Regulation of Vacuolar pH of Plant Cells: I. Isolation and Properties of Vacuoles Suitable for P NMR Studies

For the first time, the ³¹P nuclear magnetic resonance technique has been used to study the properties of isolated vacuoles of plant cells, namely the vacuolar pH and the inorganic phosphate content. Catharanthus roseus cells incubated for 15 hours on a culture medium enriched with 10 millimolar inorganic phosphate accumulated large amounts of inorganic phosphate in their vacuoles. Vacuolar phosphate ions were largely retained in the vacuoles when protoplasts were prepared from the cells and vacuoles isolated from the protoplasts. Vacuolar inorganic phosphate concentrations up to 150 millimolar were routinely obtained. Suspensions prepared with 2 to 3 × 10⁶ vacuoles per milliliter from the enriched C. roseus cells have an internal pH value of 5.50 ± 0.06 and a mean trans-tonoplast ΔpH of 1.56 ± 0.07. Reliable determinations of vacuolar and external pH could be made by using accumulation times as low as 2 minutes. These conditions are suitable to follow the kinetics of H⁺ exchanges at the tonoplast. The ³¹P nuclear magnetic resonance technique also offered the possibility of monitoring simultaneously the stability of the trans-tonoplast pH and phosphate gradients. Both appeared to be reasonably stable over several hours. The buffering capacity of the vacuolar sap around pH 5.5 has been estimated by several procedures to be 36 ± 2 microequivalents per milliliter per pH unit. The increase of the buffering capacity due to the accumulation of phosphate in the vacuoles is, in large part, compensated by a decrease of the intravacuolar malate content.     

Relationship between pH and Medium Dissolved Solids in Terms of Growth and Metabolism of Lactobacilli and Saccharomyces cerevisiae during Ethanol Production

The specific growth rates of four species of lactobacilli decreased linearly with increases in the concentration of dissolved solids (sugars) in liquid growth medium. This was most likely due to the osmotic stress exerted by the sugars on the bacteria. The reduction in growth rates corresponded to decreased lactic acid production. Medium pH was another factor studied. As the medium pH decreased from 5.5 to 4.0, there was a reduction in the specific growth rate of lactobacilli and a corresponding decrease in the lactic acid produced. In contrast, medium pH did not have any significant effect on the specific growth rate of yeast at any particular concentration of dissolved solids in the medium. However, medium pH had a significant (P < 0.001) effect on ethanol production. A medium pH of 5.5 resulted in maximal ethanol production in all media with different concentrations of dissolved solids. When the data were analyzed as a 4 (pH levels) by 4 (concentrations of dissolved solids) factorial experiment, there was no synergistic effect (P > 0.2923) observed between pH of the medium and concentration of dissolved solids of the medium in reducing bacterial growth and metabolism. The data suggest that reduction of initial medium pH to 4.0 for the control of lactobacilli during ethanol production is not a good practice as there is a reduction (P < 0.001) in the ethanol produced by the yeast at pH 4.0. Setting the mash (medium) with ≥30% (wt/vol) dissolved solids at a pH of 5.0 to 5.5 will minimize the effects of bacterial contamination and maximize ethanol production by yeast.     

Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase

A cDNA encoding the Arabidopsis thaliana uridine 5′-monophosphate (UMP)/cytidine 5′-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [K_m] = 29 μm when UMP is the other substrate and K_m = 292 μm when CMP is the other substrate) as a phosphate donor. However, both UMP (K_m = 153 μm) and CMP (K_m = 266 μm) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P¹, P⁵-di(adenosine-5′) pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.     

Uncoupling by Acetic Acid Limits Growth of and Acetogenesis by Clostridium thermoaceticum

When cells of the anaerobic thermophile Clostridium thermoaceticum grow in batch culture and homoferment glucose to acetic acid, the pH of the medium decreases until growth and then acid production cease, at about pH 5. We postulated that the end product of fermentation limits growth by acting as an uncoupling agent. Thus, when the pH of the medium is low, the cytoplasm of the cells becomes acidified below a tolerable pH. We have therefore measured the internal pH of growing cells and compared these values with those of nongrowing cells incubated in the absence of acetic acid. Growing cells maintained an interior about 0.6 pH units more alkaline than the exterior throughout most of batch growth (i.e., ΔpH = 0.6). We also measured the transmembrane electrical potential (ΔΨ), which decreased from 140 mV at pH 7 at the beginning of growth to 80 mV when the medium had reached pH 5. The proton motive force, therefore, was 155 mV at pH 7, decreasing to 120 mV at pH 5. When further fermentation acidified the medium below pH 5, both the ΔpH and the ΔΨ collapsed, indicating that these cells require an internal pH of at least 5.5 to 5.7. Cells harvested from stationary phase and suspended in citrate-phosphate buffer maintained a ΔpH of 1.5 at external pH 5.0. This ΔpH was dissipated by acetic acid (at the concentrations found in the growth medium) and other weak organic acids, as well as by ionophores and inhibitors of glycolysis and of the H⁺-ATPase. Nongrowing cells had a ΔΨ which ranged from about 116 mV at external pH 7 to about 55 mV at external pH 5 and which also was sensitive to ionophores. Since acetic acid, in its un-ionized form, diffuses passively across the cytoplasmic membrane, it effectively renders the membrane permeable to protons. It therefore seems unlikely that mutations at one or a few loci would result in C. thermoaceticum cells significantly more acetic acid tolerant than their parental type.     

Involvement of Genes on a Megaplasmid in the Acid-Tolerant Phenotype of Rhizobium leguminosarum Biovar Trifolii

The acid-tolerant Rhizobium leguminosarum biovar trifolii strain ANU1173 exhibited several new phenotypes when cured of its symbiotic (Sym) plasmid and the second largest megaplasmid. Strain P22, which has lost these two plasmids, had reduced exopolysaccharide production and cell mobility on TY medium. The parent strain ANU1173 was able to grow easily in laboratory media at pH 4.5, whereas the derivative strain P22 was unable to grow in media at a pH of <4.7. The intracellular pH of strain ANU1173 was 6.8 when the external pH was 4.5. In contrast, strain P22 had an acidic intracellular pH of <6.4 when the external pH was <5.5. Strain P22 had a dramatically increased membrane permeability to protons and decreased proton extrusion activity. Analysis with sodium dodecyl sulfate-polyacrylamide gels showed that strain P22 lacked a slow-migrating lipopolysaccharide (LPS) banding group which was present in the parent strain. Mobilization of the second largest megaplasmid of strain ANU1173 back into strain P22 restored the altered LPS structure and physiological characteristics of strain P22. Mobilization of the Sym plasmid of strain ANU1173 into strain P22 showed that the second largest megaplasmid of strain ANU1173 was required for the establishment of nitrogen-fixing nodules on Trifolium repens and Trifolium subterraneum. Furthermore, an examination of a large number of specific exopolysaccharide- or LPS-deficient Rhizobium mutants did not show a positive correlation between exopolysaccharide or LPS synthesis and acid tolerance.     

Intracellular Accumulation of Polyphosphate by the Yeast Candida humicola G-1 in Response to Acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.     

A Membrane-Bound NAD(P)+-Reducing Hydrogenase Provides Reduced Pyridine Nucleotides during Citrate Fermentation by Klebsiella pneumoniae

During anaerobic growth of Klebsiella pneumoniae on citrate, 9.4 mmol of H₂/mol of citrate (4-kPa partial pressure) was formed at the end of growth besides acetate, formate, and CO₂. Upon addition of NiCl₂ (36 μM) to the growth medium, hydrogen formation increased about 36% to 14.8 mmol/mol of citrate (6 kPa), and the cell yield increased about 15%. Cells that had been harvested and washed under anoxic conditions exhibited an H₂-dependent formation of NAD(P)H in vivo. The reduction of internal NAD(P)⁺ was also achieved by the addition of formate. In crude extracts, the H₂:NAD⁺ oxidoreductase activity was 0.13 μmol min⁻¹ mg⁻¹, and 76% of this activity was found in the washed membrane fraction. The highest specific activities of the membrane fraction were observed in 50 mM potassium phosphate, with 1.6 μmol of NADPH formed min⁻¹ mg⁻¹ at pH 7.0 and 1.7 μmol of NADH formed min⁻¹ mg⁻¹ at pH 9.5. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone and the Na⁺/H⁺ antiporter monensin, the H₂-dependent reduction of NAD⁺ by membrane vesicles decreased only slightly (about 16%). The NADP⁺- or NAD⁺-reducing hydrogenases were solubilized from the membranes with the detergent lauryldimethylamine-N-oxide or Triton X-100. NAD(P)H formation with H₂ as electron donor, therefore, does not depend on an energized state of the membrane. It is proposed that hydrogen which is formed by K. pneumoniae during citrate fermentation is recaptured by a novel membrane-bound, oxygen-sensitive H₂:NAD(P)⁺ oxidoreductase that provides reducing equivalents for the synthesis of cell material.     

