A novel delta-endotoxin gene cryIM from Bacillus thuringiensis ssp. wuhanensis (original) (raw)
Related papers
Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis
Biotechnology Letters, 2004
A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1177 amino acids compared to 1155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.
FEMS microbiology …, 2008
A new DNA sequence cry5Ad/orf2-5Ad (GenBank accession number EF219060) was isolated from Bacillus thuringiensis strain L366. This DNA sequence contains two ORFs: cry5Ad (a previously unreported member of the cry5A gene family) and orf2-5Ad. cry5Ad is unique among cry5A genes in that it encodes only the Nterminal region of a typical Cry5Ad-endotoxin. The cry5Ad sequence includes homology blocks 1-5, which are present in most B. thuringiensisd-endotoxins. The usual C-terminal region of a Cry5Ad-endotoxin (including homology blocks 6-8) is encoded by orf2-5Ad. Both proteins encoded by cry5Ad and orf2-5Ad were found in IPTG-induced Escherichia coli, after a copy of cry5Ad/orf2-5Ad was cloned into the pQE32 expression vector and transformed into pREP4 E. coli cells. Both proteins were also found in parasporal crystal inclusions of B. thuringiensis L366. Sequencing of cDNA derived from transformed E. coli cells showed that the two ORFs are transcribed as a single mRNA. Extracts prepared from the recombinant E. coli expressing Cry5Ad and Orf2-5Ad were not toxic to nematode larvae (Haemonchus contortus), indicating that these two proteins are most likely not responsible for the nematocidal activity seen previously in the B. thuringiensis strain L366.
MGG Molecular & General Genetics, 1994
A ~-endotoxin gene previously cloned from Bacillus thuringiensis subsp, galleriae has been shown by a combination of restriction mapping and DNA sequence analysis to be a cryIIB clone; in common with other cryIIB genes it was found to lack a functional promoter. Addition of a promoter resulted in expression of the gene in Bacillus thuringiensis but did not result in the formation of the crystalline inclusions normally associated with such toxins. Inclusion formation was only observed when the gene was incorporated into an operon containing a gene known to be involved in the crystallisation of another 6-endotoxin.
Pakistan Journal of Zoology, 2013
Bacillus thuringiensis (Bt) is endospore former, Gram positive bacterium and makes parasporal crystals (Cry proteins), which kill particular target pests of different crops. Bt isolated from different localities of Pakistan were screened for cry1Ca and cry1Cb domain III genes by polymerase chain reaction. Among all the screened Bt strains, five were positive for cry1C domain III gene, 3 of which were positive for cry1Ca and 2 for cry1Cb. Confirmation was done by colony PCR, restriction analysis, nucleotide sequencing and alignment on BLAST. The complete gene (3.6kb) encoding cry1Ca endotoxin of one of the Bt isolate (MS-SBS Bt1) was amplified and analyzed for the nucleotide sequence. The nucleotide and deduced amino acid sequences of endotoxin gene was compared with that of Bt strains available in the literature. The genes amplified from the positive strains had 99% similarity and had 100% same deduced amino acid with that of Bt strains reported in the Gene Bank. Full gene sequence was submitted in Gene Bank.
Journal of bacteriology, 1993
The cryIIIA gene encoding a coleopteran-specific toxin is poorly expressed in Bacillus thuringiensis when cloned in a low-copy-number plasmid. This weak expression is observed when the gene is cloned only with its promoter and its putative terminator. cryIIIA gene expression was analyzed by using deletion derivatives of a larger DNA fragment carrying the toxin gene and additional adjacent sequences. The results indicate that a 1-kb DNA fragment located 400 bp upstream of the promoter strongly enhances CryIIIA production in B. thuringiensis sporulating cells. Similar results were obtained when the low-copy-number plasmid pHT304 carrying transcriptional fusions between upstream regions of cryIIIA and the lacZ gene was used. Analysis of the start sites, the sizes, and the amounts of cryIIIA-specific mRNAs shows that the enhancement occurs at the transcriptional level by increasing the number of cryIIIA-specific transcripts from the onset of sporulation to about 6 h after the onset of s...
Fems Microbiology Letters, 2002
Expression of cry1Ia, one of the insecticidal protein genes of Bacillus thuringiensis subsp. kurstaki, was studied at the transcriptional level. By primer extension analysis, we have identified for the first time, the transcription start point of cry1Ia. Upstream from the cry1Ia transcription start point, was found a nucleotide sequence partially homologous to the consensus sequence for the E σE holoenzyme of Bacillus subtilis. Thus, it was strongly suggested that the identified cry1Ia promoter was under the control of σ35, the B. thuringiensis homolog of σE. The transcriptional activity from the promoter was detected only for the sporulating cells at T2 and T5 stages.
Protein Expression and Purification - PROTEIN EXPRESS PURIF, 2004
Achieving high-level expression of the Bacillus thuringiensis Cry4Aa mosquito-larvicidal protein was demonstrated. The 130-kDa Cry4Aa protoxin was overexpressed as an inclusion body in Escherichia coli under the control of the tac promoter together with the cry4Ba promoter. The solubility of the toxin inclusions in carbonate buffer, pH 10.0, was markedly enhanced at a cultivation temperature of 30°C. Elimination of the tryptic cleavage site at Arg-235 in the loop between helices 5 and 6 still retained the high-level toxicity of E. coli cells expressing the Cry4Aa mutant against Aedes aegypti larvae. Trypsin digestion of the R235Q mutant protoxin produced a protease-resistant fragment of ca. 65kDa. A homogeneous product of the 65-kDa trypsin-treated R203Q protein was obtained after size-exclusion chromatography that would pave the way for the further crystallisation and X-ray crystallographic studies.
Cry3Aa11: A New Cry3Aa δ-Endotoxin from a Local Isolate of Bacillus thuringiensis
Biotechnology Letters, 2005
A local isolate of Bacillus thuringiensis Mm2 had insecticidal activity against the larvae of Melolontha melolontha, Agelastica alni, Leptinotarsa decemlineata and Amphimallon solstitiale and produced a 65 kDa protein. SDS-PAGE profile of B. thuringiensis Mm2 was compared with those of 29 different Cry3Aa producers which verified Cry3Aa biosynthesis by the isolate. The cry3Aa gene of Mm2 was cloned, sequenced and the deduced amino acid sequence was compared with the cry3Aa sequences of ten different quaternary ranks. Its identity to these sequences ranged between 97.4% and 99.2%. The gene was next cloned into E. coli-Bacillus shuttle vector pNW33N and expressed at a low level in B. subtilis 168.