Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis

The three species of the group 1 bacilli, Bacillus anthracis, B. cereus, and B. thuringiensis, are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes. B. anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B. cereus and B. thuringiensis are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B. anthracis. Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from B. anthracis Ames. Ninety-three DNA sequences that were present in B. anthracis but absent from the non-anthrax-causing Bacillus genomes were isolated. Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing Bacillus species but were present in all members of a representative collection of B. anthracis strains. These sequences map to distinct loci on the B. anthracis genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of B. anthracis.     

On the adaptive nature of the dissociation process in Bacillus thuringiensis

The process of dissociation into variants differing in colony morphology occurring in batch cloned cultures of two Bacillus thuringiensis strains belonging to different subspecies was studied at optimal and elevated temperatures. An increase in the cultivation temperature to 40 degrees C resulted in an increase in the fraction of R variants to 100% after 72 h of cultivation of either of the strains. This increase was not due to the selection of forms with greater resistance to elevated temperature. The level of resistance to elevated temperature was determined by the strain genotype and did not correlate with morphological characteristics of the colonies.     

A Bacillus thuringiensis delta-endotoxin induces programmed cell death in mosquito larvae

We present evidence that a delta-endotoxin isolated from Bacillus thuringiensis subsp.israelensis induces programmed cell death in polytene midgut cells of Culex pipiens larvae. After exposure to toxin, polytene nuclei in the anterior region of the larval midgut undergo many of the morphological and physiological changes which are characteristic of apoptosis, including the ability to stain with the vital dye, acridine orange, and fragmentation of nuclear DNA as demonstrated by agarose gel electrophoresis and in situ TUNEL labeling. The temporal sequence of toxin ingestion, acridine orange staining and larval death suggests a cause and effect relationship between programmed cell death and larval death. Amino sugars that interfere with toxicity also interfere with the time course of acridine orange staining of larval polytene nuclei. The toxin first causes programmed cell death of anterior midgut and gastric caeca cells and, subsequently, posterior midgut cells. This pattern is similar to the temporal sequence of larval polytene cell death that occurs during metamorphosis. From the size and distribution of the nuclei that are stained with acridine orange, it appears that only polytene midgut cells are affected by toxin and that the diploid regenerative cell are not affected.     

Evidence that lithium induces a glutamatergic: Nitric oxide-mediated response in rat brain

Studies have indicated the involvement of a glutamatergic mechanism in lithium (Li⁺) action. Glutamatergic agonists, such as kainic acid, are known to promote the synthesis of nitric oxide (NO) and to increase cGMP, while Li⁺ has displayed a similar, yet unexplained, ability to increase cGMP. NO synthesis is regarded as the principal prodromal event leading to the activation of the guanyl cyclase-cGMP transduction mechanism. In the present study, the involvement of the NO:cGMP pathway in the action of Li⁺ was examined, while the possibility of a glutamatergic mechanism in this response was also investigated. Parameters examined included cortical accumulation of cGMP and the stable oxidative metabolites of NO, viz. NO ₂ ^– and NO ₃ ^–, collectively expressed as NO ₂ ^–. A significant positive correlation was observed in the in vivo cGMP and NO ₂ ^– data throughout all the groups. Chronic treatment of rats with LiCl (0.3% m/m) engendered a significant increase in cGMP levels which was inhibited by the NO-synthase (NOS) inhibitor, N-nitro-l-arginine methyl ester (L-NAME). Acute administration of kainic acid resulted in an increased accumulation of NO ₂ ^–, also prevented by concomitant L-NAME administration. In addition, a synergistic stimulatory response on cortical NO ₂ ^– was observed in the combination of LiCl and kainic acid. Collectively, these data implicate an involvement of a glutamatergic-mediated NO:cGMP transduction mechanism in the action of Li⁺.     

Discovery of crystalline inclusions in Bacillus licheniformis that resemble parasporal crystals of Bacillus thuringiensis

Crystalline inclusions were discovered in stationary and sporulating cells of the spore-forming bacterium Bacillus licheniformis ATCC 9945a. As detected by electron microscopy, dying or sporulating bacterial cells contain a single crystal of strikingly large size. The crystals in sporulating cells are located next to nascent spores and can be several times larger than the spores. Morphologically, most crystals are rhomboid with uniformly spaced grids. These newly discovered crystalline inclusions of B. licheniformis closely resemble parasporal crystals of Bacillus thuringiensis that are formed by insecticidal toxin proteins and used widely as biopesticides. The taxonomic identity of this strain was verified by its 16S rRNA gene sequence and its fatty acid profile. The finding of crystal proteins in B. licheniformis may lead to the discovery of new protein toxins and may expand our pool of biopesticides.     

苏云金杆菌Vip蛋白经历正向达尔文选择的证据

营养期杀虫蛋白(Vip)是苏云金杆菌在营养期所产生的一类新型杀虫蛋白,代表了第二代转基因杀虫蛋白,它能在一定程度上克服许多害虫对δ-内毒素低敏感或者不敏感的缺陷。但是,目前和已经深入研究的δ-内毒素相比较,有关Vip蛋白结构和功能关系方面的报道还甚少。本文采用最大似然方法和基于最大简约的滑窗分析对Vip蛋白的分子进化机制进行了评价。结果发现Vip蛋白在进化过程当中经历了正选择,并采用贝叶斯方法确定了16个正选择氨基酸残基。有意思的是所有这些正选择残基都位于Vip蛋白C端从705到809的区域。当把这些正选择残基定位到二级结构和三级结构时,发现绝大部分正选择残基都暴露在Vip蛋白空间结构的表面并且聚集在环的区域。推测Vip蛋白分子进化的机制应该是受到了正选择压力而不是功能约束的松弛。导致Vip蛋白C端多样性的潜在正选择压力可能是Vip蛋白为了在和目标昆虫之间竞争取得优势,或者是为了扩大Vip蛋白的杀虫范围。文中确定的经历了正选择残基很有可能是和昆虫宿主范围有关,因此可以为今后研究Vip蛋白的结构和功能提供相应的靶点。     

A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis

Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the B. anthracis capBCADE cluster were present on a plasmid (pAJ1-1). Strain BGSC 4AJ1, together with five strains of Bacillus cereus that hybridized to a PGA cap gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus multilocus sequence typing scheme. Bacillus thuringiensis BGSC 4AJ1 shared four identical alleles with B. anthracis and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typing database. The other cap+ strains were distributed among various lineages of Clade 1 of the B. cereus group.     

Experimental evidence that refuges delay insect adaptation to Bacillus thuringiensis

Theoretical projections suggest that refuges from exposure can delay insect adaptation to environmentally benign insecticides derived from Bacillus thuringiensis, but experimental tests of this approach have been limited. We tested the refuge tactic by selecting two sets of two colonies of diamondback moth (Plutella xylostella) for resistance to B. thuringiensis subsp. aizawai in the laboratory. In each set, one colony was selected with no refuge and the other with a 10 per cent refuge from exposure to B. thuringiensis subsp. aizawai. Bioassays conducted after nine selections were completed show that mortality caused by B. thuringiensis subsp. aizawai was significantly greater in the refuge colonies than in the no-refuge colonies. These results demonstrate that the refuges delayed the evolution of resistance. Relative to a susceptible colony, final resistance ratios were 19 and 8 for the two no-refuge colonies compared to 6 and 5 for the refuge colonies. The mean realized heritability of resistance to B. thuringiensis subsp. aizawai was 0.046 for colonies without refuges, and -0.002 for colonies with refuges. Selection with B. thuringiensis subsp. aizawai decreased susceptibility to B. thuringiensis toxin Cry1Ab, but not to Cry1C or B. thuringiensis subsp. kurstaki. Although the ultimate test of refuges will occur in the field, the experimental evidence reported here confirms modelling results indicating that refuges can slow the evolution of insect resistance to B. thuringiensis.     

Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis

Three crystalliferous (Cry⁺) strains of Bacillus thuringiensis subsp. israelensis (serotype 14) that produce parasporal protein crystals toxic to dipteran larvae and several acrystalliferous (Cry^?) mutants, either induced or spontaneously derived from a single Cry⁺ parent, were examined for the presence of covalently closed circular (CCC) DNA in attempts to correlate toxin production with the presence of a specific plasmid. The plasmid profiles of both Cry⁺ and Cry^? variants were analyzed by both a cleared lysate- and a modified Eckhardt lysateelectrophoresis technique. All of the Cry^? mutants derived from the Cry⁺ parental strain had lost a 4.0- to 4.4-megadalton (Mdal) plasmid. Bioassay data confirmed loss of toxin production by the Cry^? variants. All three Cry⁺ strains, including the parent of the Cry^? strains, contained CCC plasmids DNAs of the following approximate molecular weights: 4.0 to 4.4, 5.2 to 6.0, and 11.4 to 13.0 Mdal. One Cry⁺ strain contained an additional CCC plasmid of 6.7 to 7.2 Mdal. The plasmid patterns for several Cry^? derivatives differed in other respects from the pattern for their parent strain. The various Cry⁺ and Cry^? strains could be distinguished either by phenotypical differences in antibiotic sensitivity, crystal production, and toxicity, or by differences in their plasmid profiles.     

Spider mite infestations reduce Bacillus thuringiensis toxin concentration in corn leaves and predators avoid spider mites that have fed on Bacillus thuringiensis corn

Perceived benefits of insecticidal transgenic crops include reduced usage of broad‐based insecticides, and therefore lower risk to non‐target organisms. Numerous studies have documented low or no direct toxicity of Bacillus thuringiensis (Bt)‐derived toxins against non‐target organisms, but there has been less research on (a) effects of secondary pest infestations on Bt expressing in crops and (b) behavioural responses by predators feeding on host arthropods from Bt crops – both topics are investigated in this study. We quantified predation by the obligate spider mite predator Phytoseiulus persimilis of carmine spider mites (Tetranychus cinnabarinus), reared on Bt or non‐Bt corn (Zea mays). Both no‐choice and two‐choice studies were conducted. In addition, we quantified toxin levels in corn leaves with/without spider mite infestation. Under no‐choice conditions, P. persimilis consumed non‐Bt spider mites at a faster rate than Bt spider mites. Under two‐choice conditions, P. persimilis spent more time in the vicinity of non‐Bt spider mites than near Bt spider mites. Corn infested with spider mites exhibited lower toxin levels than non‐infested plants. These results suggest potentially complex interactions among non‐target herbivores, their natural enemies and Bt crops.     

Constructing Bacillus thuringiensis strain that co-expresses Cry2Aa and chitinase

A triple recombineering technique was used with plasmid pHT315 to produce pHTEC, a construct carrying chitinase and cry2Aa genes from Bacillus thuringiensis subsp. kurstaki 4.0718. Transformation of wild-type B. thuringiensis strain HD73 and the acrystalliferous strain Cry-B with pHTEC resulted in the recovery of recombinant strains that expressed Cry2Aa as cubic crystals in the cell pellet and soluble chitinase protein. The toxicity of HD73 (pHTEC) against Helicoverpa armigera larvae increased sevenfold when compared with HD73 (pHT315) harboring pHT315 vector. The triple recombineering protocol was optimized by comparing recombination efficacy mediated by RecE/RecT and Redα/Redβ and by using single-strand DNA as substrate.     

Crystalline inclusions in Bacillus thuringiensis

Crystalline inclusion bodies resembling those seen in Clostridium cochlearium were detected in cultures of Bacillus thuringiensis infected with bacteriophage.     

Transduction in Bacillus thuringiensis.

Bacteriophage CP-51, originally reported as a generalized transducing phage for Bacillus cereus and B. anthracis, has been shown to carry out generalized transduction in several strains of B. thuringiensis. A newly isolated phage, CP-54, which has a broader host range than CP-51, also mediates generalized transduction in B. thuringiensis. CP-51 and CP-54 are similar in size and morphology and are related serologically, but they are not identical. CP-54 is more cold labile than CP-51, and, as with CP-51, its stability both at 0 and 15 degrees C is enhanced by the presence of 0.02 M Mg2+. Some examples of cotransduction of linked markers in B. thuringiensis are presented, demonstrating the feasibility of chromosomal mapping in this organism. The rare occurrence of cross-transduction among strains of B. thuringiensis is probably a reflection of nonhomology rather than restriction, since phage itself did not appear to be restricted when grown on a particular host and assayed with other hosts as indicator.     

Physiological and biochemical response of Aedes aegypti tolerance to Bacillus thuringiensis

Bacillus thuringiensis subsp. israelensis (Bti) represents the only eco-friendly bio-degradable insecticide for mosquito-borne disease control. Our research aims to identify if mosquito detoxification enzymes play an important role in Bti tolerance mechanisms in the dengue vector, Aedes aegypti. Several enzymes, such as amylase, cytochromes P450, Na⁺/K⁺-ATPase, acetylcholinesterase, protease and glutathione S-transferase (GST) were analysed and level of activity determined in Ae. aegypti larvae after Bti treatment. Bti exposure significantly increased the level of amylase (183.2%) as well as the activity of cytochromes P450 (177.5%), and Na⁺/K⁺-ATPase (142.9%). On the other hand, there was a decrease of 8.6% and 11.4% in acetylcholinesterase and GST activity, and no significant effect in the total level of protease activity. We suggest that the variation in amylase, cytochromes P450, Na⁺/K⁺-ATPase, acetylcholinesterase, protease and GST activity may be associated with the Bti insecticidal mechanism. This study provides the basis of detoxifying enzymes in Bti tolerance.     

Metalloprotease from Bacillus thuringiensis

Bacillus thuringiensis var. kurstaki was shown to produce an extracellular, metal chelator-sensitive protease during the early stages of sporulation. Protease production in nutrient broth was dependent upon supplementation with Mn2+ or Ca2. The addition of Ca24 was required for enzyme stabilization...     

苏云金芽孢杆菌的一个新亚种(Btsubsp.sinensis)

198 9年自云南昆明市石林的红棕壤中分离到数株苏云金芽孢杆菌 (Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎ ｓｉｓ,Ｂｔ)菌株[1] ,对其中的一株ＹＫ30 0 4进行了生物学特性、杀虫特性研究及分类鉴定。1　材料与方法1.1　供鉴定的Ｂｔ菌株由云南昆明市石林的红棕壤中分离的苏云金芽孢杆菌ＹＫ30 0 4菌株。1.2　标准Ｂｔ菌株血清型Ｈ1 Ｈ4 1、Ｈ4 4 Ｈ55及Ｈ57 Ｈ69标准Ｂｔ菌株由法国巴斯德研究院ＤｒＬｅｃａｄｅｔ提供 ,其余为本实验室保存。1.3　生物测定用昆虫小菜蛾 (Ｐｌｕｔｅｌｌａｘｙｌｏｓｔｅｌｌａ) 3龄幼虫 ;斜纹夜盗蛾 (Ｐｒ…     

Normal human bronchial epithelial cells do not show an adaptive response after treatment with N-methyl-N'-nitro-N-nitrosoguanidine

Normal human bronchial epithelial cells cultured in serum-free medium were exposed to low doses o N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to examine whether increased cellular resistance and increased activity of the DNA-repair enzyme O6-methylguanine-DNA methyltransferase could be induced. After treatment with single doses of MNNG a dose-dependent decrease in O6-methylguanine-DNA methyltransferase activity was observed, as expected for this unique repair system. The activity recovered to the starting level in about 24 h when a dose that consumed approximately 65% of the enzyme activity (0.2 micrograms/ml) was given, but did not exceed the activity in the untreated control. Furthermore, treatment every 6 h for 4-5 days with non-toxic concentrations of MNNG (0.04-0.12 micrograms/ml) did not increase O6-methylguanine-DNA methyltransferase activity. Neither was cell survival following a range of challenge doses significantly increased. Our data suggest that human bronchial epithelial cells do not adapt to MNNG.     

Evidence of DNA rearrangements in the 128-kilobase pBtoxis plasmid of Bacillus thuringiensis israelensis

BUPM97 is a novel Tunisian isolate of Bacillus thuringiensis israelensis presenting insecticidal activity against Culex pipiens larvae. The δ-endotoxins pattern of this strain was different from that of the reference strain B. thuringiensis israelensis H14. Therefore, the study of its cry genes content was carried out by restriction-fragment-length-polymorphism (RFLP) using specific cry genes probes and by DNA sequencing. It was clearly demonstrated that in the strain BUPM97 the cry4A and cry10A genes were deleted from the B. thuringiensis israelensis 128-kb pBtoxis plasmid. In addition, a strong DNA sequence polymorphism was evidenced in the same plasmid downstream from the cry4B gene. This very particular DNA dynamic evidenced in this new strain of B. thuringiensis israelensis should be taken into consideration, regarding the strain stability during the industrial production of B. thuringiensis bioinsecticides.