In vitro propagation of Rudraksha (Elaeocarpus sphaericus (Gaertn.) K. Schum): a biotechnological approach for conservation

The species Elaeocarpus sphaericus (Rudraksha) is a religious, medicinally important threatened tree of India. An efficient micropropagation protocol has been developed from nodal explants of this plant species collected from north-east India for large scale production of planting material at favourable sites within the country. Best shoot initiation occurred in MS medium supplemented with 2.2μM BA+2.2μM Kn in combination. Addition of Casein Hydrolysate (CH) (100mg/L) increased the shoot number. Microshoots excised and subcultured in 2.0μM BA further enhanced growth and multiplication. The shoot cultures were maintained in this concentration for 2years with subculturing at 6weeks interval. MS medium containing 5.0μM NAA was most effective for rooting. Successfully acclimatized plants (80%) showed normal growth under suitable habitat conditions.     

The Two Major Types of Plant Plasma Membrane H+-ATPases Show Different Enzymatic Properties and Confer Differential pH Sensitivity of Yeast Growth

The proton-pumping ATPase (H⁺-ATPase) of the plant plasma membrane is encoded by two major gene subfamilies. To characterize individual H⁺-ATPases, PMA2, an H⁺-ATPase isoform of tobacco (Nicotiana plumbaginifolia), was expressed in Saccharomyces cerevisiae and found to functionally replace the yeast H⁺-ATPase if the external pH was kept above 5.0 (A. de Kerchove d'Exaerde, P. Supply, J.P. Dufour, P. Bogaerts, D. Thinès, A. Goffeau, M. Boutry [1995] J Biol Chem 270: 23828–23837). In the present study we replaced the yeast H⁺-ATPase with PMA4, an H⁺-ATPase isoform from the second subfamily. Yeast expressing PMA4 grew at a pH as low as 4.0. This was correlated with a higher acidification of the external medium and an approximately 50% increase of ATPase activity compared with PMA2. Although both PMA2 and PMA4 had a similar pH optimum (6.6–6.8), the profile was different on the alkaline side. At pH 7.2 PMA2 kept more than 80% of the maximal activity, whereas that of PMA4 decreased to less than 40%. Both enzymes were stimulated up to 3-fold by 100 μg/mL lysophosphatidylcholine, but this stimulation vanished at a higher concentration in PMA4. These data demonstrate functional differences between two plant H⁺-ATPases expressed in the same heterologous host. Characterization of two PMA4 mutants selected to allow yeast growth at pH 3.0 revealed that mutations within the carboxy-terminal region of PMA4 could still improve the enzyme, resulting in better growth of yeast cells.     

Nutritional Requirements of Methanosarcina sp. Strain TM-1

Methanosarcina sp. strain TM-1, an acetotrophic, thermophilic methanogen isolated from an anaerobic sludge digestor, was originally reported to require an anaerobic sludge supernatant for growth. It was found that the sludge supernatant could be replaced with yeast extract (1 g/liter), 6 mM bicarbonate-30% CO₂, and trace metals, with a doubling time on methanol of 14 h. For growth on either methanol or acetate, yeast extract could be replaced with CaCl₂ · 2H₂O (13.6 μM minimum) and the vitamin p-aminobenzoic acid (PABA, ca. 3 nM minimum), with a doubling time on methanol of 8 to 9 h. Filter-sterilized folic acid at 0.3 μM could not replace PABA. The antimetabolite sulfanilamide (20 mM) inhibited growth of and methanogenesis by Methanosarcina sp. strain TM-1, and this inhibition was reversed by the addition of 0.3 μM PABA. When a defined medium buffered with 20 mM N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid was used, it was shown that Methanosarcina sp. strain TM-1 required 6 mM bicarbonate-30% CO₂ for optimal growth and methanogenesis from methanol. Cells growing on acetate were less dependent on bicarbonate-CO₂. When we used a defined medium in which the only organic compounds present were methanol or acetate, nitrilotriacetic acid (0.2 mM), and PABA, it was possible to limit batch cultures of Methanosarcina sp. strain TM-1 for nitrogen at NH₄⁺ concentrations at or below 2.0 mM, in marked contrast with Methanosarcina barkeri 227, which fixes dinitrogen when grown under NH₄⁺ limitation.     

