Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1986 Nov;168(2):973–981. doi: 10.1128/jb.168.2.973-981.1986

Integrable alpha-amylase plasmid for generating random transcriptional fusions in Bacillus subtilis.

C O'Kane, M A Stephens, D McConnell
PMCID: PMC213579  PMID: 3096966

Abstract

An integrable plasmid, pOK4, which replicated independently in Escherichia coli was constructed for generating transcriptional fusions in vivo in Bacillus DNA. It did not replicate independently in Bacillus subtilis, but it could be made to integrate into the chromosome of B. subtilis if sequences homologous to chromosomal sequences were inserted into it. It had a selectable marker for chloramphenicol resistance and carried unique sites for EcoRI and SmaI just to the 5' side of a promoterless alpha-amylase gene from Bacillus licheniformis. When B. subtilis DNA fragments were ligated into one of these sites and the ligation mixture was used to transform an alpha-amylase-negative B. subtilis strain, chloramphenicol-resistant transformants could be isolated conveniently. Many of these were alpha-amylase positive, owing to the fusion of the plasmid amylase gene to chromosomal operons. In principle, because integration need not be mutagenic, it is possible to obtain fusions to any chromosomal operon. The site of each integration can be mapped, and the flanking sequences can be cloned into E. coli. The alpha-amylase gene can be used to detect regulated genes. We used it as an indicator to detect operons which are DNA-damage-inducible (din), and we identified insertions in both SP beta and PBSX prophages.

