Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1988 Jan;56(1):176–181. doi: 10.1128/iai.56.1.176-181.1988

Transposon Tn916 mutagenesis in Bacillus anthracis.

B E Ivins 1, S L Welkos 1, G B Knudson 1, D J Leblanc 1
PMCID: PMC259253  PMID: 2826334

Abstract

Mutagenesis of Bacillus anthracis by the streptococcal tetracycline resistance transposon Tn916 is described. Tn916 was transferred from Streptococcus faecalis DS16C1 to B. anthracis VNR-1 by conjugation in a standard filter mating procedure. Tetracycline-resistant (Tcr) transconjugants were obtained at a frequency of 1.6 X 10(-8) per donor CFU. When donor and recipient cells were treated with nafcillin before conjugation, the frequency was increased nearly 10-fold. Nafcillin pretreatment of donor and recipient strains was used in all subsequent conjugation experiments. S.faecalis CG110, containing multiple chromosomal insertions of Tn916, transferred the transposon to B. anthracis VNR-1 at a frequency of 9.3 x 10(-5). A Tcr B. anthracis transconjugant, strain VNR-1-tet-1, transferred Tn916 to B. anthracis UM23-1 and Bacillus subtilis BST1 at frequencies of 2.1 x 10(-4) and 5.8 X 10(-6), respectively. The transfer of Tn916 occurred only on membrane filters, since no Tcr transconjugants were obtained when strains VNR-1-tet-1 and UM23-1 were mixed and incubated in broth culture. The presence of the Tn916-associated tetM gene in Tcr B. anthracis and B. subtilis transconjugants was confirmed in hybridization experiments by using a 5-kilobase-pair DNA fragment containing the tetM gene as a probe. Of 3,000 B. anthracis UM23-1 Tcr transconjugants tested, 21 were phenylalanine auxotrophs and 2 were auxotrophic for phenylalanine, tyrosine, and tryptophan.

Full text

PDF
177

Images in this article

179Image
on p.179
179Image
on p.179

Selected References

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

  1. Anderson D. G., McKay L. L. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl Environ Microbiol. 1983 Sep;46(3):549–552. doi: 10.1128/aem.46.3.549-552.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Christie P. J., Korman R. Z., Zahler S. A., Adsit J. C., Dunny G. M. Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis. J Bacteriol. 1987 Jun;169(6):2529–2536. doi: 10.1128/jb.169.6.2529-2536.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clewell D. B., Gawron-Burke C. Conjugative transposons and the dissemination of antibiotic resistance in streptococci. Annu Rev Microbiol. 1986;40:635–659. doi: 10.1146/annurev.mi.40.100186.003223. [DOI] [PubMed] [Google Scholar]
  5. Clewell D. B., Tomich P. K., Gawron-Burke M. C., Franke A. E., Yagi Y., An F. Y. Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917. J Bacteriol. 1982 Dec;152(3):1220–1230. doi: 10.1128/jb.152.3.1220-1230.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dybvig K., Cassell G. H. Transposition of gram-positive transposon Tn916 in Acholeplasma laidlawii and Mycoplasma pulmonis. Science. 1987 Mar 13;235(4794):1392–1394. doi: 10.1126/science.3029869. [DOI] [PubMed] [Google Scholar]
  7. Franke A. E., Clewell D. B. Evidence for a chromosome-borne resistance transposon (Tn916) in Streptococcus faecalis that is capable of "conjugal" transfer in the absence of a conjugative plasmid. J Bacteriol. 1981 Jan;145(1):494–502. doi: 10.1128/jb.145.1.494-502.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Franke A. E., Clewell D. B. Evidence for conjugal transfer of a Streptococcus faecalis transposon (Tn916) from a chromosomal site in the absence of plasmid DNA. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):77–80. doi: 10.1101/sqb.1981.045.01.014. [DOI] [PubMed] [Google Scholar]
  9. Gawron-Burke C., Clewell D. B. A transposon in Streptococcus faecalis with fertility properties. Nature. 1982 Nov 18;300(5889):281–284. doi: 10.1038/300281a0. [DOI] [PubMed] [Google Scholar]
  10. Gawron-Burke C., Clewell D. B. Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria. J Bacteriol. 1984 Jul;159(1):214–221. doi: 10.1128/jb.159.1.214-221.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. González J. M., Jr, Brown B. J., Carlton B. C. Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of B. thuringiensis and B. cereus. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6951–6955. doi: 10.1073/pnas.79.22.6951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Green B. D., Battisti L., Koehler T. M., Thorne C. B., Ivins B. E. Demonstration of a capsule plasmid in Bacillus anthracis. Infect Immun. 1985 Aug;49(2):291–297. doi: 10.1128/iai.49.2.291-297.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hoiseth S. K., Stocker B. A. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature. 1981 May 21;291(5812):238–239. doi: 10.1038/291238a0. [DOI] [PubMed] [Google Scholar]
  14. Ivins B. E., Welkos S. L. Cloning and expression of the Bacillus anthracis protective antigen gene in Bacillus subtilis. Infect Immun. 1986 Nov;54(2):537–542. doi: 10.1128/iai.54.2.537-542.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jacob A. E., Hobbs S. J. Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J Bacteriol. 1974 Feb;117(2):360–372. doi: 10.1128/jb.117.2.360-372.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jones J. M., Yost S. C., Pattee P. A. Transfer of the conjugal tetracycline resistance transposon Tn916 from Streptococcus faecalis to Staphylococcus aureus and identification of some insertion sites in the staphylococcal chromosome. J Bacteriol. 1987 May;169(5):2121–2131. doi: 10.1128/jb.169.5.2121-2131.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kafatos F. C., Jones C. W., Efstratiadis A. Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure. Nucleic Acids Res. 1979 Nov 24;7(6):1541–1552. doi: 10.1093/nar/7.6.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lauer G., Pastrana R., Sherley J., Ptashne M. Construction of overproducers of the bacteriophage 434 repressor and cro proteins. J Mol Appl Genet. 1981;1(2):139–147. [PubMed] [Google Scholar]
  19. Ristroph J. D., Ivins B. E. Elaboration of Bacillus anthracis antigens in a new, defined culture medium. Infect Immun. 1983 Jan;39(1):483–486. doi: 10.1128/iai.39.1.483-486.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Roberts M. C., Kenny G. E. Conjugal transfer of transposon Tn916 from Streptococcus faecalis to Mycoplasma hominis. J Bacteriol. 1987 Aug;169(8):3836–3839. doi: 10.1128/jb.169.8.3836-3839.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tomich P. K., An F. Y., Damle S. P., Clewell D. B. Plasmid-related transmissibility and multiple drug resistance in Streptococcus faecalis subsp. zymogenes strain DS16. Antimicrob Agents Chemother. 1979 Jun;15(6):828–830. doi: 10.1128/aac.15.6.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tsao S. G., Brunk C. F., Pearlman R. E. Hybridization of nucleic acids directly in agarose gels. Anal Biochem. 1983 Jun;131(2):365–372. doi: 10.1016/0003-2697(83)90185-9. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES