Further studies of the sieve-element plastids of theCaryophyllales includingBarbeuia,Corrigiola, Lyallia,Microtea, Sarcobatus,andTelephium

The sieve-element plastids of 69 species of theCaryophyllales were investigated by transmission electron microscopy. All contained the specific subtype-P3 plastids characterized by a peripheral ring of protein filaments. The presence or absence of an additional central protein crystal and their shape being either polygonal or globular as well as the average sizes of the sieve-element plastids are useful features in the characterization of some families.—Barbeuia contains sieve-element plastids that confirm its placement within thePhytolaccaceae. Lyallia differs fromHectorella by including small starch grains in their sieve-element plastids, which otherwise by their globular crystals negate a closer connection to theCaryophyllaceae. The lack of a central protein crystal in its form-P3fs plastids placesMicrotea best within theChenopodiaceae. Sarcobatus, a so far uncontested member of theChenopodiaceae, contains form-P3cf plastids, i.e., including a central crystal not found elsewhere in this family.Telephium andCorrigiola, shifted back and forth betweenMolluginaceae andCaryophyllaceae, have form-P3cf(s) plastids with a polygonal crystal which favor their placement within theCaryophyllaceae.     

Responses of production and storage walnut pests to Bacillus thuringiensis insecticidal crystal protein fragments

Recent advances in genetic engineering have provided the opportunity to induce walnut plants to produce Bacillus thuringiensis Berliner insecticidal crystal protein fragments (ICPFs) for insect control. We studied the effects of two ICPFs CryIA(b) and CrylA(c) previously shown to be encoded by the cryIA(b) and cryIA(c) genes in the B. thuringiensis strains HD-1 and HD-73, respectively. The lethal effects on larvae of codling moth, Cydia pomonella (L.), navel orangeworm, Amyelois transitella (Walker), and the major postharvest pest Indianmeal moth, Plodia interpunctella (Hübner), were investigated. Both proteins were toxic to the three species tested. Indianmeal moth larvae were the most susceptible and navel orangeworm the least; CryIA(b) was generally more toxic to navel orangeworm. Similar relationships resulted when ICPFs were incorporated into the diet. Both ICPFs caused decreased rate of development of navel orangeworm. Effects on pupal weight occurred only at the highest concentration (100 ng/cm²). Neither ICPF affected frequency of mating or fecundity. In addition to the lethal effects, the extended development times observed could have considerable effects on the population dynamics of the navel orangeworm and possibly other species.     

Genes from Bacillus thuringiensis entomocidus 60.5 coding for insect-specific crystal proteins

Summary Five crystal protein genes have been isolated from DNA of Bacillus thuringiensis entomocidus 60.5, an isolate selected for its high toxicity against Spodoptera littoralis and Spodoptera exigua. Two of these genes belong to a family of well-described crystal protein genes. The toxic properties of the corresponding proteins are similar to those of isolate kurstaki HD1. The other three genes belong to gene families not described before. One of these genes codes for a protein product exhibiting a high degree of specificity towards Spodoptera species, explaining the high toxicity of isolate entomocidus 60.5 against these species. This gene product is much less toxic against larvae of Heliothis virescens and Pieris brassicae. Its coding sequence is separated from a supposed fourth crystal protein gene by a stretch of DNA of 3 kb. The crystal protein encoded by the fifth gene is mainly active against P. brassicae. Homology between the crystal protein genes is limited to the central region of the coding sequences, including the proteolytic cleavage site, except for the first two genes between which homology is extensive.     

Synergistic interaction between sublethal doses of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Campoletis chlorideae</Emphasis> in managing <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) is an important solitary larval endoparasitoid of the tomato fruit borer Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in India. The interaction between Bacillus thuringiensis subspecies kurstaki (Btk) HD-1 and C. chlorideae was studied under laboratory condition to explore their compatibility in managing H. armigera. The results had indicated that the growth and development of H. armigera was affected in a dose-dependent manner upon feeding on sublethal doses of Btk HD-1-treated diets. There were no larval survivors in lethal doses of Btk HD-1 (LC₇₀ and LC₉₀). The growth and survival of the parasitoid were normal when the host larvae were fed with sublethal doses or subjected to short time exposure to lethal doses of Btk HD-1. However, the parasitoid offsprings developed slowly and pupal as well as adult period, adult weight and adult emergence rate were reduced significantly if the parasitoid was developing inside a severely Bt intoxicated host larvae. There were no evident differences in longevity of parasitoid adults that were fed on honey solution containing different concentrations of Btk HD-1 as compared to adults fed only on honey solution. This indicates no direct adverse effect of Btk HD-1 on C. chlorideae. Further, the gravid female parasitoid did not discriminate Btk HD-1 intoxicated and normal H. armigera larvae for oviposition. The result implies that spore crystal formulation of Btk HD-1 can be effectively used in a synergistic manner along with existing natural or prereleased population of C. chlorideae in organic farming or as components in biointensive IPM module for managing H. armigera.     

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b ₆ f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.     

Bt新菌株资源的采集与鉴定

【目的】寻找对致倦库蚊高效的苏云金芽胞杆菌(Bacillus thuringiensis,Bt)杀蚊菌株新资源。【方法】从福建省的武夷山自然保护区、建阳、建瓯、浦城等多个地区采集土壤样品,采用热处理法从土壤中分离Bt菌株,并测定其对致倦库蚊活性的效果。【结果】从125份土壤样品中分离出71株Bt菌株,经生物测定得到4株对致倦库蚊有效菌株(QQ13、QQ42、QQ66和QQ92)。其中,QQ66和QQ92有较高的毒性,均有几丁质酶基因,没有检测到cry1、cry1Ⅰ、cry2、cry4、cry5、cry6、cry7、cry8、cry9、cry10和cry11基因,在75～100 ku处各有一条杀虫晶体蛋白条带。【结论】采集和鉴定到的Bt新菌株资源将对致倦库蚊的生物防治起到促进作用。     

Isolation and characterization of EG2158, a new strain of Bacillus thuringiensis toxic to coleopteran larvae, and nucleotide sequence of the toxin gene

Summary A novel strain of Bacillus thuringiensis was isolated from soybean grain dust from Kansas and found to be toxic to larvae of Leptinotarsa decemlineata (Colorado potato bectle). The strain (EG2158) synthesized two parasporal crystals: a rhomboid crystal composed of a 73115 dalton protein and a flat, diamond-shaped crystal composed of a protein of approximately 30 kDa. Plasmid transfer and gene cloning experiments demonstrated that the 73 kDa protein was encoded on an 88 MDa plasmid and that the protein was toxic to the larvae of Colorado potato beetle (CPB). The sequence of the 73 kDa protein, as deduced from the sequence of its gene (cryC), was found to have regions of similarity with several B. thuringiensis crystal proteins: the lepidopteran-toxic P1 proteins of var. kurstaki and berliner, the lepidopteran- and dipteran-toxic P2 (or CRYB1) protein of var. kurstaki, and the dipteran-toxic 130 kDa protein of var. israelensis. While B. megaterium cells harboring the cryC gene from EG2158 synthesized significant amounts of the 73 kDa CRYC protein, Escherichia coli cells did not. The cryC-containing B. megaterium cells produced rhomboid crystals that were toxic to CPB larvae.     

Comparison of the activities of Bacillus thuringiensis mosquitocidal crystal proteins on mosquito cell lines (Diptera,Culicidae), using two colorimetric methods

Two colorimetric methods were compared for assaying the cell toxicity of soluble crystal proteins from five mosquitocidal serovarieties of Bacillus thuringiensis: israelensis, jegathesan, medellin, darmstadiensis and fukuokaensis. In mosquitoes cell lines from Culex quinquefasciatus, Aedes aegypti and Anopheles gambiae, all the crystal proteins were equally toxic. Both colorimetric methods MTT (tetrazolium salt) and Alamar Blue gave similar results, but the Alamar Blue method was simpler, faster, cheaper, and could be used to take readings from the same cell population at various time points. The most active crystal proteins were those of B. thuringiensis israelensis, followed by those of B. thuringiensis jegathesan, and B. thuringiensis medellin, which were much less active.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

Molecular and Biochemical Characterization of an Endochitinase (ChiA-HD73) from Bacillus thuringiensis subsp. kurstaki HD-73

An endochitinase gene (chiA-HD73) from the insecticidal bacterium Bacillus thuringiensis subsp. kurstaki HD-73 was cloned, sequenced, and expressed in Escherichia coli DH5αF′. The chitinase activity of the encoded protein was studied in assays with different fluorogenic substrates. The chiA-HD73 gene contained an open-reading frame that encoded an endochitinase with a deduced molecular weight and an isoelectric point of, respectively, 74.5 kDa and 5.75. A putative signal peptide with cleavage sites for both Gram-positive and Gram-negative bacteria was identified. Comparison of ChiA-HD73 with other chitinases revealed a modular structure composed of a catalytic domain and a putative chitin-binding domain. ChiA-HD73 hydrolyzed both tetrameric and trimeric fluorogenic substrates, but not a chitobiose analog substrate, suggesting that the activity of ChiA-HD73 is mainly endochitinolytic. In addition, ChiA-HD73 showed high enzymatic activity within a broad pH range (pH 4–10), with a peak activity at pH 6.5. The optimal temperature for enzymatic activity was observed at 55°C. Its activity in a broad range of temperatures and pH suggests ChiA-HD73 could have biotechnological applications in insect control, particularly in synergizing the insecticidal crystal protein toxins of B. thuringiensis.     

Absence of CeCl3-detectable peroxisomal glycolate-oxidase activity in developing raphide crystal idioblasts in leaves of Psychotria punctata Vatke and roots of Yucca torreyi L.

Three peroxisomal enzymes, glycolate oxidase, urate oxidase and catalase were localized cytochemically in Psychotria punctata (Rubiaceae) leaves and Yucca torreyi (Agavaceae) seedling root tips, both of which contain developing and mature calcium-oxalate raphide crystal idioblasts. Glycolate-oxidase (EC 1.1.3.1) and catalase (EC 1.11.1.6) activities were present within leaftype peroxisomes in nonidioblastic mesophyll cells in Psychotria leaves, while urate-oxidase (EC 1.7.3.3) activity could not be conclusively demonstrated in these organelles. Unspecialized peroxisomes in cortical parenchyma of Yucca roots exhibited activities of all three enzymes. Reactionproduct deposits attributable to glycolate-oxidase activity were never observed in peroxisomes of any developing or mature crystal idioblasts of Psychotria or Yucca. Catalase localization indicates that idioblast microbodies are functional peroxisomes. The apparent absence of glycolate oxidase in crystal idioblasts of Psychotria and Yucca casts serious doubt that pathways involving this enzyme are operational in the synthesis of the oxalic acid precipitated as calcium-oxalate crystals in these cells.Abbreviations AMPD 2-amino-2-methyl-1,3-propandiol - CTEM conventional transmission electron microscopy - DAB 3,3-diaminobenzidine tetrahydrochloride - HVEM high-voltage electron microscopy     

Crystal structure and biophysical characterisation of Helicobacter pylori phosphopantetheine adenylyltransferase

Helicobacter pylori is a bacterium that causes chronic active gastritis and peptic ulcers. Drugs targeting H. pylori phosphopantetheine adenylyltransferase (HpPPAT), which is involved in CoA biosynthesis, may be useful. Herein, we report the expression in Escherichia coli and purification of recombinant HpPPAT and describe a crystal structure for an HpPPAT/CoA complex. As is the case for E. coli PPAT (EcPPAT), HpPPAT is hexameric in solution and as a crystal. Each protomer has a well-packed dinucleotide-binding fold in which CoA binds. Structural characterisation demonstrated that CoA derived from the E. coli expression system bound tightly to HpPPAT, presumably to initiate feedback inhibition. However, the interactions between the active-site residues of HpPPAT and CoA are not identical to those of other PPATs. Finally, CoA binding affects HpPPAT thermal denaturation.     

Stranding events provide indirect insights into the seasonality and persistence of jellyfish medusae (Cnidaria: Scyphozoa)

It is becoming increasingly evident that jellyfish (Cnidaria: Scyphozoa) play an important role within marine ecosystems, yet our knowledge of their seasonality and reproductive strategies is far from complete. Here, we explore a number of life history hypotheses for three common, yet poorly understood scyphozoan jellyfish (Rhizostoma octopus; Chrysaora hysoscella; Cyanea capillata) found throughout the Irish and Celtic Seas. Specifically, we tested whether (1) the bell diameter/wet weight of stranded medusae increased over time in a manner that suggested a single synchronised reproductive cohort; or (2) whether the range of sizes/weights remained broad throughout the stranding period suggesting the protracted release of ephyrae over many months. Stranding data were collected at five sites between 2003 and 2006 (n = 431 surveys; n = 2401 jellyfish). The relationship between bell diameter and wet weight was determined for each species (using fresh specimens collected at sea) so that estimates of wet weight could also be made for stranded individuals. For each species, the broad size and weight ranges of stranded jellyfish implied that the release of ephyrae may be protracted (albeit to different extents) in each species, with individuals of all sizes present in the water column during the summer months. For R. octopus, there was a general increase in both mean bell diameter and wet weight from January through to June which was driven by an increase in the variance and overall range of both variables during the summer. Lastly, we provide further evidence that rhizostome jellyfish may over-wintering as pelagic medusa which we hypothesise may enable them to capitalise on prey available earlier in the year. Handling editor: K. Martens     

三七自毒与化感作用初步研究

采用生物测定方法,对三七的自毒与化感作用进行初步研究。结果表明:(1)三七种子萌发过程中的自毒作用随其播种密度不同而有一定差异,但无明显规律性。三七种子萌发过程中的分泌物对油菜生长具有化感作用,主要表现为对油菜种子发芽率、发芽指数及苗高的抑制效应;(2)三七水浸液对不同受体植物的化感作用不尽相同,对小麦主要表现为对苗鲜重、苗干重、根鲜重、苗高及须根数有不同程度的促进或抑制作用;对油菜则表现为对其种子发芽率具有抑制作用,而对苗鲜重、苗干重、根鲜重、最长根长具有促进作用;(3)三七存在明显的化感自毒作用,其自毒物质可能为影响其连作的一个重要因素。     

苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26Aa和cry28Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26Aa和cry28Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26Aa在仅消除cry26Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26Aa所在质粒有关。     

Effects of the infection of toxigenic fungi and an antagonistic Streptomyces strain on wheat spikes

The objective of the present study was to determine the effect on infection of wheat spikes by toxigenic fungi (Aspergillus parasiticus NRRL 2999, Fusarium tricinctum NRRL 3299, Fusarium graminearum CEREMIC 136/92) and a strain of Streptomyces sp. that is antagonistic to the above-mentioned fungi. Wheat grains (variety GRANERO INTA) were sown in 8 pots containing natural soil and kept in a greenhouse chamber. In the period of the early anthesis the wheat spikes were inoculated with conidial suspensions of each of the fungi in the presence or absence of Streptomyces. Each pot was assigned a different treatment. After an incubation of 100 days and when the wheat plants had attained maturity, the spikes were separated and the following items were determined: (a) number of grains obtained with each treatment, (b) weight of the grains, (c) average weight of the grains/treatment, (d) average number and weight of the grains/spike, and (e) invasion of the caryopses by the microorganisms determined by the analysis of the caryopses in seriate histological sections.There was a significant decrease (p<0.01) in the average weight of the caryopses and in the weight and number of grains/spike in the presence F. graminearum. The wheat grains were invaded by of F. graminearum and A. parasiticus, an effect which was partially attenuated by the presence of antagonist Streptomyces sp. Nevertheless, the effect was not strong enough to prevent the degenerative consequences on the size and weight of the grains produced by F. graminearum.     

The genetic basis of host range inHeliothis virescens: larval survival and growth