Full text

PDF
973

Images in this article

976Image
on p.976
976Image
on p.976
977Image
on p.977

Selected References

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

  1. Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bellofatto V., Shapiro L., Hodgson D. A. Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1035–1039. doi: 10.1073/pnas.81.4.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bouvier J., Stragier P., Bonamy C., Szulmajster J. Nucleotide sequence of the spo0B gene of Bacillus subtilis and regulation of its expression. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7012–7016. doi: 10.1073/pnas.81.22.7012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Casadaban M. J., Cohen S. N. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530–4533. doi: 10.1073/pnas.76.9.4530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coleman J., Green P. J., Inouye M. The use of RNAs complementary to specific mRNAs to regulate the expression of individual bacterial genes. Cell. 1984 Jun;37(2):429–436. doi: 10.1016/0092-8674(84)90373-8. [DOI] [PubMed] [Google Scholar]
  8. Contente S., Dubnau D. Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation. Mol Gen Genet. 1979 Jan 2;167(3):251–258. doi: 10.1007/BF00267416. [DOI] [PubMed] [Google Scholar]
  9. Dedonder R. A., Lepesant J. A., Lepesant-Kejzlarová J., Billault A., Steinmetz M., Kunst F. Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Appl Environ Microbiol. 1977 Apr;33(4):989–993. doi: 10.1128/aem.33.4.989-993.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Duncan C. H., Wilson G. A., Young F. E. Mechanism of integrating foreign DNA during transformation of Bacillus subtilis. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3664–3668. doi: 10.1073/pnas.75.8.3664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Engebrecht J., Simon M., Silverman M. Measuring gene expression with light. Science. 1985 Mar 15;227(4692):1345–1347. doi: 10.1126/science.2983423. [DOI] [PubMed] [Google Scholar]
  12. Ferrari F. A., Nguyen A., Lang D., Hoch J. A. Construction and properties of an integrable plasmid for Bacillus subtilis. J Bacteriol. 1983 Jun;154(3):1513–1515. doi: 10.1128/jb.154.3.1513-1515.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Freifelder D., Levine E. Stimulation of nuclease activity by thymine starvation. Biochem Biophys Res Commun. 1972 Mar 10;46(5):1782–1787. doi: 10.1016/0006-291x(72)90051-4. [DOI] [PubMed] [Google Scholar]
  14. Gryczan T. J., Dubnau D. Direct selection of recombinant plasmids in Bacillus subtilis. Gene. 1982 Dec;20(3):459–469. doi: 10.1016/0378-1119(82)90215-3. [DOI] [PubMed] [Google Scholar]
  15. Gryczan T., Contente S., Dubnau D. Molecular cloning of heterologous chromosomal DNA by recombination between a plasmid vector and a homologous resident plasmid in Bacillus subtilis. Mol Gen Genet. 1980 Feb;177(3):459–467. doi: 10.1007/BF00271485. [DOI] [PubMed] [Google Scholar]
  16. Haas M., Yoshikawa H. Defective bacteriophage PBSH in Bacillus subtilis. I. Induction, purification, and physical properties of the bacteriophage and its deoxyribonucleic acid. J Virol. 1969 Feb;3(2):233–247. doi: 10.1128/jvi.3.2.233-247.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haldenwang W. G., Banner C. D., Ollington J. F., Losick R., Hoch J. A., O'Connor M. B., Sonenshein A. L. Mapping a cloned gene under sporulation control by inserttion of a drug resistance marker into the Bacillus subtilis chromosome. J Bacteriol. 1980 Apr;142(1):90–98. doi: 10.1128/jb.142.1.90-98.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Henner D. J., Band L., Shimotsu H. Nucleotide sequence of the Bacillus subtilis tryptophan operon. Gene. 1985;34(2-3):169–177. doi: 10.1016/0378-1119(85)90125-8. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Hubacek J., Glover S. W. Complementation analysis of temperature-sensitive host specificity mutations in Escherichia coli. J Mol Biol. 1970 May 28;50(1):111–127. doi: 10.1016/0022-2836(70)90108-7. [DOI] [PubMed] [Google Scholar]
  21. KORN D., WEISSBACH A. Thymineless induction in Escherichia coli K12 (lambda). Biochim Biophys Acta. 1962 Nov 26;61:775–790. doi: 10.1016/0926-6550(62)90060-9. [DOI] [PubMed] [Google Scholar]
  22. Kenyon C. J., Walker G. C. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci U S A. 1980 May;77(5):2819–2823. doi: 10.1073/pnas.77.5.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee J. H., Heffernan L., Wilcox G. Isolation of ara-lac gene fusions in Salmonella typhimurium LT2 by using transducing bacteriophage Mu d (Apr lac). J Bacteriol. 1980 Sep;143(3):1325–1331. doi: 10.1128/jb.143.3.1325-1331.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Love P. E., Lyle M. J., Yasbin R. E. DNA-damage-inducible (din) loci are transcriptionally activated in competent Bacillus subtilis. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6201–6205. doi: 10.1073/pnas.82.18.6201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Love P. E., Yasbin R. E. Genetic characterization of the inducible SOS-like system of Bacillus subtilis. J Bacteriol. 1984 Dec;160(3):910–920. doi: 10.1128/jb.160.3.910-920.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Michel B., Niaudet B., Ehrlich S. D. Intermolecular recombination during transformation of Bacillus subtilis competent cells by monomeric and dimeric plasmids. Plasmid. 1983 Jul;10(1):1–10. doi: 10.1016/0147-619x(83)90052-5. [DOI] [PubMed] [Google Scholar]
