Draft Whole-Genome Sequence of a New Variant of Vibrio cholerae O1 El Tor Strain Isolated from a Cholera Patient in Russia Nina I. Smirnova, Alexander V. Cherkasov, Yaroslav M. Krasnov, Dmitriy A. Agafonov, Vladimir V. Kutyrev Federal Government Health Institution Russian Researсh Anti-Plaque Institute Microbe, Saratov, Russia All authors contributed equally to this work.
Received 23 April 2014 Accepted 9 May 2014 Published 29 May 2014 Citation Smirnova NI, Cherkasov AV, Krasnov YM, Agafonov DA, Kutyrev VV. 2014. Draft whole-genome sequence of a new variant of Vibrio cholerae O1 El Tor strain isolated from a cholera patient in Russia. Genome Announc. 2(3):e00432-14. doi:10.1128/genomeA.00432-14. Copyright © 2014 Smirnova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Alexander V. Cherkasov, [email protected].
V
ibrio cholerae is the causative agent of cholera, a severe diarrheal disease that continues to be a major public health problem in many developing countries. Epidemic outbreaks of cholera have been caused by only two V. cholerae biotypes: classical and El Tor (1). Since the early 1990s, new variants of V. cholerae О1 El Tor that possess traits of both the classical and El Tor biotypes have emerged (2, 3). These strains cause more severe cholera, with higher fatality rates, than that caused by prototypic El Tor strains. Here, we report the first draft whole-genome sequence of a clinical strain, V. cholerae O1 El Tor L3226, isolated in 2010 in Moscow, and we compared it to the complete genome of the reference strain V. cholerae O1 El Tor N16961. Libraries for sequencing were prepared from 1 g of purified genomic DNA, according to the manufacturer’s protocol, and were sequenced on a PGM platform (Life Technologies, CA) using 318 Chip and 200-bp chemistry. Raw single-end reads (2,287,084 total) were trimmed using the Ion Torrent Suite Software and were assembled de novo using the Newbler GS Assembler version 2.6 (Roche). Gene prediction, annotation, and an RNA search were performed by Rapid Annotations using Subsystems Technology (RAST) (4). The draft genome sequence of V. cholerae L3226 was assembled into 121 contigs (86-fold coverage) with a total length of 3,991,045 bp and an average G⫹C content of 47.4%. The N50 of the contigs is 134 kb, with the longest contig being 329 kb. The annotation of the obtained contigs showed the presence of 3,917 open reading frames (ORFs), 3,852 protein-coding sequences, and 65 tRNAs. The V. cholerae L3226 contigs were mapped into two distinct chromosomes, as in typical V. cholerae strains. Chromosome I consists of 47 contigs, amounting to a total length of 2,978,616 bp. Chromosome II consists of 44 contigs, with a total length of 1,007,260 bp. The chromosome size and G⫹C content correlate well with those of other V. cholerae strains (5). The sequenced genome analysis revealed that the nucleotide sequences of virulence-associated mobile elements, such as CTX prophage, carrying the cholera toxin (CT)-coding genes ctxAB, Vibrio pathogenicity island 1 (VPI-1), and Vibrio seventh pan-
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demic island II (VSP-II) differed from those of the reference strain N16961. Simultaneously, these regions of strain L3226 have high similarity to the same genome regions of the new El Tor variants isolated in India, Bangladesh, and Haiti (6–8). Interestingly, in contrast to El Tor variants possessing ctxAB (ctxB1 allele) of the reference classical strains, the ctxB1 gene of L3226 strain carried a novel mutation, a C¡A substitution in position 20. Another peculiarity of the L3226 strain is found in a 13-kb deletion in the VSP-II. In view of the recent discovery of new variants of V. cholerae El Tor, the sequenced genome of L3226 strain might provide further opportunity to investigate the evolution of cholera agents in the modern period. Nucleotide sequence accession number. This whole-genome shotgun project for the L3226 strain has been deposited at DDBJ/ EMBL/GenBank under the accession no. JDVX01000000. The version presented in this article is the first one. ACKNOWLEDGMENTS This work was supported by the Russian Federal Target-Oriented Program “National system of chemical and biological safety of the Russian Federation (2009-2013)” (contract 22 D dated 8/8/2013).
REFERENCES 1. Kaper JB, Morris JG, Levine MM. 1995. Cholera. Clin. Microbiol. Rev. 8:48 – 86. 2. Safa A, Nair GB, Kong RY. 2010. Evolution of new variants of Vibrio cholerae O1. Trends Microbiol. 18:46 –54. http://dx.doi.org/10.1016/ j.tim.2009.10.003. 3. Zhang P, Zhou H, Kan B, Wang D. 2013. Novel ctxB variants of Vibrio cholerae O1 isolates, China. Infect. Genet. Evol. 20:84 –53. 4. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75. http://dx.doi.org/10.1186/1471-2164-9-75. 5. Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Umayam L, Gill SR, Nelson KE, Read TD, Tettelin H, Richardson D, Ermolaeva MD, Vamathevan J, Bass
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Draft whole-genome sequencing of the Vibrio cholerae О1 El Tor clinical strain L3226, isolated in Moscow in 2010, was carried out. Various mutations in the virulence-associated mobile elements were determined in its genome that differentiated this strain from the reference V. cholerae О1 El Tor strain N16961.
Smirnova et al.
S, Qin H, Dragoi I, Sellers P, McDonald L, Utterback T, Fleishmann RD, Nierman WC, White O, Salzberg SL, Smith HO, Colwell RR, Mekalanos JJ, Venter JC, Fraser CM. 2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406:477– 483. http:// dx.doi.org/10.1038/35020000. 6. Chin CS, Sorenson J, Harris JB, Robins WP, Charles RC, Jean-Charles RR, Bullard J, Webster DR, Kasarskis A, Peluso P, Paxinos EE, Yamaichi Y, Calderwood SB, Mekalanos JJ, Schadt EE, Waldor MK. 2011. The origin of the Haitian cholera outbreak strain. N. Engl. J. Med. 364:33– 42. http://dx.doi.org/10.1056/NEJMvcm0904262.
7. Grim CJ, Hasan NA, Taviani E, Haley B, Chun J, Brettin TS, Bruce DC, Detter JC, Han CS, Chertkov O, Challacombe J, Huq A, Nair GB, Colwell RR. 2010. Genome sequence of hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and comparative genomics with V. cholerae. J. Bacteriol. 192:3524 –3533. http://dx.doi.org/10.1128/JB.00040-10. 8. Son MS, Megli CJ, Kovacikova G, Qadri F, Taylor RK. 2011. Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes. J. Clin. Microbiol. 49:3739 –3749. http://dx.doi.org/10.1128/ JCM.01286-11.
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Received 23 April 2014 Accepted 9 May 2014 Published 29 May 2014 Citation Smirnova NI, Cherkasov AV, Krasnov YM, Agafonov DA, Kutyrev VV. 2014. Draft whole-genome sequence of a new variant of Vibrio cholerae O1 El Tor strain isolated from a cholera patient in Russia. Genome Announc. 2(3):e00432-14. doi:10.1128/genomeA.00432-14. Copyright © 2014 Smirnova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Alexander V. Cherkasov, [email protected].
V
ibrio cholerae is the causative agent of cholera, a severe diarrheal disease that continues to be a major public health problem in many developing countries. Epidemic outbreaks of cholera have been caused by only two V. cholerae biotypes: classical and El Tor (1). Since the early 1990s, new variants of V. cholerae О1 El Tor that possess traits of both the classical and El Tor biotypes have emerged (2, 3). These strains cause more severe cholera, with higher fatality rates, than that caused by prototypic El Tor strains. Here, we report the first draft whole-genome sequence of a clinical strain, V. cholerae O1 El Tor L3226, isolated in 2010 in Moscow, and we compared it to the complete genome of the reference strain V. cholerae O1 El Tor N16961. Libraries for sequencing were prepared from 1 g of purified genomic DNA, according to the manufacturer’s protocol, and were sequenced on a PGM platform (Life Technologies, CA) using 318 Chip and 200-bp chemistry. Raw single-end reads (2,287,084 total) were trimmed using the Ion Torrent Suite Software and were assembled de novo using the Newbler GS Assembler version 2.6 (Roche). Gene prediction, annotation, and an RNA search were performed by Rapid Annotations using Subsystems Technology (RAST) (4). The draft genome sequence of V. cholerae L3226 was assembled into 121 contigs (86-fold coverage) with a total length of 3,991,045 bp and an average G⫹C content of 47.4%. The N50 of the contigs is 134 kb, with the longest contig being 329 kb. The annotation of the obtained contigs showed the presence of 3,917 open reading frames (ORFs), 3,852 protein-coding sequences, and 65 tRNAs. The V. cholerae L3226 contigs were mapped into two distinct chromosomes, as in typical V. cholerae strains. Chromosome I consists of 47 contigs, amounting to a total length of 2,978,616 bp. Chromosome II consists of 44 contigs, with a total length of 1,007,260 bp. The chromosome size and G⫹C content correlate well with those of other V. cholerae strains (5). The sequenced genome analysis revealed that the nucleotide sequences of virulence-associated mobile elements, such as CTX prophage, carrying the cholera toxin (CT)-coding genes ctxAB, Vibrio pathogenicity island 1 (VPI-1), and Vibrio seventh pan-
