Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Aug;179(16):5000–5008. doi: 10.1128/jb.179.16.5000-5008.1997

Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons.

L Hoflack 1, J Seurinck 1, J Mahillon 1
PMCID: PMC179355  PMID: 9260939

Abstract

The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43 degrees C).

Full Text

The Full Text of this article is available as a PDF (544.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrup L., Bendixen H. H., Jensen G. B. Mobilization of Bacillus thuringiensis plasmid pTX14-3. Plasmid. 1995 May;33(3):159–167. doi: 10.1006/plas.1995.1017. [DOI] [PubMed] [Google Scholar]
  2. Andrup L., Damgaard J., Wassermann K., Boe L., Madsen S. M., Hansen F. G. Complete nucleotide sequence of the Bacillus thuringiensis subsp. israelensis plasmid pTX14-3 and its correlation with biological properties. Plasmid. 1994 Jan;31(1):72–88. doi: 10.1006/plas.1994.1008. [DOI] [PubMed] [Google Scholar]
  3. Asano Y., Ito H., Dairi T., Kato Y. An alkaline D-stereospecific endopeptidase with beta-lactamase activity from Bacillus cereus. J Biol Chem. 1996 Nov 22;271(47):30256–30262. doi: 10.1074/jbc.271.47.30256. [DOI] [PubMed] [Google Scholar]
  4. Boe L., Gros M. F., te Riele H., Ehrlich S. D., Gruss A. Replication origins of single-stranded-DNA plasmid pUB110. J Bacteriol. 1989 Jun;171(6):3366–3372. doi: 10.1128/jb.171.6.3366-3372.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boe L., Nielsen T. T., Madsen S. M., Andrup L., Bolander G. Cloning and characterization of two plasmids from Bacillus thuringiensis in Bacillus subtilis. Plasmid. 1991 May;25(3):190–197. doi: 10.1016/0147-619x(91)90012-l. [DOI] [PubMed] [Google Scholar]
  6. Carlson C. R., Caugant D. A., Kolstø A. B. Genotypic Diversity among Bacillus cereus and Bacillus thuringiensis Strains. Appl Environ Microbiol. 1994 Jun;60(6):1719–1725. doi: 10.1128/aem.60.6.1719-1725.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carlson C. R., Kolstø A. B. A small (2.4 Mb) Bacillus cereus chromosome corresponds to a conserved region of a larger (5.3 Mb) Bacillus cereus chromosome. Mol Microbiol. 1994 Jul;13(1):161–169. doi: 10.1111/j.1365-2958.1994.tb00411.x. [DOI] [PubMed] [Google Scholar]
  8. Devine K. M., Hogan S. T., Higgins D. G., McConnell D. J. Replication and segregational stability of Bacillus plasmid pBAA1. J Bacteriol. 1989 Feb;171(2):1166–1172. doi: 10.1128/jb.171.2.1166-1172.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Espinosa M., del Solar G., Rojo F., Alonso J. C. Plasmid rolling circle replication and its control. FEMS Microbiol Lett. 1995 Aug 1;130(2-3):111–120. doi: 10.1111/j.1574-6968.1995.tb07707.x. [DOI] [PubMed] [Google Scholar]
  11. Fickett J. W. Recognition of protein coding regions in DNA sequences. Nucleic Acids Res. 1982 Sep 11;10(17):5303–5318. doi: 10.1093/nar/10.17.5303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gruss A., Ehrlich S. D. The family of highly interrelated single-stranded deoxyribonucleic acid plasmids. Microbiol Rev. 1989 Jun;53(2):231–241. doi: 10.1128/mr.53.2.231-241.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Horinouchi S., Weisblum B. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J Bacteriol. 1982 May;150(2):815–825. doi: 10.1128/jb.150.2.815-825.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Horinouchi S., Weisblum B. Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies. J Bacteriol. 1982 May;150(2):804–814. doi: 10.1128/jb.150.2.804-814.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ilyina T. V., Koonin E. V. Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res. 1992 Jul 11;20(13):3279–3285. doi: 10.1093/nar/20.13.3279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jones D., Errington J. Construction of improved bacteriophage phi 105 vectors for cloning by transfection in Bacillus subtilis. J Gen Microbiol. 1987 Mar;133(3):483–492. doi: 10.1099/00221287-133-3-483. [DOI] [PubMed] [Google Scholar]
  17. Josson K., Scheirlinck T., Michiels F., Platteeuw C., Stanssens P., Joos H., Dhaese P., Zabeau M., Mahillon J. Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii. Plasmid. 1989 Jan;21(1):9–20. doi: 10.1016/0147-619x(89)90082-6. [DOI] [PubMed] [Google Scholar]
  18. Khan S. A., Novick R. P. Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus. Plasmid. 1983 Nov;10(3):251–259. doi: 10.1016/0147-619x(83)90039-2. [DOI] [PubMed] [Google Scholar]
  19. Khan S. A., Novick R. P. Structural analysis of plasmid pSN2 in Staphylococcus aureus: no involvement in enterotoxin B production. J Bacteriol. 1982 Feb;149(2):642–649. doi: 10.1128/jb.149.2.642-649.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leenhouts K. J., Tolner B., Bron S., Kok J., Venema G., Seegers J. F. Nucleotide sequence and characterization of the broad-host-range lactococcal plasmid pWVO1. Plasmid. 1991 Jul;26(1):55–66. doi: 10.1016/0147-619x(91)90036-v. [DOI] [PubMed] [Google Scholar]
  21. Leer R. J., van Luijk N., Posno M., Pouwels P. H. Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014. Mol Gen Genet. 1992 Aug;234(2):265–274. doi: 10.1007/BF00283847. [DOI] [PubMed] [Google Scholar]
  22. Lereclus D., Arantes O. spbA locus ensures the segregational stability of pTH1030, a novel type of gram-positive replicon. Mol Microbiol. 1992 Jan;6(1):35–46. doi: 10.1111/j.1365-2958.1992.tb00835.x. [DOI] [PubMed] [Google Scholar]
  23. Macaluso A., Mettus A. M. Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA. J Bacteriol. 1991 Feb;173(3):1353–1356. doi: 10.1128/jb.173.3.1353-1356.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mahillon J., Hespel F., Pierssens A. M., Delcour J. Cloning and partial characterization of three small cryptic plasmids from Bacillus thuringiensis. Plasmid. 1988 Mar;19(2):169–173. doi: 10.1016/0147-619x(88)90056-x. [DOI] [PubMed] [Google Scholar]
  25. Mahillon J., Seurinck J. Complete nucleotide sequence of pGI2, a Bacillus thuringiensis plasmid containing Tn4430. Nucleic Acids Res. 1988 Dec 23;16(24):11827–11828. doi: 10.1093/nar/16.24.11827. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McDowell D. G., Mann N. H. Characterization and sequence analysis of a small plasmid from Bacillus thuringiensis var. kurstaki strain HD1-DIPEL. Plasmid. 1991 Mar;25(2):113–120. doi: 10.1016/0147-619x(91)90022-o. [DOI] [PubMed] [Google Scholar]
  27. Oskam L., Venema G., Bron S. Plasmid maintenance in Bacillus stearothermophilus is strain-dependent. FEMS Microbiol Lett. 1992 Jun 15;72(3):203–208. doi: 10.1016/0378-1097(92)90463-x. [DOI] [PubMed] [Google Scholar]
  28. Seegers J. F., Zhao A. C., Meijer W. J., Khan S. A., Venema G., Bron S. Structural and functional analysis of the single-strand origin of replication from the lactococcal plasmid pWV01. Mol Gen Genet. 1995 Nov 1;249(1):43–50. doi: 10.1007/BF00290234. [DOI] [PubMed] [Google Scholar]
  29. Voskuil M. I., Chambliss G. H. Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis. Appl Environ Microbiol. 1993 Apr;59(4):1138–1142. doi: 10.1128/aem.59.4.1138-1142.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wertman K. F., Wyman A. R., Botstein D. Host/vector interactions which affect the viability of recombinant phage lambda clones. Gene. 1986;49(2):253–262. doi: 10.1016/0378-1119(86)90286-6. [DOI] [PubMed] [Google Scholar]
  31. Woodcock D. M., Crowther P. J., Doherty J., Jefferson S., DeCruz E., Noyer-Weidner M., Smith S. S., Michael M. Z., Graham M. W. Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 1989 May 11;17(9):3469–3478. doi: 10.1093/nar/17.9.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES