• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. Feb 1997; 63(2): 370–379.
PMCID: PMC168329

Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon.


The plasmid vectors described in this report are derived from the broad-host-range RK2 replicon and can be maintained in many gram-negative bacterial species. The complete nucleotide sequences of all of the cloning and expression vectors are known. Important characteristics of the cloning vectors are as follows: a size range of 4.8 to 7.1 kb, unique cloning sites, different antibiotic resistance markers for selection of plasmid-containing cells, oriT-mediated conjugative plasmid transfer, plasmid stabilization functions, and a means for a simple method for modification of plasmid copy number. Expression vectors were constructed by insertion of the inducible Pu or Pm promoter together with its regulatory gene xylR or xylS, respectively, from the TOL plasmid of Pseudomonas putida. One of these vectors was used in an analysis of the correlation between phosphoglucomutase activity and amylose accumulation in Escherichia coli. The experiments showed that amylose synthesis was only marginally affected by the level of basal expression from the Pm promoter of the Acetobacter xylinum phosphoglucomutase gene (celB). In contrast, amylose accumulation was strongly reduced when transcription from Pm was induced. CelB was also expressed with a very high induction ratio in Xanthomonas campestris. These experiments showed that the A. xylinum celB gene could not complement the role of the bifunctional X. campestris phosphoglucomutase-phosphomannomutase gene in xanthan biosynthesis. We believe that the vectors described here are useful for cloning experiments, gene expression, and physiological studies with a wide range of bacteria and presumably also for analysis of gene transfer in the environment.

Full Text

The Full Text of this article is available as a PDF (460K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abril MA, Michan C, Timmis KN, Ramos JL. Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J Bacteriol. 1989 Dec;171(12):6782–6790. [PMC free article] [PubMed]
  • Abril MA, Buck M, Ramos JL. Activation of the Pseudomonas TOL plasmid upper pathway operon. Identification of binding sites for the positive regulator XylR and for integration host factor protein. J Biol Chem. 1991 Aug 25;266(24):15832–15838. [PubMed]
  • Adhya S, Schwartz M. Phosphoglucomutase mutants of Escherichia coli K-12. J Bacteriol. 1971 Nov;108(2):621–626. [PMC free article] [PubMed]
  • Assinder SJ, Williams PA. The TOL plasmids: determinants of the catabolism of toluene and the xylenes. Adv Microb Physiol. 1990;31:1–69. [PubMed]
  • Bishop PE, Brill WJ. Genetic analysis of Azotobacter vinelandii mutant strains unable to fix nitrogen. J Bacteriol. 1977 May;130(2):954–956. [PMC free article] [PubMed]
  • Brautaset T, Standal R, Fjaervik E, Valla S. Nucleotide sequence and expression analysis of the Acetobacter xylinum phosphoglucomutase gene. Microbiology. 1994 May;140(Pt 5):1183–1188. [PubMed]
  • Cases I, de Lorenzo V, Pérez-Martín J. Involvement of sigma 54 in exponential silencing of the Pseudomonas putida TOL plasmid Pu promoter. Mol Microbiol. 1996 Jan;19(1):7–17. [PubMed]
  • Cereghino JL, Helinski DR, Toukdarian AE. Isolation and characterization of DNA-binding mutants of a plasmid replication initiation protein utilizing an in vivo binding assay. Plasmid. 1994 Jan;31(1):89–99. [PubMed]
  • Chung CT, Niemela SL, Miller RH. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172–2175. [PMC free article] [PubMed]
  • de Lorenzo V, Herrero M, Metzke M, Timmis KN. An upstream XylR- and IHF-induced nucleoprotein complex regulates the sigma 54-dependent Pu promoter of TOL plasmid. EMBO J. 1991 May;10(5):1159–1167. [PMC free article] [PubMed]
  • Dixon R. The xylABC promoter from the Pseudomonas putida TOL plasmid is activated by nitrogen regulatory genes in Escherichia coli. Mol Gen Genet. 1986 Apr;203(1):129–136. [PubMed]
  • Duetz WA, Marqués S, Wind B, Ramos JL, van Andel JG. Catabolite repression of the toluene degradation pathway in Pseudomonas putida harboring pWW0 under various conditions of nutrient limitation in chemostat culture. Appl Environ Microbiol. 1996 Feb;62(2):601–606. [PMC free article] [PubMed]
  • Durland RH, Toukdarian A, Fang F, Helinski DR. Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers. J Bacteriol. 1990 Jul;172(7):3859–3867. [PMC free article] [PubMed]
  • Fellay R, Frey J, Krisch H. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene. 1987;52(2-3):147–154. [PubMed]
