Molecular and morphological characterization of Bacillus thuringiensis isolates from middle Tennessee
Crystal staining is a differential staining method that is useful in the preliminary identification of Bacillus thuringiensis and was used in this project to screen for isolates of from the 57 isolates from Middle Tennessee. Crystalliferous isolates were further screened for the presence of cry genes by polymerase chain reaction (PCR). PCR-based identification of Bacillus thuringiensis toxin genes has become common place in most B. thuringiensis isolation and characterization initiatives. In this project a two-step PCR analysis of B. thuringiensis cry genes of a 57 crystalliferous stock was adapted and used for screening. Fifty seven B. thuringiensis strains were screened for the presence of cry genes. In this approach, five degenerate primers for detection of novel cry genes as well as known cry genes from Bacillus thuringiensis were used in a two-step PCR with these primers in five pair combinations. In the first step, only one of the primer pairs was used in the PCR, which allowed amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR was performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allowed both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates. Sequence analysis showed that samples 11, 26 and 34 are Bacillus thuringiensis cereus. The rest of the isolates are still undergoing evaluation for definitive identification.
Rendie Ellette McHenry,
"Molecular and morphological characterization of Bacillus thuringiensis isolates from middle Tennessee"
ETD Collection for Tennessee State University.