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FEMS Microbiol Lett ; 278(1): 115-20, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18028391

ABSTRACT

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 N-terminal region of a typical Cry5Adelta-endotoxin. The cry5Ad sequence includes homology blocks 1-5, which are present in most B. thuringiensisdelta-endotoxins. The usual C-terminal region of a Cry5Adelta-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.


Subject(s)
Antinematodal Agents , Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Cloning, Molecular , Endotoxins/genetics , Hemolysin Proteins/genetics , Amino Acid Motifs , Animals , Antinematodal Agents/chemistry , Antinematodal Agents/toxicity , Bacillus thuringiensis/chemistry , Bacillus thuringiensis Toxins , Bacterial Proteins/chemistry , Bacterial Proteins/toxicity , Bacterial Toxins/chemistry , Bacterial Toxins/toxicity , Endotoxins/chemistry , Endotoxins/toxicity , Escherichia coli/genetics , Haemonchus/drug effects , Haemonchus/growth & development , Hemolysin Proteins/chemistry , Hemolysin Proteins/toxicity , Larva/drug effects , Larva/growth & development , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/toxicity , Restriction Mapping
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