CTX Prophages in Vibrio cholerae O1 Strains
CTX Prophages in Vibrio cholerae O1 Strains
Journal of Microbiology and Biotechnology. 2014. Jun, 24(6): 725-731
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : March 31, 2014
  • Accepted : April 10, 2014
  • Published : June 28, 2014
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About the Authors
Eun Jin, Kim
Institute of Pharmacological Research, Hanyang University, Ansan 426-791, Republic of Korea
Dokyung, Lee
Institute of Pharmacological Research, Hanyang University, Ansan 426-791, Republic of Korea
Se Hoon, Moon
Institute of Pharmacological Research, Hanyang University, Ansan 426-791, Republic of Korea
Chan Hee, Lee
Institute of Pharmacological Research, Hanyang University, Ansan 426-791, Republic of Korea
Dong Wook, Kim
Institute of Pharmacological Research, Hanyang University, Ansan 426-791, Republic of Korea

The classical biotype strains of the Vibrio cholerae O1 serogroup harbor the biotype-specific cholera-toxin encoding phage (CTX) CTX cla , and the El Tor biotype strains contain CTX-1. Although the classical biotype strains have become extinct, a remnant of classical CTX phage is transferred to the El Tor biotype strains. The prototype El Tor strains, which produce the biotype-specific cholera toxin, are now being replaced by atypical El Tor variant strains producing classical biotype cholera toxin. The genome sequences of the CTX phages in atypical El Tor strains indicate that the CTX phages in atypical El Tor strains are a mosaic of CTX cla and CTX-1. Before the emergence of atypical El Tor stains in the early 1990s, unusual pre-seventh pandemic strains were isolated in the US Gulf Coast between 1973 and 1986. These strains have characteristics of atypical El Tor strains since they are El Tor biotype strains containing CTX cla , yet the genome sequence of this CTX phage indicates that it is different from CTX cla and is therefore classified separately as CTX US Gulf .
Classification of V. cholerae O1 Strains
Seven cholera pandemics have been recognized since the beginning of the 19 th century up to present. Although the identity of V. cholerae strains responsible for the first five pandemics are not well-known, the V. cholerae O1 classical biotype strains and El Tor biotype strains are renowned to have caused the sixth and seventh cholera pandemics, respectively. Strains belonging to each biotype have different characteristics in several microbiological tests [13] . Whole genome sequencing-based phylogenetic analysis shows that the two biotype strains differ by 20,000 SNPs (single nucleotide polymorphisms) and several biotype-specific genomic islands, including VSP ( Vibrio seventh pandemic island)-1, -2 [5] . In addition, two biotype strains contain biotype-specific cholera toxin phage (CTX) and produce biotype-specific cholera toxin (CT) [13] .
The CTX phage genome is composed of 10 genes, rstR, rstA, rstB, psh (putative minor coat protein), cep (core encoded pilin), orfU, ace (accessory cholera enterotoxin), zot (zonula occludens toxin), and ctxAB . RstR, RstA, and RstB are necessary for phage DNA replication and integration, whereas Psh, Cep, OrfU, Ace, and Zot are required for phage packaging and secretion. Ace and Zot are shown to possess enterotoxic activity. CtxA and CtxB are not required for the phage life cycle but encode the cholera toxin.
Classical biotype strains harbor CTX cla , and El Tor biotype strains contain CTX El Tor or CTX-1 ( Table 1 ). Two CTX phages differ by the rstR , which have entirely different DNA sequences and are therefore classified as rstR cla and rstR El Tor . Other genes are different between two phages by a number of SNPs. Notably, the ctxB s in each CTX phage differ by two amino acids at positions 39 and 68 (nucleotide positions 115 and 203). A global population change of V. cholerae O1 serogroup has been recognized, from classical biotype to prototype El Tor biotype [20] . Since the El Tor biotype strains first emerged in 1962, the classical biotype strains have diminished. The classical biotype strains haven not been isolated from cholera patients since the 1980s and are considered to be extinct.
