Overproduction of nitrogenase by nitrogen-limited cultures of Rhodopseudomonas palustris.

Rhodopseudomonas palustris cells grown on limiting nitrogen produced four- to eightfold higher nitrogenase specific activity relative to cells sparged with N2. The high activity of N-limited cells was the result of overproduction of the nitrogenase proteins. This was shown by four independent techniques: (i) titration of the Mo-Fe protein in cell-free extracts with Fe protein from Azotobacter vinelandii; (ii) direct detection of the subunits of Mo-Fe protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (iii) monitoring of the electron paramagnetic resonance spectrum of Mo-Fe protein in whole cells; and (iv) immunological assay of the Fe protein level with an antiserum against the homologous protein of Rhodospirillum rubrum. The derepressed level of nitrogenase found in N2-grown cells was not due to an increased turnover of nitrogenase. The apparent half-lives of nitrogenase in N2-grown and N-limited cells were 58 and 98 h, respectively, but were too long to account for the difference in enzyme level. Half-lives were determined by measuring nitrogenase after repression of de novo synthesis by ammonia and subsequent release of nitrogenase switch-off by methionine sulfoximine. Observations were extended to R. rubrum, Rhodopseudomonas capsulata, and Rhodomicrobium vannielii and indicated that overproduction of nitrogenase under nitrogen limitation is not an exceptional property of R. palustris, but rather a general property of phototrophic bacteria.     

具有谷氨酸脱羧酶的大肠杆菌固定化细胞

本文研究了用海藻酸钙包埋法制备含谷氨酸脱羧酶固定化细胞的方法以及研究了制备的条件和影响其制备的因素。该法具有包埋细胞活力回收高,方法简便等优点。比较研究了固定化细胞和自然细胞谷氨酸脱羧酶的一些生物化学性质。其中固定化细胞最适pH和pH稳定性增加,最适温度及热稳定性下降;表观米氏常数增大;二价金属离子Zn~(++)、Cu~(++)、Mg~(++)、Fe~(++),Sr~(++)程度不同的抑制酶活性,Ca~(++)激活固定化细胞酶活性,EDTA无抑制作用。对固定化细胞和自然细胞酶活力活化的研究中发现这两种细胞经蒸馏水保温处理后酶活性都上升,且自然细胞酶活的上升较固定化细胞大;而用底物溶液处理后,自然细胞无变化,固定化细胞酶活下降。     

Immobilized Aspartase-Containing Microbial Cells: Preparation and Enzymatic Properties

The immobilization of asparatase-containing Escherichia coli was investigated by various methods, and the most active immobilized cells were obtained by entrapment in a polyacrylamide gel lattice. Other asparatase-containing bacteria were also entrapped by the same method, and the enzymatically active immobilized cells were obtained. The aspartase activity of the immobilized E. coli cells was increased nine- to tenfold by autolysis of the cells entrapped in the gel lattice. Enzymatic properties of the immobilized E. coli cells were investigated and compared with those of the intact cells. The optimal pH was 8.5 for the immobilized cells and 10.5 for the intact cells. The aspartase activities of immobilized and intact cells were not activated by Mn(2+), which can activate the immobilized and native aspartases. The heat stability of the immobilized cells was somewhat higher than that of the intact cells. Bivalent metal ions such as Mn(2+), Mg(2+), Ca(2+) protected against thermal inactivation of the aspartase activity of the immobilized and intact cells.     

Growth of Spirillum lipoferum at constant partial pressures of oxygen, and the properties of its nitrogenase in cell-free extracts.

Spirillum lipoferum, an N2-fixing organism, was grown at constant concentrations of dissolved O2. When supplied with NH4+ aerobically, its doubling time was 1 h; when it fixed N2 microaerophilically, its doubling time was 5-5 to 7 h and the optimal PO2 for growth was 0-005 to 0-007 atm. At its optimal PO2 for growth on N2, S. lipoferum assimilated 8 to 10 mg nitrogen/g carbon substrate used; its efficiency was less at higher PO2 levels. Nitrogenase in cell-free extracts required Mg2+ and Mn2+, and the Fe-protein was activated by Rhodospirillum rubrum activating factor. The nitrogenase had an optimal pH of 7-1 to 7-4 and an apparent Km for acetylene of 0-0036 atm. Extracts of S. lipoferum lost their nitrogenase activity on storage at -18 degrees C, and activity was restored by adding purified Fe-protein from other N2-fixing bacteria.     

Nitrogen-fixing activity in peat soils from a raised bog

The nitrogenase (acetylene reductase) activity in monolithic and minced peat samples was found to be low, no more than 0.014-0.022 mg N/(kg h). Incorporation of the 15N2 isotope into organic compounds of peat soil was from 2.71-8.13 mg N/kg over 15 days. The nitrogen-fixing activity was the highest in a 10-20 cm layer of soil and much lower in the upper (under green moss) and deeper (20-30 cm) layers. The addition of glucose to soil samples stimulated nitrogen fixation considerably after 18-26 h. The maximum nitrogenase activity (3.5-3.8 mg N/(kg h)) observed after 60-70 h coincided with the peak of respiratory activity. A repeated addition of glucose after its exhaustion increased nitrogenase activity without a lag period to 8.5 mg N/(kg h). Investigation of the effect of environmental factors (temperature, pH, aeration, and light intensity) on potential nitrogen-fixing activity in peat samples revealed that nitrogen fixation could proceed in a wide range of pH values (from 3.0 to 7.5) and temperatures (from 5 to 35 degrees C). The nitrogen-fixing bacteria belonging to different trophic groups were enumerated by using nitrogen-free media with pH values and mineralization levels close to those in situ. In samples of peat soil, diazotrophic methanol-utilizing bacteria prevailed (2.0-2.5 x 10(6) cells/g); the second largest group was facultatively anaerobic bacteria of the family Enterobacteriaceae.     

Denitrification of PVA-immobilized denitrifying photosynthetic bacterium,Rhodobacter sphaeroides

A newly isolated denitrifying strain, Rhodobacter sphaeroides NII2 was immobilized in polyvinyl alcohol (PVA) gel, and the properties of the cells in the gel were examined. The immobilized cells had low or almost no denitrification activity, but the cells were activated by incubation in light with culture medium for denitrification containing 0.5% nitrate and no other nitrogen source. Cells grown in the dark were activated by incubation at an earlier stage and to a higher rate than the light-grown cells. The activation was markedly enhanced in the PVA gel with a low cell concentration. The immobilized cells consumed nitrate with a temporary accumulation of NO₂ and evolved nitrogen gas. The immobilized cells could use various organic compounds as electron donors for denitrification. Thus, the immobilized cells were applied to a continuous treatment of synthetic wastewater using an aparatus devised by this laboratory. The results showed an efficient removal of NO₃-N from the test water.     

Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum.

The nitrogenase activity of the microaerophilic bacteria Azospirillum brasilense and A. lipoferum was completely inhibited by 2.0 kPa of oxygen (approximately 0.02 atm of O2) in equilibrium with the solution. The activity could be partially recovered at optimal oxygen concentrations of 0.2 kPa. In contrast to the NH4+ switch off, no covalent modification of the nitrogenase reductase (Fe protein) was involved, as demonstrated by Western-blotting and 32P-labeling experiments. However, the inhibition of the nitrogenase activity under anaerobic conditions was correlated with covalent modification of the Fe protein. In contrast to the NH4+ switch off, no increase in the cellular glutamine pool and no modification of the glutamine synthetase occurred under anaerobic switch-off conditions. Therefore, a redox signal, independent of the nitrogen control of the cell, may trigger the covalent modification of the nitrogenase reductase of A. brasilense and A. lipoferum.     

Nitrogen fixation by the thermophilic green sulfur bacterium Chlorobium tepidum.

The thermophilic green sulfur bacterium Chlorobium tepidum grew with N2, NH4+, or glutamine as the sole nitrogen source under phototrophic (anaerobic-light) conditions. Growth on N2 required increased buffering capacity to stabilize uncharacterized pH changes that occurred during diazotrophic growth. Increased sulfide levels were stimulatory for growth on N2. Levels of nitrogenase activity (acetylene reduction) in N2-grown C. tepidum cells were very high, among the highest ever reported for anoxygenic phototrophic bacteria. Maximal acetylene reduction rates in C. tepidum cells were observed at 48 to 50 degrees C, which is about 15 degrees C higher than the optimum temperature for nitrogenase activity in mesophilic chlorobia, and nitrogenase activity in C. tepidum responded to addition of ammonia by a "switch-off/switch-on" mechanism like that in phototrophic purple bacteria. C. tepidum cells assimilated ammonia mainly via the glutamine synthetase-glutamate synthase pathway, elevated levels of both of these enzymes being present in cells grown on N2. These results show that N2 fixation can occur in green sulfur bacteria up to at least 60 degrees C and that regulatory mechanisms important in control of nitrogenase activity in mesophilic anoxygenic phototrophs also appear to regulate thermally active forms of the enzyme.     

柱孢鱼腥藻固氮酶防氧的呼吸保护

柱孢鱼腥藻生长在缺氮情况下,发现其固氮活性增加的同时也减少了对氧的敏感性。缺氮生长细胞的乙炔还原活性给氧抑制一半时的氧分压(pO_2)是0.5atm.,而有氮生长细胞的半抑制浓度为0.35atm.。这表明蓝藻有可能通过增加呼吸耗氧而提高了它的固氮酶活性。呼吸作用与固氮酶活性之间存在着密切的关系。无论在有氮、缺氮还是光诱导固氮酶形成的情况下,其固氮活性均随着呼吸速率的变化而变化。本研究结果,支持了柱孢鱼腥藻固氮酶的主要防氧手段是呼吸保护的观点。     

Repeated batch production of L-phenylalanine from phenylpyruvate and NH(4)Cl by immobilized cells of Nocardia opaca under hydrogen high pressure

Among various microbial cells examined under screening conditions, Nocardia opaca showed the highest activity for production of phenylalanine from phenylpyruvate. Here NH(4)Cl as well as amino acids were used as an amino donor for phenylalanine production. The phenylalanine production rate increased with increasing hydrogen pressure. The specific activity of phenylalanine dehydrogenase was increased by culturing N. opaca cells in nutrient broth containing 0.3% phenylalanine. As a result, the phenylalanine production rate increased from 0.69 to 4.4 mumol/min g dry cells. Immobilized cells were activated in nutrient broth containing ZnCl(2) before phenylalanine production. Phenylalanine dehydrogenase activity and cell number in the gel increased with increasing incubation time, and the maximum phenylalanine dehydrogenase activity was obtained at 36 h incubation. Then, phenylalanine was produced from phenylpyruvate, NH(4)Cl, and 100 atm H(2) with the activated immobilized cells. The rate of phenylalanine production was 0.24 mumol/min cm(3) gel. The conversion of phenylpyruvate to phenylalanine was 82%. Immobilized cells retained 76% of the initial phenylalanine production rate after 10 h reactions were repeated 11 times with two intervening reactivations.     

Nitrogenase activity in cultured Rhizobium sp. strain 32H1

Nutritional and physical conditions affecting nitrogenase activity in the strain of cowpea rhizobia, 32H1, were examined using cultures grown on agar medium. Arabinose in the basic medium (CS7) could be replaced by ribose, xylose, or glycerol, but mannitol, glucose, sucrose, or galactose only supported low nitrogenase (C₂H₂ reduction) activity. Succinate could be replaced by pyruvate, fumarate, malate, or 2-oxoglutarate, but without any carboxylic acid, nitrogenase activity was low or undetectable unless a high level of arabinose was provided. Inositol was not essential. Several nitrogen sources could replace glutamine including glutamate, urea, (NH₄)₂SO₄ and asparagine.The maximum nitrogenase activity of cultures grown in air at 30°C was observed under assay conditions of pO₂=0.20–0.25 atm and 30°C incubation. Greatest activity occurred after a period of rapid bacterial growth, when viable cell count was relatively constant.Compared with results obtained on the CS7 medium, nitrogenase activity could be substantially increased and/or sustained for longer periods of time by using 12.5 mM succinate and 100 mM arabinose, by increasing phosphate concentration from 2 to 30–50 mM, or by culturing the bacteria at 25°C.     

Nitrogen-Fixing Activity in Peat Soils from a Raised Bog

The nitrogenase (acetylene reductase) activity in monolithic and minced peat samples was found to be low, no more than 0.014–0.022 mg N/(kg h). Incorporation of the ¹⁵N₂ isotope into organic compounds of peat soil was 2.71–8.13 mg N/kg over 15 days. The nitrogen-fixing activity was the highest in a 10- to 20-cm layer of soil and much lower in the upper (under green moss) and deeper (20- to 30-cm) layers. The addition of glucose to soil samples stimulated nitrogen fixation considerably after 18–26 h. The maximum nitrogenase activity (3.5–3.8 mg N/(kg h)), observed after 60–70 h, coincided with the peak of respiratory activity. A repeated addition of glucose after its exhaustion increased nitrogenase activity, without a lag period, to 8.5 mg N/(kg h). Investigation of the effect of environmental factors (temperature, pH, aeration, and light intensity) on potential nitrogen-fixing activity in peat samples revealed that nitrogen fixation could proceed in a wide range of pH values (from 3.0 to 7.5) and temperatures (from 5 to 35°C). The nitrogen-fixing bacteria belonging to different trophic groups were enumerated by using nitrogen-free media with pH values and mineralization levels close to those in situ. In samples of peat soil, diazotrophic methanol-utilizing bacteria prevailed (2.0–2.5 × 10⁶ cells/g); the second largest group was facultatively anaerobic bacteria of the family Enterobacteriaceae.     

Effect of nitrogen compounds on nitrogenase activity in Azospirillum brasilense

Abstract The effect of certain nitrogen compounds on nitrogenase activity was studied in cells of Azospirillum brasilense strain Sp6, grown under microaerophilic conditions with nitrogenase fully derepressed. 0.5 mM NH₄Cl, 0.5 mM glutamine, 1.0 mM KNO₃ and 0.1 mM KNO₂ completely blocked nitrogenase activity. 1.0 mM asparagine, 1.0 mM aspartate, 1.0 mM histidine and 1.0 mM adenine did not caused no inhibition of nitrogenase; indeed asparagine, aspartate and histidine showed a slight stimulatory effect on N₂ fixation. The addition of 10 mM dl -methionine- dl -sulphoximine prevented the inhibitory effect of NH₄Cl and glutamine but did not counteract the effect of KNO₂. Rifampicin and chloramphenicol did not prevent the inhibition of nitrogenase by NH₄Cl.     

Oxygen Sensitive Nitrogen-Fixing Mutants of Anabaena

Two Anabaena mutants having heterocysts but incapable of fixing molecular nitrogen in air have been isolated by using ultraviolet radiation or NTG mutagenesis. Their vegetative cells differentiated into heterocysts at a higher frequency than that of the wild type. The phenotype of the mutants is stable and a low frequence of spontaneous reversion was observed. Under microaerobic condition the mutants cells can express the genetic information which encodes nitrogenase synthesis and were capable of utilizing nitrogen for growth with a low acetylene reductiop activity. The level of nitrogenase activity was correlated reciprocally with the content of cell phycocyanin and the light intensity. Both synthesis and activity of the mutant nitrogenase were very sensitive than wild type to the oxygen in vive. Introduction of 1% O2 (v/v) into the gas phase inhibited evidently acetylene reduction. Exposure of the mutant suspension to 20% O2 (v/v) resulted in total and irreversible denaturation of nitrogenase. Withdrawing of O2 in gas phase, the nitrogenase was synthesized de nero; The synthesis process was repressed by chloramphenical or ammonia. The nitrogenase activity of mutant cells increased significantly either by nitrogen- starvating to decrease the phycocyanin content or by lowering the light intensity. Specifically, during the anaerobic induction by treating the mutants filaments with diehloromethylurea which prevents photosynthetic oxygen production, the specific activity of mutant nitrogcnase was equivalent nearly to that of wild type. The ability to reduce 2, 3, 5-triphenyltetrazolium was lower in heterocysts and vegetative cells of mutants than in that of wild type. The results suggest that the oxygen sensitivity of nitrogen fixation by heterocystous bluegreen algal mutants may be duc to the defect of some enzymic systems which might play a role in scavenging oxygen toxity, so that the process of nitrogen fixation is inhibited by the active oxygen produced by vegetative cells. The mechanism of protecting nitrogenase from oxygen damage in blue-green algae is discussed.     

