Molecular mechanism of acquisition of the cholera toxin genes.
Abstract: One of the major pathogenic determinants of Vibrio cholerae, the cholera toxin, is encoded in the genome of a filamentous phage, CTX[phi]. CTX[phi] makes use of the chromosome dimer resolution system of V cholerae to integrate its single stranded genome into one, the other, or both V. cholerae chromosomes. Here, we review current knowledge about this smart integration process.

Key words dif--site-specific recombination--XerC--XerD
Article Type: Report
Subject: Cholera toxin (Genetic aspects)
Bacteriophages (Genetic aspects)
Authors: Das, Bhabatosh
Bischerour, Julien
Barre, Francois-Xavier
Pub Date: 02/01/2011
Publication: Name: Indian Journal of Medical Research Publisher: Indian Council of Medical Research Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Health Copyright: COPYRIGHT 2011 Indian Council of Medical Research ISSN: 0971-5916
Issue: Date: Feb, 2011 Source Volume: 133 Source Issue: 2
Geographic: Geographic Scope: France Geographic Code: 4EUFR France
Accession Number: 252944931
Full Text: Introduction

Most bacteriophages are detrimental to their host metabolism. However, phages also participate in the horizontal transfer of genes among bacteria because their genome can harbour other genes than those strictly required for their life cycle. This can be highly beneficial to the bacterial host. Indeed, many bacterial virulence factors are associated with phage-like DNA sequences. More strikingly, the exotoxins produced by many pathogenic bacteria are encoded in the genome of lysogenic phages. This is notably the case in Bordetella avium (1), Clostridium botulinum (2), Corynebacterium diphtheriae (3), Escherichia coli (4), Pseudomonas aeruginosa (5), Shigella dysenteriae (6), Staphylococcus aureus (7) and Streptococcus pyogenes (8). The integrated prophages harboured by these bacteria profit from the multiplication of their host in the environment, which is in turn favoured by the virulence factors they bring to their host.

The study of Vibrio cholerae, the agent of the deadly diarrhoeal disease cholera, provides a fascinating case of such a bacterium-phage co-evolution. V. cholerae is the host for a variety of phages, commonly known as vibriophages, which can be lytic, non-lytic, virulent or temperate (9). On the one hand, phage predation of V. cholerae has been reported to be a factor that influences seasonal epidemics of cholera (10). On the other hand, one of the major virulence factors of V. cholerae, cholera toxin, is encoded in the genome of an integrated prophage CTX[PHI] (11,12). Furthermore, different variants of the phage CTX[PHI] exist, which participate in the genetic diversity of epidemic causing cholera strains (13-15). Two different attachment sites were found for this family of phages on the V. cholerae genome. They correspond to the dimer resolution sites of the two V. cholerae chromosomes, dif1 and dif2 (16). Indeed, in contrast to most other lysogenic phages, such as bacteriophage [lambda] (17), CTX[PHI] does not encode its integrase, but makes use of XerC and XerD, the two host-encoded tyrosine recombinases that normally function to resolve chromosome dimers (18). This mode of integration is all the more intriguing since CTX[PHI] phages belong to the filamentous phage family, which are generally not lysogenic and which harbour a single stranded circular genome. Nevertheless, CTX[PHI]-like prophages were found integrated in the genome of several bacterial species, notably in pathogenic E. coli strains (19) and in Yersinia pestis (20). Finally, it is remarkable to observe that many filamentous phages and/or genetic elements other than CTX[PHI] seem to have hijacked the chromosome dimer resolution system of V. cholerae for integration. Thus, TLC (21), VEJ (22), VGJ (23), VSK (24), VSKK (AF452449), KSF-1[PHI] (24), fs1 (25), fs2 (26), f237 (14), were all found to be integrated at dif1 and/or dif2. Such a diversity of elements has not been observed in any other genera than the vibrios. Together, these elements participate in the dissemination of virulence factors among V. cholerae strains (11,28,29) and in the emergence of new genetic variants of epidemic strains of V. cholerae (13). We review current knowledge on the integration mechanism of filamentous vibriophages that hijack the XerCD recombinases, with a special focus on CTX[PHI].

CTX[PHI] integration mechanism: exception or new paradigm?

