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

B acteriophage can convert their bacterial host from a nonpathogenic form to a pathogenic form by providing the bacterium with virulence genes, in a process called lysogenic phage conversion. Vibrio cholerae is a bacterium prevalent in marine environments that can infect humans to cause the devastating diarrheal disease cholera, which is endemic in much of Asia and Africa. Although the health and economic burdens of cholera are enormous, the disease is sometimes overshadowed by other diseases, but cholera’s predilection for epidemic spread commands attention. Cholera toxin, an A-B type exotoxin encoded by the ctxAB genes, is the main cause of the voluminous watery diarrhea that is characteristic of cholera (1–3). V. cholerae isolates that cause cholera encode the ctxAB genes in the genome of a filamentous bacteriophage CTXφ (Fig. 1A) (4). 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). The basis of specificity and efficiency of integration of CTXφ is not known, nor is the mechanism of emergence of strains with novel CTXφ chromosomal arrangements. In PNAS, a report by Das et al. resolves many of the questions surrounding the mechanism of CTXφ integration and the capacity of variant CTXφ genomes to integrate at each of the known attachment sites (att) (8). Like many bacteriophage, CTXφ integrates its genome into the chromosome of a host V. cholerae, thereby ensuring stable vertical transmission within the bacterial host. 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). The XerCD proteins are conserved among eubacteria, as they serve to resolve chromosome dimers during cell division (10, 11). In Escherichia coli, XerCD proteins bind and catalyze recombination at homologous 28-bp dif sites, composed of two 12-bp binding sites for XerC and XerD separated by a 6-bp spacer or overlap region, which allows for XerC-XerD interactions that ensure stable synapsis (10, 11). Because V. cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II) (12, 13), two dif sites are present, dif1 in chromosome I and dif2 in chromosome II (9); similar to E. coli, the same FstKdependent mechanism coordinates dimer resolution on each chromosome with cell division (14). The dif1 site differs from dif2 at four polymorphic sites, one of which is located in the XerC binding site and the other three sites are located in the 6-bp spacer region (9, 14). The arrangement of CTXφ in the V. cholerae genome depends on whether it is integrated at one or two of the chromosome dimer resolution sites, dif1 and dif2, and the number of copies present at each site. For example, in many El Tor strains, the cause of the seventh and ongoing cholera pandemic, the El Tor phage CTXφ is arranged in tandem and interspersed with a related element, RS1, to give an RS1-CTX-RS1-CTX-RS1 arrangement on chromosome I. The El Tor Strain C6709, isolated in Peru in 1991, encodes a CTX-RS1 arrangement on chromosome I (7, 15). In V. cholerae CTXφ ssDNA 7 kb