A further population change in El Tor biotype strains had occurred in the 1990s, from the prototype El Tor strains to the atypical El Tor variants producing the ctxB of classical biotype. The El Tor biotype strains containing CTX-1 are now referred to as the prototype El Tor to discriminate from atypical El Tor variants producing classical biotype cholera toxin. The atypical strains are reported to have emerged in the early 1990s [20] . As the prototype El Tor strains completely replaced the classical biotype strain globally, the atypical strains also entirely replaced the prototype El Tor strains. No clinical isolates of prototype El Tor strains have been collected since 2000 globally [20] . Two groups of atypical strains are reported based on the unusual classical cholera toxin-encoding CTX phage they harbor. Moreover, another important population change is occurring currently among the atypical strains producing the classical biotype cholera toxin, as they are now being replaced by strains producing a new type of cholera toxin [17] . The increase in number of severe cholera incidence in the Indian subcontinent and the emergence of atypical V. cholerae strains were proposed to be associated; however, an exact mechanism correlating the clinical symptoms and genetic changes of the bacterial strains needs to be elucidated [21] .
The main cause of the population change from classical biotype to El Tor biotype is believed to be the changes of the genome of the bacteria; namely, the accumulation of SNPs and acquisition of several genomic islands [5 , 16] . The genome change within the El Tor biotype strains has been gradual, and only 250 SNPs are identified between prototype and atypical strains; therefore, alternative approaches must be considered in order to explain the population changes of El Tor biotype strains more comprehensively.
- Classical Biotype Strains
Classical biotype strains contain biotype-specific CTX phage, CTX cla , which is integrated in both chromosomes of the bacteria [7] . Two classical biotype strains, O395 and 569B, have been studied extensively, and they contain the same CTX phage integration structure [6] . A truncated CTX cla consisting of rstRcla, rstA, rstB, and a partial cep followed by an authentic CTX cla are integrated in chromosome 1 of these strains. Another CTX cla is integrated in chromosome 2. This array of CTX phage does not produce progeny phages since the replication of CTX phage requires a CTX repeat or CTX-RS1 array [8] . A number of classical biotype strains have been reported to contain various arrays of CTX cla [2] .
- Pre-Seventh Pandemic Strains
A few pre-seventh pandemic strains are reported to be phylogenetically located intermediate between the classical and El Tor biotypes [5 , 16] . A group of strains collected in Australia in the 1980s and another group of strains collected in the US Gulf Coast in the 1970s and 1980s have been analyzed to be pre-seventh pandemic strains [12 , 19] . Although the classical and El Tor biotype strains are believed to originate from Ganges Delta, US Gulf Coast and Australia are reported to be independent reservoirs of region-specific V. cholerae strains [12 , 19] . The biotype of the pre-seventh pandemic strains was determined as El Tor although they were shown to contain CTX cla . The whole genome sequence of the CTX phage of a US Gulf Coast strain has been recently available (CTX US Gulf ), and the sequence indicates that the CTX phage in this strain should be distinguished from the authentic CTX cla ( Table 1 ).
CTX phages inV. choleraeO1 strains.
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*Biotype was determined by the DNA sequence of tcpA.
- Wave 1 Seventh Pandemic Strains
The El Tor biotype strains usually produced the biotype-specific cholera toxin, and therefore, are called prototype El Tor strains or Wave 1 strains of the seventh cholera pandemic. El Tor biotype strains also contain a satellite phage, RS1, which contains rstREl Tor, rstA, rstB, and rstC . RS1 is integrated on either side of CTX-1 on chromosome 1 of El Tor biotype strains; thus various arrays of CTX-1 and RS1 are found in El Tor biotype strains. A CTX repeat or CTX-1-RS1 array allows the CTX phage replication. No prototype El Tor strain contains CTX phage or RS1 on chromosome 2. The type strain of prototype El Tor strains, N16961, contains the CTX-1-RS1 array.