Production and Excretion of Nod Metabolites by Rhizobium leguminosarum bv. trifolii Are Disrupted by the Same Environmental Factors That Reduce Nodulation in the Field

Lipooligosaccharides (Nod metabolites) have been shown to be essential for the successful nodulation of legumes. In strains of Rhizobium leguminosarum bv. trifolii, Nod metabolites were detected predominantly within the cell and to a lesser extent in the periplasmic space and the growth medium. The production, and in particular the excretion, of Nod metabolites was restricted by a range of environmental conditions which are associated with poor nodulation in the field. Lowering the medium pH from 7.0 to 5.0, reducing the phosphate concentration from 1 mM to 5 μM KH₂PO₄, and lowering the incubation temperature from 28 to 18°C affected the number and relative concentrations of the Nod metabolites made. The form and concentration of the nitrogen source affected the relative concentrations of the Nod metabolites produced and excreted. KNO₃ concentrations of >10 mM did not affect cell growth rate but substantially reduced the number of Nod metabolites released. Environmental stresses differentially altered Nod metabolite production and excretion in the same strain carrying different introduced nod regions. Strain ANU845(pWLH1) produced and excreted comparatively fewer Nod metabolites at pH 5.0 and at 18°C than strain ANU845(pRI4003). The excretion but not the production of Nod metabolites by strain ANU845(pRtO32) was dependent on the presence of both nodI and nodJ. Tn5-induced nodI and nodJ mutants did not accumulate any metabolites either outside the cell or within the outer membrane or periplasmic space. Recognition that Nod metabolite accumulation is a complex system of production and excretion, with each component responding differently to changes in environmental conditions, has many consequences, both at the molecular level and in the field. The ability of different strains to produce and release Nod metabolites is likely to be a major determinant of nodule occupancy and should be considered when screening strains suitable for adverse environments.     

Survival of Rhizobium in Acid Soils

A Rhizobium strain nodulating cowpeas did not decline in abundance after it was added to sterile soils at pH 6.9 and 4.4, and the numbers fell slowly in nonsterile soils at pH 5.5 and 4.1. A strain of R. phaseoli grew when added to sterile soils at pH 6.7 and 6.9; it maintained large, stable populations in soils of pH 4.4, 5.5, and 6.0, but the numbers fell markedly and then reached a stable population size in sterile soils at pH 4.3 and 4.4. The abundance of R. phaseoli added to nonsterile soils with pH values of 4.3 to 6.7 decreased similarly with time regardless of soil acidity, and the final numbers were less than in the comparable sterile soils. The minimum pH values for the growth of strains of R. meliloti in liquid media ranged from 5.3 to 5.9. Two R. meliloti strains, which differed in acid tolerance for growth in culture, did not differ in numbers or decline when added to sterile soils at pH 4.8, 5.2, and 6.3. The population size of these two strains was reduced after they were introduced into nonsterile soils at pH 4.8, 5.4, and 6.4, and the number of survivors was related to the soil pH. The R. meliloti strain that was more acid sensitive in culture declined more readily in sterile soil at pH 4.6 than did the less sensitive strain, and only the former strain was eliminated from nonsterile soil at pH 4.8; however, the less sensitive strain also survived better in limed soil. The cell density of the two R. meliloti strains was increased in pH 6.4 soil in the presence of growing alfalfa. The decline and elimination of the tolerant, but not the sensitive, strain was delayed in soil at pH 4.6 by roots of growing alfalfa.     

P and C-NMR Studies of the Phosphorus and Carbon Metabolites in the Halotolerant Alga, Dunaliella salina

The intracellular phosphorus and carbon metabolites in the halotolerant alga Dunaliella salina adapted to different salinities were monitored in living cells by ³¹P- and ¹³C-nuclear magnetic resonance (NMR) spectroscopy. The ¹³C-NMR studies showed that the composition of the visible intracellular carbon metabolites other than glycerol is not significantly affected by the salinity of the growth medium. The T₁ relaxation rates of the ¹³C-glycerol signals in intact cells were enhanced with increasing salinity of the growth medium, in parallel to the expected increase in the intracellular viscosity due to the increase in intracellular glycerol. The ³¹P-NMR studies showed that cells adapted to the various salinities contained inorganic phosphate, phosphomonoesters, high energy phosphate compounds, and long chain polyphosphates. In addition, cells grown in media containing up to 1 molar NaCl contained tripolyphosphates. The tripolyphosphate content was also controlled by the availability of inorganic phosphate during cell growth. Phosphate-depleted D. salina contained no detectable tripolyphosphate signal. Excess phosphate, however, did not result in the appearance of tripolyphosphate in ³¹P-NMR spectra of cells adapted to high (>1.5 molar NaCl) salinites.     

Purification and Characterization of an Extremely Thermostable Cyclomaltodextrin Glucanotransferase from a Newly Isolated Hyperthermophilic Archaeon, a Thermococcus sp.

The extremely thermophilic anaerobic archaeon strain B1001 was isolated from a hot-spring environment in Japan. The cells were irregular cocci, 0.5 to 1.0 μm in diameter. The new isolate grew at temperatures between 60 and 95°C (optimum, 85°C), from pH 5.0 to 9.0 (optimum, pH 7.0), and from 1.0 to 6.0% NaCl (optimum, 2.0%). The G+C content of the genomic DNA was 43.0 mol%. The 16S rRNA gene sequencing of strain B1001 indicated that it belongs to the genus Thermococcus. During growth on starch, the strain produced a thermostable cyclomaltodextrin glucanotransferase (CGTase). The enzyme was purified 1,750-fold, and the molecular mass was determined to be 83 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Incubation at 120°C with SDS and 2-mercaptoethanol was required for complete unfolding. The optimum temperatures for starch-degrading activity and cyclodextrin synthesis activity were 110 and 90 to 100°C, respectively. The optimum pH for enzyme activity was pH 5.0 to 5.5. At pH 5.0, the half-life of the enzyme was 40 min at 110°C. The enzyme formed mainly α-cyclodextrin with small amounts of β- and γ-cyclodextrins from starch. This is the first report on the presence of the extremely thermostable CGTase from hyperthermophilic archaea.     

Effect of phosphorus deficiency on levels of phosphorus compounds in spirodela

When Spirodela plants are transferred to a phosphate-deficient medium, growth slows down immediately, and ceases after 14 days. During this time, inorganic phosphate content falls from 30 to 0.7 μmoles/g fresh weight of tissue, phosphate ester content from 3.5 to 0.6 μmoles/g, phospholipid content from 3.5 to 1.2 μmoles/g, and residual phosphate (mainly RNA) content from 7.5 to 2.0 μmoles/g. Relative proportions of the various phosphate esters, and relative proportions of the various phospholipids, are not markedly affected by phosphate deficiency. Turnover rates of phosphate esters are somewhat higher in phosphate-deficient tissue. In control tissue, inorganic phosphate is present in 2 pools; a metabolic (12%) and a non-metabolic pool (88%). In phosphate-deficient tissues, most of the inorganic phosphate (>90%) is in the metabolic pool. Non-metabolic phosphate is presumably stored in the vacuole, and is not readily accessible to the tissue, so that growth normally occurs at the expense of external phosphate. During deficiency, growth is limited by the rate at which phosphate can be transported through the tonoplast and tissue to the growing point. Growth ceases when the supply of non-metabolic phosphate is exhausted. Metabolic phosphate is presumably located in the cytoplasm: it can not be used for growth. Nor can the plant respond to deficiency by making some phosphorus compounds at the expense of others. In this respect, phosphorus deficiency and nitrogen deficiency are dissimilar.     

Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Suspension-cultured cells of tomato (Lycopersicon esculentum) start to secrete an RNA-degrading enzyme activity during transition from logarithmic to stationary growth phase. Using affinity chromatography on agarose-5-(4-aminophenyl-phosphoryl) uridine 3′(2′) monophosphate as a powerful and final enrichment step, the enzyme was purified to homogeneity and characterized as ribonuclease I (RNase I) according to the following data: (a) it has an M_r of 22,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), a pH-optimum of pH 5.5, a pl of 3.9, and its activity was found to be insensitive to EDTA; (b) the enzyme splits single-stranded RNA endonucleolytically by a phosphotransferase reaction yielding 2′,3′-cNMPs as primary monomeric products; (c) as studied with diribonucleoside monophosphates as substrates, the enzyme exhibits a pronounced preference for 5′ purine residues adjacent to the cleavage site. Most interestingly, in vivo synthesis and secretion was found to be induced when tomato cells were specifically starved for phosphate as mineral nutrient. (a) Extracellular enzyme activity increased about tenfold after transfer of phosphate-grown cells into medium lacking only phosphate. Accordingly, this increase in activity was not detectable when cells were constantly supplied with phosphate. (b) Biosynthetically labeling of the extracellular protein with radioactive amino acids was detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography directly within the bulk of extracellular proteins. Therefore, we propose that the secreted tomato RNase I synthesized upon phosphate starvation is a component of a higher plant inducible rescue system for scavenging exogenous phosphate.     

