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2012 | Buch

Bacillus thuringiensis Biotechnology

herausgegeben von: Estibaliz Sansinenea

Verlag: Springer Netherlands

Enthalten in: Springer Professional "Wirtschaft+Technik" , Springer Professional "Technik" , Springer Professional "Wirtschaft"

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Über dieses Buch

Bacillus thuringiensis (Bt) has been used as a biopesticide in agriculture, forestry and mosquito control because of its advantages of specific toxicity against target insects, lack of polluting residues and safety to non-target organisms. The insecticidal properties of this bacterium are due to insecticidal proteins produced during sporulation. Despite these ecological benefits, the use of Bt biopesticides has lagged behind the synthetic chemicals. Genetic improvement of Bt natural strains, in particular Bt recombination, offers a promising means of improving efficacy and cost-effectiveness of Bt-based bioinsecticide products to develop new biotechnological applications. On the other hand, the different Bacillus species have important biotechnological applications; one of them is carried out by producing secondary metabolites, which are the study object of natural product chemistry. The amazing structural variability of these compounds has attracted the curiosity of chemists and the biological activities possessed by natural products have inspired the pharmaceutical industry to search for lead structures in microbial extracts. Screening of microbial extracts reveals the large structural diversity of natural compounds with broad biological activities, such as antimicrobial, antiviral, immunosuppressive, and antitumor activities that enable the bacterium to survive in its natural environment. These findings widen the target range of Bacillus spp., in special B. thuringiensis, besides insecticidal activity and help people to better understand its role in soil ecosystem.

Inhaltsverzeichnis

Frontmatter

Bacillus thuringiensis: An Environmentally Safety Alternative

Frontmatter

Chapter 1. Discovery and Description of Bacillus thuringiensis

Abstract

The use of biopesticides, as a component of integrated pest management (IPM), has been gaining acceptance over the world. An entomopathogenic organism should be highly specific and effective against the target pest and should demonstrate the potential to be successfully processed by continuous production technology. Bacillus thuringiensis (Bt) was discovered as a soil bacterium, which fulfills all these requirements and due to it has been used as a biopesticide in agriculture, forestry and mosquito control. Studies of the basic biology of Bt have shown that the insecticidal activity of Bt is due to the presence of parasporal protein inclusion bodies, also called crystals, produced during sporulation that determines its activity for insect species belonging to different orders, which act like a stomach poison causing larval death. Environmentally safe-insect control strategies based on Bt and their insecticidal crystal proteins are going to increase in the future, especially with the wide adoption of transgenic crops. In this chapter, I have summarized the discovery and the description of Bt.

Estibaliz Sansinenea

Chapter 2. Bacillus thuringiensis Applications in Agriculture

Abstract

Bacillus thuringiensis (Bt) and its insecticidal toxins have been used in agronomical pest control for decades. The mechanism of action of Bt toxins on insect pest involves specific molecular interactions which makes Bt a popular choice for pest control. The specificity of action of Bt toxins reduces the concern of adverse effects on non-target species, a concern which remains with chemical insecticides. Different strains of Bt are known to express different classes of toxins which in turn target different insects. Bt and its toxins can be formulated into powder or liquid sprays or expressed in transgenic plants. To maximize the effect of Bt toxins, multiple toxins are often combined when making Bt formulations or expressed in transgenic plants. Though Bt is a very effective biological control agent, there are concerns over the development of resistance by insect species and also the narrow spectrum of activity of individual toxins. To address these concerns, new strains of Bt expressing novel toxins are actively sought and existing toxins are genetically modified for improved activity.

