Abstract
The human pathogen Vibrio cholerae employs several adaptive mechanisms for environmental persistence, including natural transformation and type VI secretion, creating a reservoir for the spread of disease. Here, we report whole-genome sequences of 26 diverse V. cholerae isolates, significantly increasing the sequence diversity of publicly available V. cholerae genomes.
GENOME ANNOUNCEMENT
Vibrio cholerae is a Gram-negative, facultative anaerobe commonly found in salt water and can cause the fatal diarrheal disease cholera. V. cholerae is spread predominantly by fecal contamination of water and food sources in both endemic and epidemic regions (1). Governmental and nongovernmental labs worldwide collect V. cholerae isolates for standard bacterial surveillance, and many clinical isolates of V. cholerae have already been sequenced (2–4). Clinical samples are often isolated from limited geographic regions and clonally derived with few or no genetic differences (5–8). An expanded characterization of genomes from environmental isolates of V. cholerae, which tend to be far more genetically and phenotypically diverse (9), should substantially increase the available sequence diversity of this important human pathogen.
Vibrio spp. are known to encode type VI secretion systems (T6SS), which are often described as bacterial weapons designed to pierce the membranes of adjacent cells and deliver toxic effectors that can lead to lysis of target (prey) cells. In a recent survey, Bernardy et al. (10) noted key differences within a diverse set of isolates for several phenotypes, including chitinase production, contact-dependent killing indicative of T6SS activity, and natural transformation, which can promote horizontal gene transfer. Both clinical and environmental isolates were rarely naturally transformable. In contrast, the majority of environmental, but not clinical, isolates constitutively killed Escherichia coli prey. Because different regulatory schemes control the phenotypes tested (11, 12), we sought to better understand the genetics that underlie these diverse V. cholerae phenotypes by characterizing whole-genome sequences of 23 environmental and three clinical isolates from Bernardy et al.
All strains were grown overnight in LB medium (Difco) at 37°C, with shaking. Genomic DNA was isolated using a ZR fungal/bacterial DNA mini prep kit (Zymo Research), and paired-end fragment libraries were constructed using a Nextera XT DNA library preparation kit (Illumina) with a fragment length of 300 bp. Libraries were sequenced by the High Throughput Sequencing Core at Georgia Institute of Technology on an Illumina HiSeq 2500 Rapid platform, producing approximately 280 million 100-bp reads in total. Reads were trimmed using Trimmomatic (13) to remove adapters and bases with a read quality score of <20. Genomes were assembled using SPAdes version 3.5 (14) and annotated using the Rapid Annotation and Subsystem Technology (RAST) web tool provided by the National Microbial Pathogen Data Resource (15–18). T6SS genes were annotated using T6SS Predictor (A. T. Chande et al., unpublished data).
T6SS loci were annotated in all genomes in an effort to characterize the genetic basis of T6SS-mediated killing among diverse environmental V. cholerae isolates. All genomes were found to encode the previously characterized large cluster and two auxiliary clusters, which together comprise the canonical T6SS loci. In addition, two previously unreported T6SS loci were discovered in six of the isolates. Numerous examples of novel effector-immunity protein pairs, which function together to catalyze T6SS-mediated killing, were characterized among the set of environmental isolate genomes. Taken together, our genome analysis illuminates the diverse repertoire of genetic mechanisms that underlie T6SS-mediated killing in V. cholerae.
Accession number(s).
This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession numbers listed in Table 1.
TABLE 1 .
