Bacterial pathogens evolve continuously to adapt to the changing environment by adopting multiple mobile genetic elements into their compact, modularly organized mosaic genomes to help combat the environmental factors that are detrimental to their subsistence (Frost et al., 2005; Mercier et al., 2008; Hassan et al., 2010). Vibrio cholerae, the noxious enteric pathogen with extraordinary fitness competence resides in multiple niches across continents. A variety of the vibrio strains have acquired multiple genetic traits in their genomes (Heidelberg et al., 2000; Sack et al., 2004; Chun et al., 2009), with the purpose of contributing to the toxin production (Waldor and Mekalanos, 1996), intestinal colonization (Rhine and Taylor, 1994), disease development (Herrington et al., 1988), antimicrobial resistance (Mazel and Davies, 1998; Beaber et al., 2004), cell division (Hassan et al., 2010), and survival in aquatic as well as gut environments (Davies et al., 2012). In V. cholerae, most of the horizontally acquired genetic traits integrate site-specifically within a short region of sequence identity shared by the host chromosome and the integrative mobile genetic elements (IMGEs), using self- or host-encoded tyrosine recombinases (Huber and Waldor, 2002; Rajanna et al., 2003; Hazen et al., 2010; Das et al., 2013; Banerjee et al., 2014). Integration might be reversible or irreversible, depending upon the structures of pre- and post-integrative attachment sequences (Das et al., 2011a, 2013, 2014). Tyrosine recombinases can bind double stranded as well as folded single stranded DNA of the acquired exogenous genetic elements (Val et al., 2005; Mazel, 2006; Das et al., 2011b). Different acquired genetic traits reported in V. cholerae are heterogeneous, and they recognize different receptors for infection (Herrington et al., 1988; Campos et al., 2010; Das et al., 2013), follow different mechanisms to deliver DNA into the host cytoplasm (Heilpern and Waldor, 2000), have a wide range of genomic content (Heidelberg et al., 2000; Faruque and Mekalanos, 2003), and most importantly rely on catalytic activities of different recombinases for chromosomal integration (Rajanna et al., 2003; Das et al., 2010, 2011b; Midonet et al., in press). In a broader sense, these IMGEs can be classified into two different groups, depending upon the presence and absence of recombinases, the enzymes essential for their integration to the host genome. IMGEs, which encode and rely on their own recombinases for integration, are generally large in size and integrates specifically in front of tRNA or tmRNA operon (Karaolis et al., 1999; Heidelberg et al., 2000). On the other hand, IMGEs exploit host-encoded tyrosine recombinases (IMEX), are small in size and integrate at the dimer resolution sites (dif) of host chromosomes (Das et al., 2013, 2014).

The best-characterized IMEX, which exploits the host encoded tyrosine recombinases for its lysogenic conversion is CTXΦ, a temperate filamentous bacteriophage that encodes the cholera toxin in V. cholerae (Waldor and Mekalanos, 1996). The CTXΦ recognizes the toxin co-regulated pilus (TCP), a type IV pilus encoded by the vibrio pathogenic island–I (VPI-1), and subsequently introduces its ~ 7.0 × 10³ nucleotides long circular single stranded genomic DNA (+ssDNA) into the V. cholerae cytoplasm. Once in the bacterial cytoplasm, ssDNA may convert to dsDNA, and start the rolling circle replication, or it can be recognized by the chromosomally-encoded tyrosine recombinases, that enable CTXΦ integration into the dif site of host chromosome. Tandemly integrated CTXΦ can also initiate the rolling circle replication from the chromosome and produce extrachromosomal ssDNA genome that may contribute in virion production (Davis and Waldor, 2000). Compared to other episomally replicative filamentous phages, the number of phage particles produced by the CTX-prophage is very low, even upon induction, resulting in one phage particle produced per 10–100 host cells (Davis et al., 2002). The other two well-characterized IMEXs that use the same recombinases for integration at the dif1 site of V. cholerae are VGJΦ and TLCΦ (Campos et al., 2003b; Hassan et al., 2010; Das et al., 2011b). Presence of single or multiple copies of VGJΦ and TLCΦ were reported in toxigenic V. cholerae isolates. Unlike CTXΦ, the virion production of VGJΦ, and TLCΦ mainly relies on the episomal replication. Although the genomes of the two latter elements do not encode any toxins, they are nevertheless implicated in the host fitness and play important role in acquisition of the cholera-toxin-encoding genes and CTXΦ dissemination (Hassan et al., 2010; Das et al., 2011b; Midonet et al., in press).

