Purification and characterization of mosquitocidal Bacillus sphaericus BinA protein

Hire, Ramesh S.; Hadapad, Ashok B.; Dongre, T. K.; Kumar, Vinay

doi:10.1016/j.jip.2009.03.005

Cited by 31 publications

(17 citation statements)

References 41 publications

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“…Cry49Aa1 clearly shows significant similarity to Cry35Aa1, particularly in the core, β-sheet region of the C-terminal domain (Figure 7B). The high proportion of β-sheet structure in Cry35Ab1 is consistent with CD analysis of the related BinA protein that indicated a high proportion of β-sheet and little α-helix [48], [49], [50].…”

Section: Resultssupporting

confidence: 78%

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Kelker¹,

Berry

Evans³

et al. 2014

PLoS ONE

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Bacillus thuringiensis strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of ∼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a ∼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins.

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Section: Resultssupporting

confidence: 78%

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Kelker¹,

Berry

Evans³

et al. 2014

PLoS ONE

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“…Both components are co-transcribed from a single operon before the end of the exponential growth and sporulation as a crystal toxin [15]. The Bin protein sequences are extremely conserved between strains and only five variants have been reported to date with a few differences in their amino acid sequences [55]. In contrast, the 41.9-kDa-protein gene from the Spanish strains was found as a single coding sequence and its deduced amino acid sequence was poorly conserved among those of different Bt strains (34% maximum pairwise identity), in which 41.9-kDa related proteins were also found as single coding sequences.…”

Section: Discussionmentioning

confidence: 99%

Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

Palma

Muñoz

Berry

et al. 2014

Toxins

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In this work, we report the genome sequencing of two Bacillus thuringiensis strains using Illumina next-generation sequencing technology (NGS). Strain Hu4-2, toxic to many lepidopteran pest species and to some mosquitoes, encoded genes for two insecticidal crystal (Cry) proteins, cry1Ia and cry9Ea, and a vegetative insecticidal protein (Vip) gene, vip3Ca2. Strain Leapi01 contained genes coding for seven Cry proteins (cry1Aa, cry1Ca, cry1Da, cry2Ab, cry9Ea and two cry1Ia gene variants) and a vip3 gene (vip3Aa10). A putative novel insecticidal protein gene 1143 bp long was found in both strains, whose sequences exhibited 100% nucleotide identity. The predicted protein showed 57 and 100% pairwise identity to protein sequence 72 from a patented Bt strain (US8318900) and to a putative 41.9-kDa insecticidal toxin from Bacillus cereus, respectively. The 41.9-kDa protein, containing a C-terminal 6× HisTag fusion, was expressed in Escherichia coli and tested for the first time against four lepidopteran species (Mamestra brassicae, Ostrinia nubilalis, Spodoptera frugiperda and S. littoralis) and the green-peach aphid Myzus persicae at doses as high as 4.8 µg/cm2 and 1.5 mg/mL, respectively. At these protein concentrations, the recombinant 41.9-kDa protein caused no mortality or symptoms of impaired growth against any of the insects tested, suggesting that these species are outside the protein’s target range or that the protein may not, in fact, be toxic. While the use of the polymerase chain reaction has allowed a significant increase in the number of Bt insecticidal genes characterized to date, novel NGS technologies promise a much faster, cheaper and efficient screening of Bt pesticidal proteins.

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“…E. coli BL21(DE3) pLysS containing pCA24N-crp plasmid (27) (Table 1) was used to obtain the purified CRP using a protocol essentially as described in Hire et al (23). CRP protein was induced by the addition of 0.2 mM isopropyl-␤-D-thiogalactopyranoside (IPTG), and the bound protein was eluted using 50 to 500 mM imidazole gradient.…”

Section: Methodsmentioning

confidence: 99%

Cyclic AMP Receptor Protein Regulates cspE , an Early Cold-Inducible Gene, in Escherichia coli

et al. 2011

Self Cite

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cspE, a member of the cspA family of cold shock proteins in Escherichia coli, is an early cold-inducible protein.The nucleic acid melting ability and transcription antiterminator activity of CspE have been reported to be critical for growth at low temperature. Here, we show that the cyclic AMP receptor protein (CRP), a global regulator involved in sugar metabolism, upregulates cspE in E. coli. Sequence analysis of the cspE upstream region revealed a putative CRP target site centered at ؊61.5 relative to the transcription start. The binding of CRP to this target site was demonstrated using electrophoretic mobility shift assays. The presence of this site was shown to be essential for P cspE activation by CRP. Mutational analysis of the binding site indicated that the presence of an intact second core motif is more important than the first core motif for CRP-P cspE interaction. Based on the promoter architecture, we classified P cspE as a class I CRP-dependent promoter. This was further substantiated by our data demonstrating the involvement of the AR1 domain of CRP in P cspE transcription. Furthermore, the substitutions in the key residues of the RNA polymerase ␣-subunit C-terminal domain (␣-CTD), which are important for class I CRP-dependent transcription, showed the involvement of 265 and 287 determinants in P cspE transcription. In addition, the deletion of crp led to a growth defect at low temperature, suggesting that CRP plays an important role in cold adaptation.Escherichia coli K-12 contains nine paralogs of CspA, CspA to CspI, collectively known as the CspA family of cold shock proteins (CSPs). Members of this family are small proteins consisting of a nucleic acid binding domain called the cold shock domain (CSD), one of the most evolutionarily conserved domains found in various life forms, including bacteria, plants, and animals (19,47). In spite of the high degree of similarity among them, only five (cspA, cspB, cspE, cspG, and cspI) are induced during cold shock (20,42). cspC is constitutively expressed at 37°C, while cspD is induced during starvation (49).CspA and some of its homologues share nucleic acid melting ability (25) and transcription antitermination activity (3), two functions that are important for cold adaptation (35). A balanced level of these proteins seems to be important for cold adaptation (48). Among the nine members of this family, CspE performs functions which are important during both cold shock and diverse physiological conditions, including chromosome partitioning/condensation (24, 50), growth in cold (30), UV sensitivity (29), and the regulation of various cold-induced genes (36). cspE expression has been found to be regulated in a growth phase-dependent manner (2). Earlier reports suggested that interplay exists between the cellular metabolic status and cold shock response (13,26,43). Additionally, global gene expression profiling has indicated that many cold-induced genes are subject to catabolite repression (18,34). Cyclic AMP (cAMP) receptor protein (CRP), also known as cata...

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Purification and characterization of mosquitocidal Bacillus sphaericus BinA protein

Cited by 31 publications

References 41 publications

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Structural and Biophysical Characterization of Bacillus thuringiensis Insecticidal Proteins Cry34Ab1 and Cry35Ab1

Draft Genome Sequences of Two Bacillus thuringiensis Strains and Characterization of a Putative 41.9-kDa Insecticidal Toxin

Cyclic AMP Receptor Protein Regulates cspE , an Early Cold-Inducible Gene, in Escherichia coli

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