Crystallization of Nitrogenase MoFe Protein from a Mutant nifE Deleted Strain of Azotobacter vinelandii

Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from FeMoco-deficient MoFe protein (DeltanifE Avl) purified from a nifE deleted mutant DJ35 of Azotobacter vinelandii Lipmann grown in NH3-limited medium. The number, size and quality of crystals were significantly affected by either the concentration of precipitants and buffer or diffusion method. The longest sides of the largest crystal of DeltanifE Avl protein, which was obtained by vapor diffusion in the hanging drop method, were 0.12 and 0.13 mm, respectively.

Studies on Crystalline Growth of MoFe Protein from a nifZ Deleted Strain of Azotobacter vinelandii

Under a given condition of crystallization, dark brown short rhombohedron crystals could be obtained from Δ nifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann. Systematic studies on the effect of concentrations of PEG 8000,MgCl 2, NaCl,Tris and buffer pH on the crystallization and crystal growth of the protein showed that the protein could not be crystallized in lower concentrations of the chemicals and lower buffer pH. A large amount of smaller crystals of the protein appeared in a week with gradual increasing in the chemical concentrations and pH≥8.0. When the chemical concentrations were further increased, the time for crystallization was increased and a few high grade crystals of larger size were formed. If the concentrations of the chemicals were continuously increased, many crystals with smaller size, and, sometimes of poor quality appeared again and eventually ceased to produce any crystals. The optimal concentration for each of the above mentioned chemicals varies with other variable factors. Only one bigger crystal (both of the longest two sides: 0.16 mm) could be obtained in a hanging drop of protein sample when the concentrations of PEG 8000, MgCl 2, NaCl,Tris and protein were kept at 1.86%, 300 mmol/L, 400 mmol/L, 53 mmol/L and 4.64 g/L , respectively, with Tris buffer pH 8.2.

Use of Azotobacter sp.as an indicator to detect the toxicity of heavy metals in soils

A physiological strain of microorganism - Azotobacter sp. has been adopted as an indicator to detect the toxicity of heavy metals in soils. The concentration of heavy metals to which Azotobacter sp. was behaving initially to have the resistance to heavy metals is defined as the critical poisoning concentration. The method of physiological threshold adopted can have a quantitative determination with reproducible results. The determined critical poisoning concentration is basically consistent with the results of heavy metals and arsenic toxicities to the bacteria reported recently in literatures. Total 9 typical soils, including 6 zonal soils and 3 purple soils, in the whole country were determined for the toxicities of 5 heavy metals and arsenic to Azotobacter sp. that resulted in 48 critical poisoning concentrations.

Crystallization of Nitrogenase MoFe Protein (NifB Av1) from a nifB Mutated Strain UW45 of Azotobacter vinelandii

Six hundred and 28 mg of NifB(-) Av1 was obtained by a chromatography twice on DE 52 columns and Sephacryl S-300 column from the crude extract (37 677 mg) of a nifB mutated strain UW45 of Azotobacter vinelandii Lipmann. The protein was almost homogeneous as determined by Coomassie staining of SDS gels. The analysis by SDS-PAGE showed that NifB(-)Av1 was similar to Av1 from wild-type strain of A. vinelandii (OP) in the kinds of subunits (alpha and beta subunit). When complemented with Av2, NifB(-)Av1 had hardly any H-reducing activity, but could be significantly activated by FeMoco extracted from Av1. Under a suitable condition for crystallization, short dark-brown rhombohedral crystals could be obtained from NifB(-)Av1. Both of the longest sides of the biggest crystal were 0.1 mm. The time of the formation of crystals and number, size, quality and shape of crystals obviously depended not only on the kinds and concentrations of the components in the precipitant solution, but also on the methods for crystallization and technical bias, etc. The preliminary results showed that the crystal seemed to be formed from NifB(-)Av1.

Biosorption of Metal Ions by Exopolysaccharide Produced by <i>Azotobacter chroococcum</i>XU1

This paper deals with adsorption of Pb and Hg on the exopolysaccharide produced by Azotobacter chroococcum XU1. The adsorption of metal ions was significantly affected by the pH of metal solutions (Pb(NO3)2 and Hg(NO3)2), metal ions concentration and exopolysaccharide concentrations. At optimum pH the uptakes of the metals were 40.48% and 47.87% respectively. The metal ions biosorption was high at 4, 5-5.

Effects of La and Nd on the Fermentation of Alginic Acid Produced from the Strain 342 of Azotobacter Vinelandii

It is reported that fermentative liquids with various concentrations of La and Nd affect the fer- mentation of alginic acid from the strain 342 of Azotobacter vinelandii.The results are as follows:When the concentration of La or Nd was up to 100 ppm,the cell growth is stimulated and the production of alginic acid is promoted.The La or Nd in concentration higher than 200 ppm or 150 ppm inhibits the fermentation, respectively.As the concentration range of La is 0～100 ppm or that of Nd is 0～150 ppm,the yield of fixed nitrogen increases,and the ratio of c_M to c_G(c_M/c_G)decreases with the raise of the concentration of La or Nd.When the concentration range of La is 100～400 ppm and that of Nd is 150～400 ppm,the conclusion is contrary to the above mentioned result.

Removal of Silver from Aqueous Solution by <i>Azotobacter chroococcum</i>XU1 Biomass and Exopolysaccharide

Biosorption of silver ions onto A. chroococcum XU1 biomass and exopolysaccharide (EPS) was investigated. It was found that the overall biosorption process was the best when cell biomass and EPS were used together. Metal adsorption experiments showed that the final precipitate obtained by cell biomass and EPS, contained 7.85% of Ag. Based on the results presented in this study, the biomass and EPS of A. chroococcum XU1 indicated the possibility of application of them as biosorbent for removal of silver from waste waters.

Effect of Pesticide Residues (Sevin) on Carrot (Daucus carota L.) and Free Nitrogen Fixers (Azotobacter spp)

Since the pesticides are considered as an essential component for crops production through controlling pests, they have shown a negative effect on crops and soil environment when used intensively. This experiment was conducted at Wadi Soba farm (Sharq Elneel) Khartoum, Sudan. It aimed to study the effect of Sevin residuals on carrot growth and Azotobacter spp. colonies growth. Carrot planted in late February 2013, Sevin pesticide (2.5 L/ha and 5 L/ha) was added to estimate plant height, dry weight, the number of plant leaves and the number of Azotobacter spp. colonies isolated from the carrot rhizosphere (0 - 15 cm). The obtained results showed that the Sevin recommended dose (2.5 L/ha) relatively had a positive effect on the plant height, dry weight, and the number of plant leaves. The average of plant height for recommended dose was 59.67 cm compared to control (53.67 cm) and high dose (27.33 cm). The average of plant dry weight obtained by the recommended dose was 503.33 g and for control was 476.67 g and for high dose was 166.7 g, it decreased 67% from control and recommended dose. The average of plant leaves number were 25.34, 13.66 and 21.33 for recommended dose, high dose and control respectively, the number of leaves increased about 16% by recommended dose and decreased 35% by high dose. The average of Azotobacter spp. colonies obtained by high dose of Sevin demonstrated a lower numbers which were 20 × 104, 5.67 × 106 and 0.33 × 108 compared with control (78.33 × 104, 44 × 106 and 15.33 × 108) and the recommended dose (64 × 104, 33 × 106 and 7 × 108). The high dose of Sevin had a negative effect on both carrot growth and Azotobacter spp. colonies growth.

Azotobacter inoculation can enhance the resistance of Bt cotton to cotton bollworm, Helicoverpa armigera

The rising trend in the cultivation of Bacillus thuringiensis (Bt) transgenic crops may cause a destabilization of agroecosystems, thus increasing concerns about the sustainability of Bt crops as a valid pest management method. Azotobacter can be used as a biological regulator to increase environmental suitability and improve the soil nitrogen utilization efficiency of crops, especially Bt cotton. A laboratory test investigated effects on the development and food utilization of Helicoverpa armigera fed with different Cry1Ab/Cry1Ac proteins and nitrogen metabolism-related compounds from cotton (transgenic variety SCRC 37 vs non-Bt cotton cv. Yu 2067) inoculated with Azospirillum brasilense (Ab) and Azotobacter chroococcum (Ac). The findings indicate that inoculation with Azotobacter significantly decreased the partial development and food utilization indexes (pupal weight;pupation rate;adult longevity;fecundity;relative growth rate, RGR;efficiency of conversion of digested food, ECD;and efficiency of conversion of ingested food, ECI) of H. armigera fed on Bt cotton, but contrasting trends were found among these indexes in H. armigera fed on non-Bt cotton inoculated with Azotobacter, as a result of differences in Bt toxin production. Overall, the results showed that inoculation with Azotobacter had negative effects on the development and food utilization of H. armigera fed on Bt cotton, leading to enhanced target insect resistance. Presumably, Azotobacter inoculation can be used to stimulate plant soil nitrogen uptake to increase nitrogen metabolism-related compounds and promote plant growth for Bt and non-Bt cotton, simultaneously raising Bt protein expression and enhancing resistance efficacy against cotton bollworm in Bt cotton.

Purification and Characterization of a New Heme-Binding （HBP59） from the Mutant Strain DJ35 of Azotobacter vinelandii

A new protein, an approximately 59-kDa monomer containing iron atoms, was first isolated from the mutant strain DJ35 of Azotobacter vinelandii Llpmann. After analysis by matrix-assisted laser desorptlon ionization time-of-flight mass spectrometry, the protein was Identified as the product of a predicted gene. Thus, the protein was tentatively called HBP59. Its absorption spectra （ABS） In the reduced state exhibited three peaks at 421,517, and 556 nm and the maximal peak was shifted from 421 to 413 nm after exposure of HBP59 to air. The Soret circular dichrolsm （CD） spectrum of HBP59 In the reduced state displayed four positive peaks at 364, 382, 406, and 418 nm and two negative peaks at 398 and 433 nm; the △ε （CD extinction coefficient） values of these peaks were found to be 0.92, 0.58, 0.87, 0.72, -0.65 and -1.12 L/mol per cm, respectively. Titration with heme showed that the protein has 0.1 heme molecules/protein molecule. After HBP59 had fully Interacted with heme, Its maximal ABS value and Soret CD Intensity were increased by approximately 10-fold compared with values before Interaction. Therefore, It seems that one molecule of HBP59 can be interacted with only one heme. These results indicate that HBP59 contains heme with low spin and may be Involved In heme utilization or adhesion.

Activation In Vitro of Mutant MoFe Proteins from Azotobacter vinelandii by Reconstituent Solutions

nifB-MoFe protein （nifB-Av1）, AnifE MoFe protein （△nifE Av1） and AnifZ MoFe protein （△nifZ Av1） were obtained by chromatography on DE52, Sephacryl S-300 and Q-Sepharose columns from nifB point-mutated, nifE deleted and nifZ deleted mutant stains （UW45, DJ35 and DJ194） of Azotobacter vinelandii Llpmann, respectively. When complemented with nltrogenase Fe protein （Av2）, AnifZ Av1 had partial activity and both nifB-Avl and △nifE Av1 had hardly any activity, but could be obviously activated by FeMoco extracted from wild-type MoFe protein （OP Av1） or △nifZ Av1. After being Incubated with excess O-phenanthrollne （O-phen） for 150 mln at 30 ℃ and subjected to chromatography on a Sephadex G-25 column In an Ar atmosphere, nifB- Av1C, △nifE Av1C and △nifZ Av1C were obtained, respectively. Based on a calculation of Fe atoms In the Ophen-Fe compound with ε 512nm = 11 100, lost Fe atoms of nifB-Av1, △nifE Av1 and △nifZ Av1 were estimated to be 1.35, 2.89 and 8.44 per molecule of protein, respectively. As a result of the Fe loss, △nifZ Av1 loses Its original activity. In the presence of both MgATP and Av2, these Fe-loslng proteins, but not the original proteins untreated with O-phen, could be significantly activated by reconstltuent solution （RS） composed of dlthlothreltol, ferric homocltrate, Na2S and Na2MoO4, or K2CrO4, or KMnO4. But In the absence of MgATP or Av2, the activation did not occur, with the exception that △nifZ AvlC was partially activated, and the activity was only 17%. These findings Indicate that： （I） △nifZ Avl with half P-cluster content Is somewhat different from FeMoco-deflclent nifB-Avl and ,△nifE Av1 with respect to protein conformation either before or after treatment with O-phen; （11） full activation of these proteins with RS requires pretreatment with O-phen and the simultaneous presence of MgATP and Av2.

棕色固氮菌nifS敲除菌株的构建

从Azotobacter vinelandii中通过PCR扩增了5’和3’端分别缺失264bp和261bp的nifS′片段,克隆至载体pUC18,形成重组质粒pUCS,再通过同源重组的方法,将pUCS插入Azoto-bacter vinelandii的nifS中,形成nifS阻断突变体SU1,经Southern杂交和PCR扩增,证明所得确为nifS阻断突变株。SU1在外加氮源的BBGN培养基中能够快速生长,但在Burk's无氮培养基中,生长却极其缓慢,表明nifS基因的破坏,已造成SU1的固氮能力接近完全丧失。该突变体的成功构建,为进一步从中纯化固氮酶两组分,研究nifS对固氮酶结构及功能的影响及iscS与nifS之间的关系奠定了良好的基础。

Growth of the Crystals of Nitrogenase MnFe Protein

Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing but Mo- and NH3-free medium. The possibility of crystallization, and number, size and quality of crystals were obviously dependent on concentrations of NaCl, MgCl2, PEG 8000,Tris and Hepes buffer and on methods for crystallization. PEG concentration affected on the shape of the crystals. The optimal, concentrations of the chemicals for crystallization of MnFe protein were slightly different from those for crystallization of Delta nifZ MoFe protein from a nifZ deleted strain of Azotobacter vinelandii. SDS-PAGE showed that the protein from the dissolved crystals was almost the same as MnFe protein before crystallization, indicating that the crystal was formed from MnFe protein.

STRUCTURAL REQUIREMENT FOR CLUSTERS TO BE RECONSTITUTED WITH THE FeMoCo DEFICIENT MOLYBDENUM IRON PROTEIN

By incubating the reduced MoFe protein from Azotobacter vinelandii with O phenanthroline under air and chromatographying the incubated solution on Sephadex G 25 column, inactive MoFe protein could be obtained. Its acetylene reduction activity was remarkably recovered not only by incubation with the reconstituent solution composed of KMnO 4,ferric homocitrate, Na 2S and dithiothreitol,Azotobacter vinelandii with O phenanthroline under air and chromatographying the incubated solution on Sephadex G 25 column, inactive MoFe protein could be obtained. Its acetylene reduction activity was remarkably recovered not only by incubation with the reconstituent solution composed of KMnO 4,ferric homocitrate, Na 2S and dithiothreitol, with O phenanthroline under air and chromatographying the incubated solution on Sephadex G 25 column, inactive MoFe protein could be obtained. Its acetylene reduction activity was remarkably recovered not only by incubation with the reconstituent solution composed of KMnO 4,ferric homocitrate, Na 2S and dithiothreitol, but also with a mixture of 4Fe∶4S clusters and another cluster which had two structure units of 1Mo∶3Fe∶4S bridged by three -OCH 3- at the Mo atoms. Neither the reconstituent solution nor the mixture could reactivate apo MoFe proteins from the mutants deleting nifE and nifH genes and from the mutant UW 45 , which could be reactivated by the FeMoco extracted from the MoFe protein. The results indicated that the FeMoco deficient MoFe proteins from these mutants seemed to be reconstituted only by the clusters which were probably structures only similar to FeMoco. The partially metallocluster deficient MoFe protein could be reconstituted by the clusters with a certain kind of structure and composition; and was changed into different nitrogenase proteins with the ability to fix nitrogen.

Purification and Characteristics of Mn-containing Nitrogenase Component Ⅰ

A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but undergo phenotypic reversal to Nif + under Mo deficiency, was able to grow in Mo- and NH 3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW 3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C 2H 2- and H +-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰ protein.

Crystal Growth of Nitrogenase CrFe Protein and MnFe Protein in Space

Nitrogenase CrFe protein and MnFe protein were purified from a mutant strain UW3 of Azotobacter vinelandii Lipmann grown on a medium containing Cr and Mn, respectively. In order to meet the requirement for crystal growth Of O-2-susceptible proteins including nitrogenase in space, crystallization conditions were optimized for the proteins using a simple and suitable device, as a replacement for the cumbersome anaerobic box (dry box), for anaerobic addition of the protein samples. In all used precipitant and protein solutions added in the simplified plexi glass box, CrFe protein and MnFe protein could be crystallized on the spacecraft in one week by the liquid/liquid diffusion method and vapor diffusion by the sitting drop method, respectively. All formed crystals were single on the spacecraft, but under the same condition twin crystals appeared on the ground. The size of the largest crystal grown in space from CrFe protein was 2-fold larger than that on the ground. But the size of the largest crystal grown in space from MnFe protein was not larger than that on the ground. The difference in crystal growth in space between CrFe protein and MnFe protein could be resulted from the crystallization method, rather than the kind of protein.

Growth of Large Single Crystals of Nitrogenase CrFe Protein and MnFe Protein

By using the liquid/liquid diffusion method at a suitable crystallization conditions, large single and dark brown crystals (the sides of the largest crystals were 0.20 mm x 0.20 mm x 0.07 min and 0.18 mm x 0.18 mm x 0.05 mm, respectively) could be obtained from the solutions of nitrogenase CrFe protein and MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmarm grown in Cr- or Mn-containing but NH3-free medium. The time of crystal formation, as well as the number, size, shape and quality of crystals obviously depended on the concentrations of PEG, MgCl2 and NaCl. The liquid/liquid diffusion method seems to benefit CrFe protein and MnFe protein for the growth of large single crystals for X-ray diffraction analysis.

Science Letters:Alterations in seedling vigour and antioxidant enzyme activities in Catharanthus roseus under seed priming with native diazotrophs

An experiment was conducted on Catharanthus roseus to study the effect of seed treatments with native diazotrophs on its seedling growth and antioxidant enzyme activities. The treatments had significant influence on various seedling parameters. There is no significant influence on dry matter production with the diazotrophs, Azospirillum and Azotobacter. However, the vital seedling parameters such as germination percentage and vigour index were improved. Azotobacter treatment influenced maximum of 50% germination, whereas Azospirillum and Azotobacter were on par with C. roseus with respect to their vigour index. There was significant difference in the population of total diazotrophs. Azospirillum and Azotobacter between rhizosphere and non-rhizosphere soils of C. roseus had the same trend and were observed at various locations of the study. The activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) were increased to a significant extent due to the treatment with diazotrophs.

Crystalline Growth of Nitrogenase CrFe Protein

Under a suitable condition of crystallization, dark brown rhombohedron crystals (the lengths of the longest two diagonals were 0.25 and 0.12 mm, respectively) could be obtained from nitrogenase CrFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Cr-containing but NH3-free Medium, The possibility of crystallization, as well as the. number, size and quality of crystals obviously depended on the concentrations of PEG 8000, MgCl2, NaCl, Tris and Hepes buffer, and methods of crystallization. The optimum concentrations of the chemicals for crystallization of CrFe protein were slightly different from those for crystallization of MnFe protein from UW3 grown in Mn and DeltanifZ MoFe protein from a nifZ deleted strain of A. vinelandii. The crystal seemed to be formed from CrFe protein.

Influence of Diazotrophic Bacteria on Antioxidant Enzymes and Some Biochemical Characteristics of Soybean Subjected to Water Stress

Drought stress is an abiotic stress that imposes serious constraints on plants. The present investigation was carried out to determine the inter-relationship between some physiological attributes of soybeans affected by drought stress and pure isolates ofAzotobacter andAzospirillum. Drought stress and bacterial application increased catalase and glutathione peroxidase activity, whereas drought stress increased superoxide dismutase activity during the pod-filling stage. Abscisic acid and proline levels increased due to drought stress and bacterial application during the flowering stage, whereas total plant nitrogen was enhanced under well-watered conditions when plants were inoculated with bacteria. The close relationship between enzyme activity and drought stress with bacteria indicated that antioxidant enzymes play an important role in alleviating the detrimental effects of water stress. In addition, the enhancement of abscisic acid and proline could be positively linked with drought stress, and drought-induced abscisic acid could induce proline accumulation and the expression of antioxidant enzyme genes.