How posttranslational modification of nitrogenase is circumvented in Rhodopseudomonas palustris strains that produce hydrogen gas constitutively

Nitrogenase catalyzes the conversion of dinitrogen gas (N(2)) and protons to ammonia and hydrogen gas (H(2)). This is a catalytically difficult reaction that requires large amounts of ATP and reducing power. Thus, nitrogenase is not normally expressed or active in bacteria grown with a readily utilized nitrogen source like ammonium. nifA* mutants of the purple nonsulfur phototrophic bacterium Rhodopseudomonas palustris have been described that express nitrogenase genes constitutively and produce H(2) when grown with ammonium as a nitrogen source. This raised the regulatory paradox of why these mutants are apparently resistant to a known posttranslational modification system that should switch off the activity of nitrogenase. Microarray, mutation analysis, and gene expression studies showed that posttranslational regulation of nitrogenase activity in R. palustris depends on two proteins: DraT2, an ADP-ribosyltransferase, and GlnK2, an NtrC-regulated P(II) protein. GlnK2 was not well expressed in ammonium-grown NifA* cells and thus not available to activate the DraT2 nitrogenase modification enzyme. In addition, the NifA* strain had elevated nitrogenase activity due to overexpression of the nif genes, and this increased amount of expression overwhelmed a basal level of activity of DraT2 in ammonium-grown cells. Thus, insufficient levels of both GlnK2 and DraT2 allow H(2) production by an nifA* mutant grown with ammonium. Inactivation of the nitrogenase posttranslational modification system by mutation of draT2 resulted in increased H(2) production by ammonium-grown NifA* cells.     

Regulation of nitrogenase gene expression in anaerobic cultures of Anabaena variabilis.

Derepression of nitrogenase gene expression was studied at the mRNA and enzyme activity levels in anaerobic cultures of Anabaena variabilis 29413. Cells, previously grown with ammonium chloride, were incubated in the absence of fixed nitrogen compounds under an Ar atmosphere with dichlorophenyldimethyl-urea present to inhibit oxygen evolution. The appearance of nitrogenase mRNA (measured by dot blot hybridization analysis) and nitrogenase activity (measured as acetylene-reducing activity) was followed, and the cells were also observed by phase-contrast microscopy. Nitrogenase mRNA could be detected after 1.5 to 2.0 h of nitrogen starvation; enzyme activity appeared about 1 h later. Although enzyme activity increased for many hours, mRNA levels reached a steady state rapidly. Neither heterocysts nor proheterocysts formed under these conditions; however, the cells were observed to shrink and become chlorotic. When anaerobic, derepressed cultures were exposed to oxygen, nitrogenase mRNA levels decreased very rapidly.     

Photoproduction of hydrogen, nitrogenase and hydrogenase activities of free and immobilized whole cells of Rhodobacter sphaeroides O.U. 001

Abstract Photoproduction of hydrogen, nitrogenase activity (acetylene reduction) and hydrogenase activity (methylene blue dye reduction) were studied in free and alginate immobilized whole cells of a purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides O.U. 001. Four-fold increase in hydrogen production, two-fold increase in nitrogenase activity and 1.2-fold increase in the hydrogenase activity were observed in immobilized cells compared to free cells. Effect of various inhibitors (CO and C₂H₂) and electron donor (H₂) on the above three functions by free and immobilized cells has also been studied.     

Effect of oxygen on batch and continuous cultures of a nitrogen-fixing Arthrobacter sp.

Growth, acetylene reduction, and respiration rate were studied in batch and continuous cultures of Arthrobacter fluorescents at different oxygen partial pressures. The optimum pO2 values for growth and acetylene reduction were 0.05 and 0.025 atm, respectively, but microorganisms can tolerate higher pO2 values. The growth of cultures provided with combined nitrogen was dependent on oxygen availability, and strict anaerobic conditions did not support growth. Acetylene reduction of a population grown in continuous culture and adapted to low pO2 (0.02 atm) was much more sensitive to oxygenation than that of a population adapted to high pO2 (0.4 atm). Their maximum nitrogenase activity, at their optimal pO2 values, were quite different. The respiratory activity of nitrogen-fixing cultures increased with increasing oxygen tensions until a pO2 of 0.2 atm. At higher pO2 values, the respiration rate began to decrease.     

Phenotypic reversion of nitrogenase in pleiotropic mutants of Rhizobium meliloti.

In two out of three pleiotropic mutants of Rhizobium meliloti, defective in nitrate reductase induced by amino acid utilization in vegetative bacteria and in symbiotic nitrogen fixation, nitrogenase activity could be restored completely by purines and partially by the amino acids L-glutamate, L-aspartate, L-glutamine, and L-asparagine. The compounds restoring effectiveness in nitrogen fixation did not restore nitrate reductase activity in vegetative bacteria. The restoration of effectiveness supports our earlier conclusion that the mutation is not in the structural gene for a suggested common subunit of nitrogenase and nitrate reductase.