CTX[PHI] has a ~7-kb ss(+)DNA genome arranged in two modular structures, the "RS" and "core". The core region harbours seven genes, which are psh, cep, gIIICTX, ace, zot, ctxA and ctxB. While the psh, cep, gIIICTX, ace and zot encoded proteins are needed for phage morphogenesis, the products of the ctxAB genes are not strictly required for the life cycle of the phage but are responsible for the severe diarrhoea associated with cholera (11). Three proteins, designated as RstR, RstA and RstB, are encoded in RS. Genetic analyses indicated that RstA is essential for phage replication and that RstB plays a crucial role in integration (30). RstR acts as a transcriptional repressor by inhibiting the activity of [P.sub.rstA], the only phage promoter required for CTX[PHI] replication and integration (30). Several CTX[PHI] have been reported. These can be classified into four families based on the sequence of their rstR gene. These categories were designated as CTX[[PHI].sup.ET], CTX[[PHI].sup.Cl], CTX[[PHI].sup.Clc] and CTX[[PHI].sup.Env] according to the host cells in which they were originally isolated (31-33).

As mentioned earlier, the integration of CTX[PHI] into the V. cholerae genome depends on two host encoded tyrosine recombinases, XerC and XerD (18). XerC and XerD normally serve to resolve circular bacterial chromosome dimers generated by RecA mediated homologous recombination by adding a crossover at a specific 28 bp site dif on the chromosome (16). The dif sites consist of specific 11-bp binding sites for each of the two Xer recombinases, separated by a 6-bp central region (34). These are generally located opposite to the origin of replication of bacterial chromosomes (16). Two dif sites are present on the genome of V. cholerae, one for each of the two circular chromosomes of the bacterium (35). Three different chromosome dimer resolution sites (dif1, dif2 and difG) have been identified among the different V. cholerae strains characterized to date (36) (Table I).

The ssDNA (+) genome of CTX[PHI] harbours two dif like sites (attP1 and attP2). These are arranged in opposite orientation and are separated by ~90-bp DNA segment in the phage genome (37). Integration of CTX[PHI] at the dif loci of V. cholerae depends on the formation of a forked hairpin structure of 150 bp in the region encompassing attP1 and attP2 in the (+) ssDNA genome (38) (Fig.1). The hybridization of attP1 and attP2 at the stem of this hairpin unmasks the phage attachment site, attP(+). Integration occurs, XerC and XerD recombine this site with one of the two dimer resolution sites harboured by the host cell. This process only requires the catalytic activity of XerC: a single pair of strands is exchanged, which results in the formation of a pseudo-Holliday junction.

A proof of principle for this mechanism of integration was originally obtained for the El Tor variant of CTX[PHI] and dif1 based on in vivo work performed in Escherichia coli and in vitro work performed with the E. coli Xer recombinases (38). Later on, a sensitive and quantitative assay was developed to confirm the ssDNA(+) integration model of CTX[[PHI].sup.ET] into the dif1 site of a V. cholerae El Tor strain (36). This system was also used to define rules of compatibilities between the phage attachment sites harboured by the different CTX[PHI] variants characterized to date and their host dimer resolution sites (36): integration is solely determined by possibility to form Watson-Crick or Wobble base pair interactions to stabilize the exchange of strands promoted by XerC-catalysis between the phage attachment site and its target dimer resolution site (Table II and Fig. 1). These rules explain how integration of CTX[[PHI].sup.ET] is restricted to dif1, how CTX[[PHI].sup.Cl] can target both dif1 and dif2, and how a third CTX[PHI] variant targets difG (Table II). This single stranded integration model is not restricted to CTX[PHI]. Analysis of the attP sites of CUS-1[PHI] and Ypf-[PHI] phages revealed features for direct ssDNA integration into the chromosome dimer resolution site harboured by their respective host cells (38). Another family of mobile genetic element, the integrons, also integrates in the bacterial chromosome via a single stranded intermediate (39).


Integration mechanism of CTX[PHI]-associated genetics elements

Several filamentous phages other than CTX[PHI] are found to be integrated at the dif loci of V. cholerae (13,22,23). To date, there is no report about their particular integration mechanism. Like CTX[PHI], they do not encode a dedicated recombinase. In addition, a 29-bp dif like sequence can be identified in many of them (Table III). It is, therefore, very likely that these phages take control of the host XerC and XerD recombinases to integrate into the genome of their host. However, the presence of a single putative XerCD binding site on their genome makes it unlikely that the ssDNA form of their genome is directly used as a substrate for integration. We rather favour a model in which the double stranded replicative form of these phages is used for integration (Fig. 2). We are currently investigating this model using the tools we have developed for the study of CTX[PHI] (40).


