Nitrogen-deficient Medium Research Articles

Supplementation of the cultivation media with B-group vitamins enhances lovastatin biosynthesis by Aspergillus terreus

The impact of the supplementation of cultivation media with B-group vitamins on the biosynthesis of lovastatin (mevinolinic acid) by Aspergillus terreus ATCC20542 was investigated. A hypothesis was formulated that as the biosynthesis of lovastatin requires a high throughput of coenzymes in the cells, the application of its precursors in the form of B-group vitamins might positively influence the process. In a nitrogen-deficient medium the B-group vitamins, both single, especially nicotinamide, pyridoxine and calcium D-pantothenate, and a mixture of thiamine, riboflavin, pyridoxine, calcium d-pantothenate and nicotinamide increased the efficiency of lovastatin biosynthesis. The vitamin supplementation also increased both volumetric and specific production rates of mevinolinic acid, especially before 80 h of the process, when no lactose limitation had been observed yet.

A preliminary investigation of the Nostoc punctiforme proteome

Nostoc punctiforme ATCC 29133 is a filamentous terrestrial cyanobacterium (prokaryote) that expresses several different phenotypes in response to environmental cues. When grown in nitrogen-deficient media the most abundant proteins in addition to phycobiliproteins were superoxide dismutase, ATP synthase, and peptidyl-prolyl cis– trans isomerases. A methylated peptide from an akinete marker protein was also identified, suggesting that methylation could potentially play a regulatory role through signaling. C-phycocyanin α-chain was methylated at the C-terminal end of the protein and tandem mass spectrometric data also identified peptides that were deamidated. Since a significant number of putative polyketide/non-ribosomal peptide synthase genes are present in the annotated genome, an analysis of a methanolic extract of whole cells was also performed, and a series of nostopeptolides were identified.

Production of H 2 S and properties of sulfite reductase from selected strains of wine-producing yeasts

Correlation between H2S productivity (p) and sulfite reductase activity (A) was estimated for seven wine-producing yeasts. It was described by a linear function p=f(A), the slope of which depended on the yeast strain. The most intensive H2S synthesis by all the examined yeasts was observed within the early logarithmic growth phase, either in nitrogen abundance and SO2 presence, or in nitrogen deficient and SO2-free medium. Sulfite reductase from all the strains was optimally active at pH close to 7.4. The enzyme from cryophilic yeasts displayed maximum activity at approximately 16 °C, and from mesophiles at 22–26 °C. The preparations showed different affinities toward sulfite (K m of 16.7–67.5 μM). Values of activation energy in sulfite reduction depended on the yeast, and ranged from 14.07 to 67.04 kJ mol−1.

Characterization of the Prr1 response regulator with special reference to sexual development in Schizosaccharomyces pombe.

The histidine (His)-to-Aspartate (Asp) phosphorelay is a paradigm of intracellular signaling systems through protein phosphorylation in both prokaryotes and eukaryotes. The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), together with two response regulators (Mcs4 and Prr1). The results of recent extensive studies suggested that these His-to-Asp phosphorelay components are involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. It was also suggested that they were somehow implicated in control of both the mitotic and meiotic cell proliferations. Among these S. pombe His-to-Asp phosphorelay components, however, the function of Prr1 is less clear. We here characterized a mutant, named prr1-D418N, specifying an altered Prr1 protein that presumably acts as a gain-of-function (or constitutive-active) mutant, with special reference to sexual development. The mutant cells showed a striking phenotype in that they underwent mating even in a nitrogen-sufficient medium, under which conditions the wild-type cells hardly did so. Furthermore, the mutant cells underwent mating very rapidly in a nitrogen-deficient medium. Under anaerobic (or micro-aerobic) growth conditions, the wild-type cells were not capable of undergoing sexual development even in a nitrogen-deficient medium. The prr1-D418N cells underwent mating efficiently under such anaerobic growth conditions. Taken these together, it was suggested that the function of Prr1 is closely linked to the well-characterized signaling pathways for induction of the sexual development, in a way that this response regulator regulates a critical step of the initiation of meiosis through activating the transcription of ste11+, mam2+, and mei2+, in S. pombe.

Induction of the synthesis of an additional family of long-chain dolichols in the yeast Saccharomyces cerevisiae. Effect of starvation and ageing.

The yeast Saccharomyces cerevisiae strain W303 synthesizes in the early logarithmic phase of growth dolichols of 14-18 isoprene residues. The analysis of the polyisoprenoids present in the stationary phase revealed an additional family which proved to be also dolichols but of 19-24 isoprene residues, constituting 39% of the total dolichols. The transfer of early logarithmic phase cells to a starvation medium lacking glucose or nitrogen resulted in the synthesis of the longer chain dolichols. The additional family of dolichols represented 13.8% and 10.3% of total dolichols in the glucose and nitrogen deficient media, respectively. The level of dolichols in yeast cells increased with the age of the cultures. Since both families of dolichols are present in stationary phase cells we postulate that the longer chain dolichols may be responsible for the physico-chemical changes in cellular membranes allowing yeast cells to adapt to nutrient deficient conditions to maintain long-term viability.

Induction of the synthesis of an additional family of long-chain dolichols in the yeast Saccharomyces cerevisiae. Effect of starvation and ageing.

The yeast Saccharomyces cerevisiae strain W303 synthesizes in the early logarithmic phase of growth dolichols of 14-18 isoprene residues. The analysis of the polyisoprenoids present in the stationary phase revealed an additional family which proved to be also dolichols but of 19-24 isoprene residues, constituting 39% of the total dolichols. The transfer of early logarithmic phase cells to a starvation medium lacking glucose or nitrogen resulted in the synthesis of the longer chain dolichols. The additional family of dolichols represented 13.8% and 10.3% of total dolichols in the glucose and nitrogen deficient media, respectively. The level of dolichols in yeast cells increased with the age of the cultures. Since both families of dolichols are present in stationary phase cells we postulate that the longer chain dolichols may be responsible for the physico-chemical changes in cellular membranes allowing yeast cells to adapt to nutrient deficient conditions to maintain long-term viability.

Functional studies of the Synechocystis phycobilisomes organization by high performance liquid chromatography on line with a mass spectrometer.

This study was designed to yield data on the supramolecular organization of the phycobilisome apparatus from Synechocystis, and the possible effects of environmental stress on this arrangement. Phycobilisomes were dissociated in a low ionic strength solution and a quantitative estimation of the protein components present in each subcomplex was obtained using liquid chromatography coupled on-line with a mass spectrometer equipped with an electrospray ion source (ESI-MS). An advantage of this approach is that information can be collected on the initial events, which take place as this organism adapts to environmental changes. Ultracentrifugation of whole phycobilisomes revealed five subcomplexes; the lightest contained four linker proteins plus free phycocyanin, the second the core complex, while the last three bands contained the rod complexes. Four linkers were found in band 1 with higher molecular masses than those expected from the DNA sequence, indicating that they also contain linked chemical groups. UV-B irradiation specifically destroyed the beta-phycocyanin and one rod linker, which resulted in the disintegration of the rod complexes. The two bilins present in beta-phycocyanin give a greater contribution to the UV absorption than the single bilin of the other bilinproteins and probably react with atmospheric oxygen forming toxic radicals. The protein backbone is, in fact, protected from damage in anaerobic conditions and in the presence of radical scavengers. Cells grown in sulfur- and nitrogen-deficient medium contained significantly reduced levels of beta-phycocyanin and one rod linker.

His-to-Asp phosphorelay circuitry for regulation of sexual development in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), and two response regulators (Mcs4 and Prr1). The results of recent extensive studies on the S. pombe His-to-Asp phosphorelay circuitry suggested that it is involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. The functions of these histidine kinases have not yet been fully characterized. Here we characterize a homothallic (h90) mutant lacking the genes for all the histidine kinases, with special reference to sexual development. Homothallic phk1/2/3delta cells underwent mating precociously in a nitrogen-deficient medium. Surprisingly, the mutant cells underwent mating even in a nitrogen-sufficient medium, under which conditions wild-type cells did so rarely if at all. Under anaerobic (or microaerobic) growth conditions, wild-type cells did not undergo sexual development even in a nitrogen-deficient medium, but the homothallic phk1/2/3delta cells mated efficiently. Oxidative reagents such as H2O2 induced sexual development in wild-type cells grown anaerobically. On the basis of these results, we propose the novel view that the S. pombe His-to-Asp phosphorelay, initiated by the Phk histidine kinases, is crucial for regulation of sexual development. This Phk-mediated signaling pathway is linked to the well-documented canonical pathway for induction of the sexual development, in that both converge at the initiation of meiosis through activation of ste11+, mam2+, and mei2+ transcription.

Studies on the production of enzymes by white-rot fungi for the decolourisation of textile dyes

Four white- rot fungi, Bjerkandera adusta, Phlebia tremellosa, Pleurotus ostreatus and Coriolus versicolor, were tested for their ability to produce Lignin Peroxidase (LiP), Manganese Peroxidase (MnP), and Laccase in a nitrogen deficient mineral salts medium. B. adusta and P. tremellosa were selected, due to their high peak enzyme activities, for the degradation of 5 dyes in an artificial textile-effluent. Degradation experiments were carried out in N-rich (C:N ratio, 11.6:1) and N-limited, 116:1) conditions at a dye concentration of 100 mg/liter. B. adusta degraded 85% of the dyes in 7 days and P. tremellosa 79% in 9 days in N-rich media. 86% of the effluent was degraded in 9 days by B. adusta and 74% by P. tremellosa in 11 days in N-limited conditions. Addition of nitrogen had no substantial effect on percentage dye degradation by B. adusta, with a slight increase for P. tremellosa. Nitrogen supplementation did however reduce the decolourisation time. The results show the potential of using B. adusta and P. tremellosa for textile- effluent degradation, with or without nitrogen supplementation.

Isolation of a Novel Gene fromSchizosaccharomyces pombe: stm1+ Encoding a Seven-transmembrane Loop Protein That May Couple with the Heterotrimeric Gα2 Protein, Gpa2

A putative seven transmembrane protein gene, stm1(+), which is required for proper recognition of nitrogen starvation signals, was isolated as a multicopy suppressor of a ras1 synthetic lethal mutant in Schizosaccharomyces pombe. Under nitrogen-deficient conditions, transcription of the stm1 gene was induced; deletion of stm1 was associated with early entry into G(1) arrest. Under nutritionally sufficient conditions, overexpression of Stm1 inhibited vegetative cell growth, resulted in decreased intracellular cAMP levels, increased the expression of the meiosis-specific genes ste11, mei2, and mam2, and facilitated sexual development in homothallic cells. However inhibition of vegetative cell growth and reduction of cAMP levels were not observed in a deletion mutant of the heterotrimeric G protein Galpha2 gene, gpa2, that is responsible for regulating intracellular cAMP levels, a key factor in determining the sexual development in S. pombe. Stm1 protein was shown to interact with Gpa2 through its C-terminal transmembrane domains 5-7. Mutation at Lys(199) in the C-terminal domain (stm1(K199A)) abolished the Stm1 overexpression effect on lowering cAMP levels. Induction of ste11, a meiosis-specific gene transcription factor, by Stm1 overexpression was enhanced in gpa2-deleted cells but was absent in a deletion mutant of sty1, a key protein kinase that links mitotic control with environmental signals and induces stress-responsive genes. Moreover, deletion of both stm1 and ras1 caused delayed entry into G(1) arrest in S. pombe when the cells were grown in a nitrogen-deficient medium. Thus we consider that the stm1 gene can function through Gpa2-dependent and/or -independent pathways and may play a role in providing the prerequisite state for entering the pheromone-dependent differentiation cycle in which heterotrimeric Galpha1 protein, Gpa1, and Ras1 play major roles. Stm1 could function as a sentinel molecule sensing the nutritional state of the cells, stopping the proliferative cell cycle, and preparing the cell to enter meiosis under nutritionally deficient conditions.

