Control of Phage Bxb1 Excision by a Novel Recombination Directionality Factor
Figure 1
Identification of the Bxb1 RDF
(A) The hygromycin-resistance cassette, sucrose-sensitivity cassette (sacB), and a portion of the Bxb1 DNA containing attP and int were cloned into an integrative plasmid containing the plasmid ColE1 origin of E. coli (OriE) , and the resultant plasmid pPGA1 was transformed into M. smegmatis mc2155. Expression of int drives integration of the plasmid into the host attB site leading to the formation of attL and attR sites flanking the hyg and sacB cassettes. The resultant strain, designated as the excision tester strain, is resistant to hygromycin and sensitive to the presence of sucrose. An excisive recombination event between attL and attR results in the removal (and subsequent loss) of the intervening DNA containing int, hyg, and sacB; the strain consequently becomes hygS and sucR and can thereby be monitored by the appearance of colonies in the presence of sucrose.
(B) A segment of the Bxb1 genome containing genes involved in integration and DNA replication as well as the corresponding portion of the phage L5 genome is shown in the upper part; related genes are colored accordingly. The lower part of the figure shows the region of Bxb1 DNA present in plasmids exhibiting excision activity. Following isolation of plasmid pPGX1—which is active in promoting sucrose resistance in the excision tester strain shown in (A)—plasmid derivatives containing deletions from both ends of pPGX1 were constructed, introduced into the excision tester strain, and scored for the appearance of sucR colonies.
CFU, colony-forming units