Abstract
Human Lyme disease is commonly caused by several species of spirochetes in the Borrelia genus. In Eurasia these species are largely Borrelia afzelii, B. garinii, B. burgdorferi, and B. bavariensis sp. nov. Whole-genome sequencing is an excellent tool for investigating and understanding the influence of bacterial diversity on the pathogenesis and etiology of Lyme disease. We report here the whole-genome sequences of four isolates from two of the Borrelia species that cause human Lyme disease, B. afzelii isolates ACA-1 and PKo and B. garinii isolates PBr and Far04.
GENOME ANNOUNCEMENT
Human Lyme disease is the most prevalent tick-borne disease in North America, Europe, and far-eastern Asia (11). The bacteria that cause Lyme disease belong to a clade of at least 15 species called Borrelia burgdorferi sensu lato or the Lyme disease agent bacterial group. Among these species, B. burgdorferi causes human Lyme disease in North America, while in Europe and Asia, B. afzelii, B. garinii, and B. bavariensis sp. nov. are also frequent causes of this disease (9). To date, whole-genome sequences have been reported from 14 B. burgdorferi sensu stricto isolates (2, 5, 13) and one unnamed sensu lato species (3), and the chromosome and incomplete plasmid sequences have been reported for one B. afzelii and one B. bavariensis sp. nov. isolate (6, 7).
To further our understanding of the genetic variation among the Borrelia species that cause Lyme disease, we performed whole-genome sequencing to about 8-fold coverage (10) on three human isolates and one bird isolate from Europe: B. afzelii isolates PKo (human erythema migrans; Germany) (12) and ACA-1 (human acrodermatitis chronica atrophicans; Sweden) (1) and B. garinii isolates PBr (human cerebrospinal fluid; Germany) (12) and Far04 (puffin blood; Faroe Islands, Denmark) (8). Low-passage-number isolates were sequenced to minimize plasmid loss during culture growth. Constraints on funds required that the sequences of the ACA-1, PBr, and Far04 chromosomes remain in draft status, but all the plasmid sequences were closed. The ACA-1, PBr, and Far04 genome annotations were performed using the JCVI Prokaryotic Annotation Pipeline (http://www.jcvi.org/cms/research/projects/annotation-service/overview/), and the genome of strain PKo was annotated using the SOM-IGS annotation engine pipeline (http://ae.igs.umaryland.edu/cgi/ae_pipeline_outline.cgi).
These four genome sequences include 5,151,042 total bp, with an average of 1,287,760 bp/genome. Like other Borrelia species, these isolates were found to carry numerous plasmids, both linear and circular, ranging from 7 plasmids in Far04 to 17 in PKo. We note that an average of only 3.25 members of the cp32 family of plasmids were present in these isolates, while B. burgdorferi sensu stricto isolates average about 7 members/isolate (3). Plasmids cp26, cp32, and lp54 are universally present in these isolates (with the exception of Far04, which has no cp32 plasmid), as they are in B. burgdorferi sensu stricto isolates, and the overall gene contents of these plasmids are rather similar to those of the plasmids of B. burgdorferi. In addition, plasmids with predicted lp17 compatibility (4) are present in all four genomes; however, their gene contents as well as the contents of most of the other linear plasmid types vary considerably, due to apparent interplasmid DNA rearrangements (our unpublished data).
These genome sequences contribute to a solid foundation for understanding B. burgdorferi sensu lato diversity and evolution, as well as the development of species- and group-specific diagnostics and vaccines.
Nucleotide sequence accession numbers.
These sequences have been deposited in the GenBank database, and their accession numbers are listed in Table 1.
Table 1.
B. afzelii and B. garinii sequence accession numbers
| Elementa | Accession no. forb: |
Total | |||
|---|---|---|---|---|---|
|
Borrelia afzelii |
Borrelia garinii |
||||
| PKo (68149) | ACA-1 (19641) | PBr (28625) | Far04 (29573) | ||
| Chromosome | CP002933 | ABCU02000001-2c | ABJV02000001-5c | ABPZ02000001-33c | |
| lp17 | CP002942 | CP001239 | CP001309 | CP001315 | |
| lp25 | CP001301 | CP001317 | |||
| lp28-1 | CP001238 | CP001310 | CP001316 | ||
| lp28-2 | CP002943 | CP001244 | |||
| lp28-3 | CP002944 | CP001241 | CP001307 | ||
| lp28-4 | CP002945 | CP001249 | CP001304 | ||
| lp28-7 | CP002946 | CP001242 | CP001311 | ||
| lp28-8 | CP002947 | ||||
| lp36 | CP001302 | CP001314 | |||
| lp38 | CP002949 | CP001246 | |||
| lp54 | CP002950 | CP001247 | CP001308 | CP001318 | |
| cp26 | CP002934 | CP001250 | CP001305 | CP001319 | |
| cp32-1 | CP002937 | CP001243 | |||
| cp32-3 | CP002938 | CP001237 | |||
| cp32-4 | CP001240 | ||||
| cp32-5 | CP002939 | CP001248 | CP001303 | ||
| cp32-7 | CP002940 | ||||
| cp32-9 | CP002941 | ||||
| cp32-10 | CP002948d | CP001245d | CP001306 | CP001320d | |
| cp32-11 | CP002935 | ||||
| cp32-12 | CP002936 | ||||
| No. of: | |||||
| cp32s | 7 | 4 | 2 | 0 | 13 |
| Other circles | 1 | 1 | 1 | 1 | 4 |
| Linear plasmids | 9 | 9 | 8 | 6 | 32 |
| Total plasmids | 17 | 14 | 11 | 7 | 49 |
| Total bp sequenced | 1,404,232 | 1,353,779 | 1,265,591 | 1,127,440 | 5,151,042 |
Plasmids are named according to their type PFam32 partition and replication protein (4).
Numbers in parentheses are genome project ID numbers.
Draft sequence; contigs not joined.
“lp32-10” plasmid; these plasmids are apparently linear but encode a cp32-10 type PFam32 protein. Their gene content is quite different from that of canonical cp32 plasmids (our unpublished data).
Acknowledgments
This research was supported by grants AI49003, N01-AI30071, AI37256, GM083722, and RR03037 from the National Institutes of Health.
We thank Bettina Wilske for strains ACA-1 and PBr, Richard Marconi for PKo, and Sven Bergström for Far04. Sean Daugherty's help with the genome annotation of PKo is greatly appreciated.
REFERENCES
- 1. Åsbrink E., Hederstedt B., Hovmark A. 1984. A spirochetal etiology of acrodermatitis chronica atrophicans Herxheimer. Acta Dermatol. Venereol. 64:506–512 [PubMed] [Google Scholar]
- 2. Casjens S., et al. 2000. A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi. Mol. Microbiol. 35:490–516 [DOI] [PubMed] [Google Scholar]
- 3. Casjens S. R., et al. 2011. Whole genome sequence of an unusual Borrelia burgdorferi sensu lato isolate. J. Bacteriol. 193:1489–1490 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Casjens S. R., et al. 2006. Comparative genomics of Borrelia burgdorferi, p. 79–95. In Cabello F. C., Hulinska D., Godfrey H. P. (ed.), Molecular biology of spirochetes . IOS Press, Amsterdam, The Netherlands. [Google Scholar]
- 5. Fraser C. M., et al. 1997. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390:580–586 [DOI] [PubMed] [Google Scholar]
- 6. Glöckner G., et al. 2004. Comparative analysis of the Borrelia garinii genome. Nucleic Acids Res. 32:6038–6046 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Glöckner G., et al. 2006. Comparative genome analysis: selection pressure on the Borrelia vls cassettes is essential for infectivity. BMC Genomics 7:211. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Gylfe, et al. 1999. Isolation of Lyme disease Borrelia from puffins (Fratercula arctica) and seabird ticks (Ixodes uriae) on the Faeroe Islands. J. Clin. Microbiol. 37:890–896 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Margos G., et al. 2009. A new Borrelia species defined by multilocus sequence analysis of housekeeping genes. Appl. Environ. Microbiol. 75:5410–5416 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Nelson K. E., et al. 2004. Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. Nucleic Acids Res. 32:2386–2395 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Piesman J., Gern L. 2004. Lyme borreliosis in Europe and North America. Parasitology 129(Suppl.):S191–S220 [DOI] [PubMed] [Google Scholar]
- 12. Preac-Mursic V., Wilske B., Schierz G. 1986. European Borrelia burgdorferi isolated from humans and ticks culture conditions and antibiotic susceptibility. Zentralbl. Bakteriol. Mikrobiol. Hyg. A 263:112–118 [DOI] [PubMed] [Google Scholar]
- 13. Schutzer S. E., et al. 2011. Whole genome sequences of thirteen isolates Borrelia burgdorferi. J. Bacteriol. 193:1018–1020 [DOI] [PMC free article] [PubMed] [Google Scholar]