我国部分地区土壤中的苏芸金芽孢杆菌和球形芽孢杆菌

从云南、贵州、四川和陕西4省的土壤中分离到大量苏芸金芽孢杆菌(Bacillus thuringie—nsis)和球形芽孢杆菌(Bacillus sphaeticus)菌株。血清型分析表明,苏芸金芽孢杆菌分离株分属于23个血清型中的13个血清型,另有近20％的自凝型菌株及部分与所有标准菌抗血清无反应的菌株。对该两种昆虫病原细菌的生态分布规律进行了分析。研究了全部苏芸金芽孢杆菌分离株对鳞翅目、鞘翅目及双翅目的6种昆虫的毒力特性、伴孢晶体与芽孢的形态,以及晶体蛋白质成分。观察和测定了球形芽孢杆菌分离株的形态和毒力,并分析了部分菌株的晶体蛋白质成分。得到22株高效苏芸金芽孢杆菌和2株高效球形芽孢杆菌。证明苏芸金芽孢杆菌是典型的土壤微生物类群,我国西南地区土壤中的苏芸金芽孢杆菌资源十分丰富。     

苏云金杆菌伴孢晶体的蛋白质和抗蛋白酶多肽及其毒力特性

以SDS-聚丙烯酰胺凝胶电泳分析了19种苏云金杆菌伴孢晶体的蛋白质和抗胰蛋白酶多肽。以鳞翅目的家蚕和双翅目的致倦库蚊进行了毒力测定。根据蛋白质和抗酶多肽的特性,19种晶体可分为7个类型。晶体蛋白质的组成、溶解特性及抗酶多肽的性质与其对两种昆虫的毒力特性密切相关。含分子量为130—138 kD(千道尔顿)或130—138 kD及60一65kD蛋白质,抗蛋白酶多肽(PRP)为68—75kD的晶体,对家蚕高毒,但绝大部分对库蚊无毒或微毒。含3种以上蛋白质(15一138kD),抗酶多肽为35—65kD的晶体,对库蚊有强烈毒性,对家蚕则无毒。缺少135kD蛋白质的两种晶体对家蚕低毒。HD一282和L—14晶体的P1蛋白质中同时含有杀鳞翅目和杀蚊毒素。讨论了伴孢晶体的蛋白质结构及结构与毒力之间的关系、晶体蛋白质及毒性肽的性质在菌株定向筛选和遗传工程研究中的意义。     

苏芸金杆菌的一个新血清型

从土壤中分离出一株产生伴孢晶体的芽孢杆菌。该菌株具有苏芸金杆菌的典型特征,严生不规则型晶体,鞭毛抗原及生化反应与已知的苏芸金杆菌21个血清型不㈤,不产生β-外毒素。对棉铃虫、粘虫、大蜡螟及尖音库蚊幼虫均无毒性,是一个新血清型(H22),定名为苏芸金杆菌山东变种(Bacillus thuringiensis serovar shandongiensis, H22)。     

昆虫肠道蛋白酶对苏芸金杆菌晶体作用的比较

本文以粘虫Leucania separata Walker,棉铃虫Heliothis armigera Hūbner大蜡螟Gatleria mellonella Linnaeus和黄地老虎Agrotis segeium Schiffermǖller 4种幼虫的肠道蛋白酶作用于苏芸金杆菌7404肯尼亚变种(B.t.var.kenyae)、HD-1戈尔斯德变种(B.t.var.kurstaki)7216天门变种(B.t.var.tianmensis)、75023(变种未定)、007苏芸金变种(B.t.var.thurtngiensis)、006玉米螟变种(B.t.var.ostriniae)、7417松蠋变种(B.t.var.dendrolimus)及1897以色列变种(B.t.var. israelensis)的晶体,比较了其消化产物的聚丙烯酰胺凝胶电泳图谱。对粘虫肠道蛋白酶消化的晶体形态进行了电镜观察,并对晶体消化过程中光密度的变化作了测定。非病原菌的晶体亦能被肠液中的蛋白酶消化。     

紫外吸收法测定苏芸金杆菌伴孢晶体蛋白质的研究

本文介绍了用紫外光吸收法定量测定苏芸金杆菌制剂中伴孢晶体蛋白质的方法。本方法从样品处理到测定完成只需4个小时,方法简便准确。用此法俭出伴孢晶体蛋白质所指示的毒力效价,与昆虫生测之间有较好的相关性。     

苏芸金杆菌伴孢晶体的形态及抗原特性

结合苏芸金杆菌不同品系毒力差异,对伴孢晶体形态、血清学特性进行了比较研究。对分属于7个血清型的19个林系的伴孢晶体观察表明：晶体形态以品系而异与分类属性无关;晶体可归为五个类型：菱形、嵌合形、球形、立方形、不规则形。发现具有嵌合形多的菌株对鳞趔目幼虫毒力不仅高,且在有毒力的菌株中嵌合形并不罕见;不规则形无毒力。对毒力差异大的菌株采用密度梯度离心提纯了其伴孢晶体,进行双向琼脂糖免疫扩敞试验,证明了晶体形态与其对昆虫的毒力有关。     

昆虫中肠液性质对苏云金芽孢杆菌伴孢晶体毒力的影响

综述了昆虫中肠液性质对苏云金芽孢杆菌Bacillus thuringiensis伴孢晶体毒力的影响。中肠液的酸碱度和蛋白酶是影响伴孢晶体溶解与原毒素活化的两大因素。中肠液的酸碱度不仅影响到伴孢晶体的溶解速度,还影响到各种蛋白酶的活性表现;而蛋白酶则直接参与了原毒素的活化,其组成与活性影响着原毒素的活化速度和杀虫专一性。因中肠液蛋白水解能力过高而导致原毒素的过度降解是某些昆虫对苏云金芽孢杆菌低度敏感的主要原因,而中肠液对原毒素活化能力的降低则与昆虫抗性的形成有关。此外,中肠液的沉淀作用及其它生理生化特性也影响着原毒素毒力的正常发挥。     

苏芸金杆菌以色列变种对尖音库蚊的毒杀作用

苏芸金杆菌以色列变种(Bacillus thuringtensis var.tsraelensis de Barjac)对尖音库蚊(Culex pipiensvar. pallens Coq uillett)三龄幼虫具有极高的毒性,在浓度为1.5×10⁴芽孢/毫升时,死亡率达100%。其他几个变种B. T. var. thuringiensis(E—009)var. kurstaki(HD—1)、var.kenyae(7404)、var. ostriniae(006)浓度达4×10⁵芽孢/毫升时仍未见毒效。 用液体双相与等密度离心法将以色列变种的晶体与芽孢分离,分别研究晶体(纯度达99.5%以上)、芽孢(纯度达99.5%以上)对尖音库蚊幼虫的毒性。发现使蚊幼虫致死主要是晶体LC₅₀为0.023微克/毫升,芽孢也具有毒性LC₅₀为0.86微克/毫升,晶体与芽孢混用制剂的LC₅₀为(0.014微克晶体+0.014微克芽孢)/毫升。 晶体制剂在30℃和21℃的温度条件下毒力没有变化,但芽孢制剂在21℃毒力显著下降;芽孢制剂致死幼虫,立即用相差显微镜镜检,发现肠道内有大量正在萌发的芽孢,对芽孢致死的原因进行了初步分析。     

苏芸金杆菌的毒力比较试验

苏芸金杆菌(Bacillus thuringiensis)不同品系对某一种昆虫,或苏芸金杆菌的某一品系对不同种昆虫表现出毒力的差别已有报道,例如血清型H_5a5b的蜡螟杆菌对蜡螟(Galleria mellonella)的毒力比H_3a3b的HD-1菌株高150倍,但对烟草夜蛾(Heliothis virescens)的毒     

苏芸金杆菌以色列变种在不同培养基上的产毒能力

苏芸金杆菌以色列变种(Bacillus thuringiensis subsp.israelensis)对蚊幼虫的杀虫活性,主要在于蛋白质晶体,而营养条件对晶体的合成有着重要影响。据研究,苏芸金杆菌不同血清型对培养基的要求各有差异。关于适合苏芸金杆菌以色列变种产生毒力的培养基,特别是     

Potency factors in the delta-endotoxin of Bacillus thuringiensis var. aizawi and the significance of plasmids in their control

Mutants defective in delta-endotoxin crystal production from four closely related isolates of Bacillus thuringiensis var. aizawi with aizawi serotype crystals were as vigorous as the parents in terms of growth, extracellular protease production, sporulation and heat resistance of spores. Spores produced by mutants germinated faster than wild type spores possibly due to deficiency of protein, in the form of delta-endotoxin in the spore coat. Acrystalliferous (cry—) mutants were not active in Galleria mellonella or Pieris brassicae larvae. Mutants with small crystals (sm cry) lost activity or gained extra activity against either one or the other host, revealing the presence of different toxicity factors. Solubilized crystals of parent isolates were composed of two major polypeptides with Mr values of 130 000 and 138 000. Sm cry mutants lost either polypeptide irrespective of which insect potency had been lost. Some cry — and some sm cry mutants had the same plasmid pattern as the parent; others lost one plasmid sometimes gaining another of different size. No consistent correlation was found between plasmid loss in mutants and any loss or increase of potency indicated by bioassays. It is concluded that the delta-endotoxins of the isolates under investigation are composed of at least two toxins. The results suggest that genes coding for the production of toxic factors or for their expression may be carried on both the plasmids and the chromosome.     

Properties of Bacillus cereus temperature-sensitive mutants altered in spore coat formation.

Three conditional Bacillus cereus mutants altered in the assembly or formation of spore coat layers were analyzed. They all grew as well as the wild type in an enriched or minimal medium but produced lysozyme and octanol-sensitive spores at the nonpermissive temperature (35 to 38 degrees C). The spores also germinated slowly when produced at 35 degrees C. Temperature-shift experiments indicated that the defective protein or regulatory signal is expressed at the time of formation of the outer spore coat layers. Revertants regained all wild-type spore properties at frequencies consistent with initial point mutations. Spore coat defects were evident in thin sections and freeze-etch micrographs of mutant spores produced at 35 degrees C. In addition, one mutant contained an extra surface deposit, perhaps unprocessed spore coat precursor protein. A prevalent band of about 65,000 daltons (the same size as the presumptive precursor) was present in spore coat extracts of this mutant and may be incorrectly processed to mature spore coat polypeptides. Another class of mutants was defective in the late uptake of half-cystine residues into spore coats. Such a defect could lead to improper formation of the outer spore coat layers.     

Possible origin and function of the parasporal crystals in Bacillusthuringiensis

Acrystalliferous strains of $\text{Bacillus}\text{thuringiensis}$ subsp. $\text{kurstaki}$ were isolated at a high frequency following heat treatment of spores. Spores of these strains lacked a 130,000 dalton glycoprotein, the major component common to both parasporal crystals and coats and were nontoxic to tobacco hornworm larvae. Moreover, the deficiency of this glycoprotein resulted in lysozyme sensitivity of the spores of some mutants and the presence of new spore coat proteins in others. All nontoxic acrystalliferous mutants lacked the complete array of at least six plasmids present in the wild type, implying a relationship between presence of plasmid(s) and toxicity. The unique capacity of this species to alter the surface coating of spores which appears to be related to crystal formation may provide flexibility for germination and growth in diverse soil environments.     

Importance of spores,crystals, and δ-endotoxins in the pathogenicity of different varieties of Bacillus thuringiensis in Galleria mellonella and Pieris brassicae

Physical methods were used to produce spores containing impurities of 0.02–0.05% crystals and crystals containing impurities of 0.001–0.01% spores from cultures of Bacillus thuringiensis. In Galleria mellonella larvae, these preparations from varieties galleriae, aizawai, and wuhanensis were only moderately active compared to 1:1 mixtures of spores and crystals. Spores of an acrystalliferous aizawai mutant were inactive and did not contain a polypeptide of the same size as the potent M_r 138000 δ-endotoxin present in spores and crystals of all three wild-type strains. Thus, this polypeptide probably contributed to the moderate activity of wild-type spores. Spore impurities in the crystal preparation were killed by γ irradiation without harming the crystals. The crystals without live spores were virtually inactive (LC₅₀s, ca. 10¹⁰ crystals/g insect food). Addition of 10³ spores to 10⁸ crystals/g food (0.001% spores) increased the mortality of larvae from 0 to 36%, and addition of 10⁴ spores (0.01% spores) killed 64% larvae. Thus, the addition of low levels of spores increased the potency of crystals in G. mellonella from virtually zero to moderate levels, suggesting that the live spore impurities in the crystal preparations were responsible for the observed moderate potency of crystals before γ irradiation, a view supported by a reduction of potency of crystal preparations following admixture of streptomycin to the insect food. In contrast to the results with G. mellonella, crystals were ca. 30 times as active as spores in Pieris brassicae larvae. Many authors have found crystals purified by physical methods to be highly active in a range of lepidopterous hosts. The present work indicates that the role of the spore impurities in these species may need further investigation. Absence of live spores of B. thuringiensis may impair the control of some insect species feeding on spore-free products and on microorganisms or plants into which endotoxins have been introduced by genetic manipulation.     

Properties of the Bacillus subtilis spore coat.

About 70% of the protein in isolated Bacillus subtilis spore coats was solubilized by treatment with a combination of reducing and denaturing agents at alkaline pH. The residue, consisting primarily of protein, was insoluble in a variety of reagents. The soluble proteins were resolved into at least seven bands by sodium dodecyl sulfate gel electrophoresis. About one-half of the total was four proteins of 8,000 to 12,000 daltons. These were relatively tyrosine rich, and one was a glycoprotein. There was also a cluster of proteins of about 40,000 daltons and two or three in the 20,000- to 25,000-dalton range. The insoluble fraction had an amino acid composition and N-terminal pattern of amino acids very similar to those of the soluble coat proteins. A major difference was the presence of considerable dityrosine in performic acid-oxidized preparations of insoluble coats. Coat antigen including a 60,000-dalton protein not present in extracts of mature spores was detected in extracts of sporulating cells by immunoprecipitation. This large antigen turned over in a pulse-chase experiment. Antibodies to either the array of 8,000- to 12,000-dalton coat polypeptides or to the larger coat proteins reacted with this 60,000-dalton species, suggesting a common precursor for many of the mature coat polypeptides. Spore coats seem to be assembled by processing of proteins and by secondary modifications including perhaps dityrosine formation for cross-linking.     

Mutations in the gerP locus of Bacillus subtilis and Bacillus cereus affect access of germinants to their targets in spores

The gerP1 transposon insertion mutation of Bacillus cereus is responsible for a defect in the germination response of spores to both L-alanine and inosine. The mutant is blocked at an early stage, before loss of heat resistance or release of dipicolinate, and the efficiency of colony formation on nutrient agar from spores is reduced fivefold. The protein profiles of alkaline-extracted spore coats and the spore cortex composition are unchanged in the mutant. Permeabilization of gerP mutant spores by coat extraction procedures removes the block in early stages of germination, although a consequence of the permeabilization procedure in both wild type and mutant is that late germination events are not complete. The complete hexacistronic operon that includes the site of insertion has been cloned and sequenced. Four small proteins encoded by the operon (GerPA, GerPD, GerPB, and GerPF) are related in sequence. A homologous operon (yisH-yisC) can be found in the Bacillus subtilis genome sequence; null mutations in yisD and yisF, constructed by integrational inactivation, result in a mutant phenotype similar to that seen in B. cereus, though somewhat less extreme and equally repairable by spore permeabilization. Normal rates of germination, as estimated by loss of heat resistance, are also restored to a gerP mutant by the introduction of a cotE mutation, which renders the spore coats permeable to lysozyme. The B. subtilis operon is expressed solely during sporulation, and is sigma K-inducible. We hypothesize that the GerP proteins are important as morphogenetic or structural components of the Bacillus spore, with a role in the establishment of normal spore coat structure and/or permeability, and that failure to synthesize these proteins during spore formation limits the opportunity for small hydrophilic organic molecules, like alanine or inosine, to gain access to their normal target, the germination receptor, in the spore.     

Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.     

Protein filaments may initiate the assembly of the Bacillus subtilis spore coat.

The Bacillus subtilis spore coat consists of three morphological layers: a diffuse undercoat, a striated inner coat and a densely staining outer coat. These layers are comprised of at least 15 polypeptides and the absence of one in particular, CotE, had extensive pleiotropic effects. Only a partial inner coat was present on the spores which were lysozyme-sensitive. The initial rate of germination of these spores was the same as for the wild type but the overall optical density decrease was greater apparently due to the loss of the incomplete spore coat from germinated spores. Suppressors of the lysozyme-sensitive phenotype had some outer coat proteins restored as well as some novel minor polypeptides. These spores still lacked an undercoat and germinated as did those produced by the cotE deletion strain. The CotE protein was synthesized starting at stage II-III of sporulation, long before the appearance of the coat on spores at stage IV-V. Despite its apparent hydrophilic properties, this protein was present in the crude insoluble fraction from sporulating cells. CotE was not solubilized by high or low ionic strength buffers not by detergents used for the solubilization of membrane proteins. Either 8 M urea or 6 M guanidine HC1 was required and dialysis against a low ionic strength buffer resulted in aggregation into long, sticky filaments. Both the CotE and CotT spore coat proteins appeared to be necessary for the formation of these filaments. Each of these proteins contains sequences related to a bovine intermediate filament protein so their interaction could result in an analogous structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and spore formation in Bacillus thuringiensis at high substrate concentrations

The work was aimed at studying the effect exerted by elevated concentrations of glucose, yeast extract and acetate on the growth of Bacillus thuringiensis subsp. galleriae, strain 69-6, and on the formation of spores and crystals by it. Glucose concentrations from 30 to 100 g per litre did not prevent spore formation. Yeast extract inhibited spore formation to a greater extent and stopped it almost completely at a concentration of 20 g per litre. Acetate at a concentration of 1.0 to 10 g per litre delayed spore formation and produced a less action on crystal formation, so that those processes were uncoupled in time.     

Spore Coat Protein Synergizes Bacillus thuringiensis Crystal Toxicity for the Indianmeal Moth (Plodia interpunctella)

Spores from Bacillus thuringiensis serovars kurstaki and entomocidus synergized crystal protein toxicity for larvae of the Indianmeal moth (Plodia interpunctella). Preparations of spore-crystal mixtures of either serovar were more toxic for the larvae than either purified spores or crystals alone (based on dry weight). Spores lost 53% of their toxicity for the Indianmeal moth after 2 h of UV-irradiation, but remained partially toxic (28%) even after 4 h of irradiation. Spore coat protein was toxic for the Indianmeal moth and was synergistic with B. thuringiensis serovar kurstaki HD-1 crystal protein. Enhanced toxicity of the combined spore-crystal preparation was attributed to a combination of crystal and spore coat protein, and included the effects of spore germination and resulting septicemia in the larval hemolymph. Ultraviolet irradiation of spores reduced the toxicity from septicemia but not the synergism caused by spore coat protein. The potencies of spore-crystal preparations must be carefully evaluated on the basis of contributions from all three factors. Received: 15 September 1997/Accepted: 21 October 1997