-like protein (ABP-25) of the Cynops embryo: regional distribution and gene expression during early embryogenesis

A maternal protein showing a unique distribution during early Cynops embryogenesis was screened by monoclonal antibody. The antigen protein, designated as ABP-25 (animal blastomere protein, molecular weight 25,000), was distributed uniformly in the uncleaved egg and concentrated into blastomeres of the animal half during cleavage. At the blastula stage, ABP-25 was definitely localized in cells of the animal half and a polarized distribution was observed within the cytoplasm. During gastrulation, immunohistochemical analysis indicated that the reactivity of the marginal zone (presumptive mesoderm) to the monoclonal antibody ABP-25 decreased after involution. At the end of gastrulation, a polarized distribution was still clearly observed in the ventral epidermis, but not in the neuroectoderm. Both Western and Northern blots indicated that the amount of antigen protein and the intensity of gene expresion were almost constant until the neurula stage. The deduced amino acid sequence of the ABP-25 cDNA showed a strong homology (84%) with that of the pag gene associated with cell proliferation.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the accession number D37808     

Eukaryotic integral membrane protein expression utilizing the <Emphasis Type="Italic">Escherichia coli</Emphasis> glycerol-conducting channel protein (GlpF)

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).     

Asymmetric reduction of 3-chloropropiophenone to (S)-3-chloro-1-phenylpropanol using immobilized Saccharomyces cerevisiae CGMCC 2266 cells

(S)-3-Chloro-1-phenylpropanol is an important chiral precursor for numerous antidepressants such as tomoxetine. A high enantiomeric excess (e.e.) of (S)-3-chloro-1-phenylpropanol can be achieved by asymmetric reduction of 3-chloropropiophenone using Saccharomyces cerevisiae CGMCC 2266 cells immobilized in calcium alginate. Thermal pretreatment of the immobilized cells at 50 °C for 30 min resulted in high enantioselectivity (99% e.e.) and good percent conversion (80%). The effects of various conditions on the reduction reaction were investigated. The optimal conditions were found to be as follows: sodium alginate concentration, 2%; bead diameter, 2 mm; temperature, 30 °C; re-culture time, 24 h; and batch addition of the substrate. After reusing these three times, the immobilized cells retained approximately 60% of their original catalytic activity with their enantioselectivity intact.     

A metallothionein-like protein of rice (rgMT) functions in E. coli and its gene expression is induced by abiotic stresses

A metallothionein-like (rgMT) gene was isolated from a rice (Oryza sativa L.) root cDNA library that was prepared from plants grown under NaHCO₃ stress. The rgMT gene expression was induced in rice leaves and roots under several abiotic stresses from salts (NaCl and NaHCO₃), drought (PEG) and metals (CuCl_2, ZnCl₂, CdCl₂). The results suggested that the rgMT gene was expressed in response to environmental stresses. The rgMT gene was expressed in Escherichia coli, and the final yield of the purified rgMT protein was 4.8 mg g⁻¹ dry cells. Tolerance of E. coli expressing GST-rgMT fusion protein to Cu²⁺, Zn²⁺ and Cd²⁺ was enhanced, and cells dry weight increased 0.04 mg, 0.17 mg and 0.07 mg in 1 ml culture treated with either CuCl₂, ZnCl₂ or CdCl₂, respectively, compared with control after 6 h culture.