List of V. cholerae strains sequenced in this study
| Straina | Location | Source | Yr of isolation | Type VI killing activityb | NCBI accession no. |
|---|---|---|---|---|---|
| 1496-86 | United States (LA) | Moore swab | 1986 | − | MIPC00000000 |
| 2523-87 | United States (LA) | Moore swab | 1974 | + | MIPE00000000 |
| VC48 | United States (FL) | Oyster | 1981 | + | MIOT00000000 |
| 2633-78 | Brazil | Sewage | 1978 | + | MIPH00000000 |
| 857 | Bangladesh | Water | 1996 | + | MIKH00000000 |
| 3272-78 | United States (MD) | Water | 1977 | + | MIOZ00000000 |
| TP | United States (CA) | Water | 2000 | + | MIPK00000000 |
| 2559-78 | United States (LA) | Crab | 1978 | + | MIOW00000000 |
| HE46 | Haiti (center) | Gray water | 2011 | + | MIPM00000000 |
| 2479-86 | United States (LA) | Moore swab | 1986 | + | MIPB00000000 |
| 2497-86 | United States (LA) | Moore swab | 1987 | + | MIPD00000000 |
| 2512-86 | United States (LA) | Moore swab | 1986 | + | MIOY00000000 |
| 2631-78 | United States (LA) | Moore swab | 1978 | + | MIOX00000000 |
| VC22 | United States (FL) | Oyster | 1981 | + | MIKK00000000 |
| VC53 | United States (AL) | Oyster | 2009 | + | MIOU00000000 |
| VC56 | United States (AL) | Oyster | 2009 | + | MIOV00000000 |
| 3568-07 | Mexico | Queso fresco | 2007 | + | MIPL00000000 |
| 1074-78 | Brazil | Sewage | 1978 | + | MIPG00000000 |
| 3223-74 | Guam | Storm drain | 1974 | + | MIZG00000000 |
| 3225-74 | Guam | Storm drain | 1974 | + | MIPF00000000 |
| 2740-80 | United States (Gulf Coast) | Water | 1980 | + | MIKI00000000 |
| 692-79 | United States (LA) | Water | 1979 | + | MIPA00000000 |
| SIO | United States (CA) | Water | 2000 | + | MIPJ00000000 |
| C6706 | Peru | Patient | 1991 | − | MIPI00000000 |
| MZO-2 | Bangladesh | Patient | 2001 | − | MIKJ00000000 |
| V52 | Sudan | Patient | 1968 | + | MIPN00000000 |
Strains were isolated from an environmental source, except strains C6706, MZO-2, and V52.
Presence (+) or absence (−) of constitutive type VI killing activity.
ACKNOWLEDGMENTS
Strains were generously provided by D. Bartlett, Scripps Institution of Oceanography; C. Tarr and C. Bopp, Centers for Disease Control and Prevention; R. Colwell, University of Maryland; A. DePaulo, Food and Drug Administration; and J. Zhu, University of Pennsylvania. The Georgia Tech Sequencing Core assisted with genome sequencing.
Footnotes
Citation Watve SS, Chande AT, Rishishwar L, Mariño-Ramírez L, Jordan IK, Hammer BK. 2016. Whole-genome sequences of 26 Vibrio cholerae isolates. Genome Announc 4(6):e01396-16. doi:10.1128/genomeA.01396-16.
REFERENCES
- 1.Finkelstein RA. 1996. Cholera, Vibrio cholerae O1 and O139, and other pathogenic vibrios, chap. 24. In Baron S (ed), Medical microbiology, 4th ed. University of Texas Medical Branch at Galveston, Galveston, TX. [PubMed] [Google Scholar]
- 2.CDC 2008. Summary of human Vibrio cases reported to CDC, 2008. http://www.cdc.gov/nationalsurveillance/pdfs/jackson_vibrio_cste2008_final.pdf.
- 3.Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL. 2005. GenBank. Nucleic Acids Res 33:D34–D38. doi: 10.1093/nar/gki063. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Pruitt KD, Tatusova T, Maglott DR. 2007. NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35:D61–D65. doi: 10.1093/nar/gkl842. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Chatterjee S, Ghosh K, Raychoudhuri A, Chowdhury G, Bhattacharya MK, Mukhopadhyay AK, Ramamurthy T, Bhattacharya SK, Klose KE, Nandy RK. 2009. Incidence, virulence factors, and clonality among clinical strains of non-O1, non-O139 Vibrio cholerae isolates from hospitalized diarrheal patients in Kolkata, India. J Clin Microbiol 47:1087–1095. doi: 10.1128/JCM.02026-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Horwood PF, Collins D, Jonduo MH, Rosewell A, Dutta SR, Dagina R, Ropa B, Siba PM, Greenhill AR. 2011. Clonal origins of Vibrio cholerae O1 El Tor strains, Papua New Guinea, 2009–2011. Emerg Infect Dis 17:2063–2065. doi: 10.3201/eid1711.110782. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Ali A, Chen Y, Johnson JA, Redden E, Mayette Y, Rashid MH, Stine OC, Morris JG. 2011. Recent clonal origin of cholera in Haiti. Emerg Infect Dis 17:699–701. doi: 10.3201/eid1704.101973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Bakhshi B, Mahmoudi-Aznaveh A, Salimi-Khorashad A. 2015. Clonal dissemination of a single Vibrio cholerae O1 Biotype El Tor strain in Sistan-Baluchestan province of Iran during 2013. Curr Microbiol 71:163–169. doi: 10.1007/s00284-015-0806-x. [DOI] [PubMed] [Google Scholar]
- 9.Chun J, Grim CJ, Hasan NA, Lee JH, Choi SY, Haley BJ, Taviani E, Jeon YS, Kim DW, Lee JH, Brettin TS, Bruce DC, Challacombe JF, Detter JC, Han CS, Munk AC, Chertkov O, Meincke L, Saunders E, Walters RA, Huq A, Nair GB, Colwell RR. 2009. Comparative genomics reveals mechanism for short-term and long-term clonal transitions in pandemic Vibrio cholerae. Proc Natl Acad Sci U S A 106:15442–15447. doi: 10.1073/pnas.0907787106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Bernardy EE, Turnsek MA, Wilson SK, Tarr CL, Hammer BK. 2016. Diversity of clinical and environmental isolates of Vibrio cholerae in natural transformation and contact-dependent bacterial killing indicative of type VI secretion system activity. Appl Environ Microbiol 82:2833–2842. doi: 10.1128/AEM.00351-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Watve SS, Thomas J, Hammer BK. 2015. CytR is a global positive regulator of competence, type VI secretion, and chitinases in Vibrio cholerae. PLoS One 10:e0138834. doi: 10.1371/journal.pone.0138834. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Metzger LC, Stutzmann S, Scrignari T, Van der Henst C, Matthey N, Blokesch M. 2016. Independent regulation of type VI secretion in Vibrio cholerae by TfoX and TfoY. Cell Rep 15:951–958. doi: 10.1016/j.celrep.2016.03.092. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.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. doi: 10.1186/1471-2164-9-75. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, Edwards RA, Gerdes S, Parrello B, Shukla M, Vonstein V, Wattam AR, Xia F, Stevens R. 2014. The SEED and the Rapid annotation of microbial genomes using subsystems technology (RAST). Nucleic Acids Res 42:D206–D214. doi: 10.1093/nar/gkt1226. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Wattam AR, Abraham D, Dalay O, Disz TL, Driscoll T, Gabbard JL, Gillespie JJ, Gough R, Hix D, Kenyon R, Machi D, Mao C, Nordberg EK, Olson R, Overbeek R, Pusch GD, Shukla M, Schulman J, Stevens RL, Sullivan DE, Vonstein V, Warren A, Will R, Wilson MJ, Yoo HS, Zhang C, Zhang Y, Sobral BW. 2014. PATRIC, the bacterial bioinformatics database and analysis resource. Nucleic Acids Res 42:D581–D591. doi: 10.1093/nar/gkt1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, Olson R, Overbeek R, Parrello B, Pusch GD, Shukla M, Thomason JA III, Stevens R, Vonstein V, Wattam AR, Xia F. 2015. RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep 5:8365. doi: 10.1038/srep08365. [DOI] [PMC free article] [PubMed] [Google Scholar]