VGJphi integration and excision mechanisms contribute to the genetic diversity of Vibrio cholerae epidemic strains

Abstract

Most strains of Vibrio cholerae are not pathogenic or cause only local outbreaks of gastroenteritis. Acquisition of the capacity to produce the cholera toxin results from a lysogenic conversion event due to a filamentous bacteriophage, CTX. Two V. cholerae tyrosine recombinases that normally serve to resolve chromosome dimers, XerC and XerD, promote CTX integration by directly recombining the ssDNA genome of the phage with the dimer resolution site of either or both V. cholerae chromosomes. This smart mechanism renders the process irreversible. Many other filamentous vibriophages seem to attach to chromosome dimer resolution sites and participate in the rapid and continuous evolution of toxigenic V. cholerae strains. We analyzed the molecular mechanism of integration of VGJ, a representative of the largest family of these phages. We found that XerC and XerD promote the integration of VGJ into a specific chromosome dimer resolution site, and that the dsDNA replicative form of the phage is recombined. We show that XerC and XerD can promote excision of the integrated prophage, and that this participates in the production of new extrachromosomal copies of the phage genome. We further show how hybrid molecules harboring the concatenated genomes of CTX and VGJ can be produced efficiently. Finally, we discuss how the integration and excision mechanisms of VGJ can explain the origin of recent epidemic V. cholerae strains.

Fig. 1.

Two different types of lysogenic phages that hijack the dimmer resolution machinery of their host. (A) The attachment region of the genome of CTXɸ-related phages contains two dif-like sites, whereas the attachment region of the genome of VGJɸ-related phages contains a single dif-like site. The genome of filamentous phages known to integrate in the vicinity of chromosome dimer resolution sites was scanned for the presence of dif-like sites. The corresponding regions were then aligned to the sequences of the (+) strand of El Tor CTXɸ attP or to the putative VGJɸ attP region. Dots indicate identical bases. Vc, V. cholerae; Vf, V. fischeri; Vm, V. mimicus; Yp, Yersinia pestis; Ec, E. coli; Vp, V. parahaemolyticus; El Tor, El Tor variants of CTXɸ; classical, classical variants of CTXɸ; GGT, G variants of CTXɸ; TAT, CTXɸ variants found in V. fisheri and V. mimicus. References: El Tor, classical and GGT, (8); TAT (23, 24); Ypfɸ and CUS-1ɸ (9); VGJɸ (15); VEJɸ (12); VSKK (AF452449); VSK (AF453500); fs2 (25); f237 (AP000581); VfO3K6 (AB043678); Vf33 (AB012573); and Vf12 (AB012574). (B) Double-stranded scheme of V. cholerae dif1 and of the related sites found in the RF of El Tor CTXɸ (attP1-attP2), in the (+) ssDNA genome of El Tor CTXɸ [attP(+)], and in the RF of VGJɸ (VGJɸ attP). XerC cleavage positions are indicated by triangles. The bases that are involved in the stabilization of the strand exchanges catalyzed by XerC between dif1 and El Tor CTXɸ attP(+) are shown in color, with those of the strand liberated on XerC-mediated cleavage in red and those of the complementary partner strand in blue. Homologous bases found in the overlap regions of El Tor CTXɸ attP1 and attP2 and VGJɸ attP are highlighted similarly. El Tor CTXɸ attP1 and attP2 are connected by a 90-bp DNA sequence, which is indicated as a dotted line. (C) Scheme of the XerC- and XerD-dependent mechanism of lysogenic conversion of V. cholerae by CTXɸ. Chromosomal DNA strands are shown as continuous lines, and the (+) ssDNA genome of the phage is represented by a dotted line. The strands of the bacterial attachment site, dif1, and of the phage attachment site, CTXɸ attP(+), which are exchanged after XerC cleavage, are indicated in red and blue, respectively. XerD and XerC are represented as light-gray and dark-gray figures, respectively. The XerC/DNA phosphotyrosyl linkage is depicted by a black dot.

Fig. 2.

XerC- and XerD-dependent recombination of VGJɸ attP with dif1 and CTXɸ attP(+). (A) Schemes of the Watson–Crick base-pair interactions that could stabilize the strand exchange catalyzed by XerC between the overlap regions of the two dimer resolution sites of N16961, dif1 and dif2, and the dif-like site found in the RF of VGJɸ, attP^VGJ. The strand cleaved by XerC on dif1 and dif2 is shown in red, and the equivalent strain in attP^VGJ is shown in blue. The positions of the the XerC/DNA phosphotyrosyl linkages are indicated by black dots. Pairing interactions are indicated by the proximity of the bases. Numbers indicate the length of the overlap region of each of the four strands of the pseudo-HJ intermediates. (B) V. cholerae XerC- and XerD-mediated recombination of attP^VGJ with dif1 and dif2. A short radioactively labeled attP substrate was reacted with a longer cold dimer resolution substrate (Left), and short radioactively labeled dimer resolution substrates were reacted with a longer cold attP substrate (Right). Schemes of substrate and products are indicated on the side of each panel. A black triangle indicates the position of cleavage of V. cholerae XerC. A star indicates the position of the radioactive label on the probes. (C) Scheme of the XerC- and XerD-dependent mechanism of lintegration of VGJɸ into dif1. The legend is as in Fig. 1B. (D) Scheme of the Watson–Crick base-pair interactions that could stabilize the strand exchange catalyzed by XerC between the overlap regions of the attachment sites of El Tor CTXɸ attP^ET(+) and of VGJɸ attP and attP^VGJ. The legend is as in A. (E) V. cholerae XerC- and XerD-mediated recombination of attP^VGJ with attP^ET(+). Schemes of the products and of the substrates are indicated as in B.

Fig. 3.

XerC and XerD-dependent excision of the VGJɸ prophage and de novo XerC- and XerD-dependent production of its RF. (A) β-galactosidase production in cells harboring a copy of VGJɸ integrated at lacZ-dif1. (Left) Xer⁺ cells. (Right) Xer⁻ cells. (B) Scheme of the XerC- and XerD-dependent mechanism of excision of VGJɸ. The legend is as in Fig. 1B. (C) Phage DNA was extracted from strains and analyzed by agaraose gel electrophoresis and ethidium bromide staining. The genotype of the strains is indicated above the pictures of the gels. xerC and recA were successively deleted using an integration/excision strategy. The presence or absence of the endogenous xerC gene is indicated by a “+” or “−” above each lane. Complementation with a functional copy of xerC is indicated by a “+” or “−” above each lane. Integration by homologous recombination of a suicide vector was used to reintroduce xerC into the recA⁺ strain. In the recA⁻ strain, xerC had to be harbored on a replicative plasmid. VGJɸ, RF of the phage; pXerC, pSC101 vector harboring a copy of xerC under its own promoter.