• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Jul 1995; 177(14): 4121–4130.
PMCID: PMC177145

Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter.


We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.

Full Text

The Full Text of this article is available as a PDF (554K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Belder JB, Nguyen-Distèche M, Houba-Herin N, Ghuysen JM, Maruyama IN, Hara H, Hirota Y, Inouye M. Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12. Mol Microbiol. 1988 Jul;2(4):519–525. [PubMed]
  • Beverin S, Sheppard DE, Park SS. D-Fucose as a gratuitous inducer of the L-arabinose operon in strains of Escherichia coli B-r mutant in gene araC. J Bacteriol. 1971 Jul;107(1):79–86. [PMC free article] [PubMed]
  • Boyd D, Manoil C, Beckwith J. Determinants of membrane protein topology. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8525–8529. [PMC free article] [PubMed]
  • Brosius J, Erfle M, Storella J. Spacing of the -10 and -35 regions in the tac promoter. Effect on its in vivo activity. J Biol Chem. 1985 Mar 25;260(6):3539–3541. [PubMed]
  • Cagnon C, Valverde V, Masson JM. A new family of sugar-inducible expression vectors for Escherichia coli. Protein Eng. 1991 Oct;4(7):843–847. [PubMed]
  • Carra JH, Schleif RF. Variation of half-site organization and DNA looping by AraC protein. EMBO J. 1993 Jan;12(1):35–44. [PMC free article] [PubMed]
  • Carson MJ, Barondess J, Beckwith J. The FtsQ protein of Escherichia coli: membrane topology, abundance, and cell division phenotypes due to overproduction and insertion mutations. J Bacteriol. 1991 Apr;173(7):2187–2195. [PMC free article] [PubMed]
  • Cass LG, Horwitz AH, Wilcox G. The araIc mutation in Escherichia coli B/r. J Bacteriol. 1981 Jun;146(3):1098–1105. [PMC free article] [PubMed]
  • Chang AC, Cohen SN. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. [PMC free article] [PubMed]
  • Conley MP, Wolfe AJ, Blair DF, Berg HC. Both CheA and CheW are required for reconstitution of chemotactic signaling in Escherichia coli. J Bacteriol. 1989 Sep;171(9):5190–5193. [PMC free article] [PubMed]
  • Cull MG, Miller JF, Schatz PJ. Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1865–1869. [PMC free article] [PubMed]
  • Dalbey RE, Wickner W. Leader peptidase catalyzes the release of exported proteins from the outer surface of the Escherichia coli plasma membrane. J Biol Chem. 1985 Dec 15;260(29):15925–15931. [PubMed]
  • de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. [PMC free article] [PubMed]
  • de Boer PA, Crossley RE, Rothfield LI. Isolation and properties of minB, a complex genetic locus involved in correct placement of the division site in Escherichia coli. J Bacteriol. 1988 May;170(5):2106–2112. [PMC free article] [PubMed]
  • Diederich L, Roth A, Messer W. A versatile plasmid vector system for the regulated expression of genes in Escherichia coli. Biotechniques. 1994 May;16(5):916–923. [PubMed]
  • Doherty AJ, Connolly BA, Worrall AF. Overproduction of the toxic protein, bovine pancreatic DNaseI, in Escherichia coli using a tightly controlled T7-promoter-based vector. Gene. 1993 Dec 22;136(1-2):337–340. [PubMed]
  • Dong H, Nilsson L, Kurland CG. Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction. J Bacteriol. 1995 Mar;177(6):1497–1504. [PMC free article] [PubMed]
  • Elvin CM, Thompson PR, Argall ME, Hendry P, Stamford NP, Lilley PE, Dixon NE. Modified bacteriophage lambda promoter vectors for overproduction of proteins in Escherichia coli. Gene. 1990 Mar 1;87(1):123–126. [PubMed]
  • ENGLESBERG E, ANDERSON RL, WEINBERG R, LEE N, HOFFEE P, HUTTENHAUER G, BOYER H. L-Arabinose-sensitive, L-ribulose 5-phosphate 4-epimerase-deficient mutants of Escherichia coli. J Bacteriol. 1962 Jul;84:137–146. [PMC free article] [PubMed]
  • Gil D, Bouché JP. ColE1-type vectors with fully repressible replication. Gene. 1991 Aug 30;105(1):17–22. [PubMed]
  • Giza PE, Huang RC. A self-inducing runaway-replication plasmid expression system utilizing the Rop protein. Gene. 1989 May 15;78(1):73–84. [PubMed]
  • Guzman LM, Barondess JJ, Beckwith J. FtsL, an essential cytoplasmic membrane protein involved in cell division in Escherichia coli. J Bacteriol. 1992 Dec;174(23):7716–7728. [PMC free article] [PubMed]
  • Hoffmann I, Widström J, Zeppezauer M, Nyman PO. Overproduction and large-scale preparation of deoxyuridine triphosphate nucleotidohydrolase from Escherichia coli. Eur J Biochem. 1987 Apr 1;164(1):45–51. [PubMed]
  • Johnston S, Lee JH, Ray DS. High-level expression of M13 gene II protein from an inducible polycistronic messenger RNA. Gene. 1985;34(2-3):137–145. [PubMed]
  • Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. [PMC free article] [PubMed]
  • Kuhn A, Wickner W. Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage. J Biol Chem. 1985 Dec 15;260(29):15907–15913. [PubMed]
  • Larsen JE, Gerdes K, Light J, Molin S. Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter. Gene. 1984 Apr;28(1):45–54. [PubMed]
  • Lee N, Francklyn C, Hamilton EP. Arabinose-induced binding of AraC protein to araI2 activates the araBAD operon promoter. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8814–8818. [PMC free article] [PubMed]
  • Lobell RB, Schleif RF. DNA looping and unlooping by AraC protein. Science. 1990 Oct 26;250(4980):528–532. [PubMed]
  • Lopilato J, Bortner S, Beckwith J. Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives. Mol Gen Genet. 1986 Nov;205(2):285–290. [PubMed]
  • Miyada CG, Stoltzfus L, Wilcox G. Regulation of the araC gene of Escherichia coli: catabolite repression, autoregulation, and effect on araBAD expression. Proc Natl Acad Sci U S A. 1984 Jul;81(13):4120–4124. [PMC free article] [PubMed]
  • Mukherjee A, Donachie WD. Differential translation of cell division proteins. J Bacteriol. 1990 Oct;172(10):6106–6111. [PMC free article] [PubMed]
  • Murphy CK, Beckwith J. Residues essential for the function of SecE, a membrane component of the Escherichia coli secretion apparatus, are located in a conserved cytoplasmic region. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2557–2561. [PMC free article] [PubMed]
  • O'Connor CD, Timmis KN. Highly repressible expression system for cloning genes that specify potentially toxic proteins. J Bacteriol. 1987 Oct;169(10):4457–4462. [PMC free article] [PubMed]
  • Phillips GJ, Silhavy TJ. The E. coli ffh gene is necessary for viability and efficient protein export. Nature. 1992 Oct 22;359(6397):744–746. [PubMed]
  • Pogliano JA, Beckwith J. SecD and SecF facilitate protein export in Escherichia coli. EMBO J. 1994 Feb 1;13(3):554–561. [PMC free article] [PubMed]
  • Russell CB, Stewart RC, Dahlquist FW. Control of transducer methylation levels in Escherichia coli: investigation of components essential for modulation of methylation and demethylation reactions. J Bacteriol. 1989 Jul;171(7):3609–3618. [PMC free article] [PubMed]
  • San Millan JL, Boyd D, Dalbey R, Wickner W, Beckwith J. Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase. J Bacteriol. 1989 Oct;171(10):5536–5541. [PMC free article] [PubMed]
  • Schleif R. DNA looping. Annu Rev Biochem. 1992;61:199–223. [PubMed]
  • Shine J, Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. [PMC free article] [PubMed]
  • Stormo GD, Schneider TD, Gold LM. Characterization of translational initiation sites in E. coli. Nucleic Acids Res. 1982 May 11;10(9):2971–2996. [PMC free article] [PubMed]
  • Strauch KL, Beckwith J. An Escherichia coli mutation preventing degradation of abnormal periplasmic proteins. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1576–1580. [PMC free article] [PubMed]
  • Tabor S, Richardson CC. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. [PMC free article] [PubMed]
  • Wagatsuma M, Asami N, Miyachi J, Uchida S, Watanabe H, Amann E. Antibody recognition of the recombinant human nuclear antigens RNP 70 kD, SS-A, SS-B, Sm-B, and Sm-D by autoimmune sera. Mol Immunol. 1993 Nov;30(16):1491–1498. [PubMed]
  • Wilcox G, Clemetson KJ, Cleary P, Englesberg E. Interaction of the regulatory gene product with the operator site in the L-arabinose operon of Escherichia coli. J Mol Biol. 1974 Jan 5;85(4):589–602. [PubMed]
  • 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. [PubMed]
  • Yansura DG, Henner DJ. Use of Escherichia coli trp promoter for direct expression of proteins. Methods Enzymol. 1990;185:54–60. [PubMed]
  • Zagursky RJ, Berman ML. Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing. Gene. 1984 Feb;27(2):183–191. [PubMed]

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