Skip to main content
SpringerLink
Log in

Gene cloning, expression and functional characterization of a phosphopantetheinyl transferase from Vibrio anguillarum serotype O1

  • Original Paper
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins from fatty acid synthetases (FASs) in primary metabolism and polyketide synthetases (PKSs) and non-ribosomal polypeptide synthetases (NRPSs) in secondary metabolism. Bacteria typically harbor one PPTase specific for carrier proteins of primary metabolism (ACPS-type PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism (Sfp-type PPTases). Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, has been reported to be synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulent plasmid pJM1 from strain 775 of V. anguillarum serotype O1, and the PPTase, necessary for the activation of the anguibactin-NRPS, is therefore expected to lie on the pJM1 plasmid. In this work, a putative PPTase gene, angD, was first identified on pEIB1 plasmid (a pJM1-like plasmid) from a virulent strain MVM425 of V. anguillarum serotype O1. A recombinant clone carrying complete angD was able to complement an Escherichia coli entD mutant deficient in Sfp-type PPTase. angD was overexpressed in E. coli and the resultant protein, AngD, was purified. Simultaneously, two carrier proteins involved in anguibactin-NRPS, ArCP and PCP, were overproduced in E. coli and purified. The purified AngD, PCP and ArCP were used to establish an in vitro enzyme reaction, and the PPTase activity of AngD was proved through HPLC analysis to detect the conversion of inactive carrier proteins to active carrier proteins in the reaction mixture. Co-expression of AngD with PCP or ArCP showed that AngD functioned well as a PPTase in vivo in E. coli, modifying PCP and ArCP completely.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Borchert S, Stachelhaus T, Marahiel MA (1994) Induction of surfactin production in Bacillus subtilis by gsp, a gene located upstream of the gramicidin S operon in Bacillus brevis. J Bacteriol 176:2458–2462

    CAS  PubMed  Google Scholar 

  • Brautaset T, Sekurova ON, Sletta H, Ellingsen TE, Strom AR, Valla S, Zotchev SB (2000) Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway. Chem Biol 7:395–403

    Article  CAS  PubMed  Google Scholar 

  • Coderre PE, Earhart CF (1989) The entD gene of the Escherichia coli K12 enterobactin gene cluster. J Gen Microbiol 135:3043–3055

    CAS  PubMed  Google Scholar 

  • Crosa JH (1980) A plasmid associated with virulence in the marine fish pathogen Vibrio anguillarum specifies an iron-sequestering system. Nature 284:566–568

    CAS  PubMed  Google Scholar 

  • Crosa JH (1989) Genetics and molecular biology of siderophore mediated iron transport in bacteria. Microbiol Rev 53:517–530

    CAS  PubMed  Google Scholar 

  • Crosa JH, Walsh CT (2002) Genetics and assembly line enzymology of siderophore biosynthesis in bacteria. Microbiol Mol Biol Rev 66:223–249

    Article  CAS  PubMed  Google Scholar 

  • Crosa JH, Hodges LL, Schiewe MH (1980) Curing of a plasmid is correlated with an attenuation of virulence in the marine fish pathogen Vibrio anguillarum. Infect Immun 27:897–902

    CAS  PubMed  Google Scholar 

  • Elizabethe EW, Janice AS, Kelynne ER, Shelley MP (1997) Cloning of a Vibrio cholerae vibriobactin gene cluster: identification of genes required for early steps in siderophore biosynthesis. J Bacteriol 179:7055–7062

    PubMed  Google Scholar 

  • Fichtlscherer F, Wellein C, Mittag M, Schweizer E (2000) A novel function of yeast fatty acid synthase. Subunit alpha is capable of self-pantetheinylation. Eur J Biochem 267:2666–2671

    Article  CAS  PubMed  Google Scholar 

  • Finking R, Solsbacher J, Konz D, Schobert M, Schafer A, Jahn D, Marahiel MA (2002) Characterization of a new type of phosphopantetheinyl transferase for fatty acid and siderophore synthesis in Pseudomonas aeruginosa. J Biol Chem 277:50293–50302

    Article  CAS  PubMed  Google Scholar 

  • Ku J, Mirmira RG, Liu L, Santi DV (1997) Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase. Chem Biol 4:203–207

    Article  CAS  PubMed  Google Scholar 

  • Lambalot RH, Walsh CT (1997) Holo-[acyl-carrier-protein] synthase of Escherichia coli. Methods Enzymol 279:254–262

    CAS  PubMed  Google Scholar 

  • Lambalot RH, Gehring AM, Flugel RS, Zuber P, LaCelle M, Marahiel MA, Reid R, Khosla C, Walsh CT (1996) A new enzyme superfamily—the phosphopantetheinyl transferases. Chem Biol 3:923–936

    CAS  PubMed  Google Scholar 

  • Liu Q, Ma Y, Wu H, Shao M, Liu H, Zhang Y (2004) Cloning, identification and expression of an entE homologue angE from Vibrio anguillarum serotype O1. Arch Microbiol 181:287–293

