Abstract
The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli β-galactosidase, as well as a 1,4-β-glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. Theα-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibitedα-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation
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References
Ambulos NP Jr, Mongkolsuk S, Kaufmann JD, Lovett PS (1985) Chloramphenicol-induced translation ofcat-86 mRNA requires two cis-acting regulatory regions. J Bacteriol 164:696–703
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Chang S, Cohen SN (1979) High frequency transformation ofBacillus subtilis protoplasts by plasmid DNA. Mol Gen Genet 168:111–115
Chen W, Tabor S, Struhl K (1987) Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase. Cell 50:1047–1055
Conrad B, Bashkirov VI, Hofemeister J (1992) Imprecise excision of plasmid pE194 from the chromosomes ofBacillus subtilis pE194 insertion strains. J Bacteriol 174:6997–7002
Conrad B, Hoang V, Polley A, Hofemeister J (1995) HybridBacillus amyloliquefaciens × Bacillus licheniformis α-amylases: construction, properties and sequence determinants. Eur J Biochem 230:481–490
Davidson J, Chevalier N, Brunel F (1989) Bacteriophage T7 RNA polymerase-controlled, specific gene expression inPseudomonas. Gene 83:371–375
Fuerst TR, Niles EG, Studier FW, Moss B (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci USA 83:8122–8126
Gryczan TJ, Contente S, Dubnau D (1978) Characterization ofStaphylococcus aureus plasmids introduced by transformation intoBacillus subtilis. J Bacteriol 134:318–329
Hanahan D (1983) Studies on transformation ofEscherichia coli with plasmids. J Mol Biol 166:557–580
Henner D (1990) Inducible expression of regulatory genes inBacillus subtilis. Methods Enzymol 185:223–228
Hofemeister B, König S, Hoang V, Engel J, Mayer G, Hansen G, Hofemeister J (1994) The geneamyE(TV1) coding for a nonglucogenicα-amylase fromThermoactinomyces vulgaris 94-2A inBacillus subtilis. Appl Envir Microbiol 60:3381–3389
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lieber A, Kiessling U, Strauss M (1989) High-level expression in mammalian cells by a nuclear T7 phage RNA polymerase. Nucleic Acids Res 17:8485–8493
Rosenberg HA, Lade BN, Chui D, Lin S, Dunn JJ, Studier FW (1987) Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56:125–135
Rygus T, Hillen W (1991) Inducible high-level expression of heterologous genes inBacillus megaterium using the regulatory elements of the xylose-utilization operon. Appl Microbiol Biotechnol 35:594–599
Rygus T, Scheler A, Allmansberger R, Hillen W (1991) Molecular cloning, structure, promoters and regulatory elements for transcription of theBacillus megaterium-encoded regulon for xylose utilization. Arch Microbiol 155:535–542
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Steinmetz M, Kunst F, Dedender R (1976) Mapping of mutations affecting synthesis of extracellular enzymes inBacillus: identity ofsacU, amyB andpap mutations. Mol Gen Genet 148:281–285
Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
Tabor S, Richardson CC (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled, exclusive expression of specific genes. Proc Natl Acad Sci USA 82:1074–1078
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Wells JM, Wilson PW, Norton PM, Le Page WF (1993) A model system for the investigation of heterologous protein secretion pathways inLactococcus lactis. Appl Envir Microbiol 59:3954–3959
Youngman P (1987) Plasmid vectors for recovery and exploitingTn917 transpositions inBacillus and other Gram-positive bacteria. In: Hardy KG (ed) Plasmids — a practical approach. IRL Press, Oxford-Washington DC, pp 79–103
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Communicated by J. W. Lengeler
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Conrad, B., Savchenko, R.S., Breves, R. et al. A T7 promoter-specific, inducible protein expression system forBacillus subtilis . Molec. Gen. Genet. 250, 230–236 (1996). https://doi.org/10.1007/BF02174183
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DOI: https://doi.org/10.1007/BF02174183