Interestingly, the two TLC elements integrated in strain N16961 are flanked by the half of the dif sequence (TGTGCGCATTA TGTATG for one and AGTGCATATTA TGTATG for the other). It is, therefore, reasonable to argue that their integration might be linked to the activity of the Xer recombinases.

Future prospects

The particular mode of integration of CTX[PHI] raises several questions. First, the efficiency of integration of a circular single stranded DNA molecule harbouring the sole attachment site of CTX[PHI] is very low (38). However, it becomes extremely efficient when the RS region of the phage is included (36). One likely explanation is that constant production and/or stabilization of the phage single stranded circular genome compensate for the instability of single stranded DNA in bacterial cells. RstB, which has been shown to be a single stranded DNA binding protein (41), could play a role in the stabilization of the integration substrate. Accordingly, its biochemical properties and sequence differ from those of the single stranded DNA binding protein encoded in the genome of VGJ[PHI], a phage that seems to rely on double stranded DNA integration (40). Second, only one pair of strands is exchanged between the single stranded DNA genome of CTX[PHI] and the double stranded DNA genome of its host, which leaves open the question of how the resulting pseudo-Holliday junction intermediate is processed. Is it stably maintained until the next round of bacterial DNA replication or processed by some host DNA repair machinery? What occurs when the replication fork collides against this unusual structure? Finally, it is intriguing that so many phages take advantage of the Xer recombination system of vibrios as compared to other bacterial species. We wonder if it could be related to the particular life style and environment of the vibrios and/or their particular genome structure and management.

Received June 14, 2010


(1.) Shelton CB, Crosslin DR, Casey JL, Ng S, Temple LM, Orndorff PE. Discovery, purification, and characterization of a temperate transducing bacteriophage for Bordetella avium. J Bacteriol 2000; 182 : 6130-6.

(2.) Fujii N, Oguma K, Yokosawa N, Kimura K, Tsuzuki K. Characterization of bacteriophage nucleic acids obtained from Clostridium botulinum types C and D. Appl Environ Microbiol 1988; 54 : 69-13.

(3.) Holmes RK, Barksdale L. Genetic analysis of tox+ and tox-bacteriophages of Corynebacterium diphtheriae. J Virol 1969; 3 : 586-98.

(4.) Newland JW, Strockbine NA, Miller SF, O'Brien AD, Holmes RK. Cloning of Shiga-like toxin structural genes from a toxin converting phage of Escherichia coli. Science 1985; 230 : 119-81.

(5.) Hayashi T, Baba T, Matsumoto H, Terawaki Y. Phage-conversion of cytotoxin production in Pseudomonas aeruginosa. Mol Microbiol 1990; 4 : 1103-9.

(6.) McDonough MA, Butterton JR. Spontaneous tandem amplification and deletion of the shiga toxin operon in Shigella dysenteriae 1. MolMicrobiol 1999; 34 : 1058-69.

(7.) Betley MJ, Mekalanos JJ. Staphylococcal enterotoxin A is encoded by phage. Science 1985; 229 : 185-1.

(8.) Weeks CR, Ferretti JJ. The gene for type A streptococcal exotoxin (erythrogenic toxin) is located in bacteriophage T12. Infect Immun 1984; 46 : 531-6.

(9.) Guidolin A, Manning PA. Genetics of Vibrio cholerae and its bacteriophages. Microbiol Rev 1981; 51 : 285-98.

(10.) Faruque SM, Islam MJ, Ahmad QS, Faruque AS, Sack DA, Nair GB, et al. Self-limiting nature of seasonal cholera epidemics: role of host-mediated amplification of phage. Proc Natl Acad Sci USA 2005; 102 : 6119-24.

(11.) Waldor MK, Mekalanos JJ. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 1996; 272 : 1910-4.

(12.) De SN. Enterotoxicity of bacteria-free culture-filtrate of Vibrio cholerae. Nature 1959; 183 : 1533-4.

(13.) Chun J, Grim CJ, Hasan NA, Lee JH, Choi SY, Haley BJ, et al. Comparative genomics reveals mechanism for short-term and long-term clonal transitions in pandemic Vibrio cholerae. Proc Natl Acad Sci USA 2009; 106 : 15442-1.

(14.) Faruque SM, Tam VC, Chowdhury N, Diraphat P, Dziejman M, Heidelberg JF, et al. Genomic analysis of the Mozambique strain of Vibrio cholerae O1 reveals the origin of El Tor strains carrying classical CTX prophage. Proc Natl Acad Sci USA 2001; 104 : 5151-6.

(15.) Kimsey HH, Nair GB, Ghosh A, Waldor MK. Diverse CTXphis and evolution of new pathogenic Vibrio cholerae. Lancet 1998; 352 : 451-8.

(16.) Val ME, El Kennedy SP, Karoui M, Bonne L, Chevalier F, Barre FX. FtsK-dependent dimer resolution on multiple chromosomes in the pathogen Vibrio cholerae. PLoS Genet 2008; 4 : e1000201.

(17.) Azaro MA, Landy A. [lambda] Integrase and the X Int Family. In: Craig NL, Craigie R, Gellert M, Lambowitz AM, editors. Mobile DNA II, vol.1, Washington, D.C: American Society of Microbiology; 2002. p. 118-48.

(18.) Huber KE, Waldor MK. Filamentous phage integration requires the host recombinases XerC and XerD. Nature 2002; 417 : 656-9.

(19.) Gonzalez MD, Lichtensteiger CA, Caughlan R, Vimr ER. Conserved filamentous prophage in Escherichia coli O18:K1:H1 and Yersinia pestis biovar orientalis. J Bacteriol 2002; 184 : 6050-5.

(20.) Derbise A, Chenal-Francisque V, Pouillot F, Fayolle C, Prevost MC, Medigue C, et al. A horizontally acquired filamentous phage contributes to the pathogenicity of the plague bacillus. Mol Microbiol 2001; 63 : 1145-51.

(21.) Rubin EJ, Lin W, Mekalanos JJ, Waldor MK. Replication and integration of a Vibrio cholerae cryptic plasmid linked to the CTX prophage. Mol Microbiol 1998; 28 : 1241-54.

(22.) Campos J, Martinez E, Izquierdo Y, Fando R. VEJcp, a novel filamentous phage of Vibrio cholerae able to transduce the cholera toxin genes. Microbiology 2010; 156 : 108-15.

(23.) Campos J, Martinez E, Suzarte E, Rodriguez BL, Marrero K, Silva Y, et al. VGJpp, a novel filamentous phage of Vibrio cholerae, integrates into the same chromosomal site as CTXp. J Bacteriol 2003; 185 : 5685-96.

(24.) Kar S, Ghosh RK, Ghosh AN, Ghosh A. Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA. FEMS Microbiol Lett 1996; 145 : 11-22.

(25.) Nakasone N, Honma Y, Toma C, Yamashiro T, Iwanaga M. Filamentous phage fs1 of Vibrio cholerae O139. Microbiol Immunol 1998; 42 : 231-9.

(26.) Nguyen DT, Nguyen BM, Tran HH, Ngo TC, Le TH, Nguyen HT, et al. Filamentous vibriophage fs2 encoding the rstC gene integrates into the same chromosomal region as the CTX phage [corrected]. FEMSMicrobiol Lett 2008; 284 : 225-30.

(27.) Faruque AS, Alam K, Malek MA, Khan MG, Ahmed S, Saha D, et al. Emergence of multidrug-resistant strain of Vibrio cholerae O1 in Bangladesh and reversal of their susceptibility to tetracycline after two years. J Health Popul Nutr 2001; 25 : 241-3.

(28.) Campos J, Martinez E, Marrero K, Silva Y, Rodriguez BL, Suzarte E, et al. Novel type of specialized transduction for CTX phi or its satellite phage RS1 mediated by filamentous phage VGJ phi in Vibrio cholerae. J Bacteriol 2003; 185 : 1231-40.

(29.) Davis BM, Waldor MK. Filamentous phages linked to virulence of Vibrio cholerae. Curr Opin Microbiol 2003; 6 : 35-42.

(30.) Waldor MK, Rubin EJ, Pearson GD, Kimsey H, Mekalanos JJ. Regulation, replication, and integration functions of the Vibrio cholerae CTXphi are encoded by region RS2. Mol Microbiol 1991; 24 : 911-26.

(31.) Davis BM, Kimsey HH, Chang W, Waldor MK. The Vibrio cholerae O139 Calcutta bacteriophage CTXphi is infectious and encodes a novel repressor. J Bacteriol 1999; 181 : 6779-87.

(32.) Maiti D, Das B, Saha A, Nandy RK, Nair GB, Bhadra RK. Genetic organization of pre-CTX and CTX prophages in the genome of an environmental Vibrio cholerae non-O1, non-O139 strain. Microbiology 2006; 152 : 3633-41.

(33.) Mukhopadhyay AK, Chakraborty S, Takeda Y, Nair GB, Berg DE. Characterization of VPI pathogenicity island and CTXphi prophage in environmental strains of Vibrio cholerae. J Bacteriol 2001; 183 : 4131-46.

(34.) Barre F-X, Sherratt DJS. Xer Site-Specific Recombination: Promoting Chromosome Segregation. In: Craig NL, Craigie R, Gellert M, Lambowitz A, editors. Mobile DNA II. vol.1, Washington, D.C: American Society of Microbiology; 2002. p. 149-61.

(35.) Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML, Dodson RJ, et al. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 2000; 406 : 411-83.

(36.) Das B, Bischerour J, Val ME, Barre FX. Molecular keys of the tropism of integration of the cholera toxin phage. Proc Natl Acad Sci USA 2010; 107 : 4311-82.

(37.) McLeod SM, Waldor MK. Characterization of XerC- and XerD-dependent CTX phage integration in Vibrio cholerae. Mol Microbiol 2004; 54 : 935-41.

(38.) Val ME, Bouvier M, Campos J, Sherratt D, Cornet F, Mazel D, et al. The single-stranded genome of phage CTX is the form used for integration into the genome of Vibrio cholerae. Mol Cell 2005; 19 : 559-66.

(39.) Bouvier M, Demarre G, Mazel D. Integron cassette insertion: a recombination process involving a folded single strand substrate. EMBO J2005; 24 : 4356-61.

(40.) Das B, Bischerour J, Barre FX. VGJ[phi]-integration and excision mechanisms contribute to the genetic diversity of Vibrio cholerae epidemic strains. PNAS 2011; doi: 10.1013/ pnas.1011061108.

(41.) Falero A, Caballero A, Ferran B, Izquierdo Y, Fando R, Campos J. DNA binding proteins of the filamentous phages CTXphi and VGJphi of Vibrio cholerae. J Bacteriol 2009; 191 : 5813-6.

Reprint requests: Dr Francois-Xavier Barre, CNRS, Centre de Genetique Moleculaire, 91198 Gif-sur-Yvette, France e-mail:

Bhabatosh Das, Julien Bischerour & Francois-Xavier Barre

CNRS, Centre de Genetique Moleculaire, Gif-sur-Yvette & Universite Paris-Sud, Orsay, France
Table I. Sequences of the chromosome dimer resolution sites found
in V. cholerae strains

Site               Sequence


Source: Ref. 36

Table II. Sequences of the dif-like sites harboured by CTXO variant

CTX [PHI]    attP sequence


CTX [PHI]    Integration    Accession
variant      site           number

El Tor       dif1           VCU83196

Classical    dif1           AY349115
Calcutta     dif1           AF110029

G            difG           AF416590

Source: Ref. 40

Table III. Sequences of the dif-like sites harboured by other

Phage    Genome size    attP sequence

KSF1     1.1            UK
fs1      6.3            UK

Phage    Host                  Integration     Accession
                                   site         number

VEJ      V. cholerae               dif1        NC012151
VGJ      V. cholerae               dif1       AY242528.1
VSK      V. cholerae               dif1        NC003321
VSKK     V. cholerae               dif1        AF452449
KSF1     V. cholerae                UK         AY114348
fs1      V. cholerae                UK        NC004306.1
fs2      V. cholerae               dif1        AB002632
f231     V. cholerae               dif1        NC002362
         V. parahemolyticus

UK, unknow; Source: Ref. 40
Gale Copyright: Copyright 2011 Gale, Cengage Learning. All rights reserved.