Zenas George, Neil Crickmore

Chapter 3. Risk Assessment of Bt Transgenic Crops

Abstract

The global demand, likely to escalate for at least another 40 years, requires a multifaceted strategy to ensure food security. Augmentation of crop yields in a sustainable manner is essential. Pests destroy on an average 14–25% of the total global agricultural production. Biopesticides are an important component of integrated pest management (IPM) strategies employed for minimization of insect pests-incurred crop yield losses as also for reduction of use of environmentally harmful chemical pesticides. Bacillus thuringiensis (Bt) is an aerobic, gram-positive, spore-forming bacterium producing crystal proteins (Cry), which are selectively toxic to target insects. Cry proteins act by insertion into the microvillar brush-border membranes in the midgut of susceptible insects, leading to disruption of osmotic balance, lysis of epithelial cells and eventually death of insect. Some Bt strains additionally secrete vegetative insecticidal proteins (Vips), which cause toxicity in susceptible insects by midgut epithelial cell lysis and gut paralysis. Bt has been used as a microbial biopesticide for the past five decades because of the advantages of specific toxicity against target insects, lack of polluting residues and safety to non-target organisms, and accounts for 95% of the 1% market share of biopesticides in the total pesticide market. However, the use of Bt microbial biopesticide formulations has been rather limited due to the problems of narrow host range, low persistence on plants and inability of foliar application to reach the insects feeding inside the plants, notwithstanding several biotechnological approaches for the development of improved Bt biopesticides. Bt transgenic crops have been developed to overcome the problems of Bt biopesticides and for more effective insect control. Risk assessment in relation to certain concerns raised about environmental and food safety of Bt transgenic crops has been addressed in this chapter.

Sarvjeet Kaur

Chapter 4. Use and Efficacy of Bt Compared to Less Environmentally Safe Alternatives

Abstract

Bt is classified as a biopesticide. Market surveys from several states in the United States of America suggest that the main reason for using biopesticides is it is considered safe for the environment. Public perception and organic farming have risen in importance as reasons for using biopesticides. However, the main reason for not using biopesticides continues to be perceptions of low efficacy, while higher costs and lack of awareness appeared to have less significance. Even though limited by its narrow activity, Bt usage has increased significantly over the years. The narrow activity spectrum of Bt is to its advantage for it is well suited for IPM programs that preserve large segments of indigenous invertebrate predator and parasitoid populations. On the other hand, usage of broad spectrum pesticides that also kill non-target beneficial insects, may lead to pest outbreak when the pesticide loses efficacy due to pest resistance. Less environmentally safe pesticides also have their roles to play in IRM and IPM programs. When combined with practices in IPM, they will have even less effect on the environment while reducing the probability of resistance. Substantial economic returns and environmental safety will drive the usage of safe pest control measures, which Bt may play a significant role.

Mohd Amir Fursan Abdullah

Chapter 5. Protein Engineering of Bacillus thuringiensis δ-Endotoxins

Abstract

Protein engineering of insecticidal Bt δ-endotoxins is a powerful tool for designing novel Cry toxins with altered properties, including changing the toxin’s specificity. By following some elementary rules governing the structure/function relationship, it has been possible to create new toxins with modified properties including increased toxicity and binding affinity, enhanced ion-transport activity, and changes in insect specificity. These methods have also produced valuable information and have led to an improved understanding of the mode of action of these important biopesticides. The results discussed in this chapter derive from rational molecular design where protein structure is modified by incorporating single or multiple amino acid substitutions aimed at modifying specific protein functions. In this review, we analyze several protein modifications that have been successfully used for creating stable, functional proteins with minimal structural alterations. The understanding and proper use of protein engineering approaches may help in implementing appropriate pest management strategies by improving the efficacy of these toxins against insect pests.

Alvaro M. Florez, Cristina Osorio, Oscar Alzate

Genetics of Bacillus Thuringiensis

Frontmatter

Chapter 6. Evolution of the Bacillus cereus Group

Abstract

The Bacillus cereus group contains six approved bacterial species—Bacillus thuringiensis, Bacillus cereus (sensu stricto), Bacillus anthracis, Bacillus weihenstephanensis, Bacillus mycoides, and Bacillus pseudomycoides. In addition, thermotolerant B. cereus var. cytotoxis could constitute a member of a novel species in the group. Historically, Bacillus thuringiensis has been viewed as a species separate from the other members of the group, mainly based on its entomopathogenic properties and production of insecticidal crystal toxins. However, following the sequencing of more than 85 strains from the B. cereus group, whole genome phylogenies and MLST analyses of more than 1400 strains based on chromosomal markers clearly show that the Bacillus cereus group exhibits a complex population structure, in which strains of B. thuringiensis are strongly intermixed with other B. cereus group species. From the analysis of genome sequence data, it is clear that the only principal factor differentiating B. thuringiensis from B. cereus is the presence of insecticidal crystal toxin gene(s), which in the vast majority of cases are carried on plasmid elements that are frequently conjugative or mobilizable, and which occasionally could be lost by the bacterium. Also, strikingly, some B. thuringiensis strains are among the closest relatives to the B. anthracis clonal complex. Chromosomal factors potentially involved in stabilizing long-term carriage of plasmids in B. thuringiensis strains, have not yet been elucidated.

