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Anthony Costa

Advisor: Theresa Koehler, Ph.D.

B. thuringiensis subsp. kurstaki HD1-9 harbors the bacteriophage TP21 as an approximately 49-kb plasmidial prophage. B. thuringiensis is a member of the Bacillus cereus group of species and is closely related to Bacillus cereus and Bacillus anthracis. These species have been reported to exchange DNA by a conjugation-like mechanism and by bacteriophage CP-51 mediated transduction. Here we report transduction of chromosomal markers and the naturally-occurring tetracycline-resistance plasmid pBC16 by TP21 within and between these species. TP21 lysogens of B. cereus and B. anthracis were generated in strains cured of other resident phage and plasmids. Transducing lysates containing approximately 1.0×109 and 1.0×1010 plaque-forming units (PFU)/ml were obtained from B. anthracis and B. cereus lysogens respectively following induction by mitomycin C. Phage lysates from B. cereus mediated intraspecies transduction of three unlinked chromosomal markers with frequencies ranging from 1.1×10-7 to 6.6×10-6 transductants/PFU. The similarity in transduction frequencies for all markers tested suggests a generalized rather than a specialized mechanism of transduction by TP21 within B. cereus. Southern hybridization and restriction analysis of transductants did not reveal the presence of the TP21 prophage in the chromosome. Phage lysates from B. anthracis mediated interspecies transduction of two chromosomal markers to B. cereus, and lysates from B. cereus transduced one marker to B. anthracis. TP21 transduced pBC16 (4.6 kb) from B. cereus and B. anthracis donor strains to B. cereus recipients with frequencies ranging from 2.8×10-6 to 6.0×10-5 transductants/PFU, and to B. anthracis recipients with frequencies ranging from 1.6×10-7 to 3.4×10-6 transductants/PFU. Restriction enzyme digestion analysis indicated that TP21 phage DNA is linear, circularly permuted and terminally redundant. A model for packaging was developed indicating that TP21 packages concatemeric phage DNA by a headful mechanism initiated at an origin of packaging (or pac) site and that no more than three to four headfuls are packaged per concatamer. There is also evidence for the presence of a second, low-affinity pac site on the phage genome. TP21 thus exhibits structural similarities with the generalized transducing phage P1 of Escherichia coli, which is also maintained as a plasmidial prophage in its hosts.

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Research Info

Bacteriophage TP21: Studies of Generalized Transduction in Bacillus cereus Group Species and Physical Analysis of the Genome