Phosphate uptake and polyphosphate metabolism of mycorrhizal and nonmycorrhizal roots of pine and of Suillus bovinus at varying external pH measured by in vivo 31P-NMR

 Comparative in vivo ³¹P-NMR analyses of mycorrhizal and nonmycorrhizal roots of Pinus sylvestris and the fungus of Suillus bovinus in pure culture were used to investigate alterations in phosphate metabolism due to changes in external pH in the range 3.5–8.5. All control samples maintained a constant pH in both cytoplasm and vacuole. Mycorrhizal roots and pure fungus, but not nonmycorrhizal roots, transformed accumulated inorganic phosphate into mobile polyphosphate with a medium chain length. Phosphate uptake rates and polyphosphate accumulation responded differently to external pH. In all cases, maximal phosphate uptake occurred at an external pH close to 5.5. At an external pH of 8.5, both roots and fungus showed a distinct lag in phosphate uptake, which was abolished when the external pH was lowered to 7.5. An irreversible effect on phosphate uptake as a consequence of variation in external pH was also observed. The central role of the fungus in regulating mycorrhizal phosphate metabolism is discussed. Accepted: 15 April 1997     

In vivo P-31 NMR measurements of phosphate metabolism in Platymonas subcordiformis as related to external pH

The phosphate metabolism of Platymonas subcordiformis was investigated by 31P-NMR spectroscopy with special attention on the effect of external pH. Glycolyzing cells and cells energized by respiration or photosynthesis gave spectra dependent upon their metabolic state. The transition from deenergized to energized states is accompanied by a shift of cytoplasmic pH from 7.1–7.4, an increase of ATP level and-in well energized cells-the appearance of a new signal tentatively assigned to phosphoarginine.The spectra remain stable over a wide range of external pH. Cytoplasmic pH is well regulated in respiring cells for external pH in the range 5.3–12.3. The typical 0.4 units difference of internal pH in energized as compared to deenergized cells is not affected by external pH in the range 6–12. The intensity of a signal attributed to PEP is markedly increased at high external pH. pH regulation is less efficient below external pH of 6 in deenergized cells. Below pH 3.8 oxidative phosphorylation ceases. Upon raising cytoplasmic pH to 7.4 in deenergized cells polyphosphate chains start to disintegrate.Abbreviations PEP Phosphoenolpyruyate - P _i inorganic phosphate - PP _i inorganic pyrophosphate - poly P polyphosphates - PP-1, PP-2, PP-3 terminal, second, and third phosphate residue of polyphosphates - PP-4 core phosphate residues of polyphosphates - pH _i , pH _o internal (cytoplasmic) and external pH - NTP/NDP nucleotide triphosphate/-diphosphate - S/N signal to noise ratio     

Low Concentration of Inducer Favors Production of Active Form of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase inEscherichia coli

Expression of chicken and rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, inEscherichia coliencountered two common problems: the chicken enzyme was liable to proteolysis and the rat enzyme was prone to form inclusion bodies. Reducing the rate of protein synthesis by lowering either growth temperature or isopropyl-β- -thiogalactopyranoside (IPTG) concentration alleviated these two problems. Growth at 22°C was optimum for expression of both enzymes. The optimum range of IPTG concentration for expression was 0.1–1 μ for the chicken liver bifunctional enzyme and 10 μ for rat liver enzyme. The components of growth medium also influenced the production. Compared with Luria–Bertani medium, an enriched medium—tryptone–phosphate medium—tripled the production of the active enzymes. Addition of glucose (0.2%) doubled the expression level of active chicken liver enzyme, but reduced the production of active rat liver enzyme to half the maximal level, while the phosphate in tryptone–phosphate medium had no effect on the production of the two enzymes.     

Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix

An enrichment culture which converted acetate to methane at 60°C was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-1. Strain CALS-1 grew as filaments typically 2–5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct cross-walls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 m long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase cells tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-1, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60°C (doubling time=24–26 h), with no growth occurring at 70°C and 37°C. The optimum pH value for growth was near 6.5 in bicarbonate/CO₂ buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 mM phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 mM were inhibitory to the culture, and growth was more rapid with 1 mM 2-mercaptoethane sulfonate (coenzyme M) or 1 mM titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix.