Effects of zinc-smelter emissions on forest soil microflora.

Within 2 km of a zinc (Zn) smelter in Palmerton, Pennsylvania, near the Lehigh Water Gap, up to 13.5% Zn by weight has been measured in the O2 horizon of the soil, and up to 8% Zn in the A1 horizon. The total numbers of bacteria, actinomycetes, and fungi (measured by dilution plate counts) were greatly reduced in the most severely Zn-contaminated soils compared with control soils. The reduction of microbial populations may be a partial cause of the decreased rate of litter decomposition at Lehigh Gap. Growth of most bacteria from control sites was reduced by 100 to 200 muM Zn, most actinomycetes by 100 muM Zn, and most fungi by 100 to 1000 muM Zn in thin-Pablum extract agar (TPab). All the tested actinomycetes and non-spore-forming bacteria isolated from Zn-contaminated Lehigh Gap soils were Zn-tolerant, growing normally in media containing 600-2000 muM Zn. Most fungi, regardless of source, were capable of at least 50% of normal growth at 700 muM Zn. Zinc-tolerant bacteria, actinomycetes, and fungi were readily isolated from low-Zn soils, suggesting that selection for Zn tolerance may proceed rapidly. Acidophilic Mortierella species have been selectively eliminated near the smelter, apparently because of elevated soil pH. Peryronellaea glomerata (Corda) Goidanich and Coniothyrium spp. were found only in the high-Zn soils.     

Succession of indigenous Pseudomonas spp. and actinomycetes on barley roots affected by the antagonistic strain Pseudomonas fluorescens DR54 and the fungicide imazalil

In recent years, the interest in the use of bacteria for biological control of plant-pathogenic fungi has increased. We studied the possible side effects of coating barley seeds with the antagonistic strain Pseudomonas fluorescens DR54 or a commercial fungicide, imazalil. This was done by monitoring the number of indigenous Pseudomonas organisms and actinomycetes on barley roots during growth in soil, harvest after 50 days, and subsequent decomposition. Bacteria were enumerated by traditional plate spreading on Gould's S1 agar (Pseudomonas) and as filamentous colonies on Winogradsky agar (actinomycetes) and by two quantitative competitive PCR assays. For this we developed an assay targeting Streptomyces and closely related genera. DR54 constituted more than 75% of the Pseudomonas population at the root base during the first 21 days but decreased to less than 10% at day 50. DR54 was not successful in colonizing root tips. Initially, DR54 affected the number of indigenous Pseudomonas organisms negatively, whereas imazalil affected Pseudomonas numbers positively, but the effects were transient. Although plate counts were considerably lower than the number of DNA copies, the two methods correlated well for Pseudomonas during plant growth, but after plant harvest Pseudomonas-specific DNA copy numbers decreased while plate counts were in the same magnitude as before. Hence, Pseudomonas was 10-fold more culturable in a decomposition environment than in the rhizosphere. The abundance of actinomycetes was unaffected by DR54 or imazalil amendments, and CFU and quantitative PCR results correlated throughout the experiment. The abundance of actinomycetes increased gradually, mostly in numbers of DNA copies, confirming their role in colonizing old roots.     

有机物料厌氧发酵液中拮抗苹果再植障碍病原真菌的细菌筛选及其防治效果

有机物料厌氧发酵液(AFOF)能显著改善苹果再植障碍.本研究对AFOF中能拮抗苹果再植障碍主要病原菌(腐皮镰刀菌、层出镰刀菌、尖孢镰刀菌、串珠镰刀菌)的细菌进行了分离筛选,并对其作用效果进行了盆栽验证.结果表明: AFOF能显著抑制病原真菌的生长繁殖;对峙试验共得到4株具有较强拮抗作用的细菌(L11、L12、L13、L14),最高抑菌率达到57.3%,鉴定发现这4株细菌均属于芽孢杆菌属,相互之间没有明显的拮抗作用;在盆栽条件下,与连作土相比,溴甲烷熏蒸处理和拮抗菌菌液处理对平邑甜茶幼苗的生物量均有不同程度的促进作用;在幼苗的长势上,溴甲烷熏蒸处理效果要好于拮抗菌菌液处理;在根系活力上,拮抗菌菌液处理效果要好于溴甲烷熏蒸处理,根系长度、根尖数分别增加了25.1%、70.9%.与连作土处理相比,拮抗菌菌液和溴甲烷熏蒸均能显著降低土壤中的真菌数量,分别降低了71.2%和64.2%,拮抗菌菌液处理能显著增加土壤中的细菌和放线菌数量,分别增加了48.0%和140.2%,使土壤微生物结构向“细菌型”转化;而溴甲烷熏蒸处理显著降低了土壤中的细菌和放线菌数量,说明拮抗菌的确能够抑制土壤中病原真菌的生长.     

超高产夏玉米田土壤微生物与土壤酶的动态变化

为揭示超高产夏玉米田（产量>15 000 kg·hm^-2）土壤微生物与土壤酶活性动态变化特性,在国家玉米工程技术研究中心（山东）试验场进行夏玉米生长季农田土壤微生物与土壤酶活性研究. 在连续3年产量15 000 kg·hm^-2以上的超高产夏玉米田中选择一块超高产田（HF, 产量为20 322 kg·hm^-2）与常规生产田（CF, 产量为8920.1 kg·hm^-2）进行对比分析,主要测定0～20 cm土层土壤细菌、真菌与放线菌数量及脲酶和转化酶活性. 结果表明：播种后超高产田与常规生产田土壤微生物（细菌、真菌与放线菌）数量均表现出先升高后下降的趋势,超高产田在玉米生长后期土壤微生物数量低于常规生产田,细菌与放线菌表现尤其明显,收获期超高产田B/F值（细菌与真菌数量比）比播种期高2.03倍,比常规生产田高3.02倍,常规生产田收获期与播种期的B/F值变化不显著;超高产田土壤脲酶活性在播种31 d（拔节期）后低于常规生产田,转化酶活性播种58 d（开花期）后快速下降,低于常规生产田.     

The rhizosphere microflora of wheat grown under controlled conditions I. The effect of soil fertility and urea leaf treatment on the rhizoplane microflora

Summary Microbial colonization of seminal roots of seedlings and of nodal roots of tillering plants was studied on spring wheat ‘Kaspar’ cultivated in growth, chambers. Methods were developed to microbially condition the soil before seeding and to regulate soil humidity. Addition of inorganic nutrients (NPK) to the soil increased the number of rhizoplane bacteria and actinomycetes, but did not effect the number of fungi on seminal and nodal roots. Urea leaf treatments stimulated bacteria and actinomycetes 7 and 9 days after treatment. Fourteen days after urea leaf treatment, however, bacterial numbers were mostly reduced, especially on seminal roots, while numbers of actinomycetes generally equalled the control. Root types and soil fertility did not obviously interact with the effect of urea leaf treatment on rhizoplane bacteria and actinomycetes. The only effect of urea on total numbers of fungi, was a reduction of their numbers on seminal roots 9 days after treatment at both NPK-levels.     

Carbon Quality and Stocks in Organic Horizons in Boreal Forest Soils

We investigated the mechanisms that determine the quality and quantity of organic carbon (C) stocks in boreal forest soils by analyzing both qualitative and quantitative changes in the organic fractions in the soil organic matter (OM) in a vertical gradient in the decomposition continuum of the organic horizon [litter layer (L), fermentation layer (F), and humus layer (H)] in forest soils using a sequential fractionation method at two forest types along a climatic gradient in Finland. We predicted that the concentrations of water-soluble (WSE) and non-polar (NPE) extractives should decrease and those of the acid-soluble (AS) fraction and acid-insoluble residue (AIR) should increase from the L to the F, and from the F to the H layers, but the C/N ratio of soil OM should stay constant after reaching the critical quotient. We also predicted that the AIR concentrations should be higher in the south than north boreal, and in sub-xeric than mesic forests. Consistent with our hypothesis, the concentrations of WSE and NPE fractions decreased and concentrations of AIR increased in the vertical soil gradient. The highest concentrations of the AS fraction were found in the F layer. The C/N ratio was lowest in the F layer, and the highest in the H layer, indicating that soil OM is depleted in N in relation to C along the vertical soil gradient. Concentrations of WSE and NPE were lower, and concentrations of AIR were higher in the south than in north boreal forests, which is in agreement with our hypothesis that higher soil temperatures may enhance accumulation of slowly decomposable OM in the soil. The concentrations of AIR were higher in the sub-xeric than mesic forests. Contrary to our expectations, however, the differences in the chemical quality in soil OM between the site types were amplified from the L to the H layer. The size of the C storage was significantly larger in south than north boreal sites, and larger in the mesic than in the sub-xeric sites.     

The occurrence and distribution of actinomycetes in lakes of the English Lake District

Investigation of the occurrence of mesophilic actinomycetes in the lakes of the English Lake District revealed their widespread distribution in the lacustrine environment. Although only low numbers of actinomycetes occurred in the water, high numbers were recovered from all the lake muds. Total numbers of actinomycetes in the muds correlated quite well with the lakes’ productivity status. High numbers of Micro-monospora, Streptomyces and nocardioform actinomycetes were isolated from all the lakes sampled. Low numbers of Streptosporangium were isolated from all the muds but strains of Actinomadura, Actinoplanes, Dactylosporangium, Microbispora and Thermomonospora were only encountered occasionally. Micromonospora was the numerically dominant genus isolated from all the lakes sampled. This dominance was even more striking in deeper layers of mud and this was thought to reflect a more resistant spore stage in Micromonospora than in either of the other two main genera.     

The use of previous crops as sustainable and eco-friendly management to fight Fusarium oxysporum in sesame plants

Sesame (Sesamum indicum L.), the “Queen of oil seeds” is being infected with pathogens, i.e., fungi, bacteria, virus and nematodes. Fusarium oxysporum sp. sesami (Zap.), is one of the fiercest pathogens causing severe economic losses on sesame. This work aimed to evaluate the impact of the cultivation of some preceding crops and seed inoculation with antagonistic predominant rhizospheric bacteria and actinomycetes on the incidence and development of Fusarium damping-off and wilt disease. Results showed that the lowest pre and/or post-emergence damping-off and wilt of sesame were recorded after onion and garlic, followed by wheat compared to clover in both the 2019 and 2020 seasons. In vitro, soil extracts from plots where onion and garlic have been cultivated slightly decreased the conidia germination and mycelium radial growth of F. oxysporum. The numbers of sesame rhizospheric F. oxysporum and fungi were lower after the cultivation of onion and garlic than those after wheat and clover. However, the numbers of actinomycetes and bacteria were higher in the onion, garlic, and clover rhizosphere than wheat. Among all isolated bacteria and actinomycetes associated with sesame roots cultivated after preceding plants, the Tricoderma viride and Bacillus subtilis (isolate No.3) profoundly reduce F. oxysporum mycelial growth in vitro. When sesame seeds were inoculated with Tricoderma viride, Bacillus subtilis, Streptomyces rochei and Pseudomonas fluorescens, the disease incidence of damping-off and wilt significantly decreased in the greenhouse and field trials conducted in both tested growing seasons, also had highly significant on plant health and growth parameters. Therefore, the current study suggested that using the preceding onion and garlic plants could be used for eco-friendly reduction of damping-off and wilt disease of sesame.     

The influence of germinating seeds and developing roots on re-establishment of micro-organisms in fumigated soil

A laboratory study was made of re-invasion of soils fumigated in closed containers at rates of 3 ml./sq.ft. 10 in. deep with chloropicrin, methyl bromide and a 67:33 mixture of the two materials (MBC–33), using the dilution plate method of determination. Counts after aeration, 3 weeks later, showed that all three fumigants eliminated practically all fungi. Chloropicrin and MBC–33 also killed almost all bacteria and actinomycetes, whereas fumigation with methyl bromide markedly increased numbers of bacteria without materially altering numbers of actinomycetes. Subsequently, in soils fumigated with chloropicrin or MBC–33, fungi recolonized rapidly, but up to 93 % of the colonies isolated were Trichoderma viride. Increase of fungi in soils fumigated with methyl bromide was slower and Penicillium spp. predominated. Bacteria at first declined in numbers, then increased rapidly in soils fumigated with all three materials. In soils fumigated with methyl bromide, actinomycetes, usually considered to include good antagonists, increased but did not exploit their initial advantage over fungi. In soils fumigated with chloropicrin and MBC–33 actinomycetes did not recolonize in appreciable numbers. The greatest variety of fungous genera occurred in isolates from rhizo-spheres of red beet, but the greatest numbers of colonies of both fungi and bacteria were isolated from rhizospheres of pea and pumpkin. None of the crops tested greatly stimulated recolonization by actinomycetes. Compared with the drastic changes brought about by fumigants the influence of seed and root exudates on either the magnitude or the composition of re-invading micro-organisms was slight.     

青海湖水及湖滨盐化土壤放线菌的分类及耐盐性

采用3种含盐培养基(70g/L NaCl)分离青海湖水、湖泥、湖滨土壤及盐化荒地土壤中的中低温放线菌,纯化后按常规方法鉴定,并测定其耐盐性。结果表明:①湖泥、湖滨土壤及盐化荒地土壤中存在一定数量的中、低温耐盐放线菌。湖滨荒地土壤中的放线菌数量最高,其次为湖滨土壤及浅层湖水底淤泥。②鉴定出的耐盐性放线菌均为链霉菌(占供试菌总数的92.6%),且全部为白孢类群。③47.6%、19.0%和33.3%的供试放线菌的最高耐盐度分别为70、100和150g/L。     

Microbial communities of frozen soils of the Tuimaada Valley in Central Sakha

This study determined the characteristic features of microbial communities inhabiting frozen soils of Central Sakha. All groups of microorganisms were present in high numbers comparable with the microbial densities in steppe soils of Transbaikalia; their distribution along soil profiles followed a specific pattern with no decrease in abundance with depth. In summer, the dominant groups of the microbial pool were actinomycetes and oligonitrophilic bacteria, while in autumn it was heterotrophic bacteria. A typical feature of the frozen soils of Central Sakha was the gradual penetration of microorganisms into deeper soil horizons during the vegetation season and their accumulation in the suprapermafrost horizon by autumn.     

Mg~(2+)对线粒体H~+-ATP酶的F_O在脂质体重建时的影响

线粒体ATP合成酶是由具有H~+转运活性的F_0亚基,可溶性的催化中心F_1和连接二者的致寡霉素敏感蛋白(OSCP)所组成. 将纯化的猪心线粒体H—ATP酶复合体的F_0亚基,用胆酸盐透析法在有Mg~(2+)和无Mg~(2+)条件下在大豆磷脂脂质体上重建得脂酶体(L·F_0).用探剂9-AA荧光淬灭法和电权法测定了两种脂酶体的质子转运活力.由两种方法所得的实验结果均表明,在透返介质中加入1mmolmg~(2+)条件下形成的脂酶体(L·F_0)+Mg~(2+)较无Mg~(2+)者的质子转运活性明显增加.前者的荧光强度变化较后者增加约30%;由电极法测得的质子转运的初速度,前者为5nmolH~+′sF_0,后者为3nmolH~+′s·nmolF_O,质子转运活性高约一倍.这进一步支持Mg~(2+)通过调节脂的物理状态而诱导F_O具有较适合的构象,并进而将这一影响传递至F_1,使整个H~+—AhP酶具有较高活性的假设.     

Complementary perturbation of the kinetic mechanism and catalytic effectiveness of dihydrofolate reductase by side-chain interchange.

The variable residue Leu-28 of Escherichia coli dihydrofolate reductase (DHFR) and the corresponding residue Phe-31 in murine DHFR were interchanged, and the impact on catalysis was evaluated by steady-state and pre-steady-state analysis. The E. coli L28F mutant increased the pH-independent kcat from 11 to 50 s-1 but had little effect on Km(H2F). An increase in the rate constant for dissociation of H4F from E.H4F.NH (from 12 to 80 s-1) was found to be largely responsible for the increase in kcat. Unexpectedly, the rate constant for hydride transfer increased from 950 to 4000 s-1 with little perturbation of NADPH and NADP+ binding to E. Consequently, the flux efficiency of the E. coli L28F mutant rose from 15% to 48% and suggests a role in genetic selection for this variable side chain. The murine F31L mutant decreased the pH-independent kcat from 28 to 4.8 s-1 but had little effect on Km(H2F). A decrease in the rate constant for dissociation of H4F from E.H4F.NH (from 40 to 22 s-1) and E.H4F (from 15 to 0.4 s-1) was found to be mainly responsible for the decrease in kcat. The rate constant for hydride transfer decreased from 9000 to 5000 s-1 with minor perturbation of NADPH binding. Thus, the free energy differences along the kinetic pathway were generally similar in magnitude but opposite in direction to those incurred by the E. coli L28F mutant. This conclusion implies that DHFR hydrophobic active-site side chains impart their characteristics individually and not collectively.     

Monoclonal antibody-beta-lactamase conjugates for the activation of a cephalosporin mustard prodrug.

Cephalosporin mustard (CM) was designed as an anticancer prodrug that could be activated in a site-specific manner by monoclonal antibody-beta-lactamase conjugates targeted to antigens present on tumor cell surfaces. Purified beta-lactamases from Bacillus cereus (BC beta L) and Escherichia coli (EC beta L) catalyzed the release of phenylenediamine mustard (PDM) from CM through a fragmentation reaction which occurs after the beta-lactam ring of CM is hydrolyzed. The Km and Vmax values were 5.7 microM and 201 mumol/min per mg for BC beta L and 43 microM and 29 mumol/min per mg for EC beta L, respectively. Conjugates of BC beta L were prepared by combining the F(ab')2 fragments of the maleimide-substituted monoclonal antibodies L6 and 1F5 with thiolated BC beta L. The conjugates showed little loss in enzymatic activity and bound nearly as well as the unmodified F(ab')2 monoclonal antibodies to antigens expressed on the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). PDM was approximately 50-fold more cytotoxic than CM to H2981 cells. Treatment of the cells with L6-BC beta L followed by CM resulted in a level of cytotoxic activity that was comparable to that of PDM. This was most likely due to activation of CM by conjugate that bound to cell-surface antigens, since pretreatment of H2981 cells with BC beta L or 1F5-BC beta L enhanced the activity of CM to a lesser extent. Thus, we have shown that CM is a prodrug, and that it can be activated with immunological specificity by a monoclonal antibody-beta-lactamase conjugate.     

Rescue of HIV-1 broad neutralizing antibody-expressing B cells in 2F5 VH x VL knockin mice reveals multiple tolerance controls

The HIV-1 broadly neutralizing Ab (bnAb) 2F5 has been shown to be poly-/self-reactive in vitro, and we previously demonstrated that targeted expression of its VDJ rearrangement alone was sufficient to trigger a profound B cell developmental blockade in 2F5 V(H) knockin (KI) mice, consistent with central deletion of 2F5 H chain-expressing B cells. In this study, we generate a strain expressing the entire 2F5 bnAb specificity, 2F5 V(H) × V(L) KI mice, and find an even higher degree of tolerance control than observed in the 2F5 V(H) KI strain. Although B cell development was severely impaired in 2F5 V(H) × V(L) KI animals, we demonstrate rescue of their B cells when cultured in IL-7/BAFF. Intriguingly, even under these conditions, most rescued B cell hybridomas produced mAbs that lacked HIV-1 Envelope (Env) reactivity due to editing of the 2F5 L chain, and the majority of rescued B cells retained an anergic phenotype. Thus, when clonal deletion is circumvented, κ editing and anergy are additional safeguards preventing 2F5 V(H)/V(L) expression by immature/transitional B cells. Importantly, 7% of rescued B cells retained 2F5 V(H)/V(L) expression and secreted Env-specific mAbs with HIV-1-neutralizing activity. This partial rescue was further corroborated in vivo, as reflected by the anergic phenotype of most rescued B cells in 2F5 V(H) × V(L) KI × Eμ-Bcl-2 transgenic mice and significant (yet modest) enrichment of Env-specific B cells and serum Igs. The rescued 2F5 mAb-producing B cell clones in this study are the first examples, to our knowledge, of in vivo-derived bone marrow precursors specifying HIV-1 bnAbs and provide a starting point for design of strategies aimed at rescuing such B cells.     

Differential transfers of reduced flavin cofactor and product by bacterial flavin reductase to luciferase

It is believed that the reduced FMN substrate required by luciferase from luminous bacteria is provided in vivo by NAD(P)H-FMN oxidoreductases (flavin reductases). Our earlier kinetic study indicates a direct flavin cofactor transfer from Vibrio harveyi NADPH-preferring flavin reductase P (FRP(H)) to the luciferase (L(H)) from the same bacterium in the in vitro coupled luminescence reaction. Kinetic studies were carried out in this work to characterize coupled luminescence reactions using FRP(H) and the Vibrio fischeri NAD(P)H-utilizing flavin reductase G (FRG(F)) in combination with L(H) or luciferase from V. fischeri (L(F)). Comparisons of K(m) values of reductases for flavin and pyridine nucleotide substrates in single-enzyme and luciferase-coupled assays indicate a direct transfer of reduced flavin, in contrast to free diffusion, from reductase to luciferase by all enzyme couples tested. Kinetic mechanisms were determined for the FRG(F)-L(F) and FRP(H)-L(F) coupled reactions. For these two and the FRG(F)-L(H) coupled reactions, patterns of FMN inhibition and effects of replacement of the FMN cofactor of FRP(H) and FRG(F) by 2-thioFMN were also characterized. Similar to the FRP(H)-L(H) couple, direct cofactor transfer was detected for FRG(F)-L(F) and FRP(H)-L(F). In contrast, despite the structural similarities between FRG(F) and FRP(H) and between L(F) and L(H), direct flavin product transfer was observed for the FRG(F)-L(H) couple. The mechanism of reduced flavin transfer appears to be delicately controlled by both flavin reductase and luciferase in the couple rather than unilaterally by either enzyme species.     

The influence of short-term nutrient changes on follicle growth and embryo production following superovulation in beef heifers

Acute decreases in nutrient intake can improve embryo quality in sheep, although reductions in ovulation rate can also occur. In cattle, short-term nutrient restriction prior to ovulation has been shown to increase subsequent pregnancy rates. Thus, the objective was to determine the effect of a severe reduction in food intake on follicle growth and embryo quantity and quality in heifers superovulated with FSH. Beef heifers (n=61) were offered a diet of grass silage and concentrates (ratio of 5:1, on a fresh weight basis), which was adjusted to provide a predicted intake of 28.6 Mcal/kg ME/d (H) or 9.6 Mcal/kg MEld (L). Heifers were synchronized with a progesterone-releasing device for 7 d. They were allocated to oocyte recovery (n=16/treatment) after 3 (225 IU) or 8 (600 IU) injections of FSH given at 12-h intervals. Oocytes were matured, fertilized and cultured individually in vitro. The remaining heifers (n=14/treatment) were superovulated using FSH (600 IU), and embryos were recovered 7 d after breeding. The embryos were morphologically graded and subsequently cultured for 24 h before differential staining to determine inner cell mass and trophectoderm cell numbers. Follicle numbers increased following 8 (16.6±2.0) compared with 3 (6.7±0.6) injections of FSH (P<0.0001). Heifers on the L diet had more follicles than those on the H diet (13.5±2.4 vs 9.6±1.2; P<0.06), which was predominantly due to an increase in the number of 7- to 10-mm follicles. However, this effect was only evident after 8 injections of FSH. There was no nutritional effect on cleavage rates in vitro (55.6±8.1 vs 53.8±9.0 for H vs L diets, respectively). However, cleavage rates were lower in oocytes collected after 8 than after 3 injections of FSH (31.3 vs 69.2%; P<0.0001). There was no significant effect of nutrition on ovulation rate after FSH (14.4±1.9 vs 16.3±3.0 for H vs L diet, respectively). The number of embryos recovered was not different between heifers on H (10.4±1.3) and L (11.3±2.4) diets. Following culture for 24 h, a significantly higher proportion of embryos from heifers on the L diet developed to the blastocyst stage (72.9 vs 41.5%; P<0.01). Total cell numbers on Day 8 were greater in embryos from heifers on the L diet (98.3 vs 75.4; P< 0.0001); yet the inner cell mass as a percentage of total cells was not different (21 vs 20%). These data indicate that low energy intake prior to and during superovulation resulted in more follicles and in improved embryo quality, as evident from the increased number of blastocysts formed and higher cell numbers.     

Molecular engineering of myoglobin: the improvement of oxidation activity by replacing Phe-43 with tryptophan

The F43W and F43W/H64L myoglobin (Mb) mutants have been constructed to investigate effects of an electron rich oxidizable amino acid residue in the heme vicinity on oxidation activities of Mb. The Phe-43 --> Trp mutation increases the rate of one-electron oxidation of guaiacol by 3-4-fold; however, the peroxidase activity for F43W/H64L Mb is less than that of the F43W single mutant because the absence of histidine, a general acid-base catalyst, in the distal heme pocket suppresses compound I formation. More than 15-fold improvement versus wild-type Mb in the two-electron oxidation of thioanisole and styrene is observed with the Phe-43 --> Trp mutation. Our results indicate that Trp-43 in the mutants enhances both one- and two-electron oxidation activities (i.e., F43W Mb > wild-type Mb and F43W/H64L Mb > H64L Mb). The level of (18)O incorporation from H2(18)O2 into the epoxide product for the wild type is 31%; however, the values for F43W and F43W/H64L Mb are 75 and 73%, respectively. Thus, Trp-43 in the mutants does not appear to be utilized as a major protein radical site to form a peroxy protein radical in the oxygenation. The enhanced peroxygenase activity might be explained by the increase in the reactivity of compound I. However, the oxidative modification of F43W/H64L Mb in compound I formation with mCPBA prevents us from determining the actual reactivity of the catalytic species for the intact protein. The Lys-C achromobacter digestion of the modified F43W/H64L mutant followed by FPLC and mass analysis shows that the Trp-43-Lys-47 fragment gains a mass by 30 Da, which could correspond two oxygen atoms and loss of two protons.     

青海沙珠玉人工植被系统土壤放线菌生态分布及拮抗性

为探索人工植被重建对土壤放线菌的影响,采用稀释平板法及琼脂块法对青海沙珠玉地区植被重建后沙土中放线菌的生态分布及拮抗性进行了研究.结果表明：随着高寒干旱沙地植被重建和覆盖度的提高：土壤放线菌数量显著增加,其中退耕还草地土壤中放线菌总数较沙地提高145.4%,小单孢菌数量约为沙地的6倍;拮抗性放线菌平均筛出率大幅度增加,退耕还草地土壤中拮抗性放线菌、天然草地土壤中抗细菌放线菌及林地土壤中抗病原真菌放线菌的平均筛出率约分别为沙地的2、3.2及1.5倍;地表植被盖度与土壤养分对放线菌数量以及拮抗性放线菌数量有很大影响,其中有机质、碱解氮含量以及鲜草产量与放线菌总数的相关性达到极显著水平(P<0.01),有机质含量、鲜草产量与拮抗性放线菌株数的相关性达到极显著水平(P<0.01).此外,植被盖度、土壤全氮、全磷、全钾、全盐以及速效钾含量与放线菌总数、链霉菌数、小单孢菌数的相关性也达到显著水平(P<0.05).     

华西雨屏区天然常绿阔叶林土壤可培养微生物数量对模拟氮沉降的响应

为了解氮沉降对华西雨屏区天然常绿阔叶林土壤微生物数量的影响,从2013年11月至2014年12月,通过野外模拟N(NH_4NO_3)沉降,氮沉降水平分别为对照(CK 0 kg N hm~(-2)a~(-1))、低氮沉降(L 50 kg N hm~(-2)a~(-1))、中氮沉降(M 150 kg N hm~(-2)a~(-1))和高氮沉降(H 300 kg N hm~(-2)a~(-1)),研究了氮沉降对天然常绿阔叶林0—10cm和10—20cm土层土壤可培养微生物数量的影响。结果表明:华西雨屏区天然常绿阔叶林0—10cm土层的细菌、真菌和放线菌数量均显著大于10—20cm土层,氮沉降未改变原有垂直分布格局。L处理对0—10cm和10—20 cm土层土壤微生物总量无显著影响,M和H处理则显著降低了土壤微生物总量。氮沉降降低了0—10cm和10—20cm土层的细菌数量,且抑制作用随氮沉降量的增加而增强。氮沉降降低了0—10cm土层的真菌数量,但下降幅度与氮沉降量之间无明显规律;在10—20cm土层,M和H处理在夏季显著增加了真菌数量,表明适量氮沉降能有效缓解夏季土壤真菌的氮限制状态。氮沉降对0—10cm土层放线菌数量的影响表现为先促进再抑制,L和M处理增加了放线菌数量,H处理降低了放线菌数量;氮沉降增加了10—20cm土层的放线菌数量,其中M处理的促进作用最大。氮沉降对土壤微生物数量的影响随土壤深度的增加而减弱。