Characterization of XerC‐ and XerD‐dependent CTX phage integration in Vibrio cholerae

Abstract

Summary CTXφ is a filamentous bacteriophage that encodes cholera toxin and integrates site‐specifically into the larger of the two Vibrio cholerae chromosomes. The CTXφ genome lacks an integrase; instead, its integration depends on the chromosome‐encoded tyrosine recombinases XerC and XerD. During integration, recombination occurs between regions of homology in CTXφ and the V. cholerae chromosome. Here, we define the elements on the phage genome (attP ) and bacterial chromosome (attB) required for CTXφ integration. attB is a short sequence composed of one binding site for XerC and XerD spanning the site of recombination. Together, XerC and XerD bind to two sites within attP. While one XerC/D binding site in attP spans the core recombination region, the other site is ≈ 80 bp away. Although integration occurs at the core XerC/D binding site in attP, the second site is required for CTXφ integration, suggesting it performs an architectural role in the integration reaction. In vitro cleavage reactions showed that XerC and XerD are capable of cleaving attB and attP sequences; however, additional cellular processes such as DNA replication or Holliday junction resolution by a host resolvase may contribute to integration in vivo.

Molecular keys of the tropism of integration of the cholera toxin phage

“…1B). Two models have been proposed for CTXϕ integration at dif1 (8,9). In the first model, XerCD would catalyze the formation of a Holliday junction between the host dimer resolution sites and the dsDNA form of attP1, which is found on the replicative form of the phage (8).…”

“…Two models have been proposed for CTXϕ integration at dif1 (8,9). In the first model, XerCD would catalyze the formation of a Holliday junction between the host dimer resolution sites and the dsDNA form of attP1, which is found on the replicative form of the phage (8). In this model, attP2 is thought to play a structural role in stabilizing the synapse and/or the exchange; this assumption makes it difficult to explain how mutations in its overlap region could influence the specificity of integration.…”

“…1B). This observation led to the proposal of two integration models (8,9). The first model predicts that recombination occurs between dif1 and attP1 as the result of an unknown architectural function played by attP2.…”

Efficiency and specificity of CTXϕ chromosomal integration: dif makes all the difference

“…CTXϕ is a small, positive, single-stranded DNA [(+) ssDNA)] virus that can be found either in a replicative form or, more commonly, integrated sitespecifically in the host genome to form stable lysogens. Whether the ssDNA or dsDNA form of the virus is the substrate for recombination and integration between the dsDNA host genome and CTXϕ is open to debate (5,6). Many different CTXϕ types and arrangements have been observed in the host V. cholerae genome (7).…”

“…Subsequent to CTXϕ particle adsorption to the V. cholerae cell wall, viral ssDNA is injected into the cell cytoplasm and forms a circular pCTX, which then integrates into the V. cholerae genome at a site-specific attachment site (4,9). CTXϕ integration requires a number of phage-encoded and host-encoded factors (5,9). A recombinase (integrase), which ordinarily catalyzes this integration in other phages, is not present in the CTXϕ genome; instead, it commandeers two host-encoded tyrosine recombinases, XerC and XerD (9).…”