  • Fellay R, Krisch HM, Prentki P, Frey J. Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in gram-negative bacteria. Gene. 1989;76(2):215–226. [PubMed]
  • Figurski DH, Meyer RJ, Helinski DR. Suppression of Co1E1 replication properties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed in vitro. J Mol Biol. 1979 Sep 25;133(3):295–318. [PubMed]
  • Franklin FC, Bagdasarian M, Bagdasarian MM, Timmis KN. Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7458–7462. [PMC free article] [PubMed]
  • Frey J, Krisch HM. Omega mutagenesis in gram-negative bacteria: a selectable interposon which is strongly polar in a wide range of bacterial species. Gene. 1985;36(1-2):143–150. [PubMed]
  • Frey J, Mudd EA, Krisch HM. A bacteriophage T4 expression cassette that functions efficiently in a wide range of gram-negative bacteria. Gene. 1988;62(2):237–247. [PubMed]
  • Gallegos MT, Marqués S, Ramos JL. Expression of the TOL plasmid xylS gene in Pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and alpha 54-dependent tandem promoters according to the compound used for growth. J Bacteriol. 1996 Apr;178(8):2356–2361. [PMC free article] [PubMed]
  • Goncharoff P, Yip JK, Wang H, Schreiner HC, Pai JA, Furgang D, Stevens RH, Figurski DH, Fine DH. Conjugal transfer of broad-host-range incompatibility group P and Q plasmids from Escherichia coli to Actinobacillus actinomycetemcomitans. Infect Immun. 1993 Aug;61(8):3544–3547. [PMC free article] [PubMed]
  • Grasseschi HA, Minnick MF. Transformation of Bartonella bacilliformis by electroporation. Can J Microbiol. 1994 Sep;40(9):782–786. [PubMed]
  • Hanahan D, Jessee J, Bloom FR. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 1991;204:63–113. [PubMed]
  • Haugan K, Karunakaran P, Blatny JM, Valla S. The phenotypes of temperature-sensitive mini-RK2 replicons carrying mutations in the replication control gene trfA are suppressed nonspecifically by intragenic cop mutations. J Bacteriol. 1992 Nov;174(21):7026–7032. [PMC free article] [PubMed]
  • Haugan K, Karunakaran P, Tøndervik A, Valla S. The host range of RK2 minimal replicon copy-up mutants is limited by species-specific differences in the maximum tolerable copy number. Plasmid. 1995 Jan;33(1):27–39. [PubMed]
  • Holtel A, Marqués S, Möhler I, Jakubzik U, Timmis KN. Carbon source-dependent inhibition of xyl operon expression of the Pseudomonas putida TOL plasmid. J Bacteriol. 1994 Mar;176(6):1773–1776. [PMC free article] [PubMed]
  • Hötte B, Rath-Arnold I, Pühler A, Simon R. Cloning and analysis of a 35.3-kilobase DNA region involved in exopolysaccharide production by Xanthomonas campestris pv. campestris. J Bacteriol. 1990 May;172(5):2804–2807. [PMC free article] [PubMed]
  • Hugouvieux-Cotte-Pattat N, Köhler T, Rekik M, Harayama S. Growth-phase-dependent expression of the Pseudomonas putida TOL plasmid pWW0 catabolic genes. J Bacteriol. 1990 Dec;172(12):6651–6660. [PMC free article] [PubMed]
  • Ingram LC, Richmond MH, Sykes RB. Molecular characterization of the R factors implicated in the carbenicillin resistance of a sequence of Pseudomonas aeruginosa strains isolated from burns. Antimicrob Agents Chemother. 1973 Feb;3(2):279–288. [PMC free article] [PubMed]
  • Inouye S, Nakazawa A, Nakazawa T. Molecular cloning of regulatory gene xylR and operator-promoter regions of the xylABC and xylDEGF operons of the TOL plasmid. J Bacteriol. 1983 Sep;155(3):1192–1199. [PMC free article] [PubMed]
  • Keen NT, Tamaki S, Kobayashi D, Trollinger D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene. 1988 Oct 15;70(1):191–197. [PubMed]
  • Keil S, Keil H. Construction of a cassette enabling regulated gene expression in the presence of aromatic hydrocarbons. Plasmid. 1992 May;27(3):191–199. [PubMed]
  • Kessler B, de Lorenzo V, Timmis KN. Identification of a cis-acting sequence within the Pm promoter of the TOL plasmid which confers XylS-mediated responsiveness to substituted benzoates. J Mol Biol. 1993 Apr 5;230(3):699–703. [PubMed]
  • Kessler B, Timmis KN, de Lorenzo V. The organization of the Pm promoter of the TOL plasmid reflects the structure of its cognate activator protein XylS. Mol Gen Genet. 1994 Sep 28;244(6):596–605. [PubMed]
  • Köhler T, Harayama S, Ramos JL, Timmis KN. Involvement of Pseudomonas putida RpoN sigma factor in regulation of various metabolic functions. J Bacteriol. 1989 Aug;171(8):4326–4333. [PMC free article] [PubMed]
  • Köplin R, Arnold W, Hötte B, Simon R, Wang G, Pühler A. Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis. J Bacteriol. 1992 Jan;174(1):191–199. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Li X, Weinstock GM, Murray BE. Generation of auxotrophic mutants of Enterococcus faecalis. J Bacteriol. 1995 Dec;177(23):6866–6873. [PMC free article] [PubMed]
  • Marqués S, Gallegos MT, Ramos JL. Role of sigma S in transcription from the positively controlled Pm promoter of the TOL plasmid of Pseudomonas putida. Mol Microbiol. 1995 Dec;18(5):851–857. [PubMed]
  • Marqués S, Ramos JL. Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways. Mol Microbiol. 1993 Sep;9(5):923–929. [PubMed]
  • Mather MW, McReynolds LM, Yu CA. An enhanced broad-host-range vector for gram-negative bacteria: avoiding tetracycline phototoxicity during the growth of photosynthetic bacteria. Gene. 1995 Apr 14;156(1):85–88. [PubMed]
  • Mermod N, Ramos JL, Lehrbach PR, Timmis KN. Vector for regulated expression of cloned genes in a wide range of gram-negative bacteria. J Bacteriol. 1986 Aug;167(2):447–454. [PMC free article] [PubMed]
  • Metzler MC, Zhang YP, Chen TA. Transformation of the gram-positive bacterium Clavibacter xyli subsp. cynodontis by electroporation with plasmids from the IncP incompatibility group. J Bacteriol. 1992 Jul;174(13):4500–4503. [PMC free article] [PubMed]
  • Michan C, Zhou L, Gallegos MT, Timmis KN, Ramos JL. Identification of critical amino-terminal regions of XylS. The positive regulator encoded by the TOL plasmid. J Biol Chem. 1992 Nov 15;267(32):22897–22901. [PubMed]
  • Morris CJ, Kim YM, Perkins KE, Lidstrom ME. Identification and nucleotide sequences of mxaA, mxaC, mxaK, mxaL, and mxaD genes from Methylobacterium extorquens AM1. J Bacteriol. 1995 Dec;177(23):6825–6831. [PMC free article] [PubMed]
  • Norrander J, Kempe T, Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. [PubMed]
  • Osterås M, Driscoll BT, Finan TM. Molecular and expression analysis of the Rhizobium meliloti phosphoenolpyruvate carboxykinase (pckA) gene. J Bacteriol. 1995 Mar;177(6):1452–1460. [PMC free article] [PubMed]
  • Pansegrau W, Lanka E, Barth PT, Figurski DH, Guiney DG, Haas D, Helinski DR, Schwab H, Stanisich VA, Thomas CM. Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis. J Mol Biol. 1994 Jun 24;239(5):623–663. [PubMed]
  • Perri S, Helinski DR, Toukdarian A. Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2. J Biol Chem. 1991 Jul 5;266(19):12536–12543. [PubMed]
  • Perri S, Helinski DR. DNA sequence requirements for interaction of the RK2 replication initiation protein with plasmid origin repeats. J Biol Chem. 1993 Feb 15;268(5):3662–3669. [PubMed]
  • Pinkney M, Diaz R, Lanka E, Thomas CM. Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III. J Mol Biol. 1988 Oct 20;203(4):927–938. [PubMed]
  • Ramos JL, Mermod N, Timmis KN. Regulatory circuits controlling transcription of TOL plasmid operon encoding meta-cleavage pathway for degradation of alkylbenzoates by Pseudomonas. Mol Microbiol. 1987 Nov;1(3):293–300. [PubMed]
  • Ramos JL, Gonzalez-Carrero M, Timmis KN. Broad-host range expression vectors containing manipulated meta-cleavage pathway regulatory elements of the TOL plasmid. FEBS Lett. 1988 Jan 4;226(2):241–246. [PubMed]
  • Ramos JL, Michan C, Rojo F, Dwyer D, Timmis K. Signal-regulator interactions. Genetic analysis of the effector binding site of xylS, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon. J Mol Biol. 1990 Jan 20;211(2):373–382. [PubMed]
  • Roberts RC, Burioni R, Helinski DR. Genetic characterization of the stabilizing functions of a region of broad-host-range plasmid RK2. J Bacteriol. 1990 Nov;172(11):6204–6216. [PMC free article] [PubMed]
  • Roberts RC, Helinski DR. Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2. J Bacteriol. 1992 Dec;174(24):8119–8132. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Schmidhauser TJ, Helinski DR. Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bacteriol. 1985 Oct;164(1):446–455. [PMC free article] [PubMed]
  • Shingler V, Thomas CM. Analysis of the trfA region of broad host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products. J Mol Biol. 1984 May 25;175(3):229–249. [PubMed]
  • Sia EA, Roberts RC, Easter C, Helinski DR, Figurski DH. Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2. J Bacteriol. 1995 May;177(10):2789–2797. [PMC free article] [PubMed]
  • Valla S, Haugan K, Durland R, Helinski DR. Isolation and properties of temperature-sensitive mutants of the trfA gene of the broad host range plasmid RK2. Plasmid. 1991 Mar;25(2):131–136. [PubMed]
  • 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. [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...