Fatores de Virulência de Bacillus thuringiensis Berlinier: O Que Existe Além das Proteínas Cry?

As proteínas Cry produzidas pela bactéria entomopatogênica Bacillus thuringiensis Berliner são bem conhecidas devido a alta citotoxicidade que exibem a uma variedade de insetos-alvo. O modo de ação destas proteínas é específico e torna os produtos à base de B. thuringiensis os mais amplamente utilizados em programas de controle biológico de pragas na agricultura e de importantes vetores de doenças humanas. Contudo, embora as proteínas Cry sejam os fatores de virulência inseto-específico mais conhecidos, linhagens de B. thuringiensis apresentam também uma ampla gama de fatores de virulência, os quais permitem à bactéria atingir a hemolinfa e colonizar eficientemente o inseto hospedeiro. Dentre estes fatores, destacam-se as proteínas Vip, Cyt, enterotoxinas, hemolisinas, fosfolipases, proteases, enzimas de degradação, além das recentemente descritas parasporinas. Essa revisão aborda a ação desses fatores de virulência, bem como a caracterização e o controle da expressão de seus genes. Adicionalmente, são discutidos aspectos relacionados ao nicho ecológico da bactéria com ênfase nas características envolvidas com a biossegurança da utilização dos produtos à base de B. thuringiensis para o controle biológico de insetos-alvo.Virulence Factors of Bacillus thuringiensis Berliner: Something Beyond of Cry Proteins?Abstract. The Cry proteins produced by the entomopathogenic bacterium Bacillus thuringiensis Berliner are widely known due to its high toxicity against a variety of insects. The mode of action of these proteins is specific and becomes B. thuringiensis-based products the most used in biological control programs of insect pests in agriculture and of important human disease vectors. However, while the Cry proteins are the best-known insect-specific virulence factor, strains of B. thuringiensis show also a wide range of other virulence factors, which allow the bacteria to achieve the hemolymph and colonize efficiently the insect host. Among these factors, we highlight the Vip proteins, Cyt, enterotoxins, hemolysins, phospholipases, proteases and enzymes of degradation, in addition to the recently described parasporin. This review explores the action of these virulence factors, as well as, the characterization and control of expression of their genes. Additionally, we discuss aspects related to the ecological niche of the bacteria with emphasis on the characteristics involved in the biosafety of the use of B. thuringiensis-based products for biological control of target insects

Fatores de Virulência de Bacillus thuringiensisBerliner: O Que Existe Além das Proteínas Cry?

As proteínas Cry produzidas pela bactéria entomopatogênica Bacillus thuringiensis Berliner são bem conhecidas devido a alta citotoxicidade que exibem a uma variedade de insetos-alvo. O modo de ação destas proteínas é específico e torna os produtos à base de B. thuringiensis os mais amplamente utilizados em programas de controle biológico de pragas na agricultura e de importantes vetores de doenças humanas. Contudo, embora as proteínas Cry sejam os fatores de virulência inseto-específico mais conhecidos, linhagens de B. thuringiensis apresentam também uma ampla gama de fatores de virulência, os quais permitem à bactéria atingir a hemolinfa e colonizar eficientemente o inseto hospedeiro. Dentre estes fatores, destacam-se as proteínas Vip, Cyt, enterotoxinas, hemolisinas, fosfolipases, proteases, enzimas de degradação, além das recentemente descritas parasporinas. Essa revisão aborda a ação desses fatores de virulência, bem como a caracterização e o controle da expressão de seus genes. Adicionalmente, são discutidos aspectos relacionados ao nicho ecológico da bactéria com ênfase nas características envolvidas com a biossegurança da utilização dos produtos à base de B. thuringiensis para o controle biológico de insetos-alvoThe Cry proteins produced by the entomopathogenic bacterium Bacillus thuringiensis Berliner are widely known due to its high toxicity against a variety of insects. The mode of action of these proteins is specific and becomes B. thuringiensis-based products the most used in biological control programs of insect pests in agriculture and of important human disease vectors. However, while the Cry proteins are the best-known insect-specific virulence factor, strains of B. thuringiensis show also a wide range of other virulence factors, which allow the bacteria to achieve the hemolymph and colonize efficiently the insect host. Among these factors, we highlight the Vip proteins, Cyt, enterotoxins, hemolysins, phospholipases, proteases and enzymes of degradation, in addition to the recently described parasporin. This review explores the action of these virulence factors, as well as, the characterization and control of expression of their genes. Additionally, we discuss aspects related to the ecological niche of the bacteria with emphasis on the characteristics involved in the biosafety of the use of B. thuringiensis-based products for biological control of target insects

Genetic Differentiation between Sympatric Populations of Bacillus cereus and Bacillus thuringiensis

Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cereus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species—B. thuringiensis or B. cereus—were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions

Seleção e caracterização molecular de isolados de Bacillus thuringiensis para o controle de Spodoptera spp.

Resumo:O objetivo deste trabalho foi selecionar e caracterizar molecularmente isolados de Bacillus thuringiensistóxicos a Spodoptera eridaniaeS. frugiperda. Trinta e quatro isolados foram submetidos ao bioensaio, dos quais três foram selecionados e usados para a estimativa da CL50. Os isolados selecionados não diferiram da linhagem padrão HD-1. Na caracterização molecular, identificou-se a presença dos genes cry1 e cry2, nos isolados BR37 e BR94, e dos genes cry4A, cry4B, cry10, cry11 e cyt1 no isolado BR58, o que confirmou o perfil proteico obtido de 130, 70 e 65 kDa. Foram identificados cristais bipiramidais e esféricos. O isolado BR58, apesar de não conter os genes relacionados à toxicidade a Lepidoptera, causa mortalidade em ambas as espécie

Isolation, morphological and molecular characterization of Bacillus thuringiensis strains against Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae)

The coffee berry borer Hypothenemus hampei Ferrari, 1876 (Coleoptera: Curculionidae: Scolytinae) is considered the most serious pest of the coffee crop and is controlled primarily with the use of chemical insecticides. An alternative to this control method is the use of the entomopathogenic bacterium, Bacillus thuringiensis Berliner, 1911. Therefore, the objective of this work was to select strains of B. thuringiensis virulent against H. hampei and characterize them by morphological and molecular methods to identify possible genes for the production of genetically modified plants. To achieve this objective, 34 strains of B. thuringiensis underwent a selective bioassay to evaluate their toxicity to H. hampei first-instar larvae. Among the strains tested, 11 and the standard B. thuringiensis subspecies israelensis (IPS-82) caused mortality above 90%. Then, the median lethal concentration (LC50) was estimated for these strains followed by characterization using morphological, biochemical and molecular methods. The lowest LC50 was obtained for strain BR58, although this concentration did not differ significantly from that of the standard strain IPS-82 or from that of strains BR137, BR80 and BR67. The molecular characterization detected cry4A, cry4B, cry10, cry11 and cyt1 genes in 10 of the most virulent strains (BR58, BR137, BR80, BR81, BR147, BR135, BR146, BR138, BR139, BR140). Strain BR67 differed completely from the others and amplified only the cry3 gene. This strain was more virulent than BR135, BR146, BR138, BR139 and BR140, but it did not differ from BR58, BR137, BR80, BR81 and BR147. The protein profile revealed proteins of 28, 65, 70 and 130 kDa, and the morphological analysis identified spherical crystalline inclusions in all strains. The results showed that the 11 strains studied have potential for use as a gene source for insertion into coffee plants for the control H. hampei, especially the cry3, cry4A, cry4B, cry10, cry11 and cyt1 genes, that were repeated in the most virulent isolates. Keywords: Biological control, Entomopathogenic bacteria, Coffee berry borer, Cry protein