Ole Andreas Økstad, Anne-Brit Kolstø

Chapter 7. Bacillus thuringiensis Genetics and Phages—From Transduction and Sequencing to Recombineering

Abstract

Experimental mapping of genes was less intensive in the Bacillus cereus group, which includes B. thuringiensis, than in Bacillus subtilis, generally considered as the model for Gram-positive bacteria. Nevertheless the genomic sequencing equalized densities of available gene maps. Moreover, the genes responsible for such complex phenomena like virulence or psychrotolerance could only be identified using the authentic bacteria prone to possess these properties. The new experimental approaches of post-genomic genetics should therefore be considered. Phage-mediated gene transduction and recombineering perspectives for the B. cereus group are reviewed. In combination with new generation sequencing these approaches will constitute the gene identification methodologies in the post-genomics time.

Alexei Sorokin

Chapter 8. Conjugation in Bacillus thuringiensis: Insights into the Plasmids Exchange Process

Abstract

Since the discovery of the conjugation process in bacteria, many studies focusing on this issue have contributed to a better understanding of the biology, ecology, genetics and consequently to the taxonomy of bacteria. In this chapter, the mechanisms of the conjugation process in Gram-positive species were revised and detailed, including a set of events as the contact between donor and recipient cells, the DNA processing and its inter cellular transport, and the variations of the conjugal mating systems. Studies focusing on conjugative transfer in Bacillus thuringiensis, involving the detection of cry genes in large conjugative plasmids, the genetic basis of the process, the main plasmids, and methodological variations of mating systems are discussed. Nowadays conjugal mating systems are again prominence and several studies have been conducted to evaluate plasmid exchange both within and between B. thuringiensis and closely related species belonging to the Bacillus cereus group. Thus, conjugal mating systems became an important tool to understand the role of plasmids in the behavior and in genome evolution of B. thuringiensis.

Gislayne T. Vilas-Bôas, Clelton A. Santos

Chapter 9. Shuttle Vectors of Bacillus thuringiensis

Abstract

Bacillus thuringiensis is characterized by the synthesis of parasporal crystals during sporulation; these are composed of one or more highly specific insecticidal or nematocidal endotoxin proteins. Genetic manipulation of B. thuringiensis has been achieved due to the development of stable shuttle vectors and the establishment of efficient transformation systems. Shuttle vectors of B. thuringiensis have been constructed using essentially replicons from resident plasmids from this bacterium that replicate by the theta mechanism. Also, these vectors have been developed using plasmid replicons from other Gram-positive bacteria or RCR plasmids. The transformation of B. thuringiensis with these vectors has been accomplished mainly through electroporation. The development of shuttle vectors with better characteristics and protocols with high transformation efficiency have greatly facilitated basic research and engineering of B. thuringiensis.

Alejandra Ochoa-Zarzosa, Joel Edmundo López-Meza

Chapter 10. Construction and Application in Plasmid Vectors of Bacillus cereus Group

Abstract

The B. cereus group (B. cereus sense lato) is a subgroup of related Bacillus containing six species: B. thuringiensis, B. anthracis, B. cereus, B. weihenstephanensis, B. mycoides and B. pseudomycoides. The first three species are opportunistic or pathogenic to insects or mammals, whereas the last three are generally regarded as nonpathogenic. Different types of plasmid vectors play an important role in studying gene function in genetically engineered bacteria or construction of mutant library. The researchers have constructed a variety of shuttle vectors in two or more different host strains. Expression vectors are widely used in construction of highly effective engineered strains. Resolution vectors can site-specifically transfer recombinant genes. Integration vectors could integrate foreign genes into the chromosome or plasmid of host strains and are free of antibiotic resistance genes, therefore eliminating security risks. In recent years, researchers have made great progress in the development of genetic manipulation vectors. Here we summarize latest progress in vector construction and their application in Bacillus cereus group.

Chengchen Xu, Yan Wang, Chan Yu, Lin Li, Minshun Li, Jin He, Ming Sun, Ziniu Yu

Chapter 11. Recombination in Bacillus thuringiensis

Abstract

Recombination plays a critical role in maintaining gene diversification and genome stability. There are two types of recombination: the site specific recombination and the homologous recombination. In this chapter we present the genetic recombination in Bacillus thuringiensis. The first part describes the site specific recombination, including transposition by transposons and transduction by phage, in this bacterium and its exploitation in the construction of recombinant strains of B. thuringiensis improving their production as bioinsecticides and their insecticidal activities and B. thuringiensis mutagenesis. In the second part we are interested by the homologous recombination and its role in the construction of improved B. thuringiensis strains and in gene disruption.

Lobna Abdelkefi-Mesrati, Slim Tounsi

Chapter 12. Genetic Improvement of Bt Strains and Development of Novel Biopesticides

Abstract

This review describes how recombinant DNA technology has been used to improve Bacillus thuringiensis (Bt) products and overcome a number of the problems associated with Bt-based insect control measures. It will discuss how the knowledge of the genetics of Bt and of its insecticidal toxin genes, the understanding of their regulation and the development of cloning vectors has made possible the continuing improvement of first generation products. Several examples describing how biotechnology has been used to increase the production of insecticidal proteins in Bt, their persistence in the field by protecting them against UV degradation or to construct non-viable genetically modified strains, will be presented.

Vincent Sanchis

BT as Biopesticide: Applications in Biotechnology

Frontmatter

Chapter 13. Genetically Modified Bacillus thuringiensis Biopesticides

Abstract

The reduction in crop production that results from damages of pests and pathogenic microbes has been the major prohibitive factor restricting the further development of agricultural production worldwide. In the long-term practice of controlling agricultural pests, multiple pest management, including biological approaches, are generally recognized as possible solutions to control pest infestation effectively. Bacterial pesticides, which constitute a series of leading biopesticidal products made from various naturally occurring or genetically modified insecticidal bacteria, have attracted increasing attention as a specific means of controlling agricultural and forestry pests.

Lin Li, Ziniu Yu

Chapter 14. Bacillus thuringiensis Recombinant Insecticidal Protein Production

Abstract

The initial excitement of applying recombinant DNA technology to insecticidal proteins of Bacillus thuringiensis was subdued by regulatory caution about the technology and the genetic complexity of the proteins themselves. While seven biopesticide products containing recombinant proteins were eventually manufactured, expression of the proteins in Gram negative and Gram positive bacteria is predominantly for discovery and mode of action work. Regulatory studies for the registration of transgenic plants requires microbially produced insecticidal proteins in the tens of grams. Transgenic plants now dominate the production of B. thuringiensis recombinant insecticidal proteins, with expression technology yielding more than 10-fold higher levels than the earliest registered plants and worldwide use on nearly 60 million hectares.

H. Ernest Schnepf

Chapter 15. Bt Crops: Past and Future

Abstract

The development and commercialization of transgenic plants expressing insecticidal toxin genes from the bacterium Bacillus thuringiensis (Bt) has revolutionized agriculture in the past two decades. Development of this revolutionary insect pest control technology was facilitated by the identification and characterization of insecticidal Bt proteins and advancements in plant transformation and genetic engineering. While commercialization of this technology is currently limited to a number of countries, these transgenic “Bt crops” are replacing in most cases conventional crop varieties due to their insect resistance, lower spraying requirements, and higher yields. However, concerns related to the increasing adoption of this technology include gene flow to wild relatives, evolution of resistance in target pests, and unintended effects on the environment. In this chapter, we discuss key events in the history of Bt crop development and summarize current regulations aimed at reducing the risks associated with increased adoption of this technology. By analyzing the history of Bt transgenic crops and the current marketplace trends and issues, we aim to examine the outlook of current and impending Bt crops as well as potential issues that may emerge during their future use.

Anais S. Castagnola, Juan Luis Jurat-Fuentes

Chapter 16. A Review of the Food Safety of Bt Crops

Abstract

There is a 50-year history of safe use and consumption of agricultural food crops sprayed with commercial Bt (Bacillus thuringiensis) microbial pesticides and a 14 year history of safe consumption of food and feed derived from Bt crops. This review summarizes the published literature addressing the safety of Cry insect control proteins found in both Bt microbial pesticides and those introduced into Bt agricultural crops. A discussion on the species-specific mode of action of Cry proteins to control target insect pests is presented. This information provides the scientific basis for the absence of toxicity of Cry proteins towards non-target organisms that has been confirmed in numerous mammalian toxicology studies. A human dietary exposure assessment for Cry proteins has also been provided which includes information that food processing of Bt crops such as maize leads to loss of functionally active Cry proteins in processed food products. Lastly the food and feed safety benefits of Bt crops are briefly summarized including lower insecticide use and reduction in fumonisin mycotoxin contamination of grain.

Bruce G. Hammond, Michael S. Koch

Other Bacillus Species in Biotechnology

Frontmatter

Chapter 17. The Most Important Bacillus Species in Biotechnology

Abstract

Aerobic spore forming Bacillus and related bacteria have been known for their important impact on human activities. By including bacteria characterized by a high diversity with regard to the G + C content and genetic and metabolic capabilities, Bacillus and relatives encompass both pathogenic and beneficial bacteria. The second group is represented by several species that have been implicated in various industrial applications. These include industrial production of enzymes with great interest in detergent and food sectors; the production of primary metabolites such as vitamins and ribonucleosides; of secondary metabolites including bacteriocins and biosurfactants and of plant growth promoting formulations. Moreover, recent studies have shown that the aerobic spore formers can produce fine chemicals with interesting biotechnological applications like for example carotenoid pigments and a variety of biopolymers including poly-g-glutamic and lactic acids. These findings open perspectives for new biotechnological applications of Bacillus and related species. In this chapter, we discuss the evolution and ecology of spore formers within the genus Bacillus and report a literature review of the various biotechnological applications of the most important species including some now affiliated to related genera.

Noura Raddadi, Elena Crotti, Eleonora Rolli, Ramona Marasco, Fabio Fava, Daniele Daffonchio

Chapter 18. Secondary Metabolites of Bacillus: Potentials in Biotechnology

Abstract

The huge diversity characterizing the Bacillus species at the taxonomic level, is also noticeable for their metabolic features. These bacteria are able to produce a wide range of secondary metabolites with very different natures and structures and displaying broad spectra of activities. These metabolites; including antibiotics, pigments, toxins, growth promoters (animals and plants), effectors of ecological competition, pheromones, enzyme inhibitors and others bioactive compounds; are originally designed to enable the bacterium to survive in its natural environment (Stein 2005). In general, these metabolites serve as; (i) competitive weapons used against other bacteria, fungi, amoebae, plants, insects and large animals; (ii) metal transporting agents; (iii) symbiosis effectors between microbes and plants, nematodes, insects and higher animals; (iv) sexual hormones; and (v) as differentiation factors (Demain and Fang 2000). This wide variability of the structure and activity of the secondary compounds expands the potential industrial importance of the genus Bacillus and its related genera (Sansinenea and Ortiz 2011). Besides, Bacillus species form spores that can be easily formulated and have high viability compared with vegetative cells. Finally they are commonly diffused in the environment including soil (Sansinenea and Ortiz 2011).

Ines Chaabouni, Amel Guesmi, Ameur Cherif

Chapter 19. Future Challenges and Prospects of Bacillus thuringiensis

Abstract

Bacillus thuringiensis is the most important entomopathogenic microorganism, owing to its insecticidal crystal proteins (Cry) that are nontoxic to the human beings and represent the hallmark of this bacterium. Most of the studies performed with this microorganism are focused to understand the role of Cry proteins in toxicity, mainly because these components constituted the toxic molecules of its commercial products. B. thuringiensis produces several metabolites with potential applied uses, in particular, chitinolytic enzymes and bacteriocins are two types of proteins produced by different subspecies of this microorganism that could expand the perspective of application of this extraordinary bacterium. In this chapter, we review the different kinds of chitinases that are synthesized by B. thuringiensis, their roles in nature, and their applications in environment, agriculture and food industry. Additionally, we analyze bacteriocins of B. thuringiensis reported to date, how to enhance their production, and the methods for screening the bacteriocin activity. Finally, the future challenges and prospects of the antimicrobial peptides as biopreservatives, antibiotics, and nodulation factors are showed.

J. E. Barboza-Corona, N. M. de la Fuente-Salcido, M. F. León-Galván

Backmatter

Titel: Bacillus thuringiensis Biotechnology
herausgegeben von: Estibaliz Sansinenea
Verlag: Springer Netherlands
Electronic ISBN: 978-94-007-3021-2
Print ISBN: 978-94-007-3020-5
DOI: https://doi.org/10.1007/978-94-007-3021-2

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