In this review, I will update the mechanistic insights into filamentous vibriophage integration and cooperative interactions amongst IMGEs, which support the emergence of new pathogenic strains by contributing efficient acquisition and rapid dissemination of the cholera toxin genes in closely or distantly related bacterial strains.

The native function of Xer recombination system is to ensure the stable maintenance of monomeric circular replicon (Blakely et al., 1993). In V. cholerae, the principal components of the Xer recombination system are (i) two recombinases, XerC and XerD; (ii) a DNA motor protein, FtsK; (iii) a 28-bp DNA sequence, dif (Val et al., 2008) (Table 1). The Xer proteins are members of the tyrosine recombinase family (Esposito and Scocca, 1997). Members of this family share two conserved motifs, containing four highly conserved residues (R1-H-R2-Y), a catalytic tyrosine, which acts as the nucleophile during cleavage of phosphodiester bond, two arginines and a histidine, which have been implicated both the cleavage and rejoining of DNA strands (Esposito and Scocca, 1997). The two positively charged arginines are thought to stabilize the pentavalent phosphate transition state, whereas the histidine may act as a general base catalyst (Esposito and Scocca, 1997; Sherratt and Wigley, 1998).

During chromosome dimer resolution at the end of DNA replication, tetrameric XerC and XerD recombinase complex catalyzes successive strand exchanges between the two dif sites of a dimeric chromosome. The resolution is completed by a two-step transesterification reaction in which the OH- group of catalytic tyrosine residue of each recombinase is directly involved in the phosphodiester bond formation (Figure 1). In the first step of dimer resolution reaction, cleavage of a phosphodiester bond in DNA is introduced by the active enzyme, generating a covalently linked enzyme-DNA complex at the recombinase binding site and a free 5′-hydroxyl group at the end of the central region. Following the cleavage, the exposed sequence at the 5′-hydroxyl ends of the central region melt from their complementary strands and react with their recombinase-DNA phosphotyrosyl linked recombining partner. Subsequent joining between dif and attP strands requires complementary base pair interactions at the site of the phosphodiester bond formation (MacDonald et al., 2006; Das et al., 2010).

FtsK, a bifunctional protein essential for cell division and chromosome partitioning, induces the XerD function during chromosome dimer resolution (Recchia et al., 1999; Aussel et al., 2002; Demarre et al., 2013). The N-terminal part of FtsK forms a transmembrane domain and is directly linked to the forming septum. The cytoplasmic C-terminal domain is involved in the inter- and intracellular DNA transfer and activation of XerD recombinases (Bigot et al., 2006). Recent reports demonstrated that several IMGEs exploit the conserved Xer recombination system of bacteria to mediate their integration in the dimer resolution site of host chromosomes (Das et al., 2013). The integration mechanisms of such elements were studied in details in V. cholerae and revealed that the IMEXs present in the dif region of V. cholerae make use of three distinct integration mechanisms for lysogenic conversion (Das et al., 2010, 2011a,b, 2013; Midonet et al., in press).

