No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Protein Patterns and Larvicide Activity of Crystalline Inclusions of Bacillus thuringiensis ssp. kumamotoensis DSM 6070
Abstract
Morphological and electrophoretic analysis of the crystalline inclusions (parasporal crystals) of sporulated cultures of B. thuringiensis ssp. kumamotoensis DSM 6070 (Bt 6070) was conducted via phase contrast and scanning electron microscopy and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The activity of the spore-crystal suspensions against house fly (Musca domestica) larvae was also assessed. Bipyramidal and smaller, irregular shaped crystals were observed in the sporulated cultures. 130, 75 and 25 kDa bands were detected in the protein pattern. The presence of 25 kda proteins in Bt6070 has not been reported earlier. The spore-crystal suspension showed significant larvicide activity against housefly larvae. Larvicide activity of B. thuringiensis ssp. kumamotoensis against any dipteran species has not been detected yet. Further studies are needed on identifying the dipteran- active fraction.
ResearchGate has not been able to resolve any citations for this publication.
Bacillus thuringiensis subsp. israelensis (Bti) is the first Bacillus thuringiensis to be found and used as an effective biological control agent against larvae of many mosquito and black fly species around the world. Its larvicidal activity resides in four major (of 134, 128, 72 and 27 kDa) and at least two minor (of 78 and 29 kDa) polypeptides encoded respectively by cry4Aa, cry4Ba, cry11Aa, cyt1Aa, cry10Aa and cyt2Ba, all mapped on the 128 kb plasmid known as pBtoxis. These six δ-endotoxins form a complex parasporal crystalline body with remarkably high, specific and different toxicities to Aedes, Culex and Anopheles larvae. Cry toxins are composed of three domains (perforating domain I and receptor binding II and III) and create cation-selective channels, whereas Cyts are composed of one domain that acts as well as a detergent-like membrane perforator. Despite the low toxicities of Cyt1Aa and Cyt2Ba alone against exposed larvae, they are highly synergistic with the Cry toxins and hence their combinations prevent emergence of resistance in the targets. The lack of significant levels of resistance in field mosquito populations treated for decades with Bti-bioinsecticide suggests that this bacterium will be an effective biocontrol agent for years to come.
The bacterium Bacillus thuringiensis proved to be a good candidate in controlling Musca domestica, associated with poultry houses, as a carrier of a wide range of pathogens infecting man and animals. Chicken feces are good media attracting flies for breeding. The bacterium was used in commercial form and a laboratory preparation form to contaminate feces or administered orally to chicken. Reduction in the percentages of pupal and adult emergence was recorded for six days after chicken feeding by two doses of B. thuringiensis (1.00 and 5.00 mg/ kg).
The insecticidal bacterium, Bacillus thuringiensis, consists of a wide variety of subspecies, most of which are insecticidal for either lepidopteran, coleopteran, or dipteran insect larvae. Subspecies such as B. thuringiensis subsp. kurstaki have been used with remarkable safety for more than forty years to control lepidopteran pests in agriculture and forestry, and over the past thirty years, B. thruingeinsis subsp. israelensis, has proven to be a safe and effective larvicide for controlling mosquito and black fly larvae. Studies of the basic biology of B. thuringiensis have shown that it produces a variety of insecticidal proteins produced during vegetative growth and sporulation that determines its activity for insect species belonging to different orders, with the most important of these being the Cry proteins active against lepidopteran and coleopteran pests, and a combination of Cry and Cyt proteins for mosquitoes and blackflies. After intoxication by these proteins, spores typically germinate and invade larvae, contributing to insect mortality. Whereas strains of many wild type isolates have been commercialized and are now used worldwide, the use of recombinant DNA techniques, i.e., genetic engineering, has been used over the past decade to recombine the proteins of different B. thuringiensis strains with those of B. sphaericus to generate recombinant larvicides as much as ten-fold more toxic than the parental strains. In this chapter, we begin with a general overview of the basic biology of B. thuringiensis and B. sphaericus, then show how studies of its molecular genetics combined with recombinant DNA techniques have been used to generate highly improved bacterial larvicides for control of nuisance and vector mosquitoes.
Bacillus thuringiensis EG2838 and EG4961 are highly toxic to Colorado potato beetle larvae, and only strain EG4961 is toxic to southern corn rootworm larvae. To investigate the cause of the different insecticidal activities of EG2838 and EG4961, cryIII-type genes toxic to coleopterans were cloned from each strain. The cryIIIB gene, cloned as part of an 8.0-kb EcoRI fragment of EG2838 DNA, encoded a crystal protein (CryIIIB) of 74,237 Da. The cryIIIB2 gene, cloned as part of an 8.3-kb PstI-Asp718 fragment of EG4961 DNA, encoded a crystal protein (CryIIIB2) of 74,393 Da that was 94% identical to CryIIIB. Analysis of the transcriptional start sites showed that cryIIIB and cryIIIB2 were initiated from a conserved region located within 130 nucleotides upstream from the translation start sites of both genes. Although the CryIIIB and CryIIIB2 proteins were similar in sequence, they displayed distinct insecticidal activities: CryIIIB was one-third as toxic as CryIIIB2 to Colorado potato beetle larvae, and CryIIIB2, but not CryIIIB, was toxic to southern corn rootworm larvae. Genes encoding crystal proteins of approximately 32 and 31 kDa were located adjacent to the cryIIIB and cryIIIB2 genes, respectively. The 32- and 31-kDa crystal proteins failed to enhance the insecticidal activities of CryIIIB and CryIIIB2.
The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged.
In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we isolated a B. thuringiensis strain (Bn1) from Balaninus nucum (Coleoptera: Curculionidae), the most damaging hazelnut pest. Bn1 was characterized via morphological, biochemical, and molecular techniques. The isolate was serotyped, and the results showed that Bn1 was the B. thuringiensis serovar, kurstaki (H3abc). The scanning electron microscopy indicated that Bn1 has crystals with cubic and bipyramidal shapes. The Polymerase Chain Reactions (PCRs) revealed the presence of the cry1 and cry2 genes. The presence of Cry1 and Cry2 proteins in the Bn1 isolate was confirmed via SDS-PAGE, at approximately 130 kDa and 65 kDa, respectively. The bioassays conducted to determine the insecticidal activity of the Bn1 isolate were conducted with four distinct insects, using spore-crystal mixtures. We noted that Bn1 has higher toxicity as compared with the standard B. thuringiensis subsp. kurstaki (HD-1). The highest observed mortality was 90% against Malacosoma neustria and Lymantria dispar larvae. Our results show that the B. thuringiensis isolate (Bn1) may prove valuable as a significant microbial control agent against lepidopteran pests.
Bacillus thuringiensis (or Bt, as it has become generally known) is one of the oldest and widely used biological control agents and has a long history of use. Bt and a number of related bacteria produce a variety of toxins, mostly—but not exclusively- localized in the parasporal crystals, which are, together with the spores themselves, the components of the typical spore/crystal mixtures. These are used to control insect pests in agricultural crops. While Bt products quietly kept holding the first place in biological pesticide sales, interest in Bt was increased by the production and commercialization of transgenic crop plants expressing one or more Bt toxins since 1996. Here I will present a brief overview of the history, biology, and practical uses of Bt and its toxins.
Four collections comprising 507 strains of Bacillus thuringiensis have been analysed in this study. A different ecological origin characterizes each collection. Collection No. 1 was established from soil, dust, and grain samples from Spanish agricultural and non-cultivated soil, silos, and mills. Collection No. 2 is the result of a screening in olive-crop related environments in Spain. Collection No. 3 is made up of strains isolated from potato-growing areas in Bolivia. Collection No. 4 has been generated for this study and includes strains collected from diverse types of samples belonging to several habitats from Spain and Mexico. Crystal morphologies and cry1A and cry2 gene content were assessed for all isolates from each collection. In the 507 strains, the most common crystal morphology was bipyramidal crystals. Frequencies of cry1A and cry2 genes were 61.5% and 59.2%, respectively, and there was a strong correlation between the occurrence of cry1A and cry2 genes.
A classification for crystal protein genes of Bacillus thuringiensis is presented. Criteria used are the insecticidal spectra and the amino acid sequences of the encoded proteins. Fourteen genes are distinguished, encoding proteins active against either Lepidoptera (cryI), Lepidoptera and Diptera (cryII), Coleoptera (cryIII), or Diptera (cryIV). One gene, cytA, encodes a general cytolytic protein and shows no structural similarities with the other genes. Toxicity studies with single purified proteins demonstrated that every described crystal protein is characterized by a highly specific, and sometimes very restricted, insect host spectrum. Comparison of the deduced amino acid sequences reveals sequence elements which are conserved for Cry proteins. The expression of crystal protein genes is affected by a number of factors. Recently, two distinct sigma subunits regulating transcription during different stages of sporulation have been identified, as well as a protein regulating the expression of a crystal protein at a posttranslational level. Studies on the biochemical mechanisms of toxicity suggest that B. thuringiensis crystal proteins induce the formation of pores in membranes of susceptible cells. In vitro binding studies with radiolabeled toxins demonstrated a strong correlation between the specificity of B. thuringiensis toxins and the interaction with specific binding sites on the insect midgut epithelium. The expression of B. thuringiensis crystal proteins in plant-associated microorganisms and in transgenic plants has been reported. These approaches are potentially powerful strategies for the protection of agriculturally important crops against insect damage.
This report describes the identification of a new toxigenic strain of Bacillus thuringiensis specific for long-horned beetles. B. thuringiensis Bt866 encodes a cry3Aa-like gene (Bt886cry3Aa) that is 1,956 bp in length and is predicted to encode an 85.78-kDa protein. The gene is highly similar to cry3Aa1, differing in only six nucleotides and four amino acids. The four disparate amino acids occur within the conserved domains of the Cry3Aa toxin. The expression of Bt866cry3A in Escherichia coli cells resulted in a high level of toxicity toward Apriona germari Hope larvae. More than 75% of the larvae were killed; and the remaining survivors exhibited slower growth. These results indicate that the toxigenic strain Bt886cry3Aa encodes a protein that is specific against long-horned beetles. Genetic engineering of the Bt866cry3Aa gene into poplar plantations may provide resistance to long-horned beetles.
The multisegmented ovoidal inclusion of Bacillus thuringiensis subsp. israelensis was found to be composed of two structurally and biochemically distinct components. Electron microscopy of the inclusion revealed it to be composed mainly of osmiophobic or lightly stained segments crystallized in a lattice showing a repeat of approximately 4.3 nm. These light segments of the inclusions were shared by osmiophylic darkly stained segments with a crystal lattice repeat of approximately 7.8 nm. The lightly stained segments were soluble at pH 9.2 in sodium dodecyl sulfate-dithiothreitol-Tris-hydrochloride. The extracts of lightly stained segments were lytic to mammalian erythrocytes, and the precipitate obtained by lowering the pH to 5.2 was toxic to the larvae of Aedes egypti. The dark inclusion segments remaining, besides being much less toxic to larvae, were nonlytic to erythrocytes and were soluble at pH 10.5 in sodium dodecyl sulfate-dithiothreitol-Tris-hydrochloride. The light segment was composed of two major polypeptide doublets with molecular weights of 145,000 and 135,000, and 27,000 and 26,500, and the dark segments were composed of a single major polypeptide with a molecular-weight of 70,000. Hence, the inclusion of B. thuringiensis subsp. israelensis is more complex than previously reported, and we conclude that the toxin may be the polypeptide with a molecular weight of 27,000 and 26,500.
Compositions and Methods for Controlling Plant Pests
- S D Heins
- D R Jimenez
- D C Manker
- P G Marrone
- R J Mccoy
- J E Orjala
Heins, S. D., Jimenez, D. R., Manker, D. C.,
Marrone, P. G., Mccoy, R. J. and Orjala, J.
E. 2000. Compositions and Methods for
Controlling Plant Pests. US Patent
WO2000029426 A1.
Bacillus thuringiensis in Biological Control Handbook of Biological Pest Control
- B A Federici
- T S Bellows
- T W Fisher
- L E Caltagirone
- D L Dahlsten
- G Gordh
- C Huffaker
Federici, B. A. 1999. Bacillus thuringiensis
in Biological Control. In: " Handbook of
Biological Pest Control ", (Eds.): Bellows, T.
S., Fisher, T. W., Caltagirone, L. E.,
Dahlsten, D. L., Gordh, G. and Huffaker, C.
B. Elsevier Inc., PP. 575–593.
Bacillus thuringiensis in Pest Control Biological Control: Benefits and Risks
- R J C Cannon
Cannon, R. J. C. 1995. Bacillus
thuringiensis in Pest Control. In: " Biological
Control: Benefits and Risks ", (Eds.):
Hokkanen H. M. T. and Lync, J. M.
Cambridge University Press, PP. 190–197.