Analysis of Insertion into Secondary Attachment Sites by Phage λ and by int Mutants with Altered Recombination Specificity

Abstract

When phage l 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 l and by l 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.Published by Elsevier Science Ltd

Challenging a Paradigm: the Role of DNA Homology in Tyrosine Recombinase Reactions

“…All the secondary sites examined have the first three bases of the left side of the overlap region conserved. The remaining bases to the right are not conserved, probably because during phage integration into a secondary site, the HJ intermediate that is formed by top-strand exchange is resolved by chromosome replication or by RuvC instead of Int (71). Thus, it was accepted for several years that sequence identity was essential for branch migration of the junction.…”

Role of Secondary Attachment Sites in Changing the Specificity of Site-Specific Recombination

“…To test part of this prediction, we measured the effect of overlap region identity in integrative and excisive recombination involving attP and attB* sites. Although previous work strongly implies that overlap region identity promotes recombination involving secondary sites, the effect has not been quantified (5). Second, Int will adapt to the new target by accumulating mutations that increase both recombination frequency and specificity.…”

“…To test this prediction, we used Int mutants with altered specificities. These mutants increase recombination between att sites of the -related phage HK022 and change secondary site utilization by attP (3,5). Third, the attR* transducing phage will retain the ability to reinsert by Int-promoted recombination at the attB* site from which it came.…”

“…1. The chromosome jumping model (5). Phage insertion at a secondary site (attB*) is followed by the formation of an attR* transducing phage.…”

Insertion Site Occupancy by stx 2 Bacteriophages Depends on the Locus Availability of the Host Strain Chromosome

“…To achieve this, phage integrase, which is regulated by gene products activated in the lysogenic pathway, and host recombinases (e.g., integration host factors), which are not regulated by any phage gene, must recognize homologous sequences present in the phage DNA and in the bacterial genome (attP and attB, respectively) (14,26).…”