Most of the characterized filamentous vibriophages are lysogenic and integrate at the dif1 and/or dif2 sites of V. cholerae. All reported filamentous vibriophages are equipped with an autonomously replicating genetic module, with or without toxin-encoding genes (Figure 2). Some of the filamentous vibriophages are directly responsible for V. cholerae pathogenesis, others contribute to toxin acquisition and host fitness (Waldor and Mekalanos, 1996; Hassan et al., 2010). Their genomes may or may not be equipped with genes required for virion production and can exploit the virion structural and assembly genes of other filamentous phages for genome packaging (Hassan et al., 2010). Although most of the filamentous vibriophages use host-encoded Xer machinery for their integration, their attachment sites (attP), and their genomic organization are fairly distinct (Figure 2). The best-studied CTXΦ genome is organized into two structurally and functionally distinct modules called repeat sequence 2 (RS2) and core (Figure 2). The RS2 module carries genetic traits essential for CTXΦ replication, maintenance of the ssDNA genome, and transcriptional regulation from phage originated P_RSTA promoter (Waldor et al., 1997). The replicating genome of CTXΦ is detrimental to V. cholerae growth, but is tolerated upon integration into the dif sites of either one or both chromosomes (Das et al., 2010; Faruque and Mekalanos, 2012). Compared to toxigenic strains, that contain a single or multiple integrated CTXΦ genomes, V. cholerae cells carrying a replicative form of CTXΦ grow slowly and rapidly lose the replicative phage both under the standard laboratory growth conditions and in rabbit gastrointestinal tract animal model (Faruque et al., 2001).

VGJΦ, a filamentous (+)ssDNA lysogenic bacteriophage, infects both clinical and environmental isolates of cholera pathogen and exploits the host-encoded Xer recombinases to integrate at the dif1 site in the large chromosome of V. cholerae (Campos et al., 2003b, 2010; Das et al., 2011b). In contrast to complex attachment site of CTXΦ that contains two dif-like attP sites in an inverted orientation, the 7.5-kb genome of VGJΦ harbors a single dif-like 29-bp attachment sequence, called attP^VGJ and integrates specifically into the dif1 site.

TLCΦ, a satellite temperate phage with an autonomous replication module but lacking the morphogenesis and structural virion-encoding genes, is often present in the dif1 region of toxigenic V. cholerae isolates (Rubin et al., 1998). Recent reports demonstrated that, like CTXΦ, and VGJΦ, TLCΦ also relies on the host-encoded Xer machinery for establishing lysogeny (Hassan et al., 2010; Midonet et al., in press). Interestingly, although both the XerC and XerD recombinases are essential for integration of IMEXs, none of the reported IMEXs rely on FtsK for recombination.

Different vibriophages have different attP structures and therefore rely on to a certain extent of different mechanisms of interaction with the Xer recombinases and integration (Das et al., 2013).

In this review, several fundamental questions related to IMEXs integration have been addressed, such as contribution of these elements to continuous evolution of the cholera pathogenic strains. Although hypothesis for the adaptive advantage of lysogenic conversion of filamentous vibriophages was proposed, many important questions are still open and need to be addressed in order to improve understanding of the V. cholerae biology and cholera management (Box 1). Pangenomic studies revealed that IMEXs are not limited only to clinical isolates but are also widely distributed in the environmental V. cholerae isolates that are closely or distantly related to pathogenic strains. Much emphasis has been put on the biology of those IMEXs that are mostly present in the clinical isolates. Our knowledge on IMEXs present in the environmental isolates is limited. It is widely accepted that the environmental V. cholerae strains are ubiquitously distributed in aquatic environment and could serve as a reservoir of toxin-encoding genes as well as other fitness factors for clinical strains. Comprehensive studies of IMEXs present both in clinical and environmental isolates of V. cholerae and related pathogenic strains are required in order to understand the microevolution of the species pertinent to epidemiology. On the other hand, better mechanistic and structural knowledge of IMEXs will help to develop therapeutic agents and limit the emergence of the new cholera pathogenic strains and other strains that may pose a serious threat to human beings as well as animals.