Analysis of insertion into secondary attachment sites by phage lambda and by int mutants with altered recombination specificity.
When phage lambda lysogenizes a cell that lacks the primary bacterial attachment site, integrase catalyzes insertion of the phage chromosome into one of many secondary sites. Here, we characterize the secondary sites that are preferred by wild-type lambda and by lambda int mutants with altered insertion specificity. The sequences of these secondary sites resembled that of the primary site: they contained two imperfect inverted repeats flanking a short spacer. The imperfect inverted repeats of the primary site bind integrase, while the 7 bp spacer, or overlap region, swaps strands with a complementary sequence in the phage attachment site during recombination. We found substantial sequence conservation in the imperfect inverted repeats of secondary sites, and nearly perfect conservation in the leftmost three bases of the overlap region. By contrast, the rightmost bases of the overlap region were much more variable. A phage with an altered overlap region preferred to insert into secondary sites with the corresponding bases. We suggest that this difference between the left and right segments is a result of the defined order of strand exchanges during integrase-promoted recombination. This suggestion accounts for the unexpected segregation pattern of the overlap region observed after insertion into several secondary sites. Some of the altered specificity int mutants differed from wild-type in secondary site preference, but we were unable to identify simple sequence motifs that account for these differences. We propose that insertion into secondary sites is a step in the evolutionary change of phage insertion specificity and present a model of how this might occur.
Rutkai
Edit
E
Bay Zoltán Institute for Biotechnology, Derkovits Faser 2, H-6726 Szeged, Hungary.
Dorgai
László
L
Sirot
Regina
R
Yagil
Ezra
E
Weisberg
Robert A
RA
D001287:Attachment Sites, Microbiological / Q000502:physiology*; D010582:Bacteriophage lambda / Q000235:genetics / Q000502:physiology*; D014644:Genetic Variation; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011995:Recombination, Genetic
D019426:Integrases