BMC Genomics 2005, 6:103 http://www.biomedcentral.com/1471-2164/6/103 ally sequenced at the Flanders Interuniversity Institute for Biotechnology (VIB) Genetic Service Facility by double strand primer walking following standard procedures with a final accuracy of 99.98% per amplicon. The pri- mary assembly of individual amplicon sequences was per- formed using the Seqman software package. The overall assembly quality was improved by sequencing regions of minimum 500 bp in length overlapping the extremities of adjoining amplicons using the same protocol as described above. Sequence analysis and annotation The full assembly of the amplicons, overlapping frag- ments and the previously published pAW63 replicon was done manually using the Accelrys DS Gene sequence edi- tor. The coordinate start of the sequence was assigned with respect to that of pXO2 in order to facilitate future comparative analyses between the two plasmids, for which the B. anthracis 'Ames Ancestor' strain sequence of pXO2 was used [GenBank:NC_007323]. Potential coding regions were predicted with the prokaryotic gene finder EasyGene [52] using a hidden Markov model (HMM) pre- trained on the B. anthracis genome. Sequence similarity searches, alignments and further annotation were per- formed using the BioPerl open software package with standard default BLAST X and ClustalW parameters (details available on request). The BLAST score ratio anal- ysis (BSRA) was performed as described by Rasko et al. [53]. Protein analyses were done using Accelrys DS Gene. Proteome phylogeny analyses were performed using the Tree Building Method (Neighbor Joining, uncorrected) in Accelrys DS Gene. All figures included in this work were generated using the BioPython, GenomeDiagram and ReportLab open software packages. The complete sequence of pAW63 has been deposited in the GenBank database under the accession number DQ025752 [Gen- Bank: DQ025752]. Authors' contributions All authors contributed substantially to the study, and all have read and approved the final manuscript. Acknowledgements We are grateful to Lasse Smidt for his many contributions to this project, and to Daniel De Palmenaer for a fruitful collaboration on the analysis of the IS231L elements. We would like to thank Dr. Cheryl Kuske for sharing primer sequences and preliminary pAW63 sequence data prior to publica- tion. We also acknowledge the contribution of the VIB Genetic Service Facility http://www.vibgeneticservicefacility.be/ for the sequencing of the plasmid amplicons. This work was supported by the FRIA (Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture) (grant to G. Van der Auwera), the Carlsberg Foundation (grant to L. Andrup), the National Fund for Scientific Research, and the Université catholique de Louvain. References 1. Helgason E, økstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, Hegna I, Kolstø AB: Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence. Appl Environ Microbiol 2000, 66(6):2627-2630. 2. Schoeni JL, Wong AC: Bacillus cereus food poisoning and its toxins. J Food Prot 2005, 68(3):636-648. 3. David DB, Kirkby GR, Noble BA: Bacillus cereus endophthalmitis. Br J Ophthalmol 1994, 78(7):577-580. 4. Mock M, Fouet A: Anthrax. Annu Rev Microbiol 2001, 55:647-671. 5. Gill SS, Cowles EA, Pietrantonio PV: The mode of action of Bacil- lus thuringiensis endotoxins. Annu Rev Entomol 1992, 37:615-636. 6. Fouet A, Mock M: Differential influence of the two Bacillus anthracis plasmids on regulation of virulence gene expression. Infect Immun 1996, 64(12):4928-4932. 7. Hoton FM, Andrup L, Swiecicka I, Mahillon J: The cereulide genetic determinants of emetic Bacillus cereus are plasmid-borne. Microbiology in press. 8. Christie PJ: Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems. Biochim Biophys Acta 2004, 1694(1-3):219-234. 9. Berg T, Firth N, Apisiridej S, Hettiaratchi A, Leelaporn A, Skurray RA: Complete nucleotide sequence of pSK41: evolution of sta- phylococcal conjugative multiresistance plasmids. J Bacteriol 1998, 180(17):4350-4359. 10. Morton TM, Eaton DM, Johnston JL, Archer GL: DNA sequence and units of transcription of the conjugative transfer gene complex (trs) of Staphylococcus aureus plasmid pGO1. J Bacteriol 1993, 175(14):4436-4447. 11. Schwarz FV, Perreten V, Teuber M: Sequence of the 50-kb conju- gative multiresistance plasmid pRE25 from Enterococcus fae- calis RE25. Plasmid 2001, 46(3):170-187. 12. Dougherty BA, Hill C, Weidman JF, Richardson DR, Venter JC, Ross RP: Sequence and analysis of the 60 kb conjugative, bacteri- ocin-producing plasmid pMRC01 from Lactococcus lactis DPC3147. Mol Microbiol 1998, 29(4):1029-1038. 13. Thompson JK, Collins MA: Completed sequence of plasmid pIP501 and origin of spontaneous deletion derivatives. Plas- mid 2003, 50(1):28-35. 14. Grohmann E, Muth G, Espinosa M: Conjugative plasmid transfer in gram-positive bacteria. Microbiol Mol Biol Rev 2003, 67(2):277-301, table of contents. 15. Wilcks A, Jayaswal N, Lereclus D, Andrup L: Characterization of plasmid pAW63, a second self-transmissible plasmid in Bacil- lus thuringiensis subsp. kurstaki HD73. Microbiology 1998, 144 ( Pt 5):1263-1270. 16. Okinaka R, Cloud K, Hampton O, Hoffmaster A, Hill K, Keim P, Koehler T, Lamke G, Kumano S, Manter D, Martinez Y, Ricke D, Svensson R, Jackson P: Sequence, assembly and analysis of pX01 and pX02. J Appl Microbiol 1999, 87(2):261-262. 17. Hernandez E, Ramisse F, Ducoureau JP, Cruel T, Cavallo JD: Bacillus thuringiensis subsp. konkukian (serotype H34) superinfection: case report and experimental evidence of pathogenicity in immunosuppressed mice. J Clin Microbiol 1998, 36(7):2138-2139. 18. Wilcks A, Smidt L, økstad OA, Kolstø AB, Mahillon J, Andrup L: Rep- lication mechanism and sequence analysis of the replicon of pAW63, a conjugative plasmid from Bacillus thuringiensis. J Bacteriol 1999, 181(10):3193-3200. 19. Riley M: Systems for categorizing functions of gene products. Curr Opin Struct Biol 1998, 8(3):388-392. 20. Pannucci J, Okinaka RT, Williams E, Sabin R, Ticknor LO, Kuske CR: DNA sequence conservation between the Bacillus anthracis pXO2 plasmid and genomic sequence from closely related bacteria. BMC Genomics 2002, 3(1):34. 21. Rasko DA, Altherr MR, Han CS, Ravel J: Genomics of the Bacillus cereus group of organisms. FEMS Microbiol Rev 2005, 29(2):303-329. 22. Francia MV, Fujimoto S, Tille P, Weaver KE, Clewell DB: Replication of Enterococcus faecalis pheromone-responding plasmid pAD1: location of the minimal replicon and oriV site and RepA involvement in initiation of replication. J Bacteriol 2004, 186(15):5003-5016. 23. Hedberg PJ, Leonard BA, Ruhfel RE, Dunny GM: Identification and characterization of the genes of Enterococcus faecalis plas- mid pCF10 involved in replication and in negative control of pheromone-inducible conjugation. Plasmid 1996, 35(1):46-57. Page 13 of 14 (page number not for citation purposes)