- Wave 2 Seventh Pandemic Strains
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Genetic structure of CTX phages. Block arrows indicate the direction of transcription of each gene (not shown to scale). CTX phage genes are shown in grey or white based on the SNPs of CTXcla (grey) or CTX-1 (white). ctxB genotype 7 of CTX-3b is shown in black.
An atypical CTX phage was discovered in V. cholerae clinical isolates of cholera outbreaks in Mozambique in 2004 and was later found to have existed in South Asian countries since the early 1990s [1 , 15] . This atypical CTX phage contains classical biotype-specific rstR ( rstR cla ) and ctxB ( ctxB cla ) and was thus considered a CTX cla ( Fig. 1 ). However, this phage was revealed to contain other genes of the CTX-1 and was later renamed CTX-2. The strains containing CTX-2 harbor a tandem repeat of CTX-2 on chromosome 2 and various genetic elements on chromosome 1 and are categorized as Wave 2 strains of the seventh cholera pandemic [16] . Strains containing a repeat of RS1 or CTX-2, or no element on chromosome 1 have been reported [5 , 14 , 16] . Wave 2 strains have been reported on the Indian subcontinent, Southeast Asia, Papua New Guinea, and Mozambique, indicating they are wide spread [10 , 11 , 18] .
- Wave 3 Seventh Pandemic Strains
A cholera outbreak in northern Vietnam, where cholera had not been endemic for decades, caused thousands of cholera cases in late 2007. The cholera outbreak was caused by a Wave 3 strain. Since then, the cholera has been endemic in this area by an expansion of a single strain introduced in 2007 [18 , 22] . Wave 3 atypical El Tor strains contain the RS1-CTX array on chromosome 1 and lack an integrate element on chromosome 2. The CTX phage in Wave 3 strains has rstR El Tor and ctxB El Tor and is distinguished from CTX-1 of Wave 1 strains and CTX-2 of Wave 2 strains. This CTX phage therefore has been designated as CTX-3 [16] . Strains belonging to Wave 3 have been reported as early as 1991 in India [4] . A derivative of CTX-3 was recognized in 2006, containing the identical DNA sequence as CTX-3, except for the 58 th nucleotide of ctxB . This ctxB is classified as ctxB genotype 7 ( ctxB genotype 1 is of CTX cla , genotype 2 is of an El Tor strain collected in Australia, genotype 3 is of CTX-1, and genotypes 4, 5, and 6 have been reported in O139 strains). Currently, most of the clinical isolates of V. cholerae are Wave 3 strains. The cholera outbreak in Haiti in 2010 was reported to be caused by a single introduction of a Wave 3 strain that has the ctxB genotype 7 [3] .
Sequence Variations in CTX Phages
- CTXcla, CTX-1, and RS1
Number of SNPs in each gene in CTXclaand CTX-1.
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Number of SNPs in each gene in CTXcla and CTX-1.
SNPs inrstRclain CTXcla, CTX-2, and CTXUS Gulf.
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SNPs in rstRcla in CTXcla, CTX-2, and CTXUS Gulf.
CTX cla and CTX-1 are biotype-specific CTX phages in classical and prototype El Tor strains, respectively. Besides the biotype-specific gene rstR , two phages are different by a number of SNPs ( Table 2 ). The SNPs in each phage can be used to identify the origin of CTX phage genomes in atypical V. cholerae strains. CTX-1 and RS1 contain three common genes, rstREl Tor, rstA, and rstB . The rstR El Tor genes of CTX-1 and RS1 are identical; however, rstA and rstB have sequence variations between CTX-1 and RS1. The sequence variations of RS1 and CTX-1 are found in nucleotide positions 927, 933, and 942 in rstA ( Table 3 ). In addition to the seven SNPs, rstB of RS1 is 9 bp shorter than rstB of CTX-1 (the 74-76, 366-368, 371-372, and 381 st nucleotides are absent in rstB of RS1). The sequence variations among CTX cla , CTX-1, and RS1 can be utilized to identify the origin of CTX phages in atypical El Tor strains as described below.
- CTXUS Gulf
Although the US Gulf Coast strains can be phylogenetically located between classical and El Tor biotype strains, the biotype of US Gulf Coast strains is clearly determined as El Tor [12 , 13] . The CTX phage they harbor was known as CTX cla since it contains rstR cla and ctxB cla . The full sequence of the CTX phage of a US Gulf Coast strain reveals that there are many sequence variations between CTX US Gulf and CTX cla (Tables 3–9). The rstR cla of CTX US Gulf also contains a SNP at the 45 th nucleotide position, although it is a synonymous change ( Table 3 ).
- CTX-2
SNPs inrstAin CTX phages.
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Dots indicate the identical nucleotides as N16961 and Δ indicates nucleotide(s) deletion.
SNPs inrstBin CTX phages.
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SNPs in rstB in CTX phages.
CTX-2 was initially considered a CTX cla since it contained rstR cla and ctxB cla ; however, the SNPs of CTX-2 indicate that it is rather a complex mosaic. The first four SNPs of rstA in CTX-2 are identical to those of CTX cla , whereas the other six SNPs are of CTX-1 ( Table 4 ). The first five SNPs of rstB in CTX-2 are identical to RS1 and the rest of the rstB is the same as that of CTX-1 ( Table 5 ). The rest of the phage genome, except for ctxB , is the same as that of CTX-1 ( Tables 6 - 9 ), indicating that CTX-2 is generated by a complicated process. It could be a mosaic of CTX-1, CTX cla , and RS1 generated by at least two recombination events – one between CTX-1 and RS1 and the other between the CTX phage generated by the first recombination and CTX cla . However, experimental evidence to explain how the CTX-2 was generated is lacking entirely. Wave 2 atypical strains contain a tandem repeat of CTX-2 on chromosome 2, which can produce the progeny phages according to the replication mechanism of CTX phage. However, Wave 2 strains have been reported to be unable to produce progeny phages in vitro [9] .
SNPs incepin CTX phages.
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SNPs in cep in CTX phages.
SNPs inorfUin CTX phages.
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SNPs in orfU in CTX phages.
SNPs inacein CTX phages.
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SNPs in ace in CTX phages.
SNPs inzotandctxBin CTX phages.
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SNPs in zot and ctxB in CTX phages.
- CTX-3 and CTX-3b
CTX-3 was first reported from the cholera outbreaks in Vietnam in 2007 and was thought to contain the same genetic structure and sequence of CTX-1, except for the ctxB ( Fig. 1 ) [18] . Strains containing CTX-3 were found to have existed since the early 1990s on the Indian subcontinent [20] . However, detailed sequence analysis shows that three SNPs exist in rstA of CTX-3 ( Table 4 ). These SNPs are identical to the SNPs of rstA of RS1 compared with CTX-1 ( Table 4 ), implying that CTX is a mosaic between CTX-1 and RS1. CTX-3b was found in India in 2006. In addition to two SNPs of the ctxB cla of CTX-3 compared with CTX-1, the CTX-3b contained an additional SNP at the 20 th amino acid position [4] . CTX-3b has identical DNA sequence as CTX-3 throughout the genome, indicating that it is a derivative of CTX-3 generated by a point mutation in ctxB . Most of the current global clinical isolates of V. cholerae are atypical El Tor variants containing CTX-3 or CTX-3b [20] , and a recent surveillance study in India shows that strains containing CTX-3b have been gradually replacing strains harboring CTX-3, as isolates containing CTX-3b comprised 93.3% of the total isolates collected in 2011 [17] . Thus far, Wave 3 atypical strains only contain the RS1-CTX array on chromosome 1.
Concluding Remarks
Atypical El Tor strains are now believed to have completely replaced the prototype El Tor strains globally. Genome sequencing and CTX phage analyses show that atypical strains arose from prototype El Tor strains by lateral transfer of ctxB cla from classical biotype strains. An understanding of the changes of CTX phages would be the starting point to reveal the driving forces of the population changes of V. cholerae .
This work was supported by the research fund of Hanyang University (HY-2011-N).
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