Isolation and Characterization of Phages from Bacillus thuringiensis (Bt) Type Strains and Bt 1-3

Abstract

Bacillus thuringiensis (Bt) is a Gram-positive, soil-dwelling, spore-forming and rod-shaped bacterium that produces crystal proteins, which demonstrate toxicity towards Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera, and Mallophaga insect orders and to nematodes, mites, and protozoa. Bt is the most common environment-friendly insecticide, substituting chemical insecticides. However, the number of Bt subspecies infected by lysogenic phages is approximately 83% of the total subspecies. During the fermentation of Bt for producing spores and crystals, lysogenic phages result in the reduction of the formation of spores and crystals, up to 15-30% of the phage-free Bts yield, sometimes 50-80%. At worst, no spores and crystals can be observed due to the infection of lysogenic phages. To examine the existence of phages, phage DNAs were extracted from 65 Bt type strains by a ZnCl2-mediated precipitation method or boiling Bt cultures. Detection of phage DNAs was carried out by PCR with terminase small subunit (TerS) specific-primers. Ten phage DNAs were detected in Bt type strains by the PCR. The alignment of the deduced amino acid sequence of these phages revealed that they had >70% homology, possibly belong to Bacillus phage. The other 12 phage DNAs were not amplified by the PCR although their DNAs were observed in the electrophoresis. The phages from 22 Bt type strains were concentrated by ultracentrifugation, stained with 1% phosphotungstic acid, and observed a transmission electron microscope (TEM). They had a long tail and symmetrical head. Among Bt type stains, Bt subsp. aizawai has been widely used in many countries. To investigate if Bt subsp. aizawai (Bta) isolates from 3 countries (United States, Japan and South Korea) were infected by phages, a PCR was conducted with the TerS specipic-primers. Phage DNAs were amplified by the PCR in all the Bta isolates. These results showed that possibly Bta isolates from United States, Japan and South Korea were infected by phages. Phage 1-3 was isolated from Bt 1-3 strain that belongs to Bta. Phage 1-3 had a linear dsDNA, from the analysis of the DNA with the treatment of Exonuclease III and S1 nuclease. Phage 1-3 had a symmetrical head (60 × 50 nm) and long tail (140 × 10 nm). To prove the hypothesis that phage 1-3 DNA may be existed in circular form in the host, circular DNA from Bt 1-3 was cloned by plasmid capture system (PCS). Twenty one phage clones were acquired by the PCS. It suggested that the DNA existed in a form of plasmid in Bt 1-3. Phage 1-3 genome was 46, 517 bp long with 35.43% G + C content and 83% coding region. Sixty five open reading frames (ORFs) were found in the phage 1-3 genome. Sixty five ORFs included 9 ORFs for structural proteins, 16 ORFs for infection and replication and 5 ORFs for scaffold proteins. A phylogenetic tree of the phage 1-3 was constructed based on the DNA sequences of complete genome from the 11 Bacillus phages. Phage 1-3, TP21-L and phi29 were in the same linkage. Among them, phi29 was the nearest to phage 1-3. It suggests that phage 1-3, TP21-L and phi29 have a common ancestor or the same transition in the process of evolution. A dot plaque assay showed the infectivity of phage 1-3 toward two Bt type strains, Bt subsp. galleriae and japonensis. These phages from Bt type strains and Bt 1-3 strain will be expect to have various applications (vector, antibacterial agent, etc) in near the future.