May/June 2014 Volume 2 Issue 3 e00432-14
demic island II (VSP-II) differed from those of the reference strain N16961. Simultaneously, these regions of strain L3226 have high similarity to the same genome regions of the new El Tor variants isolated in India, Bangladesh, and Haiti (6–8). Interestingly, in contrast to El Tor variants possessing ctxAB (ctxB1 allele) of the reference classical strains, the ctxB1 gene of L3226 strain carried a novel mutation, a C¡A substitution in position 20. Another peculiarity of the L3226 strain is found in a 13-kb deletion in the VSP-II. In view of the recent discovery of new variants of V. cholerae El Tor, the sequenced genome of L3226 strain might provide further opportunity to investigate the evolution of cholera agents in the modern period. Nucleotide sequence accession number. This whole-genome shotgun project for the L3226 strain has been deposited at DDBJ/ EMBL/GenBank under the accession no. JDVX01000000. The version presented in this article is the first one. ACKNOWLEDGMENTS This work was supported by the Russian Federal Target-Oriented Program “National system of chemical and biological safety of the Russian Federation (2009-2013)” (contract 22 D dated 8/8/2013).
REFERENCES 1. Kaper JB, Morris JG, Levine MM. 1995. Cholera. Clin. Microbiol. Rev. 8:48 – 86. 2. Safa A, Nair GB, Kong RY. 2010. Evolution of new variants of Vibrio cholerae O1. Trends Microbiol. 18:46 –54. http://dx.doi.org/10.1016/ j.tim.2009.10.003. 3. Zhang P, Zhou H, Kan B, Wang D. 2013. Novel ctxB variants of Vibrio cholerae O1 isolates, China. Infect. Genet. Evol. 20:84 –53. 4. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O. 2008. The RAST server: Rapid Annotations using Subsystems Technology. BMC Genomics 9:75. http://dx.doi.org/10.1186/1471-2164-9-75. 5. Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD, Umayam L, Gill SR, Nelson KE, Read TD, Tettelin H, Richardson D, Ermolaeva MD, Vamathevan J, Bass
Genome Announcements
genomea.asm.org 1
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Draft whole-genome sequencing of the Vibrio cholerae О1 El Tor clinical strain L3226, isolated in Moscow in 2010, was carried out. Various mutations in the virulence-associated mobile elements were determined in its genome that differentiated this strain from the reference V. cholerae О1 El Tor strain N16961.
Smirnova et al.
S, Qin H, Dragoi I, Sellers P, McDonald L, Utterback T, Fleishmann RD, Nierman WC, White O, Salzberg SL, Smith HO, Colwell RR, Mekalanos JJ, Venter JC, Fraser CM. 2000. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406:477– 483. http:// dx.doi.org/10.1038/35020000. 6. Chin CS, Sorenson J, Harris JB, Robins WP, Charles RC, Jean-Charles RR, Bullard J, Webster DR, Kasarskis A, Peluso P, Paxinos EE, Yamaichi Y, Calderwood SB, Mekalanos JJ, Schadt EE, Waldor MK. 2011. The origin of the Haitian cholera outbreak strain. N. Engl. J. Med. 364:33– 42. http://dx.doi.org/10.1056/NEJMvcm0904262.
7. Grim CJ, Hasan NA, Taviani E, Haley B, Chun J, Brettin TS, Bruce DC, Detter JC, Han CS, Chertkov O, Challacombe J, Huq A, Nair GB, Colwell RR. 2010. Genome sequence of hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and comparative genomics with V. cholerae. J. Bacteriol. 192:3524 –3533. http://dx.doi.org/10.1128/JB.00040-10. 8. Son MS, Megli CJ, Kovacikova G, Qadri F, Taylor RK. 2011. Characterization of Vibrio cholerae O1 El Tor biotype variant clinical isolates from Bangladesh and Haiti, including a molecular genetic analysis of virulence genes. J. Clin. Microbiol. 49:3739 –3749. http://dx.doi.org/10.1128/ JCM.01286-11.
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