Isolation and characterization of Bacillus thuringiensis bacteriophages using electron microscope and RAPD•PCR [electronic resource].

Includes references.

Five bacteriophages have been isolated from soil able to propagate 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 witl:llong 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.Q for• ampicilline resistance gene. The phages were able also to perform transduction between B. thuringiensis strains, Random amplified polymorphic DNA polymerase chain reaction (RAPo-PCR) was performed to produce unique and reproducible band patterns in the five different baderiophages. Among using 20 RAPO primers, only seven produced polymorphic fragments with an average of 7.9 fragment! 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-OS produced the lowest number. The oligonucleotide OPD-07 presented the highest number of unique fragment! (8) in all isolated bacteriophages while, OPC-08 and OPD-05 primers presented the lowest number (one fragment). All isolated baderiophages except for R3 baderiophage were distinguishable by unique RAPO markers. The highest number of unique fragments (11) after using all primers was deteded in AS baderiophage followed by A1baderiophage which deteded by 10 unique fragments. A2 baderiophage was deteded by two unique fragments: approximately 170bp with OPC-20 and 1200bp with OPO-05 and R8 baderiophage was detected by two unique fragments with OPO-07; approximately 345bp and 570bp. The highest similarity value (0.828) was found between A2 and R8 baderiophages and the lowest value (0.313) was found between A1 and R8 baderiophages. This study demonstrates the effediveness of RAPO as a technique for identifying characterization isolated baderiophage from nature and may be useful in fingerprinting. Moreover, the phages isolated from soil in this study can be used as potential cloning vedors.

Summary in Arabic.

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