    Article  CAS  PubMed  Google Scholar 

  • Lorenzo MD, Stork M, Tolmasky ME, Actis LA, Farrell D, Welch TJ, Crosa LM, Wertheimer AM, Chen Q, Salinas P, Waldbeser L, Crosa JH (2003) Complete sequence of virulence plasmid pJM1 from the marine fish pathogen Vibrio anguillarum strain 775. J Bacteriol 185:5822–5830

    Article  PubMed  Google Scholar 

  • Mootz HD, Finking R, Marahiel MA (2001) 4′-Phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis. J Biol Chem 276:37289–37298

    Article  CAS  PubMed  Google Scholar 

  • Nakano MM, Corbell N, Besson J, Zuber P (1992) Isolation and characterization of sfp: a gene that functions in the production of the lipopeptide biosurfactant, surfactin, in Bacillus subtilis. Mol Gen Genet 232:313–321

    CAS  PubMed  Google Scholar 

  • Pedersen K, Larsen JL (1995) Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes. Appl Environ Microbiol 61:2292–2296

    CAS  PubMed  Google Scholar 

  • Quadri LE, Weinreb P H, Lei M, Nakano MM, Zuber P, Walsh CT (1998) Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37:1585–1595

    Article  CAS  PubMed  Google Scholar 

  • Quadri LEN, Sello J, Keating TA, Weinreb PH, Walsh CT (1998) Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin. Chem Biol 5:631–645

    Article  CAS  PubMed  Google Scholar 

  • Sanchez C, Du L, Edwards DJ, Toney MD, Shen B (2001) Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide-polyketide antitumor drug bleomycin. Chem Biol 8:725–738

    Article  CAS  PubMed  Google Scholar 

  • Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47–56

    CAS  PubMed  Google Scholar 

  • Suo Z, Tseng CC, Walsh CT (2001) Purification, priming, and catalytic acylation of carrier protein domains in the polyketide synthase and nonribosomal peptidyl synthetase modules of the HMWP1 subunit of yersiniabactin synthetase. Proc Natl Acad Sci U S A 98:99–104

    Article  CAS  PubMed  Google Scholar 

  • Tolmasky ME, Actis LA, Crosa JH (1988) Genetic analysis of the iron uptake region of the Vibrio anguillarum plasmid pJM1: molecular cloning of genetic determinants encoding a novel trans activator of siderophore biosynthesis. J Bacteriol 160:860–866

    Google Scholar 

  • Walsh CT, Gehring AM, Weinreb PH, Quadri LEN, Flugel RS (1997) Post-translational modification of polyketide and nonribosomal peptide synthases. Curr Opin Chem Biol 1:309–315

    Article  CAS  PubMed  Google Scholar 

  • Weissman KJ, Hong H, Oliynyk M, Siskos AP, Leadlay PF (2004) Identification of a phosphopantetheinyl transferase for erythromycin biosynthesis in Saccharopolyspora erythraea. ChemBioChem 5:116 −125

    Article  CAS  PubMed  Google Scholar 

  • Welch TJ, Chai S, Crosa JH (2000) The overlapping angB and angG genes are encoded within the transacting factor region of the virulence plasmid in Vibrio anguillarum: essential role in siderophore biosynthesis. J Bacteriol 182:6762–6773

    Article  CAS  PubMed  Google Scholar 

  • Westrich L, Domann S, Faust B, Bedford D, Hopwood DA, Bechthold A (1999) Cloning and characterization of a gene cluster from Streptomyces cyanogenus S136 probably involved in landomycin biosynthesis. FEMS Microbiol Lett 170:381–387

    Article  CAS  PubMed  Google Scholar 

  • Wu H, Zhang H, Lu C, Liang N, Jin H, Ma Y, Zhang Y (2003) DNA sequencing of a plasmid with virulence from marine fish pathogen Vibrio anguillarum. Acta Biochim Biophys Sin 35:956–959

    CAS  PubMed  Google Scholar 

  • Wu H, Ma Y, Zhang Y, Zhang H (2004) Complete sequence of virulence plasmid pEIB1 from the marine fish pathogen Vibrio anguillarum strain MVM425 and location of its replication region. J Appl Microbiol 97(5):1021–1028

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We especially thank Dr. Charles Earhart (University of Texas) for the gift of E. coli mutant AN90. This work was supported by the grants from the National High-Tech Research Programs of China (No. 2003AA622020) and Shanghai Key Discipline of Biochemical Engineering.

Author information

Authors and Affiliations

  1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China

    Qin Liu, Yue Ma, Lingyun Zhou & Yuanxing Zhang

Authors
  1. Qin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yue Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lingyun Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuanxing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yuanxing Zhang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, Q., Ma, Y., Zhou, L. et al. Gene cloning, expression and functional characterization of a phosphopantetheinyl transferase from Vibrio anguillarum serotype O1. Arch Microbiol 183, 37–44 (2005). https://doi.org/10.1007/s00203-004-0745-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00203-004-0745-6

Keywords

Search

Navigation