The effect of lowered concentrations of carbon, nitrogen and phosphorus sources on the growth dynamics of the R, S, and M dissociants of Pseudomonas aeruginosa

The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate μ of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosaK-2, culture pH, and the population composition was studied. Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants have virtually identical μ. The maximal values of μ were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant). The R and M dissociants showed the most rapid decrease in μ in the nitrogen-deficient medium containing 0.55% NaNO3. By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively. In the case of the carbon-deficient medium, the drop in the culture pH was lower. After a 2.5-day incubation of the S dissociant in the phosphorus- deficient medium containing 0.028% NaH2PO4· 2H2O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.

Alternative pathways of glucose transport in Prevotella bryantii B(1)4(1).

Prevotella bryantii B(1)4 grew faster on glucose than mannose (0.70 versus 0.45 h(-1)), but these sugars were used simultaneously rather than diauxically. 2-deoxy-glucose (2DG) decreased the growth rate of cells that were provided with either glucose or mannose, but 2DG did not completely prevent growth. Cells grown on glucose or mannose transported both (14)C-glucose and (14)C-mannose, but cells grown on glucose had over three-fold higher rates of (14)C-glucose transport than cells grown on mannose. The (14)C-mannose transport rates of glucose- and mannose-grown cells were similar. Woolf-Augustinsson-Hofstee plots were not linear, and it appeared that the glucose/mannose/2DG carrier acted as a facilitated diffusion system at high substrate concentrations. When cultures were grown on nitrogen-deficient (excess sugar) medium, isolates had three-fold lower (14)C-glucose transport, but the (14)C-mannose transport did not change significantly. (14)C-glucose and (14)C-mannose transport rates could be inhibited by 2DG and either mannose or glucose, respectively. The (14)C-glucose transport of mannose-grown cells was inhibited more strongly by mannose and 2DG than those grown on glucose. Cells grown on glucose or mannose had similar ATP-dependent glucokinase activity, and 2DG was a competitive inhibitor (K(i)=0.75 mM). Thin layer chromatography indicated that cell extracts also had ATP-dependent mannose phosphorylation, but only a small amount of phosphorylated 2DG was detected. Glucose, mannose or 2DG were not phosphorylated in the presence of PEP. Based on these results, it appeared that P. bryantii B(1)4 had: (1) two mechanisms of glucose transport, a constitutive glucose/mannose/2DG carrier and an alternative glucose carrier that was regulated by glucose availability, (2) an ATP-dependent glucokinase that was competitively inhibited by 2DG but was unable to phosphorylate 2DG at a rapid rate, and (3) virtually no PEP-dependent glucose, mannose or 2DG phosphorylation activities.

Alternative pathways of glucose transport in Prevotella bryantii B 14

Prevotella bryantii B 14 grew faster on glucose than mannose (0.70 versus 0.45 h −1), but these sugars were used simultaneously rather than diauxically. 2-deoxy-glucose (2DG) decreased the growth rate of cells that were provided with either glucose or mannose, but 2DG did not completely prevent growth. Cells grown on glucose or mannose transported both 14C-glucose and 14C-mannose, but cells grown on glucose had over three-fold higher rates of 14C-glucose transport than cells grown on mannose. The 14C-mannose transport rates of glucose- and mannose-grown cells were similar. Woolf-Augustinsson-Hofstee plots were not linear, and it appeared that the glucose/mannose/2DG carrier acted as a facilitated diffusion system at high substrate concentrations. When cultures were grown on nitrogen-deficient (excess sugar) medium, isolates had three-fold lower 14C-glucose transport, but the 14C-mannose transport did not change significantly. 14C-glucose and 14C-mannose transport rates could be inhibited by 2DG and either mannose or glucose, respectively. The 14C-glucose transport of mannose-grown cells was inhibited more strongly by mannose and 2DG than those grown on glucose. Cells grown on glucose or mannose had similar ATP-dependent glucokinase activity, and 2DG was a competitive inhibitor ( K i=0.75 mM). Thin layer chromatography indicated that cell extracts also had ATP-dependent mannose phosphorylation, but only a small amount of phosphorylated 2DG was detected. Glucose, mannose or 2DG were not phosphorylated in the presence of PEP. Based on these results, it appeared that P. bryantii B 14 had: (1) two mechanisms of glucose transport, a constitutive glucose/mannose/2DG carrier and an alternative glucose carrier that was regulated by glucose availability, (2) an ATP-dependent glucokinase that was competitively inhibited by 2DG but was unable to phosphorylate 2DG at a rapid rate, and (3) virtually no PEP-dependent glucose, mannose or 2DG phosphorylation activities.

Oxygen implication in the diazotrophic growth of Plectonema boryanum in dark–light cycles

The non-heterocystous cyanobacteria Plectonema boryanum fixes atmospheric dinitrogen by separating it temporally from oxygenic photosynthesis. The diazotrophic growth requires continuous illumination, microaerobic milieu and nitrogen-deficient medium. When the culture was incubated in alternating cycles of light and dark, the cyanobacterium failed to fix dinitrogen. Kinetics of oxygen uptake suggested the presence of complex respiratory pathways in the cyanobacterium. Nitrogenase activity was supported by respiratory activity with high affinity for oxygen. As dissolved oxygen dropped below 10 μM with the onset of the dark phase, the nitrogenase activity failed to appear and organism could not grow under diazotrophic conditions. Raising the steady-state level of dissolved oxygen during the dark phase resulted in the appearance of the nitrogenase activity, predominantly during the dark phase of the light/dark cycles. This also stimulated photosynthesis in the subsequent light phase and diazotrophic growth of the cyanobacterium. This result suggests that light-driven reactions of the two photosystems and aerobic dark respiration participate in the total nitrogen fixation capacity in the cyanobacterium.

Analysis of light signal transduction affecting morphogenesis of perithecia in Neurospora crassa

The development of perithecia in Neurospora crassa is under the control of light, nutritional condition such as nitrogen starvation and temperature. Blue light can stimulate(i)the formation of protoperithecia on nitrogen deficient media, (ii)positive bending of perithecial beak, and(iii)the ejection of ascospores from asci in the perithecia. We found that the position of the formation of perithecial beak was dependent on light.(iv)Light stimulated the positioning of perithecial beak on the upperward of it, which we defined as“ light induced polarity of perithecia”. We developed in vitro systems to analyse molecular mechanism of light signal transduction that used the membrane and soluble fractions of mycelial extract. UV-A light induced the binding of ATP/GTP to 58kDA, 77kDa, 83kDa, and 129kDa proteins in the membrane fraction. Blue light stimulated the ADP-ribosylation of 38kDa and 56kDa proteins in the soluble fraction. Blue light also stimulated the phosphorylation of 15kDa protein in the membrane fraction. We isolated a mutant missing completely the phosphorylation of 15kDa protein in the membrane fraction. The mutation designated as psp(phosphorylation of small protein)was mapped on LGVR between al-3and his-6. The psp mutats showed blindness in the light induced polarity of perithecia. The 15kDa protein was purified and the cDNA was cloned. It was identified as nucleoside diphosphate(NDP)kinase. The cDNA for NDP kinase prepared from psp mutants included a mutation of amino acid replacement from Pro 72to His. The putative function of NDP kinase in the light signal transduction to perithecial morphogenesis was discussed.

Flower-Inducing Activity of Lysine in Lemna paucicostata 6746

Flowering of Lemna paucicostata 6746, a typical short-day plant, was induced by culture for 96 or 120 h in nitrogen-free medium under continuous illumination. To examine the effects of lysine, we homogenized entire plants of L. paucicostata 151 in a solution of lysine and the supernatant obtained after centrifugation of the homogenate was added to the medium to give various concentrations of lysine in the medium. Flowering of strain 6746 in nitrogenfree or nitrogen-deficient culture medium was effectively promoted by the addition of a lysine-containing supernatant to the medium. The suppressive effect of elastatinal, a protease inhibitor, on the induction of flowering was almost completely reversed by the simultaneous application of a lysine-containing supernatant to the medium. During nitrogen-free culture, the level of endogenous free lysine, expressed on the basis of the amount of total free amino acids, increased. Lysine-containing supernatants also induced flowering of plants in nitrogen-rich medium under continuous illumination. These findings suggest that endogenous lysine is involved in the induction of flowering in L. paucicostata 6746 on nitrogen-free or nitrogen-deficient medium, as it is in the induction of flowering in L. paucicostata 151.

Involvement of an ATP-dependent carboxylase in a CO2-dependent pathway of acetone metabolism by Xanthobacter strain Py2.

The metabolism of acetone by the aerobic bacterium Xanthobacter strain Py2 was investigated. Cell suspensions of Xanthobacter strain Py2 grown with propylene or glucose as carbon sources were unable to metabolize acetone. The addition of acetone to cultures grown with propylene or glucose resulted in a time-dependent increase in acetone-degrading activity. The degradation of acetone by these cultures was prevented by the addition of rifampin and chloramphenicol, demonstrating that new protein synthesis was required for the induction of acetone-degrading activity. In vivo and in vitro studies of acetone-grown Xanthobacter strain Py2 revealed a CO2-dependent pathway of acetone metabolism for this bacterium. The depletion of CO2 from cultures grown with acetone, but not glucose or n-propanol, prevented bacterial growth. The degradation of acetone by whole-cell suspensions of acetone-grown cells was stimulated by the addition of CO2 and was prevented by the depletion of CO2. The degradation of acetone by acetone-grown cell suspensions supported the fixation of 14CO2 into acid-stable products, while the degradation of glucose or beta-hydroxybutyrate did not. Cultures grown with acetone in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C-label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. Cell extracts prepared from acetone-grown cells catalyzed the CO2- and ATP-dependent carboxylation of acetone to form acetoacetate as a stoichiometric product. ADP or AMP were incapable of supporting acetone carboxylation in cell extracts. The sustained carboxylation of acetone in cell extracts required the addition of an ATP-regenerating system consisting of phosphocreatine and creatine kinase, suggesting that the carboxylation of acetone is coupled to ATP hydrolysis. Together, these studies provide the first demonstration of a CO2-dependent pathway of acetone metabolism for a strictly aerobic bacterium and provide direct evidence for the involvement of an ATP-dependent carboxylase in bacterial acetone metabolism.

In Situ Glutamine Synthetase Activity in a Marine Unicellular Alga (Development of a Sensitive Colorimetric Assay and the Effects of Nitrogen Status on Enzyme Activity).

A malachite green colorimetric assay for glutamine synthetase is described. Glutamine synthetase activity was determined in situ in the marine diatom Phaeodactylum tricornutum Bohlin using cells permeabilized by freeze/thawing. Higher activities were obtained with cells permeabilized in N-2-hydroxyethylpiperazine-N[prime]-2-ethanesulfonic acid compared with N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid, tris(hydroxymethyl)aminomethane, or imidazole, and the optimum pH was 7.9. Activities were higher in cells permeabilized in the presence of reductant, particularly dithiothreitol. Glutamine synthetase activities were markedly decreased in the presence of methionine sulfoximine. In the presence of saturating concentrations of glutamate and ATP, the apparent Km for ammonia was 320 [mu]M, but this value decreased to 110 [mu]M with subsaturating concentrations of glutamate and ATP. The apparent Km values for glutamate and ATP, in the presence of saturating concentrations of ammonia, were 9.7 and 2.9 mM, respectively. Ammonia-grown cells had lower glutamine synthetase activities than did nitrate-grown cells. During nitrogen starvation of both ammonia- and nitrate-grown cells, glutamine synthetase activities increased rapidly during the first 8 h, reaching maximum values after 24 to 48 h. Moreover, the time course for the increases in glutamine synthetase activities and rate of methylamine uptake following the transfer of nitrate-grown cells to nitrogen-deficient medium were very similar. In nitrate-grown cells and cells deprived of combined nitrogen, glutamine synthetase activities and maximum rates of ammonia uptake gave comparable values when measured at the same temperature (20[deg]C).

Carbon dioxide fixation in the metabolism of propylene and propylene oxide by Xanthobacter strain Py2.

Evidence for a requirement for CO2 in the productive metabolism of aliphatic alkenes and epoxides by the propylene-oxidizing bacterium Xanthobacter strain Py2 is presented. In the absence of CO2, whole-cell suspensions of propylene-grown cells catalyzed the isomerization of propylene oxide (epoxypropane) to acetone. In the presence of CO2, no acetone was produced. Acetone was not metabolized by suspensions of propylene-grown cells, in either the absence or presence of CO2. The degradation of propylene and propylene oxide by propylene-grown cells supported the fixation of 14CO2 into cell material, and the time course of 14C fixation correlated with the time course of propylene and propylene oxide degradation. The degradation of glucose and propionaldehyde by propylene-grown or glucose-grown cells did not support significant 14CO2 fixation. With propylene oxide as the substrate, the concentration dependence of 14CO2 fixation exhibited saturation kinetics, and at saturation, 0.9 mol of CO2 was fixed per mol of propylene oxide consumed. Cultures grown with propylene in a nitrogen-deficient medium supplemented with NaH13CO3 specifically incorporated 13C label into the C-1 (major labeled position) and C-3 (minor labeled position) carbon atoms of the endogenous storage compound poly-beta-hydroxybutyrate. No specific label incorporation was observed when cells were cultured with glucose or n-propanol as a carbon source. The depletion of CO2 from cultures grown with propylene, but not glucose or n-propanol, inhibited bacterial growth. We propose that propylene oxide metabolism in Xanthobacter strain Py2 proceeds by terminal carboxylation of an isomerization intermediate, which, in the absence of CO2, is released as acetone.

Temperature-sensitive photosynthesis-deficient mutants of the cyanobacteriumAnabœna variabilis impaired in nitrogen assimilation

Temperature-sensitive (Ts) mutants ofAnabœna variabilis exhibited differences in the rates of oxygen evolution at 28 and 40°C. They were unable to perform photosynthesis and nitrogen fixation at 40°C beyond a period of 3 d in a nitrogen-deficient medium. However, the addition of combined nitrogen sources enhanced the growth of all the Ts mutants at both temperatures. Studies on nitrate uptake ty Ts mutants revealed the existence of a low affinity system, whereKm values were found to be lower than 1 mmol/L. The activity of nitrate reductase gradually increased with time at 28°C with the exception of Ts-161 where the activity decreased with time at 40°C. The rate of ammonia uptake byA. variabilis and its Ts mutants greatly differed and the results suggest the existence of a single phase of uptake. The activity of glutamate-ammonia ligase (GAL) of the parent and Ts mutants was slightly higher in cells from nitrogen-deficient medium when compared to nitrate grown cells at 28°C. At 40°C, the GAL activity decreased after 3 d. The inability of the Ts mutants to grow at 40°C appears to be due to an impairment in nitrogen asimilation.