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Volume 135, Issue 5

Protoplast Transformation of NUB36 by Plasmid DNA Free

Abstract

An efficient protoplast transformation system was established for NUB3621 using thermophilic plasmid pTHT15 Tc (4·5 kb) and mesophilic plasmid pLW05 Cm (3 kb), a spontaneous deletion derivative of pPL401 Cm Km. The efficiency of transformation of NUB3621 with pLW05 and pTHT15 was 2 x 10 to 4 x 10 transformants per µg DNA. The transformation frequency (transformants per regenerant) was 0·5 to 1·0. Chloramphenicol-resistant and tetracycline-resistant transformants were obtained when competent cells of were transformed with pLW05 [2·5 x 10 transformants (µg DNA)] and pTHT15 [1·8 x 10 transformants (µg DNA)], respectively. Thus, these plasmids are shuttle vectors for mesophilic and thermophilic bacilli. Plasmid pLW05 Cm was not stably maintained in cultures growing at temperatures between 50 and 65 °C but the thermostable chloramphenicol acetyltransferase was active at temperatures up to 70 °C. In contrast, thermophilic plasmid pTHT15 Tc was stable in cultures growing at temperatures up to 60 °C but the tetracycline resistance protein was relatively thermolabile at higher temperatures. The estimated copy number of pLW05 in cells of NUB3621 growing at 50,60, and 65 °C was 69,18, and 1 per chromosome equivalent, respectively. The estimated copy number of pTHT15 in cells of NUB3621 growing at 50 or 60 °C was about 41 to 45 per chromosome equivalent and 12 in cells growing at 65 °C.

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© Society for General Microbiology 1989
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1989-05-01
2024-04-26
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References

  1. Aiba S., Koizumi J.-I. 1984; Effects of temperature on plasmid stability and penicillinase productivity in a transformant of Bacillus stearothermophilus . Biotechnology and Bioengineering 26:1026–1031
     [Google Scholar]
  2. Aiba S., Kitai K., Imanaka T. 1983; Cloning and expression of thermostable α-amylase gene from Bacillus stearothermophilus and Bacillus subtilis . Applied and Environmental Microbiology 46:1059–1065
     [Google Scholar]
  3. Aiba S., Zhang M., Koizuma J.-I. 1987a; Construction of a vector plasmid that can be maintained stably at higher temperatures in Bacillus stearothermophilus and its application. Annals of the New York Academy of Sciences 506:67–75
     [Google Scholar]
  4. Aiba S., Zhang M., Ohnishi M., Koizumi J.-I. 1987b; Production of α-amylase in transformant of Bacillus stearothermophilus — improvement of recombinant plasmid that can be used at higher temperatures. Biotechnology and Bioengineering 30:978–982
     [Google Scholar]
  5. Ambulos N.P. Jr Chow J.H., Mongkolsuk S., Preis L.H., Vollmar W.R. II Lovett P.S. 1984; Constitutive variants of the PC194 cat gene exhibit DNA alterations in the vicinity of the ribosome binding site sequence. Gene 28:171–176
     [Google Scholar]
  6. Ano T., Imanaka T., Aiba S. 1985; De novo synthesis of poly(dAT) by the cell extract of a thermophile Bacillus stearothermophilus carrying a plasmid pTB913. FEMS Microbiology Letters 26:131–134
     [Google Scholar]
  7. Bingham A.H.A., Burton C.J., Atkinson T. 1979; Isolation and partial characterization of four plasmids from antibiotic-resistant thermophilic bacilli. Journal of General Microbiology 114:401–408
     [Google Scholar]
  8. Bott K., Wilson G.A. 1967; Development of competence in the Bacillus subtilis transformation system. Journal of Bacteriology 94:562–570
     [Google Scholar]
  9. Chang S., Cohen S.N. 1979; High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Molecular and General Genetics 168:111–115
     [Google Scholar]
  10. Chen Z.-F., Wojcik S.F., Welker N.E. 1986; Genetic analysis of Bacillus stearothermophilus by protoplast fusion. Journal of Bacteriology 165:994–1001
     [Google Scholar]
  11. Fujii M., Imanaka T., Aiba S. 1982; Molecular cloning and expression of penicillinase genes from Bacillus licheniformis in the thermophilic Bacillus stearothermophilus . Journal of General Microbiology 128:2997–3000
     [Google Scholar]
  12. Fujii M., Takagi M., Imanaka T., Aiba S. 1983; Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expression in Bacillus stearothermophilus and Bacillus subtilis . Journal of Bacteriology 154:831–837
     [Google Scholar]
  13. Gryczan T.J., Contente S., Dubnau D. 1978; Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis . Journal of Bacteriology 134:318–329
     [Google Scholar]
  14. Hoshino T., Ikeda T., Furukawa K., Tomizuka N. 1985a; Genetic relationship between pUBllO and antibiotic-resistant plasmids obtained from thermophilic bacilli. Canadian Journal of Microbiology 31:614–619
     [Google Scholar]
  15. Hoshino T., Ikeda T., Narushima H., Tomizuka N. 1985b; Isolation and characterization of antibiotic-resistance plasmids in thermophilic bacilli. Canadian Journal of Microbiology 31:339–345
     [Google Scholar]
  16. Hoshino T., Ikeda T., Tomizuka N., Furukawa K. 1985c; Nucleotide sequence of the tetracycline resistance gene of pTHT15, a thermophilic Bacillusplasmid: comparison with staphylococcal Tcr controls. Gene 37:131–138
     [Google Scholar]
  17. Hoshino T., Sohma A., Ikeda T., Tomizuka N. 1985d; Temperature dependency of plasmid maintenance and gene expression in Bacillus subtilis and Bacillus stearothermophilus. Report of the Fermentation Research Institute, Japan no. 64: pp 29–41
     [Google Scholar]
  18. Imanaka T., Fujii M., Aiba S. 1981; Isolation and characterization of antibiotic resistance plasmids from thermophilic Bacilli and construction of deletion plasmids. Journal of Bacteriology 146:1091–1097
     [Google Scholar]
  19. Imanaka T., Fujii M., Aramori I., Aiba S. 1987; Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis . Journal of Bacteriology 149:824–830
     [Google Scholar]
  20. Kieser T., Hopwood D.A., Wright H.M., Thompson C.J. 1982; pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors. Molecular and General Genetics 185:223–238
     [Google Scholar]
  21. Koizumi J.-I., Min Z., Imanaka T., Aiba S. 1986; Temperature-dependent plasmid integration into and excision from the chromosome of Bacillus stearothermophilus . Journal of General Microbiology 132:1951–1958
     [Google Scholar]
  22. Koyama Y., Hoshino T., Tomizuka N., Furukawa K. 1986; Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp. Journal of Bacteriology 166:338–340
     [Google Scholar]
  23. Kubo M., Imanaka T. 1988; Cloning and nucleotide sequences of the highly thermostable neutral protease gene from Bacillus stearothermophilus . Journal of General Microbiology 134:1883–1892
     [Google Scholar]
  24. Liao H., McKenzie T., Hageman R. 1986; Isolation of a thermostable enzyme variant by cloning and selection in a thermophile. Proceedingsof the National Academy of Sciences of the United States of America 83:576–580
     [Google Scholar]
  25. McKenzie T., Hoshino T., Tanaka T., Sueoka N. 1986; The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. Plasmid 15:93–103
     [Google Scholar]
  26. Maniatis T., Fritsch E.F., Sambrook J. 1982 Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory;
     [Google Scholar]
  27. Matsumura M., Aiba S. 1985; Screening for thermostable mutant of kanamycin nucleotidyltransferase by the use of a transformation system for thermophile, Bacillus stearothermophilus . Journal of Biological Chemistry 260:15298–15303
     [Google Scholar]
  28. Matsumura M., Katakura Y., Imanaka T., Aiba S. 1984; Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUBl10. Journal of Bacteriology 160:413–420
     [Google Scholar]
  29. Min Z., Kozumi J.-I., Aiba S. 1987; Emergence of thermostable plasmid pTRZ117 and pTRA117 via temperature-dependent integration and excision is dependent on host property of Rec in Bacillus stearothermophilus . FEMS Microbiology Letters 44:77–80
     [Google Scholar]
  30. Pidcock K.A., Montenecourt B.S., Sands J.A. 1985; Genetic recombination and transformation in protoplasts of Thermomonospora fusca . Applied and Environmental Microbiology 50:693–695
     [Google Scholar]
  31. Rodriguez R.L., Tait R.C. 1983 Recombinant DNA Techniques: an Introduction. Reading, Mass.:: Addison-Wesley.;
     [Google Scholar]
  32. Sakaguchi R., Shishido K., Hoshino T., Furukawa K. 1986; The nucleotide sequence of the tetracycline resistance gene of plasmid NS1981 from Bacillus subtilis differs from pTHTl5 from a thermophilic Bacillus by two base pairs. Plasmid 16:72–73
     [Google Scholar]
  33. Scheer-Abramowitz J., Gryczan T.J., Dubnau D. 1981; Origin and mode of replication of plasmids pE194 and pUBl10. Plasmids 6:67–77
     [Google Scholar]
  34. Shaw W.V., Brodsky R.F. 1968; Characterization of chloramphenicol acetyltransferase from chloramphenicol-resistant Staphylococcus aureus . Journal of Bacteriology 95:28–36
     [Google Scholar]
  35. Soutschek-Bauer E., Staudenbauer W.L. 1987; Synthesis and secretion of a heat-stable carboxy- methylcellulase from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus . Molecular and General Genetics 208:537–541
     [Google Scholar]
  36. Soutschek-Bauer E., Harti L., Staudenbauer W.L. 1985; Transformation of Clostridium thermohydrosulfuricum DSM 568 with plasmid DNA. Biotechnology Letters 7:705–710
     [Google Scholar]
  37. Soutschek-Bauer E., Scholz W., Grill E., Staudenbauer W.L. 1987; Thermostability and superhelicity of plasmid DNA in Bacillus stearothermophilus . Molecular and General Genetics 20 9:575–579
     [Google Scholar]
  38. Sullivan M.A., Yasbin R.E., Young F.E. 1984; New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments. Gene 29:21–26
     [Google Scholar]
  39. Tanaka T., Sueoka N. 1983; Site-specific in vitrobinding of plasmid pUBllO to Bacillus subtilismembrane fraction. Journal of Bacteriology 154:1184–1194
     [Google Scholar]
  40. Welker N.E. 1988; Transduction in Bacillus stearothermophilus . Journal of Bacteriology 170:3761–3764
     [Google Scholar]
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Protoplast Transformation of Bacillus stearothermophilus NUB36 by Plasmid DNA
Microbiology 135, 1315 (1989); https://doi.org/10.1099/00221287-135-5-1315
/content/journal/micro/10.1099/00221287-135-5-1315
/content/journal/micro/10.1099/00221287-135-5-1315
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