固氮酶CrFe蛋白和MnFe蛋白的空间晶体生长

从分别生长于含Mn和Cr培养基中的棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3分离纯化出MnFo和CrFe蛋白.为适应包括固氮酶在内的氧敏感蛋白的空间晶体生长的要求,应用简易而适用的厌氧加样装置代替固氮酶实验室所用的笨重厌氧箱(dry box),在地面进行厌氧加样.在充满氮气的简便有机玻璃箱内厌氧加样的所有样品中,分别用液/液扩散法和汽相扩散的坐滴法都可在一周内使MnFe和CrFe蛋白在宇宙飞船上从溶液中结晶出来.在所用的数种蛋白沉淀剂中,飞船上形成的所有晶体都为单晶,而地面上在多数沉淀剂中部生成大量孪晶.在相同沉淀剂中用液/液扩散法,飞船上生成CrFe蛋白的最大晶体比地面生成的最大晶体大1倍.而在相同沉淀剂中用汽相扩散的坐滴法,飞船上生成的MnFe蛋白最大晶体却没有地面生成的最大晶体大.这种差异也许是由不同结晶方法而不是不同蛋白所引起的.     

Relationship Between nifZ and the Synthesis of P cluster in Nitrogenase MoFe Protein of Azotobacter vinelandii

In comparison with OP MoFe protein from wild type strain Azotobacter vinelandii Lipmann, the C2H2-reduction activity and atom ratio of Fe to Mo of △nifZ MoFe protein from a nifZ deletion strain of A. vinelandii were remarkably decreased. FeMoco, which were extracted from these two proteins under the same condition, were almost similar to each other in activity and metal composition, and the circular dichroism (CD) spectra of these proteins were significantly different from each other. In the visible region except 540 750 nm, the △ε at 380 - 540 nm of △nifZ MoFe protein decreased and had a peculiar sharp negative peak around 430 nm; and in the ultraviolet region, the peaks at 208 nm and 222 nm were higher than those of OP MoFe protein. △nifZ MoFe protein could be crystallized in a suitable concentration of PEG 8000 and MgCl2, the size of crystals and amount of precipitation seemed to be related to the above-mentioned negative peaks. The results showed that △nifZ of Azotobacter vinelanclii might be related to the synthesis of P-cluster, rather than to that of FeMoco, which resulted in its conformation, stability and process of crystallization.     

Characteristics and Crystallization of Nitrogenase MoFe Protein from a nif Z Deleted Strain of Azotobacter vinelandii

△nifZ MoFe protein purified from a nifZ deleted strain of Azotobacter vinelandii (DJ194) was shown to be pure by SDS-Polyacrylamide gel electrophoresis. The protein contained 1.5 Mo atoms and 15.9 Fe atoms per molecule, the ratio of Fe to Mo was lower than that of the MoFe protein purified from the wild type strain of A. vinelandii; and Call2, H+ -reduction activity and their ratio (C2H4/H2 (Ar)) were 16.6%, 21.7% and 77.2% of those of the wild type MoFe protein, respectively. Under a somewhat different condition from that for the crystallization of the wild type MoFe protein dark brown rhombohedron crystals of △nifZ MoFe protein were obtained. It indicated that the deletion of the △nif Z resulted in the decrease of number or change in the structure of P-cluster in the mutant MoFe protein, which caused the significant structured and function of change of the protein.     

固氮酶CrFe蛋白和MnFe蛋白的空间晶体生长

从分别牛长于含Mn和Cr培养基中的棕色固氮菌(Azotobacter vinelandii Lipmann)突变种UW3分离纯化出MnFe和CrFe蛋白。为适应包括固氮酶在内的氧敏感蛋白的空间晶体生长的要求,应用简易而适用的厌氧加样装置代替固氮酶实验室所用的笨重厌氧箱(dry box),在地面进行厌氧加样。在充满氮气的简便有机玻璃箱内厌氧加样的所有样品中,分别用液／液扩散法和汽相扩散的坐滴法都可在一周内使MnFe和CrFe蛋白在宇宙飞船上从溶液中结晶出来。在所用的数种蛋白沉淀剂中,飞船上形成的所有晶体都为单品,而地面上在多数沉淀剂中都生成大量挛晶。在相同沉淀剂中用液／液扩散法,飞船上生成CrFe蛋白的最大晶体比地面生成的最大晶体大1倍。而在相同沉淀剂中用汽相扩散的坐滴法,飞船上生成的MnFe蛋白最大晶体却没有地面生成的最大晶体大。这种差异也许是由不同结晶方法而不是不同蛋白所引起的。     

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 ΔnifZ MoFe protein purified from a nifZ deleted mutant strain of Azotobacter vinelandii Lipmann. Systematic studies on the effect of concentrations of PEG 8000,MgCl2, 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, MgCl2, 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.     

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.     

Characterization of Azotobacter vinelandii nifZ deletion strains. Indication of stepwise MoFe protein assembly

The nifZ gene product (NifZ) of Azotobacter vinelandii has been implicated in MoFe protein maturation. However, its exact function in this process remains largely unknown. Here, we report a detailed biochemical/biophysical characterization of His-tagged MoFe proteins purified from A. vinelandii nifZ and nifZ/nifB deletion strains DJ1182 and YM6A (Delta nifZ and Delta nifZ Delta nifB MoFe proteins, respectively). Our data from EPR, metal, activity, and stability analyses indicate that one alpha beta subunit pair of the Delta nifZ MoFe protein contains a P cluster ([8Fe-7S]) and an iron-molybdenum cofactor (FeMoco) ([Mo-7Fe-9S-X-homocitrate]), whereas the other contains a presumed P cluster precursor, possibly comprising a pair of [4Fe-4S]-like clusters, and a vacant FeMoco site. Likewise, the Delta nifZ Delta nifB MoFe protein has the same composition as the Delta nifZ MoFe protein except for the absence of FeMoco, an effect caused by the deletion of the nifB gene. These results suggest that the MoFe protein is likely assembled stepwise, i.e. one alpha beta subunit pair of the tetrameric MoFe protein is assembled prior to the other, and that NifZ might act as a chaperone in the assembly of the second alpha beta subunit pair by facilitating a conformational rearrangement that is required for the formation of the P cluster through the condensation of two [4Fe-4S]-like clusters. The possibility of NifZ exercising its effect through the Fe protein was ruled out because the Fe proteins from nifZ and nifZ/nifB deletion strains are not defective in their normal functions. However, the detailed mechanism of how NifZ carries out its exact function in MoFe protein maturation awaits further investigation.     

Quantitative profiling of the membrane proteome in a halophilic archaeon

We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotope-coded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small.     

固氮酶铬铁蛋白的晶体生长

在合适的结晶条件下,从含Cr无氨培养基中生长的固氮菌(Azotobacter vinelandii Lipmann)突变种UW3中纯化出的CrFe蛋白可从溶液中析出深棕色斜四棱柱晶体,晶体最大的两条对角线长度分别可达0.25 mm和0.12 mm.PEG 8000、MgCl2、NaCl、Tris 和Hepes 缓冲液的浓度及结晶方法等对该蛋白的出晶率、晶核数目、晶体大小和质量都有明显影响.CrFe蛋白结晶所需的上述化合物的最适浓度与在Mn中生长的固氮菌突变种UW3的MnFe蛋白和缺失nifZ固氮菌突变种的ΔnifZ MoFe蛋白结晶所需的最适浓度有所不同.结果表明,该蛋白晶体可能为CrFe蛋白的晶体.     

Growth of the Crystals of Nitrogenase MnFe Protein (in English)

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 ΔnifZ 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.     

Growth characteristics on cellobiose of three different anaerobic fungi isolated from the ovine rumen.

Three morphologically different anaerobic fungi, a Neocallimastix sp. strain (LM-1), a Piromonas sp. strain (SM-1), and a Sphaeromonas sp. strain (NM-1), were isolated from the rumens of sheep. Growth studies were conducted with each isolate in batch cultures by using an anaerobic semidefined medium that lacked ruminal fluid and contained 0.5% cellobiose. Cultures were incubated for periods of up to 10 days, and fungal growth was assessed at regular intervals by dry weight measurements. Samples of fungal biomass were also analyzed for cell-associated protein and, after acid hydrolysis, for chitin as hexosamine. The isolates produced similar yields of dry weight and contained similar amounts of protein. However, strain LM-1 grew at a higher rate and contained less than half the level of chitin compared with the other two isolates. There were high positive correlations between chitin and protein for all three fungi, but comparisons of these parameters with dry weights were affected by the presence of variable amounts of storage carbohydrate. The amount of storage carbohydrate reached maximum levels in strain LM-1 during mid-growth phase and then quickly declined thereafter. When dry weight yields for strain LM-1 were adjusted for changes in storage carbohydrate, high positive correlations were obtained between dry weight and protein or chitin. The storage carbohydrate was probably an alpha-1,4-glucan with alpha-1,6 branches.     

Growth characteristics on cellobiose of three different anaerobic fungi isolated from the ovine rumen

Three morphologically different anaerobic fungi, a Neocallimastix sp. strain (LM-1), a Piromonas sp. strain (SM-1), and a Sphaeromonas sp. strain (NM-1), were isolated from the rumens of sheep. Growth studies were conducted with each isolate in batch cultures by using an anaerobic semidefined medium that lacked ruminal fluid and contained 0.5% cellobiose. Cultures were incubated for periods of up to 10 days, and fungal growth was assessed at regular intervals by dry weight measurements. Samples of fungal biomass were also analyzed for cell-associated protein and, after acid hydrolysis, for chitin as hexosamine. The isolates produced similar yields of dry weight and contained similar amounts of protein. However, strain LM-1 grew at a higher rate and contained less than half the level of chitin compared with the other two isolates. There were high positive correlations between chitin and protein for all three fungi, but comparisons of these parameters with dry weights were affected by the presence of variable amounts of storage carbohydrate. The amount of storage carbohydrate reached maximum levels in strain LM-1 during mid-growth phase and then quickly declined thereafter. When dry weight yields for strain LM-1 were adjusted for changes in storage carbohydrate, high positive correlations were obtained between dry weight and protein or chitin. The storage carbohydrate was probably an alpha-1,4-glucan with alpha-1,6 branches.     

A Disposable Anaerobic System Designed for Field and Laboratory Use

A disposable anaerobic system which is characterized by its light weight and its compactness is described. The system consists of a multilayer plastic bag with a unique sealing device. A collapsible impregnated cardboard container is fitted with a catalyst and holders for a disposable hydrogen generator and an anaerobic indicator. The catalyst is active at room temperature and requires no heat activation. This system, which lends itself readily to compact storage, quick assembly, and ease of operation, is disposable after use.     

Isolation of Anaerobic Bacteria from Human Gingiva and Mouse Cecum by Means of a Simplified Glove Box Procedure

An anaerobic glove box constructed of clear flexible vinyl plastic is described. It is sufficiently inexpensive and simple in operation to be used not only in research but also in a clinical laboratory by technicians without special training. Conventional bacteriological techniques may be used inside the glove box for culturing and transferring anaerobic bacteria. The box may be heated to 37 C and thus serve as an anaerobic incubator as well, permitting inspection of cultures at any time. Media may be prepared and agar plates may be poured on the laboratory bench in the conventional manner. An overlay of trace amounts of palladium black catalyst over plated agar media reduces the medium to an oxidation-reduction (O-R) potential of - 300 mv within 2 days after introduction into the glove box. In spite of its greater simplicity, the system matched or excelled the roll tube method with respect to all parameters tested, including O-R potential obtainable in the media, O(2) concentration in the gas phase, and efficiency in isolating anaerobic bacteria from the mouse cecum. Comparative studies indicate that the conventional anaerobic jar method was inadequate for the isolation of strict anaerobes from human gingival specimens and from the mouse cecum. This was due to the exposure of specimens and media to air during plating on the open laboratory bench. Anaerobic jars were adequate for maintaining the proper conditions for growth of anaerobic bacteria once these had been established in the glove box.     

Production of Soy Sauce Koji Mold Spore Inoculum in Plastic Bags

An innovation is described for producing soy sauce koji mold spore inoculum by using inexpensive autoclavable plastic bags and reuseable plastic enclosures to make culture vessels. After growth, the spore mass could be dried and packaged in the same bag after removing the enclosure. Broken rice was used as the substrate for mold cultivation. Viable spore counts of 10⁹ spores per g were obtained under optimal conditions. After drying at 50°C for 6 h, the moisture content of the spore mass decreased from 35.22 to 6.32% with no significant effect on spore viability. The dry spores could be stored in the refrigerator or at room temperature for at least 3 months.     

ISOLATION AND CHARACTERIZATION OF CHLORELLA SOROKINIANA GXNN01 (CHLOROPHYTA) WITH THE PROPERTIES OF HETEROTROPHIC AND MICROAEROBIC GROWTH1

Heterotrophic and anaerobic microalgae are of significance in both basic research and industrial application. A microalga strain was isolated from a wastewater treatment pond and identified as Chlorella sorokiniana Shihira et W. R. Krauss GXNN01 in terms of morphology, physiology, and phylogeny. The strain grows rapidly in heterotrophic or mixotrophic conditions with addition of various carbon sources, and even in anaerobic conditions. The maximum growth rate reached 0.28 d^?1 when using d,l ‐malate as the carbon source, and the protein content of the microalgae was 75.32% in cell dry weight. The strain was shown to be capable of (1) utilizing d,l ‐malate only with light, (2) inhibiting photosynthesis in mixotrophic growth, and (3) growing in anaerobic conditions with regular photosynthesis and producing oxygen internally. This study demonstrates the influence of oxygen (aerobic vs. anaerobic) and metabolic regime (autotrophy, mixotrophy, heterotrophy) on the physiological state of the cell.     

Expression, purification, crystallization and preliminary x-ray analysis of Cyan fluorescent protein CyPet

The technique of fluorescence (or F?rster) resonance energy transfer (FRET) is widely used to observe bimolecular interaction in living cells. Cyan and yellow fluorescent proteins are the most widely used pair in FRET analysis. CyPet and YPet are two newly optimized fluorescent proteins that have much better dynamic range and sensitivity than CFP/YFP pair, although the crystallographic structure and the mechanism of better fluorescent characteristics of CyPet are still unknown. We have expressed the cyan fluorescent protein CyPet using pT7 prokaryocyte expression system in Escherichia coli strain Rosetta (DE3) pLysS by auto-induction. After purification, the recombinant CyPet protein was crystallized by hanging drop vapor diffusion technique and could diffract to 2.55A resolution. The data showed that the orthorhombic CyPet crystal was in space group P212121 with unit cell parameters (51.55, 61.53, 63.36) and contained one molecule in one asymmetric unit.     

Isolation of a Citrobacter species able to grow on malonate under strictly anaerobic conditions

An anaerobic enrichment from lake mud yielded a pure culture of a facultatively anaerobic bacterium able to grow on malonate under strictly anaerobic conditions. Strain 16mal1 was identified as a member of the family Enterobacteriaceae, and assigned to the genus Citrobacter on the basis of morphological, metabolic and biochemical characteristics. Malonate was fermented under strictly anaerobic (sulphide-reduced) conditions to acetate and CO2 concomitant with growth. A maximum growth rate of 1.88 generations h-1 (mu = 1.30 h-1) was measured. The dry weight yield of cells from malonate was estimated at 2.5 g mol-1. Yeast extract was required for growth on malonate: other additives, or a vitamin solution, could not replace this requirement. Other dicarboxylic acids were not degraded in the absence or presence of malonate. Malonate was degraded under anaerobic, but not aerobic conditions. Malonate-decarboxylating activity was inducible by malonate under both anaerobic and aerobic conditions, and was not expressed in glucose- or citrate-grown anaerobic cultures. Monensin had no effect on malonate degradation, while 2,4-dinitrophenol decreased the rate of malonate degradation. This, with the lack of a sodium requirement for anaerobic growth on malonate, suggested that ATP generation may not be mediated by a sodium-pumping mechanism.