Larval feeding was assayed in a generalist caterpillar (Heliothis virescens (F.)), a specialist caterpillar (Heliothis subflexa (Gn.)), their F₁ hybrids and a backcross withH. s. to gain a preliminary understanding of the genetic basis of host use inH. v. Plants used in these experiments were tobacco (Nicotiana tabaccum), cotton (Gossypium hirsutum), soybean (Glycine max) (hosts ofH. v.) and ground cherry (Physalis pubescens) (host ofH.s.). A feeding study ofH.v., H.s. and their reciprocal hybrids showed that, after the first eight days of feeding,H.v. had its highest survival and weight gain on soybean and had lowest survival and weight gain on its non-host,Physalis. H.s. had its highest survival and weight gain on its host plant,Physalis, and performed very poorly on the three non-host plants. The two F₁ hybrids were SV₀ (offspring ofH.s. female andH.v. male) and VS₀ (offspring ofH.v. female andH.s. male). The hybrids did not differ from each other, indicating no sex linkage or maternal effects, except that VS₀ had greater weight gain on tobacco than did SV₀. The hybrids, unlike their parents, survived well on all four host plants and their weight gain was intermediate on all four host plants. In a separate experiment the VS₀ hybrid was mated to the specialist to produce the VS₁ backcross. In contrast to the F₁, the backcross had significantly lower survival than the generalist on soybean, cotton, tobacco and weight gain was lower on soybean, cotton and tobacco but higher onPhysalis. Survival and weight gain on cotton and tobacco were inherited as partially dominant traits; onPhysalis there was overdominance for survival and complete dominance for weight gain; on soybean both survival and weight gain were additive. Survival on cotton,Physalis, soybean and tobacco and weight gain onPhysalis could not be completely explained by a model that included only dominance and additive effects. These traits may be influenced by epistatic and/or environmental effects.     

Expression of parasporal crystal protein (δ—endotoxin) gene(s) ofBacillus thuringiensis var.israelensis in sporogenic and asporogenic mutant strains ofBacillus cereus

The expression of parasporal crystal protein (δ-endotoxin) coding gene(s) ofBacillus thuringlensis var.israelensis and its association, if any, with sporulation was studied in sporogenicBacillus cereus and its asporogenic mutant strains. Five asporogenous mutants ofBacillus cereus blocked at different stages of sporulation, were isolated from a streptomycin-resistant strain, The transconjugants isolated from the plasmid transfer experiments betweenBacillus thuringiensis var.israelensis and streptomycin resistantBacillus cereus and its asporogenous mutants, showed larvicidal activity. The crystal protein gene(s) are, therefore, expressed both in sporulating and in non-sporulating mutant strains ofBacillus cereus suggesting that the expression of crystal protein gene(s), is independent of sporulation specific functions inBacillus cereus. Part of the work was carried out at Biotechnology Programme, Jadavpur University, Calcutta 700 032, India.     

野生兜兰菌根真菌对带叶兜兰生长和生理指标的效应

为探究兰科菌根真菌对带叶兜兰(Paphiopedilum hirsutissimum)生长的影响,用4个分离自广西野生兜兰根部的兰科菌根真菌(Cladosporium perangustum、Kirschsteiniothelia tectonae、Phialophora sp.和Cyphellophora sp.)与带叶兜兰试管苗和营养钵苗进行菌-苗共生试验,对其生长和生理指标的变化进行了研究。结果表明,接菌处理具有明显的促进生长作用和生理效应,Cladosporium perangustum和Phialophora sp.对试管苗的接种效果最佳,鲜质量增量、3种保护酶活性和叶绿素总量均与对照存在显著或极显著差异,鲜质量增加了360%～380%。Kirschsteiniothelia tectonae、Phialophora sp.对营养钵苗的接种效果最佳,鲜质量增量、叶面积及3种保护酶活性均与对照存在显著或极显著差异,鲜质量增加了261%～330%。因此,实际生产中可在带叶兜兰不同生长阶段接种适当菌株,Phialophora sp.对带叶兜兰表现出较好的促生效应,可研发为带叶兜兰育...     

Improvement of a crystallization process for the purification of vancomycin

We evaluated the effects of the sample feeding and mixing methods on the efficiency of vancomycin crystallization and developed a method to dramatically shorten the time required for crystal formation by increasing the available surface area inside the reactor. The highest purity (97%) and yield (99%) of vancomycin could be obtained with an initial one-step feeding and mixing method, resulting in the formation and growth of spherical fan-shaped crystals. The yield of vancomycin increased when the surface area per working volume (S/V) was increased to 0.428 mm⁻¹ using glass beads (0.5 mm), the cation exchange resin Amberlite 200, or the anion exchange resin Amberlite IRA-400. In particular, most of the vancomycin (>99%) could be recovered after 6 h of crystallization using Amberlite 200, resulting in a dramatic reduction in the time required for crystallization. On the other hand, increasing S/V had almost no effect on vancomycin purity and crystal size.