Proteinaceous and genomic characterization of Bacillus thuringiensis isolates collected in the Tennessee geographical region.

摘要

Bacillus thuringiensis is a soil growing bacterium that has one or more delta-endotoxin (cry) gene. These genes encode for multi-domain globular proteins which are entomopathogenic. In this study, molecular and physiological properties of cry genes and their products are being analyzed. Characterization of the different subspecies of Bacillus thuringiensis depends essentially on PCR profiles and Polyacrylamide gel studies. It is believed, that there is a direct correlation between the PCR analysis and crystal protein profile. Over 25 isolates of this bacterium were obtained from Middle Tennessee by the sodium acetate medium technique. DNA was extracted by phenol/chloroform/isoamyl alcohol method, precipitated with ice-cold ethanol, and quantified by a spectrophotometer. The genes were amplified by polymerase chain reaction (PCR), cloned and then sequenced. Alignment studies were performed on the genes using the National Center of Biotechnological Information database (NCBI). This sequencing data aligned to specific locations within the genome of previously collected Bacillus thuringiensis strains, suggesting genesis. Crystal shapes were studied by growing the cells to stationary phase at the agitation speeds of 250 rpm in Luria Bertani medium, a semi synthetic medium. The spore/crystal mixtures were harvested by centrifugation at 10,000g at 4°C, and six 10 minutes wash cycles in ice-cold deionized water. Spores and crystals were separated on a discontinuous sucrose gradient by ultracentrifugation at 80,000g. The separation was confirmed by examination of smears by polarized light microscopy. Protein concentration was determined spectrophotometrically by the biuret reagent method using Bovine Serum Albumin (BSA) as the standard. The separated crystals at 100mg were subjected to three states: solubilized, insolubilized, and native. Each state was analyzed by electrophoresis techniques to identify band patterns. These two techniques showed that there were correlation between gene presence and protein profile.; The compilation of the data suggest that 14 of the 25 resembled Bacillus thuringiensis subspecies kurstaki, 4 of the 25 resembled Bacillus thuringiensis subspecies israelensis while just 1 of the 25 resembled Bacillus thuringiensis subspecies tenebrionis.