ISOLATION AND CHARACTERIZATION OF Bacillus thuringiensis BACTERIOPHAGES USING ELECTRON MICROSCOPE AND RAPD-PCR

Five bacteriophages have been isolated from soil able to propagate on Bacillus thuringiensis  bacteria, designated A1, A2, A8, R3 and R8. Phages A2, A8, R3 and R8 produced small turbid plaques but phage A1 produced relatively large clear plaques. Transmission electron microscopy showed great differences in the morphology of these phages. Four phages have icosahedral heads with long tails (A1, A8, R3 and R8). Phage A2 has icosahedral head with tailless. The phages have a broad host range, where successfully forming plaques on B. thuringiensis(6 isolates) and  Bacillus cereus bacteria. Five phages were able to mediate transduction between B. thurimgiensis and B. cereus with frequencies ranged from 1.32 to 1.8x10^-9 for ampicilline resistance gene. The phages were able also to perform transduction between B. thuringiensis strains. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was performed to produce unique and reproducible band patterns in the five different bacteriophages. Among using 20 RAPD primers, only seven produced polymorphic fragments with an average of 7.9 fragments per primer (ranging from approximately 135 to 1500 bp). The number of polymorphic fragments through each primer ranged from 6 to 14 fragments per primer. Primer OPD-07 produced the highest number of polymorphic fragments among the used primers while, primer OPC-08 produced the lowest number. The oligonucleotide OPD-07 presented the highest number of unique fragments (8) in all isolated bacteriophages while, OPC-08 and OPD-05 primers presented the lowest number (one fragment). All isolated bacteriophages except for R3 bacteriophage were distinguishable by unique RAPD markers. The highest number of unique fragments (11) after using all primers was detected in A8 bacteriophage followed by A1 bacteriophage which detected by 10 unique fragments. A2 bacteriophage was detected by two unique fragments; approximately 170bp with OPC-20 and 1200bp with OPD-05 and R8 bacteriophage was detected by two unique fragments with OPD-07; approximately 345bp and 570bp. The highest similarity value (0.828) was found between A2 and R8 bacteriophages and the lowest value (0.313) was found between A1 and R8 bacteriophages. This study demonstrates the effectiveness of RAPD as a technique for identifying characterization isolated bacteriophage from nature and may be useful in fingerprinting. Moreover, the phages isolated from soil in this study can be used as potential cloning vectors.