  27. Michel B., Palla E., Niaudet B., Ehrlich S. D. DNA cloning in Bacillus subtilis. III. Efficiency of random-segment cloning and insertional inactivation vectors. Gene. 1980 Dec;12(1-2):147–154. doi: 10.1016/0378-1119(80)90025-6. [DOI] [PubMed] [Google Scholar]
  28. Mizuno T., Chou M. Y., Inouye M. A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci U S A. 1984 Apr;81(7):1966–1970. doi: 10.1073/pnas.81.7.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Niaudet B., Goze A., Ehrlich S. D. Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloning. Gene. 1982 Oct;19(3):277–284. doi: 10.1016/0378-1119(82)90017-8. [DOI] [PubMed] [Google Scholar]
  30. Okamoto K., Mudd J. A., Mangan J., Huang W. M., Subbaiah T. V., Marmur J. Properties of the defective phage of Bacillus subtilis. J Mol Biol. 1968 Jun 28;34(3):413–428. doi: 10.1016/0022-2836(68)90169-1. [DOI] [PubMed] [Google Scholar]
  31. Ortlepp S. A., Ollington J. F., McConnell D. J. Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase. Gene. 1983 Sep;23(3):267–276. doi: 10.1016/0378-1119(83)90017-3. [DOI] [PubMed] [Google Scholar]
  32. Piggot P. J., Curtis C. A., de Lencastre H. Use of integrational plasmid vectors to demonstrate the polycistronic nature of a transcriptional unit (spoIIA) required for sporulation of Bacillus subtilis. J Gen Microbiol. 1984 Aug;130(8):2123–2136. doi: 10.1099/00221287-130-8-2123. [DOI] [PubMed] [Google Scholar]
  33. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simons R. W., Kleckner N. Translational control of IS10 transposition. Cell. 1983 Sep;34(2):683–691. doi: 10.1016/0092-8674(83)90401-4. [DOI] [PubMed] [Google Scholar]
  35. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  36. Stachel S. E., An G., Flores C., Nester E. W. A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J. 1985 Apr;4(4):891–898. doi: 10.1002/j.1460-2075.1985.tb03715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stephens M. A., Ortlepp S. A., Ollington J. F., McConnell D. J. Nucleotide sequence of the 5' region of the Bacillus licheniformis alpha-amylase gene: comparison with the B. amyloliquefaciens gene. J Bacteriol. 1984 Apr;158(1):369–372. doi: 10.1128/jb.158.1.369-372.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. TAKAHASHI I. Transducing phages for Bacillus subtilis. J Gen Microbiol. 1963 May;31:211–217. doi: 10.1099/00221287-31-2-211. [DOI] [PubMed] [Google Scholar]
  39. Thurm P., Garro A. J. Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX. J Virol. 1975 Jul;16(1):184–191. doi: 10.1128/jvi.16.1.184-191.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tomich P. K., An F. Y., Clewell D. B. Properties of erythromycin-inducible transposon Tn917 in Streptococcus faecalis. J Bacteriol. 1980 Mar;141(3):1366–1374. doi: 10.1128/jb.141.3.1366-1374.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tomizawa J. Control of ColE1 plasmid replication: the process of binding of RNA I to the primer transcript. Cell. 1984 Oct;38(3):861–870. doi: 10.1016/0092-8674(84)90281-2. [DOI] [PubMed] [Google Scholar]
  42. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  43. West R. W., Jr, McConnell D., Rodriguez R. L. Isolation of E.coli promoters from the late region of bacteriophage T7 DNA. Mol Gen Genet. 1980;180(2):439–447. doi: 10.1007/BF00425860. [DOI] [PubMed] [Google Scholar]
  44. Yasbin R. E. DNA repair in Bacillus subtilis. I. The presence of an inducible system. Mol Gen Genet. 1977 Jun 8;153(2):211–218. [PubMed] [Google Scholar]
  45. Yasbin R. E., Fields P. I., Andersen B. J. Properties of Bacillus subtilis 168 derivatives freed of their natural prophages. Gene. 1980 Dec;12(1-2):155–159. doi: 10.1016/0378-1119(80)90026-8. [DOI] [PubMed] [Google Scholar]
  46. Youngman P. J., Perkins J. B., Losick R. Genetic transposition and insertional mutagenesis in Bacillus subtilis with Streptococcus faecalis transposon Tn917. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2305–2309. doi: 10.1073/pnas.80.8.2305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Youngman P., Zuber P., Perkins J. B., Sandman K., Igo M., Losick R. New ways to study developmental genes in spore-forming bacteria. Science. 1985 Apr 19;228(4697):285–291. doi: 10.1126/science.228.4697.285. [DOI] [PubMed] [Google Scholar]
  48. Zahler S. A., Korman R. Z., Rosenthal R., Hemphill H. E. Bacillus subtilis bacteriophage SPbeta: localization of the prophage attachment site, and specialized transduction. J Bacteriol. 1977 Jan;129(1):556–558. doi: 10.1128/jb.129.1.556-558.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zuber P., Losick R. Use of a lacZ fusion to study the role of the spoO genes of Bacillus subtilis in developmental regulation. Cell. 1983 Nov;35(1):275–283. doi: 10.1016/0092-8674(83)90230-1. [DOI] [PubMed] [Google Scholar]

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

RESOURCES