In vitro and in vivo proteolysis of the Bacillus thuringiensis subsp. israelensis CryIVD protein by Culex quinquefasciatus larval midgut proteases

1993 ◽  
Vol 23 (2) ◽  
pp. 273-283 ◽  
Author(s):  
Shu-mei Dai ◽  
Sarjeet S. Gill
Keyword(s):  
Bacillus Thuringiensis ◽  
Culex Quinquefasciatus ◽  
Larval Midgut ◽  
Bacillus Thuringiensis Subsp

THE PRODUCTION BY BACILLUS THURINGIENSIS BERLINER OF A HEAT-STABLE SUBSTANCE TOXIC FOR INSECTS

1959 ◽  
Vol 5 (2) ◽  
pp. 161-168 ◽  
Author(s):  
Ellicott McConnell ◽  
A. Glenn Richards
Keyword(s):  
Bacillus Thuringiensis ◽  
Inclusion Bodies ◽  
Galleria Mellonella ◽  
Culture Media ◽  
Water Soluble ◽  
Broth Culture ◽  
Toxic Principle ◽  
Heat Stable

Bacillus thuringiensis Berliner produces in vitro a heat-stable, dialyzable substance which is toxic for insects when injected. The same or a similar substance is produced in vivo. The toxic principle is of unknown composition. It is heat-stable, water-soluble, dialyzable, and resistant to low temperatures. It is probably neither a protein nor a lipid. Clearly it is distinct from the heat-labile inclusion bodies and from lecithinase. Growth-curve studies showed that the heat-stable toxin appeared in liver broth cultures during the active growth phase, prior to the formation of spores or inclusion bodies. An attempt to produce the toxic principle from culture media in the absence of bacteria was unsuccessful from sterile inocula both from in vivo and in vitro sources. The LD50 for larvae of Galleria mellonella injected with autoclaved supernatant from a 10-day-old liver broth culture of B. thuringiensis was determined to be 0.00036 ml per larva or 0.002 ml per gram of larvae. Approximately the same level of toxicity was found for another caterpillar, a fly larva, and cockroaches. After larvae of Galleria or Pyrausla have been dead for more than 2 days from infection with B. thuringiensis the bacillus could no longer be recovered. A sublethal amount of the heat-stable toxin injected into old larvae of Galleria delayed emergence of the adults by 30 to 40%. The non-pathogenic Bacillus cereus was found to produce a similar-acting, heat-stable toxin under the same conditions that one is produced by B. thuringiensis.

scholarly journals A Bacillus thuringiensis Chitin-Binding Protein is Involved in Insect Peritrophic Matrix Adhesion and Takes Part in the Infection Process

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 252
Author(s):  
Jiaxin Qin ◽  
Zongxing Tong ◽  
Yiling Zhan ◽  
Christophe Buisson ◽  
Fuping Song ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Galleria Mellonella ◽  
Binding Protein ◽  
Insect Pest ◽  
Infection Process ◽  
Peritrophic Matrix ◽  
Limited Information ◽  
Chitin Binding Protein

Bacillus thuringiensis (Bt) is used for insect pest control, and its larvicidal activity is primarily attributed to Cry toxins. Other factors participate in infection, and limited information is available regarding factors acting on the peritrophic matrix (PM). This study aimed to investigate the role of a Bt chitin-binding protein (CBPA) that had been previously shown to be expressed at pH 9 in vitro and could therefore be expressed in the alkaline gut of lepidopteron larvae. A ∆cbpA mutant was generated that was 10-fold less virulent than wild-type Bt HD73 towards Ostrinia furnacalis neonate larvae, indicating its important role in infection. Purified recombinant Escherichia coli CBPA was shown to have a chitin affinity, thus indicating a possible interaction with the chitin-rich PM. A translational GFP–CBPA fusion elucidated the localization of CBPA on the bacterial surface, and the transcriptional activity of the promoter PcbpA was immediately induced and confirmed at pH 9. Next, in order to connect surface expression and possible in vivo gut activity, last instar Galleria mellonella (Gm) larvae (not susceptible to Bt HD-73) were used as a model to follow CBPA in gut expression, bacterial transit, and PM adhesion. CBPA-GFP was quickly expressed in the Gm gut lumen, and more Bt HD73 strain bacteria adhered to the PM than those of the ∆cbpA mutant strain. Therefore, CBPA may help to retain the bacteria, via the PM binding, close to the gut surface and thus takes part in the early steps of Bt gut interactions.

scholarly journals Identification and Characterization of the Bacillus thuringiensis phaZ Gene, Encoding New Intracellular Poly-3-Hydroxybutyrate Depolymerase

2006 ◽  
Vol 188 (21) ◽  
pp. 7592-7599 ◽  
Author(s):  
Chi-Ling Tseng ◽  
Hui-Ju Chen ◽  
Gwo-Chyuan Shaw
Keyword(s):  
Bacillus Thuringiensis ◽  
Wild Type ◽  
Gene Encoding ◽  
Significant Similarity ◽  
Phb Depolymerase ◽  
New Type ◽  
Identification And Characterization

ABSTRACTA gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome ofBacillus thuringiensissubsp.israelensisATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designatedphaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD ofPseudomonas putidawas unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. TheB. thuringiensisPhaZ was inactive againstp-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-typeB. thuringiensiscells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from aphaZmutant lost this activity. ThephaZmutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S102-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate thatB. thuringiensisharbors a new type of intracellular PHB depolymerase.

scholarly journals Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae

2012 ◽  
Vol 443 (3) ◽  
pp. 711-717 ◽  
Author(s):  
Claudia Rodríguez-Almazán ◽  
Esmeralda Z. Reyes ◽  
Fernando Zúñiga-Navarrete ◽  
Carlos Muñoz-Garay ◽  
Isabel Gómez ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Aedes Aegypti ◽  
Lipid Bilayers ◽  
Insect Pests ◽  
Cry Toxins ◽  
Bacillus Thuringiensis Subsp ◽  
Limiting Step ◽  
Cry Toxin ◽  
Receptor Fragment

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7–11 (cadherin repeats 7–11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7–11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7–11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7–11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.

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